CA3176245A1 - Use of bone morphogenetic proteins and their receptors for aesthetics and cosmetics - Google Patents
Use of bone morphogenetic proteins and their receptors for aesthetics and cosmeticsInfo
- Publication number
- CA3176245A1 CA3176245A1 CA3176245A CA3176245A CA3176245A1 CA 3176245 A1 CA3176245 A1 CA 3176245A1 CA 3176245 A CA3176245 A CA 3176245A CA 3176245 A CA3176245 A CA 3176245A CA 3176245 A1 CA3176245 A1 CA 3176245A1
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Abstract
Provided herein are methods for preventing or reducing skin wrinkles and/or enhancing or preserving facial contours using one or more BMPs and/or one or more proteins associated with a BMP.
Description
USE OF BONE MORPHOGENETIC PROTEINS AND THEIR RECEPTORS FOR
AESTHETICS AND COSMETICS
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. 119(e) of U.S.
Provisional Application No. 63/078,174, filed September 14, 2020, entitled "USE OF BONE
MORPHOGENETIC PROTEINS AND THEIR RECEPTORS FOR AESTHETICS AND
COSMETICS," the entire disclosure of which is hereby incorporated by reference.
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA
EFS-WEB
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 7, 2021, is named G091970069W000-SEQ-SXT, and is 19,220 bytes in size.
FIELD OF THE INVENTION
The present disclosure relates to the use of bone morphogenetic proteins (BMPs) and/or proteins associated with BMPs and their cellular receptors in aesthetic and cosmetic applications such as enhancing or preserving desirable facial contours and/or preventing or reducing skin wrinkles and improving skin texture.
BACKGROUND
Anti-aging strategies have been widespread in the industries of dermatology and aesthetics for decades. Traditionally, plastic surgeries, invasive procedures, and/or high intensity chemical treatments have been implemented for reversing or delaying the inevitable signs of aging of the skin and improving attractive appearance. However, these traditional strategies can be cost prohibitive, intolerably invasive, and/or potentially unsafe to human subjects, especially given that multiple rounds of treatments and/or long-term use are frequently required.
SUMMARY
Aspects of the disclosure relate to methods of preventing or reducing skin wrinkles or improving skin texture or evenness of skin tone, comprising administering to a subject in need thereof an effective amount of a composition comprising one or more bone morphogenetic proteins (BMPs), or variants or derivatives thereof.
Further aspects of the disclosure relate to methods of enhancing or preserving facial contours or improving the attractiveness of facial contours, comprising administering to a subject in need thereof an effective amount of a composition comprising one or more bone morphogenetic proteins (BMPs), or variants or derivatives thereof.
In some embodiments, the BMP or variant or derivative thereof, is selected from the group consisting of BMP2, BMP6, BMP7, and BMP9, or variants or derivatives thereof. In some embodiments, the BMP or variant or derivative thereof, is BMP2, or a variant or derivative thereof. In some embodiments, the BMP or variant or derivative thereof, is BMP6, or a variant or derivative thereof. In some embodiments, the BMP or variant or derivative thereof, is BMP7, or a variant or derivative thereof. In some embodiments, the BMP or variant or derivative thereof, is BMP9, or a variant or derivative thereof. In some embodiments, the composition comprises BMP2, BMP7, BMP6, and BMP9, or variants or derivatives thereof. In some embodiments, the composition further comprises one or more proteins associated with a BMP, or a variant or derivative thereof. In some embodiments, the protein associated with a BMP is Noggin, or a variant or derivative thereof.
In some embodiments, the protein associated with a BMP is a BMP peptide, or a variant or derivative thereof. In some embodiments, the protein associated with a BMP is Activin, or a variant or derivative thereof.
In some embodiments, the subject is a human subject having, suspected of having, or at risk of developing, skin wrinkles. In some embodiments, the subject is a human subject who desires improving skin texture or evenness of skin tone. In some embodiments, the skin wrinkles are facial and/or neck wrinkles. In some embodiments, the subject is a human subject having, suspected of having, or at risk of having, loss of natural facial contours. In some embodiments, the subject is a human subject who desires a change in facial contours to improve attractiveness. In some embodiments, the subject is elderly. In some embodiments, the subject is not elderly.
In some embodiments, enhancing or preserving facial contours comprises inhibiting or reversing bone resorption. In some embodiments, the one or more BMPs, or variants or
AESTHETICS AND COSMETICS
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. 119(e) of U.S.
Provisional Application No. 63/078,174, filed September 14, 2020, entitled "USE OF BONE
MORPHOGENETIC PROTEINS AND THEIR RECEPTORS FOR AESTHETICS AND
COSMETICS," the entire disclosure of which is hereby incorporated by reference.
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA
EFS-WEB
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 7, 2021, is named G091970069W000-SEQ-SXT, and is 19,220 bytes in size.
FIELD OF THE INVENTION
The present disclosure relates to the use of bone morphogenetic proteins (BMPs) and/or proteins associated with BMPs and their cellular receptors in aesthetic and cosmetic applications such as enhancing or preserving desirable facial contours and/or preventing or reducing skin wrinkles and improving skin texture.
BACKGROUND
Anti-aging strategies have been widespread in the industries of dermatology and aesthetics for decades. Traditionally, plastic surgeries, invasive procedures, and/or high intensity chemical treatments have been implemented for reversing or delaying the inevitable signs of aging of the skin and improving attractive appearance. However, these traditional strategies can be cost prohibitive, intolerably invasive, and/or potentially unsafe to human subjects, especially given that multiple rounds of treatments and/or long-term use are frequently required.
SUMMARY
Aspects of the disclosure relate to methods of preventing or reducing skin wrinkles or improving skin texture or evenness of skin tone, comprising administering to a subject in need thereof an effective amount of a composition comprising one or more bone morphogenetic proteins (BMPs), or variants or derivatives thereof.
Further aspects of the disclosure relate to methods of enhancing or preserving facial contours or improving the attractiveness of facial contours, comprising administering to a subject in need thereof an effective amount of a composition comprising one or more bone morphogenetic proteins (BMPs), or variants or derivatives thereof.
In some embodiments, the BMP or variant or derivative thereof, is selected from the group consisting of BMP2, BMP6, BMP7, and BMP9, or variants or derivatives thereof. In some embodiments, the BMP or variant or derivative thereof, is BMP2, or a variant or derivative thereof. In some embodiments, the BMP or variant or derivative thereof, is BMP6, or a variant or derivative thereof. In some embodiments, the BMP or variant or derivative thereof, is BMP7, or a variant or derivative thereof. In some embodiments, the BMP or variant or derivative thereof, is BMP9, or a variant or derivative thereof. In some embodiments, the composition comprises BMP2, BMP7, BMP6, and BMP9, or variants or derivatives thereof. In some embodiments, the composition further comprises one or more proteins associated with a BMP, or a variant or derivative thereof. In some embodiments, the protein associated with a BMP is Noggin, or a variant or derivative thereof.
In some embodiments, the protein associated with a BMP is a BMP peptide, or a variant or derivative thereof. In some embodiments, the protein associated with a BMP is Activin, or a variant or derivative thereof.
In some embodiments, the subject is a human subject having, suspected of having, or at risk of developing, skin wrinkles. In some embodiments, the subject is a human subject who desires improving skin texture or evenness of skin tone. In some embodiments, the skin wrinkles are facial and/or neck wrinkles. In some embodiments, the subject is a human subject having, suspected of having, or at risk of having, loss of natural facial contours. In some embodiments, the subject is a human subject who desires a change in facial contours to improve attractiveness. In some embodiments, the subject is elderly. In some embodiments, the subject is not elderly.
In some embodiments, enhancing or preserving facial contours comprises inhibiting or reversing bone resorption. In some embodiments, the one or more BMPs, or variants or
2 derivatives thereof, stimulates the formation of adipocytes in the skin. In some embodiments, the one or more BMPs, or variants or derivatives thereof, stimulates dedifferentiation of keratinocytes. In some embodiments, the one or more BMPs, or variants or derivatives thereof, reduces or modulates melanin formation by melanocytes. In some embodiments, the presence of Noggin, or a variant or derivative thereof, reduces potential side effects of the one or more BMPs, or variants or derivatives thereof.
In some embodiments, the composition is administered topically. In some embodiments, the composition is administered in a cream or serum. In some embodiments, the composition is administered through subdermal injection. In some embodiments, the subdermal injection is near periosteum.
In some embodiments, the concentration of the one or more BMPs, or variants or derivatives thereof, in the composition is less than about 3%. In some embodiments, the concentration of Noggin, or a variant or derivative thereof, in the composition is less than about 3%. In some embodiments, the concentration of the BMP peptide, or a variant or derivative thereof, in the composition is less than about 10%. In some embodiments, the one or more BMPs, or variants or derivatives thereof, are administered at a dose of about 100-1,000 ng/mL per administration site. In some embodiments, the method further comprises administering Noggin, or a variant or derivative thereof, at a dose of about 100-1,000 ng/mL
per administration site.
Further aspects of the disclosure relate to compositions comprising one or more bone morphogenetic proteins (BMPs), or variants or derivatives thereof, in a pharmaceutically acceptable carrier for use in preventing or reducing skin wrinkles or improving skin texture or evenness of skin tone.
Further aspects of the disclosure relate to compositions comprising one or more bone morphogenetic proteins (BMPs), or variants or derivatives thereof, in a pharmaceutically acceptable carrier for use in enhancing or preserving facial contours or improving the attractiveness of facial contours.
In some embodiments, the BMP, or variant or derivative thereof, is selected from the group consisting of BMP2, BMP7, and BMP9, or variants or derivatives thereof.
In some embodiments, the BMP, or variant or derivative thereof, is BMP2, or a variant or derivative thereof. In some embodiments, the BMP, or variant or derivative thereof, is BMP6, or a variant or derivative thereof. In some embodiments, the BMP, or variant or derivative
In some embodiments, the composition is administered topically. In some embodiments, the composition is administered in a cream or serum. In some embodiments, the composition is administered through subdermal injection. In some embodiments, the subdermal injection is near periosteum.
In some embodiments, the concentration of the one or more BMPs, or variants or derivatives thereof, in the composition is less than about 3%. In some embodiments, the concentration of Noggin, or a variant or derivative thereof, in the composition is less than about 3%. In some embodiments, the concentration of the BMP peptide, or a variant or derivative thereof, in the composition is less than about 10%. In some embodiments, the one or more BMPs, or variants or derivatives thereof, are administered at a dose of about 100-1,000 ng/mL per administration site. In some embodiments, the method further comprises administering Noggin, or a variant or derivative thereof, at a dose of about 100-1,000 ng/mL
per administration site.
Further aspects of the disclosure relate to compositions comprising one or more bone morphogenetic proteins (BMPs), or variants or derivatives thereof, in a pharmaceutically acceptable carrier for use in preventing or reducing skin wrinkles or improving skin texture or evenness of skin tone.
Further aspects of the disclosure relate to compositions comprising one or more bone morphogenetic proteins (BMPs), or variants or derivatives thereof, in a pharmaceutically acceptable carrier for use in enhancing or preserving facial contours or improving the attractiveness of facial contours.
In some embodiments, the BMP, or variant or derivative thereof, is selected from the group consisting of BMP2, BMP7, and BMP9, or variants or derivatives thereof.
In some embodiments, the BMP, or variant or derivative thereof, is BMP2, or a variant or derivative thereof. In some embodiments, the BMP, or variant or derivative thereof, is BMP6, or a variant or derivative thereof. In some embodiments, the BMP, or variant or derivative
3 thereof, is BMP7, or a variant or derivative thereof. In some embodiments, the BMP, or variant or derivative thereof, is BMP9, or a variant or derivative thereof. In some embodiments, the composition comprises BMP2, BMP6, BMP7 and BMP9, or variants or derivatives thereof.
In some embodiments, the composition further comprises one or more proteins associated with a BMP, or a variant or derivative thereof. In some embodiments, the protein associated with a BMP is Noggin, or a variant or derivative thereof. In some embodiments, the protein associated with BMP is a BMP peptide, or a variant or derivative thereof. In some embodiments, the protein associated with a BMP is Activin, or a variant or derivative thereof.
In some embodiments, the composition is a cream or serum. In some embodiments, the concentration of the one or more BMPs, or variants or derivatives thereof, in the composition is less than about 3%. In some embodiments, the concentration of Noggin, or a variant or derivative thereof, in the composition is less than about 3%. In some embodiments, the concentration of the BMP peptide, or a variant or derivative thereof, in the composition is less than about 10%. In some embodiments, the composition is formulated for subdermal injection. In some embodiments, the composition further comprises a dermal filler. In some embodiments, the composition further comprises one or more agents that enhance penetration.
In some embodiments, the one or more BMPs, or variants or derivatives thereof, are recombinantly produced. In some embodiments, the one or more BMPs, or variants or derivatives thereof, are recombinantly produced in a mammalian cell. In some embodiments, the mammalian cell is a HEK 293 cell or a CHO cell. In some embodiments, the one or more BMPs, or variants or derivatives thereof, are recombinantly produced in a yeast cell. In some embodiments, the yeast cell is a Pichia pastoris cell. In some embodiments, the one or more BMPs, or variants or derivatives thereof, are recombinantly produced in an E.
coli cell.
Further aspects of the disclosure relate to kits comprising a composition comprising one or more bone morphogenetic proteins (BMPs), or variants or derivatives thereof, and instructions for use in administering the composition to prevent or reduce skin wrinkles or to improve skin texture or evenness of skin tone.
Further aspects of the disclosure relate to kits comprising a composition comprising one or more bone morphogenetic proteins (BMPs), or variants or derivatives thereof, and
In some embodiments, the composition further comprises one or more proteins associated with a BMP, or a variant or derivative thereof. In some embodiments, the protein associated with a BMP is Noggin, or a variant or derivative thereof. In some embodiments, the protein associated with BMP is a BMP peptide, or a variant or derivative thereof. In some embodiments, the protein associated with a BMP is Activin, or a variant or derivative thereof.
In some embodiments, the composition is a cream or serum. In some embodiments, the concentration of the one or more BMPs, or variants or derivatives thereof, in the composition is less than about 3%. In some embodiments, the concentration of Noggin, or a variant or derivative thereof, in the composition is less than about 3%. In some embodiments, the concentration of the BMP peptide, or a variant or derivative thereof, in the composition is less than about 10%. In some embodiments, the composition is formulated for subdermal injection. In some embodiments, the composition further comprises a dermal filler. In some embodiments, the composition further comprises one or more agents that enhance penetration.
In some embodiments, the one or more BMPs, or variants or derivatives thereof, are recombinantly produced. In some embodiments, the one or more BMPs, or variants or derivatives thereof, are recombinantly produced in a mammalian cell. In some embodiments, the mammalian cell is a HEK 293 cell or a CHO cell. In some embodiments, the one or more BMPs, or variants or derivatives thereof, are recombinantly produced in a yeast cell. In some embodiments, the yeast cell is a Pichia pastoris cell. In some embodiments, the one or more BMPs, or variants or derivatives thereof, are recombinantly produced in an E.
coli cell.
Further aspects of the disclosure relate to kits comprising a composition comprising one or more bone morphogenetic proteins (BMPs), or variants or derivatives thereof, and instructions for use in administering the composition to prevent or reduce skin wrinkles or to improve skin texture or evenness of skin tone.
Further aspects of the disclosure relate to kits comprising a composition comprising one or more bone morphogenetic proteins (BMPs), or variants or derivatives thereof, and
4 instructions for use in administering the composition to enhance or preserve facial contours or to improve the attractiveness of facial contours.
Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention.
This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including,"
"comprising," or "having," "containing," "involving," and variations of thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The accompanying drawings are not intended to be drawn to scale. The drawings are illustrative only and are not required for enablement of the disclosure. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:
FIG. 1 shows a diagram depicting mechanisms of BMP signaling. The diagram is adapted from Wang et al., Genes and Diseases, DOT:
10.1016/j.gendis.2014.07.005.
DETAILED DESCRIPTION OF THE INVENTION
This disclosure provides methods and compositions for the use of BMPs and/or proteins associated with BMPs in aesthetic and cosmetic indications. For example, BMPs and/or proteins associated with BMPs described herein can be used for preventing or reducing skin wrinkles, improving skin texture or evenness of skin tone, enhancing or preserving facial contours, and/or improving the attractiveness of facial contours. This
Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention.
This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including,"
"comprising," or "having," "containing," "involving," and variations of thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The accompanying drawings are not intended to be drawn to scale. The drawings are illustrative only and are not required for enablement of the disclosure. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:
FIG. 1 shows a diagram depicting mechanisms of BMP signaling. The diagram is adapted from Wang et al., Genes and Diseases, DOT:
10.1016/j.gendis.2014.07.005.
DETAILED DESCRIPTION OF THE INVENTION
This disclosure provides methods and compositions for the use of BMPs and/or proteins associated with BMPs in aesthetic and cosmetic indications. For example, BMPs and/or proteins associated with BMPs described herein can be used for preventing or reducing skin wrinkles, improving skin texture or evenness of skin tone, enhancing or preserving facial contours, and/or improving the attractiveness of facial contours. This
5 disclosure describes recombinant production of BMPs and/or proteins associated with BMPs in host cells and the use of recombinantly produced BMPs and/or proteins associated with BMPs for administration to human subjects for preventing or reversing various signs of skin aging. A strategy that uses BMPs and/or associated proteins and receptors may provide an advantageous approach to meet the increasing anti-aging demand in the aesthetic and cosmetic industries.
Bone Morphogenetic Proteins (BMPs) and Proteins Associated with BMPs Bone morphogenetic proteins (BMPs) are a large subclass (more than 20 members) of the TGF-beta (Transforming Growth Factor-Beta) super family that is active in many tissues under normal physiologic conditions. BMPs are growth factors that are known as cytokines and as metabologens. They play a crucial role in bone and cartilage formation as well as in adult homeostasis of bone function. Initially discovered for their ability to induce bone formation, BMPs are now known to play crucial roles in all organ systems. BMPs are also considered to provide a group of pivotal morphogenetic signals, orchestrating tissue architecture throughout the body. Without wishing to be bound by any theory, BMPs can be regulated through reversible interactions with extracellular antagonists, including Noggin, Chordin, Follistatin and Gremlin. These interactions determine the bioavailability of different BMPs for binding to their cognate receptors and activation of downstream responses. The biological functions and signaling mechanisms of BMPs are further described, for example, in Wang et al. (Bone Morphogenetic Protein (BMP) signaling in development and human diseases; Genes and Diseases, (2014) 1, 87-105) and Katagiri and Watabe (Bone Morphogenetic Proteins; Cold Spring Harb Perspect Biol. 2016, 8)6): a021899), which are incorporated herein by reference in their entireties.
Typically, BMPs interact with specific receptors on the cell surface, such as bone morphogenetic protein receptors (BMPRs). As illustrated in FIG. 1, signal transduction through BMPRs results in mobilization of members of the SMAD family of proteins (e.g., Smad1/5/8 and Smad 4). Currently, more than 15 known BMPs are structurally related and can be further categorized into subgroups based on amino acid or nucleotide similarity. For example, BMPs include but are not limited to BMP1, BMP 2, BMP 3, BMP 4, BMP 5, BMP
Bone Morphogenetic Proteins (BMPs) and Proteins Associated with BMPs Bone morphogenetic proteins (BMPs) are a large subclass (more than 20 members) of the TGF-beta (Transforming Growth Factor-Beta) super family that is active in many tissues under normal physiologic conditions. BMPs are growth factors that are known as cytokines and as metabologens. They play a crucial role in bone and cartilage formation as well as in adult homeostasis of bone function. Initially discovered for their ability to induce bone formation, BMPs are now known to play crucial roles in all organ systems. BMPs are also considered to provide a group of pivotal morphogenetic signals, orchestrating tissue architecture throughout the body. Without wishing to be bound by any theory, BMPs can be regulated through reversible interactions with extracellular antagonists, including Noggin, Chordin, Follistatin and Gremlin. These interactions determine the bioavailability of different BMPs for binding to their cognate receptors and activation of downstream responses. The biological functions and signaling mechanisms of BMPs are further described, for example, in Wang et al. (Bone Morphogenetic Protein (BMP) signaling in development and human diseases; Genes and Diseases, (2014) 1, 87-105) and Katagiri and Watabe (Bone Morphogenetic Proteins; Cold Spring Harb Perspect Biol. 2016, 8)6): a021899), which are incorporated herein by reference in their entireties.
Typically, BMPs interact with specific receptors on the cell surface, such as bone morphogenetic protein receptors (BMPRs). As illustrated in FIG. 1, signal transduction through BMPRs results in mobilization of members of the SMAD family of proteins (e.g., Smad1/5/8 and Smad 4). Currently, more than 15 known BMPs are structurally related and can be further categorized into subgroups based on amino acid or nucleotide similarity. For example, BMPs include but are not limited to BMP1, BMP 2, BMP 3, BMP 4, BMP 5, BMP
6, BMP 7, BMP 8a, BMP 8b, BMP 9, BMP10, BMP11, BMP12, BMP13, BMP14, and BMP15. Specifically, BMP2/4, BMP5/6/7/8, BMP9/BMP10, and BMP12/13/14 (GDF5/6/7) have been reported to be subgroups based on phylogenetic analysis. BMPs typically have a domain structure that includes at least a secretory leader sequence at the amino terminus, a protease domain, and three internal repeat Clr/Cls domains, wherein the second and the third internal repeat C lr/Cls domain are separated by an EGF domain. The structure of BMPs and their related biological functions is discussed further, for example, in Wozney (Bone Morphogenetic Proteins and Their Gene Expression; Cellular and Molecular Biology of Bone, 1993), which is incorporated herein by reference in its entirety.
Without wishing to be bound by any theory, each of the BMPs may have distinct biological functions. For example, experimental knockout of BMP3 can result in increased bone density, whereas experimental knockout of BMP7 is associated with defects in skeletal patterning and decreased brown fat. BMPs are also linked to activation of adipocyte transcription factors due to neogenic hair follicle formation during myofibroblast reprogramming when the tissues are undergoing wound healing. Mechanisms of adipocyte formation triggered by BMPs are further described, for example, in Plikus et al.
(Regeneration of fat cells from myofibroblasts during wound healing; Science, 2017, 355(6326): 748-752), which is incorporated herein by reference in its entirety. In addition, BMP2 and BMP4 have been reported to be regulators of skin stem cell proliferation and differentiation.
Methods and compositions described herein can also include one or more proteins associated with a BMP. As used in the present disclosure, "protein associated with a BMP" or "BMP associated protein," which are used interchangeably, includes any protein that interacts with a BMP or that impacts, directly or indirectly, a signaling pathway involving a BMP
and/or the biological function of a BMP. A protein associated with a BMP may be capable of regulating (e.g., inhibiting, stimulating, activating, delaying) the signaling pathways of a BMP. Proteins associated with BMPs can include, for example, Activin, Noggin, Chordin, Folistatin, Gremlin, and BMP receptor 1 a-extracellular domain. Others may include BMP
peptides, BMP receptors, and BMP or receptor decoys.
In some embodiments, methods and compositions described herein can also include modulation of one or more BMP receptors. BMP receptors are hetero-tetrameric complex transmembrane receptors and generally contain at least an N-terminal extracellular ligand binding domain, a transmembrane domain and an intracellular region. Non-limiting examples of BMP receptors include but are not limited to ALK1, ALK2, ALK3, ALK6, ACVR2A, ACVR2B, BMPR2, and AMHR2.
In some embodiments, receptor availability can be regulated. As non-limiting examples, BMP receptors may be regulated by BMP3, LEFTYA/B monomers and/or activin 13/inhibin a heterodimers. Without wishing to be bound by any theory, these ligands may
Without wishing to be bound by any theory, each of the BMPs may have distinct biological functions. For example, experimental knockout of BMP3 can result in increased bone density, whereas experimental knockout of BMP7 is associated with defects in skeletal patterning and decreased brown fat. BMPs are also linked to activation of adipocyte transcription factors due to neogenic hair follicle formation during myofibroblast reprogramming when the tissues are undergoing wound healing. Mechanisms of adipocyte formation triggered by BMPs are further described, for example, in Plikus et al.
(Regeneration of fat cells from myofibroblasts during wound healing; Science, 2017, 355(6326): 748-752), which is incorporated herein by reference in its entirety. In addition, BMP2 and BMP4 have been reported to be regulators of skin stem cell proliferation and differentiation.
Methods and compositions described herein can also include one or more proteins associated with a BMP. As used in the present disclosure, "protein associated with a BMP" or "BMP associated protein," which are used interchangeably, includes any protein that interacts with a BMP or that impacts, directly or indirectly, a signaling pathway involving a BMP
and/or the biological function of a BMP. A protein associated with a BMP may be capable of regulating (e.g., inhibiting, stimulating, activating, delaying) the signaling pathways of a BMP. Proteins associated with BMPs can include, for example, Activin, Noggin, Chordin, Folistatin, Gremlin, and BMP receptor 1 a-extracellular domain. Others may include BMP
peptides, BMP receptors, and BMP or receptor decoys.
In some embodiments, methods and compositions described herein can also include modulation of one or more BMP receptors. BMP receptors are hetero-tetrameric complex transmembrane receptors and generally contain at least an N-terminal extracellular ligand binding domain, a transmembrane domain and an intracellular region. Non-limiting examples of BMP receptors include but are not limited to ALK1, ALK2, ALK3, ALK6, ACVR2A, ACVR2B, BMPR2, and AMHR2.
In some embodiments, receptor availability can be regulated. As non-limiting examples, BMP receptors may be regulated by BMP3, LEFTYA/B monomers and/or activin 13/inhibin a heterodimers. Without wishing to be bound by any theory, these ligands may
7
8 occupy and therefore reduce or eliminate the availability of the receptors but not activate the receptors (e.g., ACVR2A or ACVR2B). The negative regulation may diminish Activin and BMP signaling pathways since ACVR2A and ACVR2B are shared receptors for these two ligand subtypes.
In general, activated BMP receptors promote the activation of Smad-dependent or independent pathways. Without wishing to be bound by any theory, BMPs described herein, including chimeric BMPs, may have increased binding specificity for receptors, so that the receptors are less likely to be negatively regulated by BMP antagonists (e.g., blocking BMP
signal pathway). BMPs described herein, including chimeric BMPs, may have increased binding affinity for receptors, which can result in increasing the potency of the BMPs by increasing activation of the BMP signal pathway. BMP receptors are discussed further, for example, in Carreira et al., (Bone Morphogenetic Proteins: Structure, biological function and therapeutic applications; Archives of Biochemistry and Biophysics 561 (2014) 64-73), which is incorporated herein by reference in its entirety.
Without wishing to be bound by any theory, Activin may be able to act as an "on"
switch for BMP signaling. Activin may include but is not limited to activin PA, activin f3B, activin PC, activin PE, myostatin, GDF1, GDF3, GDF11, and GDF15. Noggin, also known as NOG, on the contrary, may be able to act as an "off' switch for BMP signaling by binding to a BMP receptor. Other examples of proteins associated with BMPs that may function as endogenous BMP antagonists include Chordin, Cerberus, Glypixan-3 and Follistatin. Proteins associated with BMPs also include BMP peptides that may be able to function as antagonists or agonists for BMP signal transduction. Other examples of proteins associated with BMPs include receptor decoys, which may act as inhibitors of BMP signaling.
Proteins associated with BMPs include BMP, type 1, which includes BMP receptor type la and BMP
receptor type lb, as well as BMP, type 2.
BMPs, including recombinant BMPs, have previously been used in areas such as surgical operations, orthopedics, dental implants, and dental tissue regeneration. For instance, BMP2 and BMP7 have been approved by the FDA. Discussion of the use of recombinant human BMP2 can be found, for example, in Lykissas and Gkiatas (World J Orthop.
2017.18;
8(7): 531-535), which is incorporated herein by reference in its entirety.
Methods of Use of BMPs and BMP-Associated Proteins for Aesthetics and Cosmetics Aspects of the present disclosure relate to administering one or more proteins that regulate morphogenesis to preserve or improve appearance (e.g., appearance of human skin).
For example, proteins that regulate morphogenesis disclosed herein can be used for preventing or reducing skin wrinkles or improving skin texture or evenness of skin tone.
Proteins that regulate morphogenesis disclosed herein can be used for enhancing or preserving facial contours or improving the attractiveness of facial contours.
In some examples, proteins that regulate morphogenesis disclosed herein can be used for any desired improvements of appearance of the skin. In some embodiments, proteins that regulate morphogenesis are multi-functional growth factors. In some embodiments, proteins that regulate morphogenesis are cytokines. In some embodiments, proteins that regulate morphogenesis are growth and differentiation factors.
In some embodiments, proteins that regulate morphogenesis are BMPs, including but not limited to, BMP1, BMP 2, BMP 3, BMP 4, BMP 5, BMP 6, BMP 7, BMP 8a, BMP
8b, BMP 9, BMP10, BMP11, BMP12, BMP13, BMP14, and BMP15. In some embodiments, the BMP is BMP2, BMP6, BMP7, or BMP9. In some embodiments, the BMP is BMP2. In some embodiments, the BMP is BMP6. In some embodiments, the BMP is BMP7. In some embodiments, the BMP is BMP9. The BMP may be any BMP known in the art that is suitable for the compositions and methods disclosed herein.
In some embodiments, compositions comprise more than one BMP. In some embodiments, compositions comprise BMP2 and BMP7. In some embodiments, compositions comprise BMP2 and BMP6. In some embodiments, compositions comprise BMP2 and BMP9. In some embodiments, compositions comprise BMP6 and BMP7. In some embodiments, compositions comprise BMP6 and BMP9. In some embodiments, compositions comprise BMP7 and BMP9. In some embodiments, compositions comprise BMP2, BMP7 and BMP9. In some embodiments, compositions comprise BMP2, BMP6 and BMP7. In some embodiments, compositions comprise BMP6, BMP7 and BMP9. In some embodiments, compositions comprise BMP2, BMP6, BMP7 and BMP9. In some embodiments, compositions comprise any combination of BMPs that is able to prevent or reduce skin wrinkles and/or improve skin texture or evenness of skin tone. In some embodiments, compositions comprise any combination of BMPs that is able to enhance or preserve facial contours or improve the attractiveness of facial contours.
In some embodiments, proteins that regulate morphogenesis can be one or more proteins associated with BMPs. In some embodiments, proteins associated with BMPs include Activin, Noggin, Chordin, Cerberus, Gremlin, Glypixan-3, Follistatin, BMP peptides, and receptor decoys. In some embodiments, a protein associated with BMPs is Noggin. In
In general, activated BMP receptors promote the activation of Smad-dependent or independent pathways. Without wishing to be bound by any theory, BMPs described herein, including chimeric BMPs, may have increased binding specificity for receptors, so that the receptors are less likely to be negatively regulated by BMP antagonists (e.g., blocking BMP
signal pathway). BMPs described herein, including chimeric BMPs, may have increased binding affinity for receptors, which can result in increasing the potency of the BMPs by increasing activation of the BMP signal pathway. BMP receptors are discussed further, for example, in Carreira et al., (Bone Morphogenetic Proteins: Structure, biological function and therapeutic applications; Archives of Biochemistry and Biophysics 561 (2014) 64-73), which is incorporated herein by reference in its entirety.
Without wishing to be bound by any theory, Activin may be able to act as an "on"
switch for BMP signaling. Activin may include but is not limited to activin PA, activin f3B, activin PC, activin PE, myostatin, GDF1, GDF3, GDF11, and GDF15. Noggin, also known as NOG, on the contrary, may be able to act as an "off' switch for BMP signaling by binding to a BMP receptor. Other examples of proteins associated with BMPs that may function as endogenous BMP antagonists include Chordin, Cerberus, Glypixan-3 and Follistatin. Proteins associated with BMPs also include BMP peptides that may be able to function as antagonists or agonists for BMP signal transduction. Other examples of proteins associated with BMPs include receptor decoys, which may act as inhibitors of BMP signaling.
Proteins associated with BMPs include BMP, type 1, which includes BMP receptor type la and BMP
receptor type lb, as well as BMP, type 2.
BMPs, including recombinant BMPs, have previously been used in areas such as surgical operations, orthopedics, dental implants, and dental tissue regeneration. For instance, BMP2 and BMP7 have been approved by the FDA. Discussion of the use of recombinant human BMP2 can be found, for example, in Lykissas and Gkiatas (World J Orthop.
2017.18;
8(7): 531-535), which is incorporated herein by reference in its entirety.
Methods of Use of BMPs and BMP-Associated Proteins for Aesthetics and Cosmetics Aspects of the present disclosure relate to administering one or more proteins that regulate morphogenesis to preserve or improve appearance (e.g., appearance of human skin).
For example, proteins that regulate morphogenesis disclosed herein can be used for preventing or reducing skin wrinkles or improving skin texture or evenness of skin tone.
Proteins that regulate morphogenesis disclosed herein can be used for enhancing or preserving facial contours or improving the attractiveness of facial contours.
In some examples, proteins that regulate morphogenesis disclosed herein can be used for any desired improvements of appearance of the skin. In some embodiments, proteins that regulate morphogenesis are multi-functional growth factors. In some embodiments, proteins that regulate morphogenesis are cytokines. In some embodiments, proteins that regulate morphogenesis are growth and differentiation factors.
In some embodiments, proteins that regulate morphogenesis are BMPs, including but not limited to, BMP1, BMP 2, BMP 3, BMP 4, BMP 5, BMP 6, BMP 7, BMP 8a, BMP
8b, BMP 9, BMP10, BMP11, BMP12, BMP13, BMP14, and BMP15. In some embodiments, the BMP is BMP2, BMP6, BMP7, or BMP9. In some embodiments, the BMP is BMP2. In some embodiments, the BMP is BMP6. In some embodiments, the BMP is BMP7. In some embodiments, the BMP is BMP9. The BMP may be any BMP known in the art that is suitable for the compositions and methods disclosed herein.
In some embodiments, compositions comprise more than one BMP. In some embodiments, compositions comprise BMP2 and BMP7. In some embodiments, compositions comprise BMP2 and BMP6. In some embodiments, compositions comprise BMP2 and BMP9. In some embodiments, compositions comprise BMP6 and BMP7. In some embodiments, compositions comprise BMP6 and BMP9. In some embodiments, compositions comprise BMP7 and BMP9. In some embodiments, compositions comprise BMP2, BMP7 and BMP9. In some embodiments, compositions comprise BMP2, BMP6 and BMP7. In some embodiments, compositions comprise BMP6, BMP7 and BMP9. In some embodiments, compositions comprise BMP2, BMP6, BMP7 and BMP9. In some embodiments, compositions comprise any combination of BMPs that is able to prevent or reduce skin wrinkles and/or improve skin texture or evenness of skin tone. In some embodiments, compositions comprise any combination of BMPs that is able to enhance or preserve facial contours or improve the attractiveness of facial contours.
In some embodiments, proteins that regulate morphogenesis can be one or more proteins associated with BMPs. In some embodiments, proteins associated with BMPs include Activin, Noggin, Chordin, Cerberus, Gremlin, Glypixan-3, Follistatin, BMP peptides, and receptor decoys. In some embodiments, a protein associated with BMPs is Noggin. In
9 some embodiments, a protein associated with BMPs is a BMP peptide. In some embodiments, a protein associated with BMPs is Activin.
BMPs associated with the disclosure may comprise wildtype sequences or may be engineered. BMPs associated with the disclosure may comprise one or more amino acid substitutions, additions, deletions, or insertions. BMPs associated with the disclosure may be naturally occurring or may be synthetic. BMPs associated with the disclosure can be in the form of fusion proteins. BMPs associated with the disclosure can be in the form of chimeric proteins. BMPs associated with the disclosure can be fragments or peptides of BMPs, such as fragments or peptides that preserve some or all of the activity of a full-length BMP. BMPs associated with the disclosure can be truncated forms of BMPs, such as truncated forms that preserve some or all of the activity of a full-length BMP.
The sequence of human BMP2 is provided by UniProt Accession number P12643:
MVAGTRCLLALLLPQVLLGGAAGLVPELGRRKFAAASSGRPSSQPSDEVLSEFELRLLSM
FGLKQRPTPSRDAVVPPYMLDLYRRHSGQPGSPAPDHRLERAASRANTVRSFHHEESLEE
LPETSGKTTRRFFFNLS SIPTEEFITSAELQVFREQMQDALGNNS S FHHRINIYEIIKPA
TANSKFPVTRLLDTRLVNQNASRWESFDVTPAVMRWTAQGHANHGFVVEVAHLEEKQGVS
KRHVRISRS LHQDEHSWS QIRPLLVTFGHDGKGHPLHKREKRQAKHKQRKRLKS SCKRHP
LYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKAC
CVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR (SEQ ID NO: 1) The sequence of human BMP4 is provided by UniProt Accession number P12644:
MIPGNRMLMVVLLCQVLLGGASHASLIPETGKKKVAEIQGHAGGRRSGQSHELLRDFEAT
LLQMFGLRRRPQPSKS AVIPDYMRDLYRLQSGEEEEEQIHSTGLEYPERPASRANTVRSF
HHEEHLENIPGTSENS AFRFLFNLSSIPENEVISSAELRLFREQVDQGPDWERGFHRINI
YEVMKPPAEVVPGHLITRLLDTRLVHHNVTRWETFDVSPAVLRWTREKQPNYGLAIEVTH
LHQTRTHQGQHVRISRSLPQGSGNWAQLRPLLVTFGHDGRGHALTRRRRAKRSPKHHSQR
ARKKNKNCRRHS LYVDFS DVGWNDWIVAPPGYQAFYCHGDCPFPLADHLNS TNHAIVQTL
VNSVNSSIPKACCVPTELSAISMLYLDEYDKVVLKNYQEMVVEGCGCR (SEQ ID NO: 2) The sequence of human BMP6 is provided by Uniprot Accession number P22004:
MPGLGRRAQWLCWWWGLLCSCCGPPPLRPPLPAAAAAAAGGQLLGDGGSPGRTEQPPPSPQSSSGFL
YRRLKTQEKREMQKEILS VLGLPHRPRPLHGLQQPQPPALRQQEEQQQQQQLPRGEPPPGRLKSAPLFM
LDLYNALSADNDEDGASEGERQQSWPHEAASSSQRRQPPPGAAHPLNRKSLLAPGSGSGGASPLTSAQ
DSAFLNDADMVMSFVNLVEYDKEFSPRQRHHKEFKFNLSQIPEGEVVTAAEFRIYKDCVMGSFKNQTF
LISIYQVLQEHQHRDSDLFLLDTRVVWASEEGWLEFDITATSNLWVVTPQHNMGLQLSVVTRDGVHV
HPRAAGLVGRDGPYDKQPFMVAFFKVSEVHVRTTRSASSRRRQQSRNRSTQSQDVARVS SASDYNS SE
LKTACRKHELYVSFQDLGWQDWIIAPKGYAANYCDGECSFPLNAHMNATNHAIVQTLVHLMNPEYVP
KPCCAPTKLNAISVLYFDDNSNVILKKYRNMVVRACGCH (SEQ ID NO: 3) The sequence of human BMP7 is provided by UniProt Accession number P18075:
MHVRSLRAAAPHSFVALWAPLFLLRS ALADFSLDNEVHSSFIHRRLRSQERREMQREILS
ILGLPHRPRPHLQGKHNSAPMFMLDLYNAMAVEEGGGPGGQGFSYPYKAVFSTQGPPLAS
LQDSHFLTDADMVMSFVNLVEHDKEFFHPRYHHREFRFDLSKIPEGEAVTAAEFRIYKDY
IRERFDNETFRIS VYQVLQEHLGRESDLFLLD SRTLWASEEGWLVFDITATSNHWVVNPR
HNLGLQLSVETLDGQSINPKLAGLIGRHGPQNKQPFMVAFFKATEVHFRSIRSTGSKQRS
QNRSKTPKNQEALRMANVAENSS SDQRQACKKHELYVSFRDLGWQDWIIAPEGYAAYYCE
GECAFPLNSYMNATNHAIVQTLVHFINPETVPKPCCAPTQLNAIS VLYFDDS SNVILKKY
RNMVVRACGCH (SEQ ID NO: 4) The sequence of BMP9, which is also called Growth/differentiation factor 2 (GDF2) is provided by UniProt Accession number Q9UK05:
MCPGALWVALPLLSLLAGSLQGKPLQSWGRGSAGGNAHSPLGVPGGGLPEHTFNLKMFLE
NVKVDFLRS LNLS GVPSQDKTRVEPPQYMIDLYNRYTSDKS TTPASNIVRSFSMEDAISI
TATEDFPFQKHILLFNISIPRHEQITRAELRLYVSCQNHVDPSHDLKGS V VIYDVLDGTD
AWDSATETKTFLVSQDIQDEGWETLEVSS AVKRWVRSDSTKSKNKLEVTVESHRKGCDTL
DISVPPGSRNLPFFVVFSNDHSSGTKETRLELREMISHEQESVLKKLSKDGSTEAGESSH
EEDTDGHVAAGSTLARRKRS AGAGSHCQKTSLRVNFEDIGWDSWIIAPKEYEAYECKGGC
FFPLADDVTPTKHAIVQTLVHLKFPTKVGKACCVPTKLSPIS VLYKDDMGVPTLKYHYEG
MSVAECGCR (SEQ ID NO: 5) Aspects of the present disclosure provide methods and compositions comprising one or more BMPs in combination with one or more proteins associated with a BMP.
For example, the composition can comprise: BMP2, BMP6, BMP7 and/or BMP9 and Noggin;
BMP2, BMP6, BMP7 and/or BMP9 and Activin; or BMP2, BMP6, BMP7 and/or BMP9 and one or more BMP peptides. In some embodiments, compositions may include a fusion protein derived from sequences of two or more proteins or peptides (e.g., comprising portions of BMP2 and BMP6), or a chimeric protein (e.g., comprising portions of BMP7 and Activin).
In some embodiments, chimeric proteins can comprise portions of different BMPs. In some embodiments, chimeric proteins may be heterodimers. In some embodiments, chimeric proteins can comprise portions of proteins associated with BMPs. In some embodiments, chimeric proteins can comprise portions of BMPs and portions of proteins associated with BMPs. For example, chimeric proteins can comprise portions of a BMP and an Activin. In some embodiments, chimeric proteins can comprise portions of BMP2 and BMP7.
Chimeric proteins encompassed by the present disclosure can comprise portions of any BMP and/or any protein associated with BMPs. In some embodiments, chimeric proteins may have one or more improved properties relative to native BMPs and/or native proteins associated with BMPs. In some embodiments, chimeric proteins may have one or more amino acid modifications such as amino acid substitutions, insertions, additions or deletions. Without wishing to be bound by any theory, heterodimers, chimeric proteins, and amino acid modifications may have increased binding affinity or specificity to BMP
receptors, which may prohibit BMP antagonists from negatively regulating the BMP signaling pathways. Any methods known in the art for producing chimeric proteins can be used for generating BMP
chimeric proteins. BMP chimeric proteins and their biological effects are discussed further in, for example, Seeherman et al., (A BMP/activin A chimera is superior to native BMPs and induces bone repair in nonhuman primates when delivered in a composite matrix;
Sci. Transl.
Med. 11, eaar4953 (2019)), which is incorporated herein by reference in its entirety.
In some embodiments, compositions described herein comprise one or more proteins associated with a BMP and do not comprise one or more BMPs. For example, a composition can comprise Noggin, Activin, and/or one or more BMP peptides, or any other protein associated with a BMP disclosed herein.
Compositions described herein comprising one or more BMPs and/or one or more proteins associated with BMPs can be administered to enhance or preserve facial contours or improve the attractiveness of facial contours. In some embodiments, enhancing or preserving facial contours or improving the attractiveness of facial contours involves inhibiting or reversing bone resorption. Bone resorption refers to resorption of bone tissues by which osteoclasts break down the tissue in bones. Bone structure and mineral density is gradually lost during this process. Inhibiting or reversing bone resorption can re-build or strengthen the natural facial contours. In some embodiments, enhancing or preserving facial contours or improving the attractiveness of facial contours involves bone augmentation. In some embodiments, enhancing or preserving facial contours or improving the attractiveness of facial contours involves cartilage augmentation. Bone augmentation or cartilage augmentation in the face can result in a fuller appearance of the face. In some embodiments, bone augmentation or cartilage augmentation can result in effects similar to implanting facial bones or cartilage tissues. In some embodiments, compositions described herein comprising one or more BMPs and/or one or more proteins associated with BMPs can be administered to produce bone augmentation and/or cartilage augmentation effects with or without enhancing or preserving facial contours.
In some embodiments, "enhancing" facial contours involves restoring facial contours during or after bone resorption. In some embodiments, "preserving" facial contours involves maintaining facial contours before actual or noticeable bone resorption has occurred.
Compositions described herein comprising one or more BMPs can be administered to prevent or reduce skin wrinkles and/or improve skin texture or evenness of skin tone. As used herein, skin texture refers to skin surface conditions in general. Skin texture can be generally categorized into even or uneven skin texture. Even skin texture in some embodiments can be considered as soft, smooth, firm, or hydrated. Uneven skin texture in some embodiments can be considered as coarse, rough, bumpy, dull, or dry. Uneven skin texture can also include the appearance of "macro-texture" (e.g., fine lines and wrinkles) as disclosed in, for example, EP2640347A2, which is incorporated herein by reference in its entirety. In some embodiments, skin "evenness" can be determined based at least in part on the criteria listed above ¨ the degree of softness, smoothness, firmness, and/or hydration, or coarseness, roughness, bumpiness, dullness, and/or dryness.
Any technique known in the art can be used for assessing and determining skin texture or the evenness of the skin. As an example, a digital camera equipped with a suitable lens for facial imaging (e.g., 60mm Nikor lens) can be used for capturing facial images to determine skin texture. The region of interest (ROT) can be marked manually based on predefined facial landmarks such as corners of the eyes and bridge of the nose. The degree of textured skin in the ROT can be quantified using image analysis algorithms based on, for example, an Optimus software platform. Other suitable software platforms can also be used.
The software platforms can automatically locate each surface feature and can quantify the total number, length and area of facial features, based on a predetermined standard (e.g., shorter than 5 mm and less than 0.16 mm wide), known magnification used to convert pixel data to actual length and area data. Thresholds can be based on clinically important facial texture. For example, in some embodiments, lines greater than 5 mm and broader than 0.16 mm may be excluded. Because the ROT varies in shape and size, total textured area can be normalized to total ROT size to yield a Texture Area Fraction (TAF). In some embodiments, skin evenness can also be measured by analyzing the luminance homogeneity with the applications of Haralick homogeneity or chromaticity. Methods for measuring skin evenness are discussed further in, for example, Batres et al. (Cosmetics increase skin evenness:
Evidence from perceptual and physical measures; Skin Res Technol, 2019, 25(5):
672-676), which is incorporated herein by reference in its entirety.
In some embodiments, compositions described herein including one or more BMPs and/or one or more proteins associated with BMPs reduce the thickness of the stratum corneum, and/or increase the thickness of the living epidermis, resulting in less wrinkled, smoother surface texture. In some embodiments, compositions described herein including one or more BMPs and/or one or more proteins associated with BMPs stimulate the formation of adipocytes in the skin, which may contribute to fuller, less wrinkled skin, or improved texture or evenness of skin tone. In some embodiments, compositions described herein including one or more BMPs and/or one or more proteins associated with BMPs stimulate dedifferentiation of keratinocytes. Keratinocytes are the primary type of cell found in the epidermis, which is the outermost layer of the skin. Keratinocytes function in the formation of a barrier against environmental damages such as UV light and radiation. Keratinocyte dedifferentiation has biological significance during wound healing when epidermis must rapidly regenerate barrier function. In some embodiments, compositions described herein including one or more BMPs and/or one or more proteins associated with BMPs reduces, evens out, or modulates melanin formation by melanocytes. Without wishing to be bound by any theory, BMPs may be capable of affecting or regulating melanin synthesis (or skin pigmentation), so that administration of BMPs can improve the skin tone or the evenness of the skin tone (e.g., reduced pigmentation in the skin). In some embodiments, compositions described herein including one or more BMPs and/or one or more proteins associated with BMPs contributes to the appearance of more youthful or attractive skin.
In some embodiments, the presence of Noggin in a composition comprising one or more BMPs can reduce potential side effects associated with the one or more BMPs. The presence of Noggin as an inhibitor of BMP can ensure that the anti-wrinkle and/or pro-facial contouring effects of BMPs can be appropriately regulated. In some embodiments, Noggin can be formulated with or without the presence of one or more BMPs. For example, a Noggin formulation can be administered to a subject separately from administration of one or more BMPs, such as a few days after the administration of one or more BMPs.
Subjects associated with the disclosure include human and non-human subjects.
In some embodiments, a non-human subject is a non-human primate. In some embodiments, a non-human subject is a companion animal or a farm animal. In some embodiments, the subject is a subject that has exposed skin.
In some embodiments, the subject is a human subject having, suspected of having, or at risk of developing skin wrinkles. In some embodiments, the skin wrinkles are facial wrinkles. In some embodiments, the skin wrinkles are neck wrinkles. Skin wrinkles include any types of wrinkles, such as fine lines and creases that can be found or observed on human skins particularly facial and neck regions. Skin wrinkles include raised portions of the epithelial layer which are elevated above a normal surface of skin. Skin wrinkles can be caused by a variety of factors such as aging, UV light exposure, lifestyle, environmental factors, genetics, and medical treatments. In general, wrinkles start to form at the dermis layer of the skin, which constitutes fibroblast cells that synthesize the structural proteins collagens and elastin. During the formation of wrinkles, the dermis layer loses its ability to effectively repair and restore. Therefore, the skin's strength deteriorates and begins to wrinkle.
Without wishing to be bound by any theory, wrinkles can in some embodiments be classified as crinkle lines, permanent elastotic creases, dynamic expression lines, and/or gravitational folds. Crinkle lines are also known as atrophic crinkling rhytids, which are relatively shallow. They typically run in parallel to each other, often on the forehead, and disappear when the skin is stretched. Permanent elastotic creases can be associated with pale complexions and heavy sun exposure. Characterized by deep lines in the skin, they can occur at points where the skin creases naturally, such as the base of the neck, the lips, and the cheeks. Dynamic expression lines can be caused by habitual facial expressions, combined with the skin's loss of elasticity; for example, frown lines on the brow, crow's feet around the eyes, and laugh lines above the mouth. Gravitational folds can be associated with the losses of firmness of the skin with age during which the skin begins to sag, pulling away from the underlying fat and muscle. This can create folds that are particularly prominent on the neck, chin, and jowls.
A subject having, suspected of having, or at risk of developing skin wrinkles can be identified by routine examination, e.g., visual inspection, laboratory tests, physical exams, and/or dermatoscopy, as would be understood by one of ordinary skill in the art. Numbers, sites and depths of the wrinkles can be measured. The non-invasive digital fringe profilometry, which utilizes phase-shifted fringes being projected to the skin, can be used.
Digital fringe profilometry is discussed further in Sari et al (Measurement of skin wrinkles differences using novel optical skin imaging system of digital fringes profilometry; Proc.
SPIE 11044, Third International Seminar on Photonics, Optics, and Its Applications (ISPh0A
2018), 1104406 (2019)), which is incorporated herein by reference in its entirety.
In some embodiments, a subject is a human subject who desires to improve skin texture or evenness of skin tone. In some embodiments, a human subject who desires to improve skin texture or evenness of skin tone does not have visible signs of aging such as wrinkles or uneven skin tone.
In some embodiments, a subject is a human subject having, suspected of having, or at risk of having a loss of natural facial contours. Facial contours can include the shape and size of the facial skeleton and structure of a subject. With aging, natural facial contours may be lost due to facial bone resorption. Many plastic surgery or aesthetic procedures are used for preserving or improving facial contours, such as implant insertion by facial skeletal augmentation surgery, e.g., to make the face more balanced by adding fullness, making the cheekbones higher, the chin more prominent, and/or the jawline stronger and wider.
In some embodiments, a subject is a human subject who desires a change in his or her current facial contours to improve attractiveness of facial contours. A human subject may want to change his or her facial contours at least in part due to personal choices. In some embodiments, a human subject has never received any previous procedures or treatments to change facial contours. In other embodiments, a human subject has previously received one or more procedures or treatments to change facial contours.
Improved attractiveness can be assessed by subjective and/or objective methods. In some embodiments, improved attractiveness can be assessed by the human subject who is administered a composition disclosed herein. In other embodiments, improved attractiveness can be assessed by another human subject. In other embodiments, improved attractiveness can be assessed by a machine, such as a computer program or algorithm that can predict attractiveness, such as facial attractiveness. For example, a machine can comprise a computer-based system and an auto-encoder, creating a machine learning system that can automatically determine how attractive a certain facial image is. A set of facial images that are considered "attractive" or "non (less) attractive" can be provided to the system for modeling. After a composition associated with the disclosure is administered to a human subject, one or more facial images of the human subject can be uploaded to the machine learning system, which will assess whether the facial images are attractive and/or whether the facial images are more attractive than a control, such as facial images of the same subject before being administered the composition. In some embodiments, assessing attractiveness can be done by reviewing whether a facial image contains attractive or unattractive features.
A facial image that lacks pre-determined unattractive features may be considered as attractive. A facial image that has less unattractive features relative to a control may be considered to have increased attractiveness relative to the control. Solely by way of example, unattractive features can include but are not limited to, acne, age spots/sun damage, bruises, bumps, cellulite, light spots, pitting, scars, freckles, including damaged freckles, and wrinkles. In some embodiments, unattractive features can be particularly associated with the appearance of facial contours. For example, such features may include sunken cheeks, facial asymmetry, or sunken eyebrow area. In some embodiments, attractiveness is based on the observations or opinions of a population (e.g., social standards and human behaviors).
Methods and approaches for determining attractiveness of facial appearance are further described, for example, in US 10,726,601 and US 10,486,174, which are incorporated herein by reference in their entireties.
In some embodiments, a subject has been identified as having skin wrinkles or loss of natural facial contours. In other embodiments, a subject has not been identified as having skin wrinkles or loss of natural facial contours. A subject suspected of having skin wrinkles or loss of natural facial contours may be a subject that exhibits one or more symptoms or signs of skin wrinkles or loss of natural facial contours, e.g., small creases on the skin, loss of elasticity. In other embodiments, the subject has not exhibited any symptoms or signs of skin wrinkles or loss of natural facial contours and/or has no history of skin wrinkles or loss of natural facial contours. In other embodiments, the subject desires a change in skin texture or evenness of skin tone or facial contour in the belief that the change produces a more attractive appearance. A subject at risk for skin wrinkles or loss of natural facial contours may be a subject having one or more of the risk factors associated with the development of skin wrinkles and/or loss of natural facial contours. For example, risk factors associated with skin wrinkles or loss of natural facial contours can include in some embodiments:
(a) genetic factors; (b) age; (c) family history; (d) habitual exposure to UV light;
and/or (e) smoking.
In some embodiments, a subject is a human subject who has undergone, is undergoing, or will undergo gender reassignment procedures and/or surgeries.
Gender reassignment procedures and/or surgeries refers to any procedures or surgeries that change the sex assigned at birth of a subject. In some embodiments, the human subject is male assigned at birth and undergoes gender reassignment procedures and/or surgeries to change the gender to female. In some embodiments, the human subject is female assigned at birth and undergoes gender reassignment procedures and/or surgeries to change the gender to male. For example, a male subject who wants to undergo gender reassignment procedures and/or surgeries can be administered a composition disclosed herein with the goal of achieving softer and more feminine facial features as part of facial feminization surgery during the male-to-female transition. In some embodiments, before and after facial images can verify whether administration of compositions disclosed herein achieves the desired effects.
In some embodiments, a subject is elderly (e.g., 65 years old or older).
Elderly subjects are more prone to develop skin wrinkles and to suffer from the loss of facial contours. For instance, the quantities and the types of skin wrinkles may be more prominent in an elderly subject than in a subject who is younger than 65 years old. In other embodiments, a subject is not elderly (e.g., a subject is younger than 65 years old). A subject who is not elderly may also be prone to developing skin wrinkles and to suffering from the loss of facial contours. As disclosed in the present application, many factors other than aging can cause or accelerate the appearance of skin wrinkles and undesirable facial contours. In some embodiments, a subject is not prone to developing skin wrinkles or to suffering from the loss of facial contours but is administered compositions disclosed herein to prevent or inhibit the formation of skin wrinkles and/or to prevent or inhibit the loss of facial contours.
In some embodiments, compositions described here that contain one or more BMPs and/or one or more proteins associated with BMPs prevent or reduce skin wrinkles by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. In some embodiments, the one or more BMP used in the methods described in the present application enhances or preserves facial contours by at least 5%,
BMPs associated with the disclosure may comprise wildtype sequences or may be engineered. BMPs associated with the disclosure may comprise one or more amino acid substitutions, additions, deletions, or insertions. BMPs associated with the disclosure may be naturally occurring or may be synthetic. BMPs associated with the disclosure can be in the form of fusion proteins. BMPs associated with the disclosure can be in the form of chimeric proteins. BMPs associated with the disclosure can be fragments or peptides of BMPs, such as fragments or peptides that preserve some or all of the activity of a full-length BMP. BMPs associated with the disclosure can be truncated forms of BMPs, such as truncated forms that preserve some or all of the activity of a full-length BMP.
The sequence of human BMP2 is provided by UniProt Accession number P12643:
MVAGTRCLLALLLPQVLLGGAAGLVPELGRRKFAAASSGRPSSQPSDEVLSEFELRLLSM
FGLKQRPTPSRDAVVPPYMLDLYRRHSGQPGSPAPDHRLERAASRANTVRSFHHEESLEE
LPETSGKTTRRFFFNLS SIPTEEFITSAELQVFREQMQDALGNNS S FHHRINIYEIIKPA
TANSKFPVTRLLDTRLVNQNASRWESFDVTPAVMRWTAQGHANHGFVVEVAHLEEKQGVS
KRHVRISRS LHQDEHSWS QIRPLLVTFGHDGKGHPLHKREKRQAKHKQRKRLKS SCKRHP
LYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKAC
CVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR (SEQ ID NO: 1) The sequence of human BMP4 is provided by UniProt Accession number P12644:
MIPGNRMLMVVLLCQVLLGGASHASLIPETGKKKVAEIQGHAGGRRSGQSHELLRDFEAT
LLQMFGLRRRPQPSKS AVIPDYMRDLYRLQSGEEEEEQIHSTGLEYPERPASRANTVRSF
HHEEHLENIPGTSENS AFRFLFNLSSIPENEVISSAELRLFREQVDQGPDWERGFHRINI
YEVMKPPAEVVPGHLITRLLDTRLVHHNVTRWETFDVSPAVLRWTREKQPNYGLAIEVTH
LHQTRTHQGQHVRISRSLPQGSGNWAQLRPLLVTFGHDGRGHALTRRRRAKRSPKHHSQR
ARKKNKNCRRHS LYVDFS DVGWNDWIVAPPGYQAFYCHGDCPFPLADHLNS TNHAIVQTL
VNSVNSSIPKACCVPTELSAISMLYLDEYDKVVLKNYQEMVVEGCGCR (SEQ ID NO: 2) The sequence of human BMP6 is provided by Uniprot Accession number P22004:
MPGLGRRAQWLCWWWGLLCSCCGPPPLRPPLPAAAAAAAGGQLLGDGGSPGRTEQPPPSPQSSSGFL
YRRLKTQEKREMQKEILS VLGLPHRPRPLHGLQQPQPPALRQQEEQQQQQQLPRGEPPPGRLKSAPLFM
LDLYNALSADNDEDGASEGERQQSWPHEAASSSQRRQPPPGAAHPLNRKSLLAPGSGSGGASPLTSAQ
DSAFLNDADMVMSFVNLVEYDKEFSPRQRHHKEFKFNLSQIPEGEVVTAAEFRIYKDCVMGSFKNQTF
LISIYQVLQEHQHRDSDLFLLDTRVVWASEEGWLEFDITATSNLWVVTPQHNMGLQLSVVTRDGVHV
HPRAAGLVGRDGPYDKQPFMVAFFKVSEVHVRTTRSASSRRRQQSRNRSTQSQDVARVS SASDYNS SE
LKTACRKHELYVSFQDLGWQDWIIAPKGYAANYCDGECSFPLNAHMNATNHAIVQTLVHLMNPEYVP
KPCCAPTKLNAISVLYFDDNSNVILKKYRNMVVRACGCH (SEQ ID NO: 3) The sequence of human BMP7 is provided by UniProt Accession number P18075:
MHVRSLRAAAPHSFVALWAPLFLLRS ALADFSLDNEVHSSFIHRRLRSQERREMQREILS
ILGLPHRPRPHLQGKHNSAPMFMLDLYNAMAVEEGGGPGGQGFSYPYKAVFSTQGPPLAS
LQDSHFLTDADMVMSFVNLVEHDKEFFHPRYHHREFRFDLSKIPEGEAVTAAEFRIYKDY
IRERFDNETFRIS VYQVLQEHLGRESDLFLLD SRTLWASEEGWLVFDITATSNHWVVNPR
HNLGLQLSVETLDGQSINPKLAGLIGRHGPQNKQPFMVAFFKATEVHFRSIRSTGSKQRS
QNRSKTPKNQEALRMANVAENSS SDQRQACKKHELYVSFRDLGWQDWIIAPEGYAAYYCE
GECAFPLNSYMNATNHAIVQTLVHFINPETVPKPCCAPTQLNAIS VLYFDDS SNVILKKY
RNMVVRACGCH (SEQ ID NO: 4) The sequence of BMP9, which is also called Growth/differentiation factor 2 (GDF2) is provided by UniProt Accession number Q9UK05:
MCPGALWVALPLLSLLAGSLQGKPLQSWGRGSAGGNAHSPLGVPGGGLPEHTFNLKMFLE
NVKVDFLRS LNLS GVPSQDKTRVEPPQYMIDLYNRYTSDKS TTPASNIVRSFSMEDAISI
TATEDFPFQKHILLFNISIPRHEQITRAELRLYVSCQNHVDPSHDLKGS V VIYDVLDGTD
AWDSATETKTFLVSQDIQDEGWETLEVSS AVKRWVRSDSTKSKNKLEVTVESHRKGCDTL
DISVPPGSRNLPFFVVFSNDHSSGTKETRLELREMISHEQESVLKKLSKDGSTEAGESSH
EEDTDGHVAAGSTLARRKRS AGAGSHCQKTSLRVNFEDIGWDSWIIAPKEYEAYECKGGC
FFPLADDVTPTKHAIVQTLVHLKFPTKVGKACCVPTKLSPIS VLYKDDMGVPTLKYHYEG
MSVAECGCR (SEQ ID NO: 5) Aspects of the present disclosure provide methods and compositions comprising one or more BMPs in combination with one or more proteins associated with a BMP.
For example, the composition can comprise: BMP2, BMP6, BMP7 and/or BMP9 and Noggin;
BMP2, BMP6, BMP7 and/or BMP9 and Activin; or BMP2, BMP6, BMP7 and/or BMP9 and one or more BMP peptides. In some embodiments, compositions may include a fusion protein derived from sequences of two or more proteins or peptides (e.g., comprising portions of BMP2 and BMP6), or a chimeric protein (e.g., comprising portions of BMP7 and Activin).
In some embodiments, chimeric proteins can comprise portions of different BMPs. In some embodiments, chimeric proteins may be heterodimers. In some embodiments, chimeric proteins can comprise portions of proteins associated with BMPs. In some embodiments, chimeric proteins can comprise portions of BMPs and portions of proteins associated with BMPs. For example, chimeric proteins can comprise portions of a BMP and an Activin. In some embodiments, chimeric proteins can comprise portions of BMP2 and BMP7.
Chimeric proteins encompassed by the present disclosure can comprise portions of any BMP and/or any protein associated with BMPs. In some embodiments, chimeric proteins may have one or more improved properties relative to native BMPs and/or native proteins associated with BMPs. In some embodiments, chimeric proteins may have one or more amino acid modifications such as amino acid substitutions, insertions, additions or deletions. Without wishing to be bound by any theory, heterodimers, chimeric proteins, and amino acid modifications may have increased binding affinity or specificity to BMP
receptors, which may prohibit BMP antagonists from negatively regulating the BMP signaling pathways. Any methods known in the art for producing chimeric proteins can be used for generating BMP
chimeric proteins. BMP chimeric proteins and their biological effects are discussed further in, for example, Seeherman et al., (A BMP/activin A chimera is superior to native BMPs and induces bone repair in nonhuman primates when delivered in a composite matrix;
Sci. Transl.
Med. 11, eaar4953 (2019)), which is incorporated herein by reference in its entirety.
In some embodiments, compositions described herein comprise one or more proteins associated with a BMP and do not comprise one or more BMPs. For example, a composition can comprise Noggin, Activin, and/or one or more BMP peptides, or any other protein associated with a BMP disclosed herein.
Compositions described herein comprising one or more BMPs and/or one or more proteins associated with BMPs can be administered to enhance or preserve facial contours or improve the attractiveness of facial contours. In some embodiments, enhancing or preserving facial contours or improving the attractiveness of facial contours involves inhibiting or reversing bone resorption. Bone resorption refers to resorption of bone tissues by which osteoclasts break down the tissue in bones. Bone structure and mineral density is gradually lost during this process. Inhibiting or reversing bone resorption can re-build or strengthen the natural facial contours. In some embodiments, enhancing or preserving facial contours or improving the attractiveness of facial contours involves bone augmentation. In some embodiments, enhancing or preserving facial contours or improving the attractiveness of facial contours involves cartilage augmentation. Bone augmentation or cartilage augmentation in the face can result in a fuller appearance of the face. In some embodiments, bone augmentation or cartilage augmentation can result in effects similar to implanting facial bones or cartilage tissues. In some embodiments, compositions described herein comprising one or more BMPs and/or one or more proteins associated with BMPs can be administered to produce bone augmentation and/or cartilage augmentation effects with or without enhancing or preserving facial contours.
In some embodiments, "enhancing" facial contours involves restoring facial contours during or after bone resorption. In some embodiments, "preserving" facial contours involves maintaining facial contours before actual or noticeable bone resorption has occurred.
Compositions described herein comprising one or more BMPs can be administered to prevent or reduce skin wrinkles and/or improve skin texture or evenness of skin tone. As used herein, skin texture refers to skin surface conditions in general. Skin texture can be generally categorized into even or uneven skin texture. Even skin texture in some embodiments can be considered as soft, smooth, firm, or hydrated. Uneven skin texture in some embodiments can be considered as coarse, rough, bumpy, dull, or dry. Uneven skin texture can also include the appearance of "macro-texture" (e.g., fine lines and wrinkles) as disclosed in, for example, EP2640347A2, which is incorporated herein by reference in its entirety. In some embodiments, skin "evenness" can be determined based at least in part on the criteria listed above ¨ the degree of softness, smoothness, firmness, and/or hydration, or coarseness, roughness, bumpiness, dullness, and/or dryness.
Any technique known in the art can be used for assessing and determining skin texture or the evenness of the skin. As an example, a digital camera equipped with a suitable lens for facial imaging (e.g., 60mm Nikor lens) can be used for capturing facial images to determine skin texture. The region of interest (ROT) can be marked manually based on predefined facial landmarks such as corners of the eyes and bridge of the nose. The degree of textured skin in the ROT can be quantified using image analysis algorithms based on, for example, an Optimus software platform. Other suitable software platforms can also be used.
The software platforms can automatically locate each surface feature and can quantify the total number, length and area of facial features, based on a predetermined standard (e.g., shorter than 5 mm and less than 0.16 mm wide), known magnification used to convert pixel data to actual length and area data. Thresholds can be based on clinically important facial texture. For example, in some embodiments, lines greater than 5 mm and broader than 0.16 mm may be excluded. Because the ROT varies in shape and size, total textured area can be normalized to total ROT size to yield a Texture Area Fraction (TAF). In some embodiments, skin evenness can also be measured by analyzing the luminance homogeneity with the applications of Haralick homogeneity or chromaticity. Methods for measuring skin evenness are discussed further in, for example, Batres et al. (Cosmetics increase skin evenness:
Evidence from perceptual and physical measures; Skin Res Technol, 2019, 25(5):
672-676), which is incorporated herein by reference in its entirety.
In some embodiments, compositions described herein including one or more BMPs and/or one or more proteins associated with BMPs reduce the thickness of the stratum corneum, and/or increase the thickness of the living epidermis, resulting in less wrinkled, smoother surface texture. In some embodiments, compositions described herein including one or more BMPs and/or one or more proteins associated with BMPs stimulate the formation of adipocytes in the skin, which may contribute to fuller, less wrinkled skin, or improved texture or evenness of skin tone. In some embodiments, compositions described herein including one or more BMPs and/or one or more proteins associated with BMPs stimulate dedifferentiation of keratinocytes. Keratinocytes are the primary type of cell found in the epidermis, which is the outermost layer of the skin. Keratinocytes function in the formation of a barrier against environmental damages such as UV light and radiation. Keratinocyte dedifferentiation has biological significance during wound healing when epidermis must rapidly regenerate barrier function. In some embodiments, compositions described herein including one or more BMPs and/or one or more proteins associated with BMPs reduces, evens out, or modulates melanin formation by melanocytes. Without wishing to be bound by any theory, BMPs may be capable of affecting or regulating melanin synthesis (or skin pigmentation), so that administration of BMPs can improve the skin tone or the evenness of the skin tone (e.g., reduced pigmentation in the skin). In some embodiments, compositions described herein including one or more BMPs and/or one or more proteins associated with BMPs contributes to the appearance of more youthful or attractive skin.
In some embodiments, the presence of Noggin in a composition comprising one or more BMPs can reduce potential side effects associated with the one or more BMPs. The presence of Noggin as an inhibitor of BMP can ensure that the anti-wrinkle and/or pro-facial contouring effects of BMPs can be appropriately regulated. In some embodiments, Noggin can be formulated with or without the presence of one or more BMPs. For example, a Noggin formulation can be administered to a subject separately from administration of one or more BMPs, such as a few days after the administration of one or more BMPs.
Subjects associated with the disclosure include human and non-human subjects.
In some embodiments, a non-human subject is a non-human primate. In some embodiments, a non-human subject is a companion animal or a farm animal. In some embodiments, the subject is a subject that has exposed skin.
In some embodiments, the subject is a human subject having, suspected of having, or at risk of developing skin wrinkles. In some embodiments, the skin wrinkles are facial wrinkles. In some embodiments, the skin wrinkles are neck wrinkles. Skin wrinkles include any types of wrinkles, such as fine lines and creases that can be found or observed on human skins particularly facial and neck regions. Skin wrinkles include raised portions of the epithelial layer which are elevated above a normal surface of skin. Skin wrinkles can be caused by a variety of factors such as aging, UV light exposure, lifestyle, environmental factors, genetics, and medical treatments. In general, wrinkles start to form at the dermis layer of the skin, which constitutes fibroblast cells that synthesize the structural proteins collagens and elastin. During the formation of wrinkles, the dermis layer loses its ability to effectively repair and restore. Therefore, the skin's strength deteriorates and begins to wrinkle.
Without wishing to be bound by any theory, wrinkles can in some embodiments be classified as crinkle lines, permanent elastotic creases, dynamic expression lines, and/or gravitational folds. Crinkle lines are also known as atrophic crinkling rhytids, which are relatively shallow. They typically run in parallel to each other, often on the forehead, and disappear when the skin is stretched. Permanent elastotic creases can be associated with pale complexions and heavy sun exposure. Characterized by deep lines in the skin, they can occur at points where the skin creases naturally, such as the base of the neck, the lips, and the cheeks. Dynamic expression lines can be caused by habitual facial expressions, combined with the skin's loss of elasticity; for example, frown lines on the brow, crow's feet around the eyes, and laugh lines above the mouth. Gravitational folds can be associated with the losses of firmness of the skin with age during which the skin begins to sag, pulling away from the underlying fat and muscle. This can create folds that are particularly prominent on the neck, chin, and jowls.
A subject having, suspected of having, or at risk of developing skin wrinkles can be identified by routine examination, e.g., visual inspection, laboratory tests, physical exams, and/or dermatoscopy, as would be understood by one of ordinary skill in the art. Numbers, sites and depths of the wrinkles can be measured. The non-invasive digital fringe profilometry, which utilizes phase-shifted fringes being projected to the skin, can be used.
Digital fringe profilometry is discussed further in Sari et al (Measurement of skin wrinkles differences using novel optical skin imaging system of digital fringes profilometry; Proc.
SPIE 11044, Third International Seminar on Photonics, Optics, and Its Applications (ISPh0A
2018), 1104406 (2019)), which is incorporated herein by reference in its entirety.
In some embodiments, a subject is a human subject who desires to improve skin texture or evenness of skin tone. In some embodiments, a human subject who desires to improve skin texture or evenness of skin tone does not have visible signs of aging such as wrinkles or uneven skin tone.
In some embodiments, a subject is a human subject having, suspected of having, or at risk of having a loss of natural facial contours. Facial contours can include the shape and size of the facial skeleton and structure of a subject. With aging, natural facial contours may be lost due to facial bone resorption. Many plastic surgery or aesthetic procedures are used for preserving or improving facial contours, such as implant insertion by facial skeletal augmentation surgery, e.g., to make the face more balanced by adding fullness, making the cheekbones higher, the chin more prominent, and/or the jawline stronger and wider.
In some embodiments, a subject is a human subject who desires a change in his or her current facial contours to improve attractiveness of facial contours. A human subject may want to change his or her facial contours at least in part due to personal choices. In some embodiments, a human subject has never received any previous procedures or treatments to change facial contours. In other embodiments, a human subject has previously received one or more procedures or treatments to change facial contours.
Improved attractiveness can be assessed by subjective and/or objective methods. In some embodiments, improved attractiveness can be assessed by the human subject who is administered a composition disclosed herein. In other embodiments, improved attractiveness can be assessed by another human subject. In other embodiments, improved attractiveness can be assessed by a machine, such as a computer program or algorithm that can predict attractiveness, such as facial attractiveness. For example, a machine can comprise a computer-based system and an auto-encoder, creating a machine learning system that can automatically determine how attractive a certain facial image is. A set of facial images that are considered "attractive" or "non (less) attractive" can be provided to the system for modeling. After a composition associated with the disclosure is administered to a human subject, one or more facial images of the human subject can be uploaded to the machine learning system, which will assess whether the facial images are attractive and/or whether the facial images are more attractive than a control, such as facial images of the same subject before being administered the composition. In some embodiments, assessing attractiveness can be done by reviewing whether a facial image contains attractive or unattractive features.
A facial image that lacks pre-determined unattractive features may be considered as attractive. A facial image that has less unattractive features relative to a control may be considered to have increased attractiveness relative to the control. Solely by way of example, unattractive features can include but are not limited to, acne, age spots/sun damage, bruises, bumps, cellulite, light spots, pitting, scars, freckles, including damaged freckles, and wrinkles. In some embodiments, unattractive features can be particularly associated with the appearance of facial contours. For example, such features may include sunken cheeks, facial asymmetry, or sunken eyebrow area. In some embodiments, attractiveness is based on the observations or opinions of a population (e.g., social standards and human behaviors).
Methods and approaches for determining attractiveness of facial appearance are further described, for example, in US 10,726,601 and US 10,486,174, which are incorporated herein by reference in their entireties.
In some embodiments, a subject has been identified as having skin wrinkles or loss of natural facial contours. In other embodiments, a subject has not been identified as having skin wrinkles or loss of natural facial contours. A subject suspected of having skin wrinkles or loss of natural facial contours may be a subject that exhibits one or more symptoms or signs of skin wrinkles or loss of natural facial contours, e.g., small creases on the skin, loss of elasticity. In other embodiments, the subject has not exhibited any symptoms or signs of skin wrinkles or loss of natural facial contours and/or has no history of skin wrinkles or loss of natural facial contours. In other embodiments, the subject desires a change in skin texture or evenness of skin tone or facial contour in the belief that the change produces a more attractive appearance. A subject at risk for skin wrinkles or loss of natural facial contours may be a subject having one or more of the risk factors associated with the development of skin wrinkles and/or loss of natural facial contours. For example, risk factors associated with skin wrinkles or loss of natural facial contours can include in some embodiments:
(a) genetic factors; (b) age; (c) family history; (d) habitual exposure to UV light;
and/or (e) smoking.
In some embodiments, a subject is a human subject who has undergone, is undergoing, or will undergo gender reassignment procedures and/or surgeries.
Gender reassignment procedures and/or surgeries refers to any procedures or surgeries that change the sex assigned at birth of a subject. In some embodiments, the human subject is male assigned at birth and undergoes gender reassignment procedures and/or surgeries to change the gender to female. In some embodiments, the human subject is female assigned at birth and undergoes gender reassignment procedures and/or surgeries to change the gender to male. For example, a male subject who wants to undergo gender reassignment procedures and/or surgeries can be administered a composition disclosed herein with the goal of achieving softer and more feminine facial features as part of facial feminization surgery during the male-to-female transition. In some embodiments, before and after facial images can verify whether administration of compositions disclosed herein achieves the desired effects.
In some embodiments, a subject is elderly (e.g., 65 years old or older).
Elderly subjects are more prone to develop skin wrinkles and to suffer from the loss of facial contours. For instance, the quantities and the types of skin wrinkles may be more prominent in an elderly subject than in a subject who is younger than 65 years old. In other embodiments, a subject is not elderly (e.g., a subject is younger than 65 years old). A subject who is not elderly may also be prone to developing skin wrinkles and to suffering from the loss of facial contours. As disclosed in the present application, many factors other than aging can cause or accelerate the appearance of skin wrinkles and undesirable facial contours. In some embodiments, a subject is not prone to developing skin wrinkles or to suffering from the loss of facial contours but is administered compositions disclosed herein to prevent or inhibit the formation of skin wrinkles and/or to prevent or inhibit the loss of facial contours.
In some embodiments, compositions described here that contain one or more BMPs and/or one or more proteins associated with BMPs prevent or reduce skin wrinkles by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. In some embodiments, the one or more BMP used in the methods described in the present application enhances or preserves facial contours by at least 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. In some embodiments, a control is a subject or a skin sample that is not administered a composition that contains one or more BMPs and/or one or more proteins associated with BMPs.
In some embodiments, compositions described here that contain one or more BMPs and/or one or more proteins associated with BMPs improve skin texture or evenness of skin tone by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. In some embodiments, compositions described here that contain one or more BMPs and/or one or more proteins associated with BMPs improve the attractiveness of facial contours by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control.
In some embodiments, a control is a subject or a skin sample that is not administered a composition that contains one or more BMPs and/or one or more proteins associated with BMPs.
In some embodiments, the control is the skin from the same subject at an earlier time period such as prior to the administration of a composition comprising a BMP
or a protein associated with a BMP. In some embodiments, a control is a subject having the same signs of skin wrinkles but without administration of a composition comprising a BMP
or a protein associated with a BMP. The changes to formation of skin wrinkles can be determined with methods known to one of skill in the art.
The term "effective amount" or "amount effective" in the context of a composition or dose for administration to a subject refers to an amount of the composition or dose that produces one or more desired responses in the subject, e.g., preventing or reducing skin wrinkles, improving skin texture or evenness of skin tone, enhancing or preserving facial contours, or improving the subject's perception of their own attractiveness.
In some embodiment, one or more desired responses in the subject is the change of facial appearance during a male-to-female transition or a female-to-male transition. Therefore, in some embodiments, an effective amount is any amount of a composition or dose provided in the present application that produces one or more of the desired effects and/or preventative responses as provided in the present application. The amount can be one that a clinician would believe may have a clinical benefit for a subject in need thereof, or an amount that a clinician would believe would produce an improvement in attractiveness of the subject in need thereof, or in the subject's perception of their own attractiveness. Any one of the compositions or doses as provided in the present application can be in an amount effective.
The amount can be one that is disclosed in the present application.
An effective amount can involve reducing the level of an undesired response, although in some embodiments, it involves preventing an undesired response altogether. An effective amount can also involve delaying the occurrence of an undesired response. An amount that is effective can also be an amount that produces a desired therapeutic endpoint or a desired therapeutic result. In other embodiments, the amounts effective can involve enhancing the level of a desired response, such as a therapeutic endpoint or result. An effective amount, preferably, results in a preventative result or therapeutic result or endpoint with respect to signs of skin aging such as skin wrinkles and fine lines as well as the changes of facial contours in a subject. The achievement of any of the foregoing can be monitored by routine methods and the methods as disclosed in the present application.
Effective amounts will depend, of course, on the particular subject being treated; the severity of a condition; the individual patient parameters including age, physical condition, size and weight; the duration of the treatment; the nature of concurrent therapy (if any); the specific route of administration and like factors; the subject's perception of their need for improvement in appearance and the amount of change that would satisfy that need.
Compositions, Kits, and Administration The present disclosure provides compositions, including pharmaceutical or cosmetic compositions, comprising one or more BMPs and/or BMP-associated proteins, or pharmaceutically acceptable salts thereof, and optionally a pharmaceutically acceptable excipient.
In certain embodiments, a BMP described in this application is provided in an effective amount in a composition, such as a pharmaceutical or cosmetic composition. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount. In certain embodiments, the effective amount is a cosmetically effective amount.
Compositions, such as pharmaceutical or cosmetic compositions, described in this application can be prepared by any method known in the art. In general, such preparatory methods include bringing a protein or peptide (e.g., an active ingredient) described in this application into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single-or multi-dose unit.
Pharmaceutical or cosmetic compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. A
"unit dose" is a discrete amount of a pharmaceutical or cosmetic composition comprising a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.
Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical or cosmetic composition described in this application will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
The composition may comprise, e.g., between 0.1% and 100% (w/w) active ingredient.
The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" means a pharmacologically inactive material used together with a pharmacologically active material to formulate the compositions. Pharmaceutically acceptable excipients comprise a variety of materials known in the art, including but not limited to saccharides (such as glucose, lactose, and the like), preservatives such as antimicrobial agents, reconstitution aids, colorants, saline (such as phosphate buffered saline), and buffers. Any one of the compositions provided in the present application may include a pharmaceutically acceptable excipient or carrier Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical or cosmetic compositions can include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition. Exemplary excipients include diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils (e.g., synthetic oils, semi-synthetic oils).
The term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated by reference.
Pharmaceutically acceptable salts of the compounds disclosed in this application include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2¨hydroxy¨ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3¨phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.
Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4 alky1)4- salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.
Exemplary diluents can include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.
Exemplary granulating and/or dispersing agents can include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.
Exemplary surface active agents and/or emulsifiers can include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan (Tween 60), polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60), sorbitan tristearate (Span 65), glyceryl monooleate, sorbitan monooleate (Span 80), polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Soluto1 ), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor ), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij 30)), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F-68, poloxamer P-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or mixtures thereof.
Exemplary binding agents can include starch (e.g., cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum ), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.
Exemplary preservatives can include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.
Exemplary antioxidants can include alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.
Exemplary chelating agents can include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives can include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.
Exemplary antifungal preservatives can include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.
Exemplary alcohol preservatives can include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
Exemplary acidic preservatives can include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.
Other preservatives can include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip , methylparaben, German 115, Germaben II, Neolone , Kathon , and Euxyl .
Exemplary buffering agents can include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.
Exemplary lubricating agents can include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.
Exemplary natural oils can include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic or semi-synthetic oils include, but are not limited to, butyl stearate, medium chain triglycerides (such as caprylic triglyceride and capric triglyceride), cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof. In certain embodiments, exemplary synthetic oils comprise medium chain triglycerides (such as caprylic triglyceride and capric triglyceride).
Carriers used in the manufacture of pharmaceutical or cosmetic compositions can include human collagen, recombinant collagen, collagen sponges, hyaluronic acid, or composite matrices. Carriers used in the manufacture of pharmaceutical or cosmetic compositions can include liposomes, polymeric micelles, or microspheres. In some embodiments, carriers used in the manufacture of pharmaceutical or cosmetic compositions can include any known ingredients or materials that are suitable for compositions described in the present disclosure.
In some embodiments, compositions comprising one or more BMPs and/or BMP-associated proteins are formulated for subdermal injection. In some embodiments, compositions comprising one or more BMPs and/or BMP-associated proteins are formulated for subcutaneous injection. In some embodiments, compositions comprising one or more BMPs and/or BMP-associated proteins are formulated for supraperiostial injection. In some embodiments, the subdermal injection is near periosteum. Periosteum is a dense layer of vascular connective tissue enveloping the bones except at the surfaces of the joints. It is a membrane that covers the outer surface of all bones. An injection around periosteum promotes localized effects of the BMPs and/or the proteins associated with a BMP.
Compositions described herein can be administered via any route that is suitable for the composition and the subject in need thereof.
In some embodiments, the composition comprises a dermal filler. Liquid dosage forms for subdermal, subcutaneous or supraperiostial administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients (e.g., the BMPs), the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, agents such as BMPs can be mixed with solubilizing agents such as Cremophor , alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.
Injectable preparations, for example sterile injectable aqueous or oleaginous suspensions, can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed, including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
Dermal fillers may include but are not limited to hyaluronic acid, calcium hydroxylapatite, poly-L-lactic acid, polymethylmethacrylate (PMMA), and autologous fat injections (facial fat grafting). In some embodiments, dermal fillers may include sugar threading (e.g., sugar thread lift work). Any of the dermal fillers that are approved by the FDA and are appropriate for the composition as disclosed in the present application can be used for aspects of the disclosure.
In some embodiments, compositions associated with the disclosure comprise one or more agents that enhance penetration. In some embodiments, the penetration enhancers may include but are not limited to ethanol, dimethyl sulfoxide, dimethyl isosorbide, isopropyl myristate and propylene glycol. In some embodiments, compositions can be formulated as nanoemulsions for enhancing the penetration of BMPs. In some embodiments, encapsulating compositions can be used for enhancing penetration, which includes the use of polymeric substances, waxes and amphipathic lipids capable of forming liposomes. Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like. The active ingredient can be in a micro-encapsulated form with one or more excipients as noted above.
Dosage forms for topical administration of a protein or peptide described in this application may include ointments, pastes, creams, serums, lotions, gels, powders, solutions, and/or patches. In some embodiments, the dosage form for topical administration is a cream or serum. Generally, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier or excipient and/or any needed preservatives and/or buffers as can be required. Additionally, the present disclosure contemplates the use of dermal patches for providing controlled delivery of an active ingredient into the skin. A
dermal patch, skin patch, or the like as used herein refers to a medicated adhesive patch that is placed on the skin to deliver a specific dose of a composition into the skin. Dermal or skin patches can include but are not limited to single-layer drug-in-adhesive, multi-layer drug-in-adhesive, reservoir, matrix, and vapour patches. Dosage forms, including for dermal or skin patches, can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium. Alternatively, or additionally, the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel. In some embodiments, permeation enhancers can be used for enhancing the permeation of active ingredients in the patch (e.g., BMP proteins and/or proteins associated with BMPs). In some embodiments, hypodermic needles can be used for dermal administration. In some embodiments, microneedles can be used for dermal administration.
In some embodiments, different types of administration methods can be used.
For instance, in some embodiments, compositions disclosed herein can be administered via dermal patch in combination with hyaluronic acid microneedle injection. In some embodiments, the administration is not systemic administration (e.g., the active ingredient(s) being delivered do not enter the bloodstream). In some embodiments, administration of compositions disclosed herein delivers active ingredients only to the skin surface and/or to deeper layers of the skin.
Formulations suitable for topical administration include, but are not limited to, liquid and/or semi-liquid preparations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, serums, ointments, and/or pastes, and/or solutions and/or suspensions. Topically administrable formulations may, for example, comprise various concentrations (w/w) of active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described in this application.
Although the descriptions of pharmaceutical compositions provided in this application are principally directed to pharmaceutical or cosmetic compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts.
Modification of pharmaceutical or cosmetic compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.
Proteins and peptides provided in this application are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described in this application can be decided by a physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the volumes of skin wrinkles; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, and gender of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; proteins or peptides used in combination or coincidental with the specific active ingredient employed;
and like factors well known in the medical arts.
In some embodiments, proteins or compositions disclosed in this application are formulated and/or administered in nanoparticles. Nanoparticles are particles in the nanoscale.
In some embodiments, nanoparticles are less than 1 p.m in diameter. In some embodiments, nanoparticles are between about 1 and 100 nm in diameter. Nanoparticles include organic nanoparticles, such as dendrimers, liposomes, or polymeric nanoparticles.
Nanoparticles also include inorganic nanoparticles, such as fullerenes, quantum dots, and gold nanoparticles.
Compositions may comprise an aggregate of nanoparticles. In some embodiments, the aggregate of nanoparticles is homogeneous, while in other embodiments the aggregate of nanoparticles is heterogeneous.
The exact amount of a protein or peptide, or composition comprising a protein or peptide, required to achieve an effective amount will vary from subject to subject, depending, for example, on age, and general condition of a subject, identity of the particular protein, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, any two doses of the multiple doses include different or substantially the same amounts of a protein described in this application. Dosage forms may be administered at a variety of frequencies.
In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks, or less frequent than every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue, or cell. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, tissue, or cell. In some embodiments, dose ranging studies can be conducted to establish optimal therapeutic or effective amounts of the component(s) (e.g., proteins or peptides) to be present in dosage forms. In embodiments, the component(s) are present in dosage forms in an amount effective to generate a preventative or therapeutic response to various signs of aging.
In some embodiments, the composition is provided to a subject in need thereof preventatively; e.g., prior to the subject experiencing one or more signs of aging. In some embodiments, the composition is provided to a subject who perceives the need thereof to improve their own appearance. In some embodiments, the composition is provided to a subject about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 years before the onset of one or more signs of aging. In some embodiments, the composition is provided to a subject therapeutically, i.e., after the subject has one or more signs of aging. In some embodiments, the composition is provided to a subject about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 years after the onset of the one or more signs of aging. In some embodiments, the composition is provided both preventatively and, if necessary, therapeutically (e.g., the composition is administered prior to and following the onset of the one or more signs of aging). In some embodiments, the composition is provided to a subject about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 years before the onset of one or more signs of aging and about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 years after the onset of one or more signs of aging.
In some embodiments, more than one composition associated with the disclosure is administered to a subject. In some embodiments, the compositions are administered concomitantly. In other embodiments, the compositions are not administered concomitantly.
In some embodiments, the first composition is not administered within 1 month, 1 week, 6 days, 5, days, 4 days, 3 days, 2 days, 1 day, 12 hour, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour of the second composition.
The term "concomitantly" refers to administering two or more materials/agents to a subject in a manner that is correlated in time, preferably sufficiently correlated in time such that a first composition has an effect on a second composition, such as increasing the efficacy of the second composition, preferably the two or more materials/agents are administered in combination. In some instance, a second composition has an effect on a first composition, such as regulating the efficacy of the first composition. In some embodiments, concomitant administration may encompass administration of two or more compositions within a specified period of time. In some embodiments, the two or more compositions are administered within 1 month, within 1 week, within 1 day, or within 1 hour. In some embodiments, concomitant administration encompasses simultaneous administration of two or more compositions. In some embodiments, when two or more compositions are not administered concomitantly, there is little to no effect of the first composition on the second composition.
The compositions provided in the present application may be administered according to a dosing schedule. As an example, any one of the subjects provided herein may be treated with a composition comprising one or more BMPs or proteins associated with a BMP, such as BMP2, BMP7, BMP9 and/or Noggin, according to any dosage schedule disclosed herein.
In some embodiments, the concentration of the one or more BMPs in a composition for topical administration described in this application is less than about 1%, less than about 2%, less than about 3%, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, or less than about 80%, of the composition, inclusive of all ranges and subranges therebetween. In certain embodiments, the concentration of the one or more BMPs in the composition described in this application is less than about 3% of the composition. In some embodiments, the concentration of the one or more BMPs in the composition described in this application can be any concentration suitable for the method.
In some embodiments, the concentration of Noggin in a composition for topical administration described in this application is less than about 1%, less than about 2%, less than about 3%, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, of the composition, inclusive of all ranges and subranges therebetween. In certain embodiments, the concentration of Noggin in the composition for topical administration described in this application is less than about 3% of the composition. In some embodiments, the concentration of Noggin in the composition for topical administration described in this application can be any concentration suitable for the method.
In some embodiments, the concentration of one or more BMP peptides in a composition for topical administration described in this application is less than about 1%, less than about 2%, less than about 3%, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, of the composition, inclusive of all ranges and subranges therebetween. In some embodiments, the concentration of the BMP peptide in the composition for topical administration described in this application is less than about 10% of the composition. In some embodiments, the concentration of the BMP peptide in the composition for topical administration described in this application can be any concentration suitable for the method.
In some embodiments, the concentration of a protein associated with a BMP in a composition for topical administration described in this application is less than about 1%, less than about 2%, less than about 3%, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, of the composition, inclusive of all ranges and subranges therebetween. In some embodiments, the concentration of proteins associated with a BMP in the composition for topical administration described in this application is less than about 10% of the composition. In some embodiments, the concentration of a protein associated with a BMP in the composition for topical administration described in this application can be any concentration suitable for the method.
In some embodiments, the one or more BMPs described in this application are subdermally administered at a dose of about 50-1,500 ng/mL, about 100-1,000 ng/mL, about 150-950 ng/mL, about 200-900 ng/mL, about 250-850 ng/mL, about 300-800 ng/mL, about 350-400 ng/mL, about 100-950 ng/mL, about 50-400 ng/mL, about 200-1,500 ng/mL, about 500-600 ng/mL, about 550-850 ng/mL, or about 650-1,000 ng/mL per administration site, inclusive of all ranges and subranges therebetween, to a subject in need thereof. In some embodiments, the one or more BMPs described in this application are subdermally administered at a dose of about 100-1,000 ng/mL per administration site to a subject in need thereof. In some embodiments, the one or more BMPs described in this application are subdermally administered at any suitable dose per administration site to a subject in need thereof. In some embodiments, the one or more BMPs described in this application are subcutaneouslly or supraperiostially administered at any suitable dose per administration site to a subject in need thereof.
In some embodiments, Noggin is subdermally administered at a dose of about 50-1,500 ng/mL, about 100-1,000 ng/mL, about 150-950 ng/mL, about 200-900 ng/mL, about 250-850 ng/mL, about 300-800 ng/mL, about 350-400 ng/mL, about 100-950 ng/mL, about 50-400 ng/mL, about 200-1,500 ng/mL, about 500-600 ng/mL, about 550-850 ng/mL, or about 650-1,000 ng/mL per administration site, inclusive of all ranges and subranges therebetween, to a subject in need thereof. In some embodiments, Noggin is subdermally administered at a dose of about 100-1,000 ng/mL per administration site to a subject in need thereof. In some embodiments, Noggin is subdermally administered at any suitable dose per administration site to a subject in need thereof. In some embodiments, Noggin is subcutaneouslly or supraperiostially administered at any suitable dose per administration site to a subject in need thereof.
In some embodiments, other proteins associated with a BMP described in this application are subdermally administered at a dose of about 50-1,500 ng/mL, about 100-1,000 ng/mL, about 150-950 ng/mL, about 200-900 ng/mL, about 250-850 ng/mL, about 300-800 ng/mL, about 350-400 ng/mL, about 100-950 ng/mL, about 50-400 ng/mL, about 200-1,500 ng/mL, about 500-600 ng/mL, about 550-850 ng/mL, or about 650-1,000 ng/mL
per administration site, inclusive of all ranges and subranges therebetween, to a subject in need thereof. In some embodiments, other proteins associated with a BMP
described in this application are subdermally administered at a dose of about 100-1,000 ng/mL
per administration site to a subject in need thereof. In some embodiments, other proteins associated with a BMP described in this application are subdermally administered at any suitable dose per administration site to a subject in need thereof. In some embodiments, a protein associated with a BMP is subcutaneouslly or supraperiostially administered at any suitable dose per administration site to a subject in need thereof.
Dose ranges as described in this application provide guidance for the administration of provided pharmaceutical or cosmetic compositions to an adult. The amount to be administered to, for example, an elderly subject can be determined by a medical practitioner or person skilled in the art and can, in some embodiments, be lower or higher than the amount that is administered to a younger subject.
Compositions as described in this application can be administered in combination with one or more additional pharmaceutical or cosmetic agents (e.g., therapeutically and/or prophylactically active agents). The compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their activity, improve bioavailability, improve safety, reduce and/or modify metabolism, inhibit excretion, and/or modify distribution in a subject or cell. It will also be appreciated that the therapy employed may achieve a desired effect for the same purpose such as skin wrinkle reduction, and/or it may achieve different effects. In certain embodiments, a pharmaceutical or cosmetic composition described in this application, including a protein or peptide described in this application and an additional pharmaceutical agent, shows a synergistic effect that is absent in a pharmaceutical or cosmetic composition including one of the proteins or peptides described in this application and the additional pharmaceutical agent, but not both.
The composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical or cosmetic agents, which may be useful as, e.g., combination therapies. Pharmaceutical agents include therapeutically active agents.
Pharmaceutical agents also include prophylactically active agents.
Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In certain embodiments, the additional pharmaceutical or cosmetic agent is a pharmaceutical or cosmetic agent useful for preventing or alleviating signs of aging (e.g., preventing or reducing skin wrinkles, enhancing or preserving facial contours). Each additional pharmaceutical or cosmetic agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical or cosmetic agent. The additional pharmaceutical or cosmetic agents may also be administered together with each other and/or with the composition described in this application in a single dose or administered separately in different doses. The particular combination to employ in a regimen will take into account compatibility of the protein or peptide described in this application with the additional pharmaceutical or cosmetic agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical or cosmetic agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.
In some embodiments, one or more additional pharmaceutical or cosmetic agents can be any agents that are useful for anti-aging purposes. A non-limiting list of pharmaceutical or cosmetic agents can include botulinum toxin (Botox), fillers, sunscreens, skincare products, antioxidants such as polyphenols, flavonoids, Vitamins C, B3, E, and K, hormone replacement therapy, collagens, cell regulators, such as retinols and their derivates (retinyl palmitate, retinyl propionate, retinyl linoleate and other retinyl complex), peptides and growth factors, superficial peels (a-f3-, lipo-hydroxy acids (HA), trichloroacetic acid (TCA) 10-30%), medium-depth peels (TCA above 30 to 50%), deep peels (TCA > 50%, phenol), placental extract and polycyclic aromatic hydrocarbons (PAHs), natural plant such as tragacanth, natural oils extracts such as pomegranate oil, fennel oil, citrus oil, rosemary oil, chamomile oil, jojoba oil, rosehip oil, biological active ingredients such as acetyl hexapeptide-8, aspartic acid, minerals such as Zn or Mg Silicates, platelet-rich plasma (PRP), and other natural additives such as glutathione, hyaluronic acid. One or more aesthetic procedures (invasive or non-invasive) can be implemented in combination with compositions disclosed herein comprising one or more BMPs and/or proteins associated with a BMP. A
non-limiting list of aesthetic procedures can include visible light devices, intense pulsed light (HT), ablative and nonablative laser photo¨rejuvenation, radiofrequency (RF), injectable skin biostimulation and rejuvenation, prevention of dynamic wrinkles, correction of static, anatomical wrinkles, restoration (redistribution) of fat and volume loss, and skin augmentation and contouring.
In some embodiments, compositions can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical or cosmetic agents by using devices for enhanced penetration through the skin. For example, devices that rely on ultrasound, microcurrent, or microneedles can be used. In some embodiments, the devices are also used for delivering the one or more additional pharmaceutical or cosmetic agents.
Also encompassed by the disclosure are kits (e.g., pharmaceutical or cosmetic packs) comprising a composition comprising one or more BMPs and/or one or more proteins associated with a BMP for use in administering the composition to prevent or reduce skin wrinkles and/or to enhance or preserve facial contours. The kits provided may comprise a composition, such as a pharmaceutical or cosmetic composition, or a protein or peptide described in this application, and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical or cosmetic composition or a protein or peptide described in this application. In some embodiments, the pharmaceutical or cosmetic composition or a protein or peptide described in this application provided in a first container and a second container are combined to form one unit dosage form. Thus, in one aspect, provided are kits including a first container comprising a composition, protein, or peptide described in this application. In certain embodiments, the kits are useful for alleviating signs of aging in a subject in need thereof. In certain embodiments, the kits are useful for preventing or reducing the appearance of skin wrinkles in a subject in need thereof. In certain embodiments, the kits are useful for enhancing or preserving facial contours in a subject in need thereof.
In certain embodiments, a kit described in this application further includes instructions for using the kit. A kit described in this application may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information. In certain embodiments, the kits and instructions provide for alleviating signs of aging in a subject in need thereof. In certain embodiments, the kits and instructions provide for preventing or reducing the appearance of skin wrinkles in a subject in need thereof. In certain embodiments, the kits and instructions provide for improving skin texture or evenness of skin tone in a subject in need thereof. In certain embodiments, the kits and instructions provide for enhancing or preserving facial contours in a subject in need thereof. In certain embodiments, the kits and instructions provide for improving the attractiveness of facial contours in a subject in need thereof. A kit described in this application may include one or more additional pharmaceutical agents described in this application as a separate composition or one or more additional aesthetic procedures as a separate instruction (e.g., recommendation for receiving intense pulsed light).
Host cells The term "host cell" refers to a cell that can be used to express a polynucleotide, such as a polynucleotide that encodes a BMP or a protein associated with a BMP. The terms "genetically modified host cell," "recombinant host cell," and "recombinant strain" are used interchangeably and refer to host cells that have been genetically modified by, e.g., cloning and transformation methods, or by other methods known in the art (e.g., selective editing methods). Thus, the terms include a host cell (e.g., bacterial cell, yeast cell, fungal cell, insect cell, plant cell, mammalian cell, human cell, etc.) that has been genetically altered, modified, or engineered, so that it exhibits an altered, modified, or different genotype and/or phenotype, as compared to the naturally-occurring cell from which it was derived. It is understood that the term "cell," as used in this application, may refer to a single cell or a population of cells, such as a population of cells belonging to the same cell line or strain. Use of the singular term "cell" should not be construed to refer explicitly to a single cell rather than a population of cells.
The term "heterologous" with respect to a polynucleotide, such as a polynucleotide comprising a gene, is used interchangeably with the term "exogenous" and the term "recombinant" and refers to: a polynucleotide that has been artificially supplied to a biological system; a polynucleotide that has been modified within a biological system, or a polynucleotide whose expression or regulation has been manipulated within a biological system. A heterologous polynucleotide that is introduced into or expressed in a host cell may be a polynucleotide that comes from a different organism or species than the host cell, or may be a synthetic polynucleotide, or may be a polynucleotide that is also endogenously expressed in the same organism or species as the host cell. For example, a polynucleotide that is endogenously expressed in a host cell may be considered heterologous when it is situated non-naturally in the host cell; expressed recombinantly in the host cell, either stably or transiently; modified within the host cell; selectively edited within the host cell; expressed in a copy number that differs from the naturally occurring copy number within the host cell; or expressed in a non-natural way within the host cell, such as by manipulating regulatory regions that control expression of the polynucleotide. In some embodiments, a heterologous polynucleotide is a polynucleotide that is endogenously expressed in a host cell but whose expression is driven by a promoter that does not naturally regulate expression of the polynucleotide. In other embodiments, a heterologous polynucleotide is a polynucleotide that is endogenously expressed in a host cell and whose expression is driven by a promoter that does naturally regulate expression of the polynucleotide, but the promoter or another regulatory region is modified. In some embodiments, the promoter is recombinantly activated or repressed. For example, gene-editing based techniques may be used to regulate expression of a polynucleotide, including an endogenous polynucleotide, from a promoter, including an endogenous promoter. See, e.g., Chavez et al., Nat Methods. 2016 Jul; 13(7):
563-567. A heterologous polynucleotide may comprise a wild-type sequence or a mutant sequence as compared with a reference polynucleotide sequence.
Suitable host cells include, but are not limited to: yeast cells, bacterial cells, algal cells, plant cells, fungal cells, insect cells, and animal cells, including mammalian cells.
Suitable yeast host cells include, but are not limited to: Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, and Yarrowia. In some embodiments, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccaromyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Komagataella phaffii, formerly known as Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica.
In some embodiments, the yeast strain is an industrial polyploid yeast strain.
Other non-limiting examples of fungal cells include cells obtained from Aspergillus spp., Penicillium spp., Fusarium spp., Rhizopus spp., Acremonium spp., Neurospora spp., Sordaria spp., Magnaporthe spp., Allomyces spp., Ustilago spp., Botrytis spp., and Trichoderma spp.
In certain embodiments, the host cell is an algal cell such as, Chlamydomonas (e.g., C.
Reinhardtii) and Phormidium (P. sp. ATCC29409).
In other embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include gram positive, gram negative, and gram-variable bacterial cells. The host cell may be a species of, but not limited to: Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Saccharopolyspora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermos ynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia, and Zymomonas.
In some embodiments, the bacterial host strain is an industrial strain.
Numerous bacterial industrial strains are known and suitable for the methods and compositions described in this application. In some embodiments, the bacterial host cell is of the Agrobacterium species (e.g., A. radiobacter, A. rhizogenes, A. rubi), the Arthrobacterspecies (e.g., A. aurescens, A. citreus, A. globformis, A.
hydrocarboglutamicus, A. mysorens, A. nicotianae, A. paraffineus, A.
protophonniae, A.
roseoparaffinus, A. sulfureus, A. ureafaciens), the Bacillus species (e.g., B.
thuringiensis, B.
anthracis, B. megaterium, B. subtilis, B. lentus, B. circulars, B. pumilus, B.
lautus, B.
coagulans, B. brevis, B. firmus, B. alkaophius, B. lichernformis, B. clausii, B.
stearothermophilus, B. halodurans and B. amyloliquefaciens. In particular embodiments, the host cell will be an industrial Bacillus strain including but not limited to B. subtilis, B.
pumilus, B. lichernformis, B. megaterium, B. clausii, B. stearothermophilus and B.
amyloliquefaciens. In some embodiments, the host cell will be an industrial Clostridium species (e.g., C. acetobutylicum, C. tetani E88, C. lituseburense, C.
saccharobutylicum, C. perfringens, C. beijerinckii). In some embodiments, the host cell will be an industrial Corynebacterium species (e.g., C. glutamicum, C.
acetoacidophilum). In some embodiments, the host cell will be an industrial Escherichia species (e.g., E. coli). In some embodiments, the host cell will be an industrial Erwinia species (e.g., E. uredovora, E.
carotovora, E. ananas, E. herbicola, E. punctata, E. terreus). In some embodiments, the host cell will be an industrial Pantoea species (e.g., P. citrea, P. agglomerans).
In some embodiments, the host cell will be an industrial Pseudomonas species, (e.g., P. putida, P.
aeruginosa, P. mevalonii). In some embodiments, the host cell will be an industrial Streptococcus species (e.g., S. equisimiles, S. pyo genes, S.
uberis). In some embodiments, the host cell will be an industrial Streptomyces species (e.g., S. ambofaciens, S.
achromo genes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S.
fun gicidicus, S.
griseus, S. lividans). In some embodiments, the host cell will be an industrial Zymomonas species (e.g., Z. mobilis, Z. lipolytica), and the like.
The present disclosure is also suitable for use with a variety of animal cell types, including mammalian cells, for example, human (including HEK 293, HEK 293T, A549, HepG2, HeLa, WI38, PER.C6 and Bowes melanoma cells), non-human primate (including COS-1, COS-7) mouse (including 3T3, C2C12, ROS 17/2.8 (osteosarcoma cells), NSO, NS1, Sp2/0), hamster (CHO, BHK), monkey (COS, FRhL, Vero), insect cells, for example fall armyworm (including Sf9 and Sf21), silkmoth (including BmN), cabbage looper (including BTI-Tn-5B1-4) and common fruit fly (including Schneider 2), and hybridoma cell lines.
In some embodiments, the effect of BMPs and/or proteins associated with BMPs is tested in one or more cell types such as mesenchymal stem cells, preosteoblast cells, and/or osteoblast cells.
In various embodiments, strains that may be used in the practice of the disclosure including both prokaryotic and eukaryotic strains, and are readily accessible to the public from a number of culture collections such as American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
Culturing of Host Cells Any of the cells disclosed in this application can be cultured in media of any type (rich or minimal) and any composition prior to, during, and/or after contact and/or integration of a nucleic acid. The conditions of the culture or culturing process can be optimized through routine experimentation as would be understood by one of ordinary skill in the art. In some embodiments, the selected media is supplemented with various components. In some embodiments, the concentration and amount of a supplemental component is optimized. In some embodiments, other aspects of the media and growth conditions (e.g., pH, temperature, etc.) are optimized through routine experimentation. In some embodiments, the frequency that the media is supplemented with one or more supplemental components, and the amount of time that the cell is cultured, is optimized.
Culturing of the cells described in this application can be performed in culture vessels known and used in the art. In some embodiments, an aerated reaction vessel (e.g., a stirred tank reactor) is used to culture the cells. In some embodiments, a bioreactor or fermenter is used to culture the cell. Thus, in some embodiments, the cells are used in fermentation. As used in this application, the terms "bioreactor" and "fermenter" are interchangeably used and refer to an enclosure, or partial enclosure, in which a biological, biochemical and/or chemical reaction takes place that involves a living organism or part of a living organism. A "large-scale bioreactor" or "industrial-scale bioreactor" is a bioreactor that is used to generate a product on a commercial or quasi-commercial scale. Large scale bioreactors typically have volumes in the range of liters, hundreds of liters, thousands of liters, or more.
Non-limiting examples of bioreactors include: stirred tank fermenters, bioreactors agitated by rotating mixing devices, chemostats, bioreactors agitated by shaking devices, airlift fermenters, packed-bed reactors, fixed-bed reactors, fluidized bed bioreactors, bioreactors employing wave induced agitation, centrifugal bioreactors, roller bottles, and hollow fiber bioreactors, roller apparatuses (for example benchtop, cart-mounted, and/or automated varieties), vertically-stacked plates, spinner flasks, stirring or rocking flasks, shaken multi-well plates, MD bottles, T-flasks, Roux bottles, multiple-surface tissue culture propagators, modified fermenters, and coated beads (e.g., beads coated with serum proteins, nitrocellulose, or carboxymethyl cellulose to prevent cell attachment).
In some embodiments, the bioreactor includes a cell culture system where the cell (e.g., yeast cell) is in contact with moving liquids and/or gas bubbles. In some embodiments, the cell or cell culture is grown in suspension. In other embodiments, the cell or cell culture is attached to a solid phase carrier. Non-limiting examples of a carrier system includes microcarriers (e.g., polymer spheres, microbeads, and microdisks that can be porous or non-porous), cross-linked beads (e.g., dextran) charged with specific chemical groups (e.g., tertiary amine groups), 2D microcarriers including cells trapped in nonporous polymer fibers, 3D carriers (e.g., carrier fibers, hollow fibers, multicartridge reactors, and semi-permeable membranes that can comprising porous fibers), microcarriers having reduced ion exchange capacity, encapsulation cells, capillaries, and aggregates. In some embodiments, carriers are fabricated from materials such as dextran, gelatin, glass, or cellulose.
In some embodiments, industrial-scale processes are operated in continuous, semi-continuous or non-continuous modes. Non-limiting examples of operation modes are batch, fed batch, extended batch, repetitive batch, draw/fill, rotating-wall, spinning flask, and/or perfusion mode of operation. In some embodiments, a bioreactor allows continuous or semi-continuous replenishment of the substrate stock, for example a carbohydrate source and/or continuous or semi-continuous separation of the product, from the bioreactor.
In some embodiments, the bioreactor or fermenter includes a sensor and/or a control system to measure and/or adjust reaction parameters. Non-limiting examples of reaction parameters include biological parameters (e.g., growth rate, cell size, cell number, cell density, cell type, or cell state, etc.), chemical parameters (e.g., pH, redox-potential, concentration of reaction substrate and/or product, concentration of dissolved gases, such as oxygen concentration and CO2 concentration, nutrient concentrations, metabolite concentrations, concentration of an oligopeptide, concentration of an amino acid, concentration of a vitamin, concentration of a hormone, concentration of an additive, serum concentration, ionic strength, concentration of an ion, relative humidity, molarity, osmolarity, concentration of other chemicals, for example buffering agents, adjuvants, or reaction by-products), physical/mechanical parameters (e.g., density, conductivity, degree of agitation, pressure, and flow rate, shear stress, shear rate, viscosity, color, turbidity, light absorption, mixing rate, conversion rate, as well as thermodynamic parameters, such as temperature, light intensity/quality, etc.). Sensors to measure the parameters described in this application are well known to one of ordinary skill in the relevant mechanical and electronic arts. Control systems to adjust the parameters in a bioreactor based on the inputs from a sensor described in this application are well known to one of ordinary skill in the art in bioreactor engineering.
In some embodiments, the method involves batch fermentation (e.g., shake flask fermentation). General considerations for batch fermentation (e.g., shake flask fermentation) include the level of oxygen and glucose. For example, batch fermentation (e.g., shake flask fermentation) may be oxygen and glucose limited, so in some embodiments, the capability of a strain to perform in a well-designed fed-batch fermentation is underestimated.
In some embodiments, the cells of the present disclosure are adapted to produce BMPs or proteins associated with a BMP in vivo. In some embodiments, the cells are adapted to secrete one or more BMPs or proteins associated with a BMP. In some embodiments, the cells of the present disclosure are lysed, and the remaining lysates are recovered for subsequent use. In some embodiments, any of the methods described in this application may include isolation and/or purification of BMPs or proteins associated with a BMP. For example, the isolation and/or purification can involve one or more of cell lysis, centrifugation, extraction, column chromatography, distillation, crystallization, and lyophilization.
Expression of Nucleic Acids in Host Cells Aspects of the present disclosure relate to recombinant proteins, functional modifications and variants thereof, as well as their uses. For example, the methods described in this application may be used to produce BMPs or proteins associated with a BMP. The methods may comprise using a host cell comprising a protein or peptide disclosed in this application, cell lysate, isolated protein or peptide, or any combination thereof. Methods comprising recombinant expression of genes encoding a protein or peptide disclosed in this application in a host cell are encompassed by the present disclosure.
A nucleic acid encoding any of the recombinant polypeptides (e.g., BMP2, BMP9, Noggin) described in this application may be incorporated into any appropriate vector through any method known in the art. For example, the vector may be an expression vector, including but not limited to a viral vector (e.g., a lentiviral, retroviral, adenoviral, or adeno-associated viral vector), any vector suitable for transient expression, any vector suitable for constitutive expression, or any vector suitable for inducible expression (e.g., a galactose-inducible or doxycycline-inducible vector).
A vector encoding any of the recombinant polypeptides (e.g., BMP2, BMP9, Noggin) described in this application may be introduced into a suitable host cell using any method known in the art. Non-limiting examples of yeast transformation protocols are described in Gietz et al., Yeast transformation can be conducted by the LiAc/SS Carrier DNA/PEG
method. Methods Mol Biol. 2006;313:107-20, which is hereby incorporated by reference in its entirety. Host cells may be cultured under any conditions suitable as would be understood by one of ordinary skill in the art. For example, any media, temperature, and incubation conditions known in the art may be used. For host cells carrying an inducible vector, cells may be cultured with an appropriate inducible agent to promote expression.
In some embodiments, a vector replicates autonomously in the cell. In some embodiments, a vector integrates into a chromosome within a cell. A vector can contain one or more endonuclease restriction sites that are cut by a restriction endonuclease to insert and ligate a nucleic acid containing a gene described in this application to produce a recombinant vector that is able to replicate in a cell. Vectors are typically composed of DNA, although RNA vectors are also available. Cloning vectors include, but are not limited to: plasmids, fosmids, phagemids, virus genomes and artificial chromosomes. As used in this application, the terms "expression vector" or "expression construct" refer to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. In some embodiments, the nucleic acid sequence of a gene described in this application is inserted into a cloning vector so that it is operably joined to regulatory sequences and, in some embodiments, expressed as an RNA transcript. In some embodiments, the vector contains one or more markers, such as a selectable marker as described in this application, to identify cells transformed or transfected with the recombinant vector. In some embodiments, a host cell has already been transformed with one or more vectors. In some embodiments, a host cell that has been transformed with one or more vectors is subsequently transformed with one or more vectors.
In some embodiments, a host cell is transformed simultaneously with more than one vector.
In some embodiments, a cell that has been transformed with a vector or an expression cassette incorporates all or part of the vector or expression cassette into its genome. In some embodiments, the nucleic acid sequence of a gene described in this application is codon-optimized. Codon optimization may increase production of the gene product by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, including all values in between) relative to a reference sequence that is not codon-optimized.
In some embodiments, the nucleic acid encoding any of the proteins described in this application is under the control of regulatory sequences (e.g., enhancer sequences). In some embodiments, a nucleic acid is expressed under the control of a promoter. The promoter can be a native promoter, e.g., the promoter of the gene in its endogenous context, which provides normal regulation of expression of the gene. Alternatively, a promoter can be a promoter that is different from the native promoter of the gene, e.g., the promoter is different from the promoter of the gene in its endogenous context.
In some embodiments, the promoter is a eukaryotic promoter. Non-limiting examples of eukaryotic promoters include TDH3, PGK1, PKC1, PDC1, TEF1, TEF2, RPL18B, SSA1, TDH2, PYK1, TPI1, GAL1, GAL10, GAL7, GAL3, GAL2, MET3, MET25, HXT3, HXT7, ACT1, ADH1, ADH2, CUP1-1, EN02, and SOD1, as would be known to one of ordinary skill in the art (see, e.g., Addgene website: blog.addgene.org/plasmids-101-the-promoter-region). In some embodiments, the promoter is a prokaryotic promoter (e.g., bacteriophage or bacterial promoter). Non-limiting examples of bacteriophage promoters include Pls icon, T3, T7, SP6, and PL. Non-limiting examples of bacterial promoters include Pbad, PmgrB, Ptrc2, Plac/ara, Ptac, and Pm.
In some embodiments, the promoter is an inducible promoter. As used in this application, an "inducible promoter" is a promoter controlled by the presence or absence of a molecule. Non-limiting examples of inducible promoters include chemically regulated promoters and physically regulated promoters. For chemically regulated promoters, the transcriptional activity can be regulated by one or more compounds, such as methanol, alcohol, tetracycline, galactose, a steroid, a metal, an amino acid, or other compounds. For physically regulated promoters, transcriptional activity can be regulated by a phenomenon such as light or temperature. Non-limiting examples of tetracycline-regulated promoters include anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems (e.g., a tetracycline repressor protein (tetR), a tetracycline operator sequence (tet0) and a tetracycline transactivator fusion protein (tTA)). Non-limiting examples of steroid-regulated promoters include promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily. Non-limiting examples of metal-regulated promoters include promoters derived from metallothionein (proteins that bind and sequester metal ions) genes. Non-limiting examples of pathogenesis-regulated promoters include promoters induced by salicylic acid, ethylene or benzothiadiazole (BTH). Non-limiting examples of temperature/heat-inducible promoters include heat shock promoters.
Non-limiting examples of light-regulated promoters include light responsive promoters from plant cells. In certain embodiments, the inducible promoter is a galactose-inducible promoter. In some embodiments, the inducible promoter is induced by one or more physiological conditions (e.g., pH, temperature, radiation, osmotic pressure, saline gradients, cell surface binding, or concentration of one or more extrinsic or intrinsic inducing agents). Non-limiting examples of an extrinsic inducer or inducing agent include amino acids and amino acid analogs, saccharides and polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal containing compounds, salts, ions, enzyme substrate analogs, hormones or any combination.
In some embodiments, the promoter is a constitutive promoter. As used in this application, a "constitutive promoter" refers to an unregulated promoter that allows continuous transcription of a gene. Non-limiting examples of a constitutive promoter include TDH3, PGK1, PKC1, PDC1, TEF1, TEF2, RPL18B, SSA1, TDH2, PYK1, TPI1, HXT3, HXT7, ACT1, ADH1, ADH2, EN02, and SOD1. Other inducible promoters or constitutive promoters, including synthetic promoters, that may be known to one of ordinary skill in the art are also contemplated.
The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but generally include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. In particular, such 5' non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene.
Regulatory sequences may also include enhancer sequences or upstream activator sequences.
The vectors disclosed may include 5' leader or signal sequences. The regulatory sequence may also include a terminator sequence. In some embodiments, a terminator sequence marks the end of a gene in DNA during transcription. The choice and design of one or more appropriate vectors suitable for inducing expression of one or more genes described in this application in a heterologous organism is within the ability and discretion of one of ordinary skill in the art. Expression vectors containing the necessary elements for expression are commercially available and known to one of ordinary skill in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012).
Variants Aspects of the disclosure relate to nucleic acids encoding any of the polypeptides (e.g., BMP2, BMP7, BMP9, Noggin) described in this application. In some embodiments, a nucleic acid encompassed by the disclosure is a nucleic acid that hybridizes under high or medium stringency conditions to a nucleic acid encoding a protein or peptide (e.g., BMP2, BMP7, BMP9, Noggin) and is biologically active. For example, high stringency conditions of 0.2 to 1 x SSC at 65 C followed by a wash at 0.2 x SSC at 65 C can be used.
In some embodiments, a nucleic acid encompassed by the disclosure is a nucleic acid that hybridizes under low stringency conditions to a nucleic acid encoding a protein or peptide (e.g., BMP2, BMP7, BMP9, Noggin) and is biologically active. For example, low stringency conditions of 6 x SSC at room temperature followed by a wash at 2 x SSC at room temperature can be used. Other hybridization conditions include 3 x SSC at 40 or 50 C, followed by a wash in 1 or 2 x SSC at 20, 30, 40, 50, 60, or 65 'C.
Hybridizations can be conducted in the presence of formaldehyde, e.g., 10%, 20%, 30% 40% or 50%, which further increases the stringency of hybridization.
Theory and practice of nucleic acid hybridization is described, e.g., in S. Agrawal (ed.) Methods in Molecular Biology, volume 20; and Tijssen (1993) Laboratory Techniques in biochemistry and molecular biology-hybridization with nucleic acid probes, e.g., part I
chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays,"
Elsevier, New York provide a basic guide to nucleic acid hybridization.
Variants of proteins or peptides (or derivatives thereof) described in this application (e.g., BMP2, BMP7, BMP9, Noggin) are also encompassed by the present disclosure. A
variant may share at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a reference sequence, including all values in between.
Unless otherwise noted, the term "sequence identity," which is used interchangeably in this disclosure with the term "percent identity," as known in the art, refers to a relationship between the sequences of two polypeptides or polynucleotides, as determined by sequence comparison (alignment). In some embodiments, sequence identity is determined across the entire length of a sequence (e.g., BMP2, BMP7, BMP9, Noggin sequence). In some embodiments, sequence identity is determined over a region (e.g., a stretch of amino acids or nucleic acids, e.g., the sequence spanning an active site) of a sequence (e.g., BMP2, BMP7, BMP9, Noggin). For example, in some embodiments, sequence identity is determined over a region corresponding to at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or over 100% of the length of the reference sequence.
Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model, algorithm, or computer program. Identity of related polypeptides or nucleic acid sequences can be readily calculated by any of the methods known to one of ordinary skill in the art. The percent identity of two sequences (e.g., nucleic acid or amino acid sequences) may, for example, be determined using the algorithm of Karlin and Altschul Proc. Natl.
Acad. Sci.
USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad.
Sci. USA
90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST
programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990.
BLAST protein searches can be performed, for example, with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins described in this application. Where gaps exist between two sequences, Gapped BLAST can be utilized, for example, as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST ) can be used, or the parameters can be adjusted appropriately as would be understood by one of ordinary skill in the art.
Another local alignment technique which may be used, for example, is based on the Smith-Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981) "Identification of common molecular subsequences." J. Mol. Biol. 147:195-197). A general global alignment technique which may be used, for example, is the Needleman¨Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol.
48:443-453), which is based on dynamic programming. More recently, a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) was developed that purportedly produces global alignment of nucleic acid and amino acid sequences faster than other optimal global alignment methods, including the Needleman¨Wunsch algorithm. In some embodiments, the identity of two polypeptides is determined by aligning the two amino acid sequences, calculating the number of identical amino acids, and dividing by the length of one of the amino acid sequences. In some embodiments, the identity of two nucleic acids is determined by aligning the two nucleotide sequences and calculating the number of identical nucleotide and dividing by the length of one of the nucleic acids. For multiple sequence alignments, computer programs including Clustal Omega (Sievers et al., Mol Syst Biol. 2011 Oct 11;7:539) may be used.
In preferred embodiments, a sequence, including a nucleic acid or amino acid sequence, is found to have a specified percent identity to a reference sequence, when sequence identity is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad.
Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl.
Acad. Sci. USA
90:5873-77, 1993 (e.g., BLAST , NBLAST , XBLAST or Gapped BLAST programs, using default parameters of the respective programs).
In some embodiments, a sequence, including a nucleic acid or amino acid sequence, is found to have a specified percent identity to a reference sequence when sequence identity is determined using the Smith-Waterman algorithm (Smith, T.F. & Waterman, M.S.
(1981) "Identification of common molecular subsequences." J. Mol. Biol. 147:195-197) or the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol.
Biol. 48:443-453) using default parameters.
In some embodiments, a sequence, including a nucleic acid or amino acid sequence, is found to have a specified percent identity to a reference sequence when sequence identity is determined using a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) using default parameters.
In some embodiments, a sequence, including a nucleic acid or amino acid sequence, is found to have a specified percent identity to a reference sequence when sequence identity is determined using Clustal Omega (Sievers et al., Mol Syst Biol. 2011 Oct
In some embodiments, compositions described here that contain one or more BMPs and/or one or more proteins associated with BMPs improve skin texture or evenness of skin tone by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control. In some embodiments, compositions described here that contain one or more BMPs and/or one or more proteins associated with BMPs improve the attractiveness of facial contours by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control.
In some embodiments, a control is a subject or a skin sample that is not administered a composition that contains one or more BMPs and/or one or more proteins associated with BMPs.
In some embodiments, the control is the skin from the same subject at an earlier time period such as prior to the administration of a composition comprising a BMP
or a protein associated with a BMP. In some embodiments, a control is a subject having the same signs of skin wrinkles but without administration of a composition comprising a BMP
or a protein associated with a BMP. The changes to formation of skin wrinkles can be determined with methods known to one of skill in the art.
The term "effective amount" or "amount effective" in the context of a composition or dose for administration to a subject refers to an amount of the composition or dose that produces one or more desired responses in the subject, e.g., preventing or reducing skin wrinkles, improving skin texture or evenness of skin tone, enhancing or preserving facial contours, or improving the subject's perception of their own attractiveness.
In some embodiment, one or more desired responses in the subject is the change of facial appearance during a male-to-female transition or a female-to-male transition. Therefore, in some embodiments, an effective amount is any amount of a composition or dose provided in the present application that produces one or more of the desired effects and/or preventative responses as provided in the present application. The amount can be one that a clinician would believe may have a clinical benefit for a subject in need thereof, or an amount that a clinician would believe would produce an improvement in attractiveness of the subject in need thereof, or in the subject's perception of their own attractiveness. Any one of the compositions or doses as provided in the present application can be in an amount effective.
The amount can be one that is disclosed in the present application.
An effective amount can involve reducing the level of an undesired response, although in some embodiments, it involves preventing an undesired response altogether. An effective amount can also involve delaying the occurrence of an undesired response. An amount that is effective can also be an amount that produces a desired therapeutic endpoint or a desired therapeutic result. In other embodiments, the amounts effective can involve enhancing the level of a desired response, such as a therapeutic endpoint or result. An effective amount, preferably, results in a preventative result or therapeutic result or endpoint with respect to signs of skin aging such as skin wrinkles and fine lines as well as the changes of facial contours in a subject. The achievement of any of the foregoing can be monitored by routine methods and the methods as disclosed in the present application.
Effective amounts will depend, of course, on the particular subject being treated; the severity of a condition; the individual patient parameters including age, physical condition, size and weight; the duration of the treatment; the nature of concurrent therapy (if any); the specific route of administration and like factors; the subject's perception of their need for improvement in appearance and the amount of change that would satisfy that need.
Compositions, Kits, and Administration The present disclosure provides compositions, including pharmaceutical or cosmetic compositions, comprising one or more BMPs and/or BMP-associated proteins, or pharmaceutically acceptable salts thereof, and optionally a pharmaceutically acceptable excipient.
In certain embodiments, a BMP described in this application is provided in an effective amount in a composition, such as a pharmaceutical or cosmetic composition. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount. In certain embodiments, the effective amount is a cosmetically effective amount.
Compositions, such as pharmaceutical or cosmetic compositions, described in this application can be prepared by any method known in the art. In general, such preparatory methods include bringing a protein or peptide (e.g., an active ingredient) described in this application into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single-or multi-dose unit.
Pharmaceutical or cosmetic compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. A
"unit dose" is a discrete amount of a pharmaceutical or cosmetic composition comprising a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.
Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical or cosmetic composition described in this application will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
The composition may comprise, e.g., between 0.1% and 100% (w/w) active ingredient.
The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" means a pharmacologically inactive material used together with a pharmacologically active material to formulate the compositions. Pharmaceutically acceptable excipients comprise a variety of materials known in the art, including but not limited to saccharides (such as glucose, lactose, and the like), preservatives such as antimicrobial agents, reconstitution aids, colorants, saline (such as phosphate buffered saline), and buffers. Any one of the compositions provided in the present application may include a pharmaceutically acceptable excipient or carrier Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical or cosmetic compositions can include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition. Exemplary excipients include diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils (e.g., synthetic oils, semi-synthetic oils).
The term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated by reference.
Pharmaceutically acceptable salts of the compounds disclosed in this application include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2¨hydroxy¨ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3¨phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.
Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4 alky1)4- salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.
Exemplary diluents can include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.
Exemplary granulating and/or dispersing agents can include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.
Exemplary surface active agents and/or emulsifiers can include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan (Tween 60), polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60), sorbitan tristearate (Span 65), glyceryl monooleate, sorbitan monooleate (Span 80), polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Soluto1 ), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor ), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij 30)), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F-68, poloxamer P-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or mixtures thereof.
Exemplary binding agents can include starch (e.g., cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum ), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.
Exemplary preservatives can include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.
Exemplary antioxidants can include alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.
Exemplary chelating agents can include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives can include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.
Exemplary antifungal preservatives can include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.
Exemplary alcohol preservatives can include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
Exemplary acidic preservatives can include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.
Other preservatives can include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip , methylparaben, German 115, Germaben II, Neolone , Kathon , and Euxyl .
Exemplary buffering agents can include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.
Exemplary lubricating agents can include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.
Exemplary natural oils can include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic or semi-synthetic oils include, but are not limited to, butyl stearate, medium chain triglycerides (such as caprylic triglyceride and capric triglyceride), cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof. In certain embodiments, exemplary synthetic oils comprise medium chain triglycerides (such as caprylic triglyceride and capric triglyceride).
Carriers used in the manufacture of pharmaceutical or cosmetic compositions can include human collagen, recombinant collagen, collagen sponges, hyaluronic acid, or composite matrices. Carriers used in the manufacture of pharmaceutical or cosmetic compositions can include liposomes, polymeric micelles, or microspheres. In some embodiments, carriers used in the manufacture of pharmaceutical or cosmetic compositions can include any known ingredients or materials that are suitable for compositions described in the present disclosure.
In some embodiments, compositions comprising one or more BMPs and/or BMP-associated proteins are formulated for subdermal injection. In some embodiments, compositions comprising one or more BMPs and/or BMP-associated proteins are formulated for subcutaneous injection. In some embodiments, compositions comprising one or more BMPs and/or BMP-associated proteins are formulated for supraperiostial injection. In some embodiments, the subdermal injection is near periosteum. Periosteum is a dense layer of vascular connective tissue enveloping the bones except at the surfaces of the joints. It is a membrane that covers the outer surface of all bones. An injection around periosteum promotes localized effects of the BMPs and/or the proteins associated with a BMP.
Compositions described herein can be administered via any route that is suitable for the composition and the subject in need thereof.
In some embodiments, the composition comprises a dermal filler. Liquid dosage forms for subdermal, subcutaneous or supraperiostial administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients (e.g., the BMPs), the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, agents such as BMPs can be mixed with solubilizing agents such as Cremophor , alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.
Injectable preparations, for example sterile injectable aqueous or oleaginous suspensions, can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed, including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
Dermal fillers may include but are not limited to hyaluronic acid, calcium hydroxylapatite, poly-L-lactic acid, polymethylmethacrylate (PMMA), and autologous fat injections (facial fat grafting). In some embodiments, dermal fillers may include sugar threading (e.g., sugar thread lift work). Any of the dermal fillers that are approved by the FDA and are appropriate for the composition as disclosed in the present application can be used for aspects of the disclosure.
In some embodiments, compositions associated with the disclosure comprise one or more agents that enhance penetration. In some embodiments, the penetration enhancers may include but are not limited to ethanol, dimethyl sulfoxide, dimethyl isosorbide, isopropyl myristate and propylene glycol. In some embodiments, compositions can be formulated as nanoemulsions for enhancing the penetration of BMPs. In some embodiments, encapsulating compositions can be used for enhancing penetration, which includes the use of polymeric substances, waxes and amphipathic lipids capable of forming liposomes. Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like. The active ingredient can be in a micro-encapsulated form with one or more excipients as noted above.
Dosage forms for topical administration of a protein or peptide described in this application may include ointments, pastes, creams, serums, lotions, gels, powders, solutions, and/or patches. In some embodiments, the dosage form for topical administration is a cream or serum. Generally, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier or excipient and/or any needed preservatives and/or buffers as can be required. Additionally, the present disclosure contemplates the use of dermal patches for providing controlled delivery of an active ingredient into the skin. A
dermal patch, skin patch, or the like as used herein refers to a medicated adhesive patch that is placed on the skin to deliver a specific dose of a composition into the skin. Dermal or skin patches can include but are not limited to single-layer drug-in-adhesive, multi-layer drug-in-adhesive, reservoir, matrix, and vapour patches. Dosage forms, including for dermal or skin patches, can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium. Alternatively, or additionally, the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel. In some embodiments, permeation enhancers can be used for enhancing the permeation of active ingredients in the patch (e.g., BMP proteins and/or proteins associated with BMPs). In some embodiments, hypodermic needles can be used for dermal administration. In some embodiments, microneedles can be used for dermal administration.
In some embodiments, different types of administration methods can be used.
For instance, in some embodiments, compositions disclosed herein can be administered via dermal patch in combination with hyaluronic acid microneedle injection. In some embodiments, the administration is not systemic administration (e.g., the active ingredient(s) being delivered do not enter the bloodstream). In some embodiments, administration of compositions disclosed herein delivers active ingredients only to the skin surface and/or to deeper layers of the skin.
Formulations suitable for topical administration include, but are not limited to, liquid and/or semi-liquid preparations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, serums, ointments, and/or pastes, and/or solutions and/or suspensions. Topically administrable formulations may, for example, comprise various concentrations (w/w) of active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described in this application.
Although the descriptions of pharmaceutical compositions provided in this application are principally directed to pharmaceutical or cosmetic compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts.
Modification of pharmaceutical or cosmetic compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.
Proteins and peptides provided in this application are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described in this application can be decided by a physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the volumes of skin wrinkles; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, and gender of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; proteins or peptides used in combination or coincidental with the specific active ingredient employed;
and like factors well known in the medical arts.
In some embodiments, proteins or compositions disclosed in this application are formulated and/or administered in nanoparticles. Nanoparticles are particles in the nanoscale.
In some embodiments, nanoparticles are less than 1 p.m in diameter. In some embodiments, nanoparticles are between about 1 and 100 nm in diameter. Nanoparticles include organic nanoparticles, such as dendrimers, liposomes, or polymeric nanoparticles.
Nanoparticles also include inorganic nanoparticles, such as fullerenes, quantum dots, and gold nanoparticles.
Compositions may comprise an aggregate of nanoparticles. In some embodiments, the aggregate of nanoparticles is homogeneous, while in other embodiments the aggregate of nanoparticles is heterogeneous.
The exact amount of a protein or peptide, or composition comprising a protein or peptide, required to achieve an effective amount will vary from subject to subject, depending, for example, on age, and general condition of a subject, identity of the particular protein, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, any two doses of the multiple doses include different or substantially the same amounts of a protein described in this application. Dosage forms may be administered at a variety of frequencies.
In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks, or less frequent than every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue, or cell. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, tissue, or cell. In some embodiments, dose ranging studies can be conducted to establish optimal therapeutic or effective amounts of the component(s) (e.g., proteins or peptides) to be present in dosage forms. In embodiments, the component(s) are present in dosage forms in an amount effective to generate a preventative or therapeutic response to various signs of aging.
In some embodiments, the composition is provided to a subject in need thereof preventatively; e.g., prior to the subject experiencing one or more signs of aging. In some embodiments, the composition is provided to a subject who perceives the need thereof to improve their own appearance. In some embodiments, the composition is provided to a subject about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 years before the onset of one or more signs of aging. In some embodiments, the composition is provided to a subject therapeutically, i.e., after the subject has one or more signs of aging. In some embodiments, the composition is provided to a subject about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 years after the onset of the one or more signs of aging. In some embodiments, the composition is provided both preventatively and, if necessary, therapeutically (e.g., the composition is administered prior to and following the onset of the one or more signs of aging). In some embodiments, the composition is provided to a subject about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 years before the onset of one or more signs of aging and about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 years after the onset of one or more signs of aging.
In some embodiments, more than one composition associated with the disclosure is administered to a subject. In some embodiments, the compositions are administered concomitantly. In other embodiments, the compositions are not administered concomitantly.
In some embodiments, the first composition is not administered within 1 month, 1 week, 6 days, 5, days, 4 days, 3 days, 2 days, 1 day, 12 hour, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour of the second composition.
The term "concomitantly" refers to administering two or more materials/agents to a subject in a manner that is correlated in time, preferably sufficiently correlated in time such that a first composition has an effect on a second composition, such as increasing the efficacy of the second composition, preferably the two or more materials/agents are administered in combination. In some instance, a second composition has an effect on a first composition, such as regulating the efficacy of the first composition. In some embodiments, concomitant administration may encompass administration of two or more compositions within a specified period of time. In some embodiments, the two or more compositions are administered within 1 month, within 1 week, within 1 day, or within 1 hour. In some embodiments, concomitant administration encompasses simultaneous administration of two or more compositions. In some embodiments, when two or more compositions are not administered concomitantly, there is little to no effect of the first composition on the second composition.
The compositions provided in the present application may be administered according to a dosing schedule. As an example, any one of the subjects provided herein may be treated with a composition comprising one or more BMPs or proteins associated with a BMP, such as BMP2, BMP7, BMP9 and/or Noggin, according to any dosage schedule disclosed herein.
In some embodiments, the concentration of the one or more BMPs in a composition for topical administration described in this application is less than about 1%, less than about 2%, less than about 3%, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, or less than about 80%, of the composition, inclusive of all ranges and subranges therebetween. In certain embodiments, the concentration of the one or more BMPs in the composition described in this application is less than about 3% of the composition. In some embodiments, the concentration of the one or more BMPs in the composition described in this application can be any concentration suitable for the method.
In some embodiments, the concentration of Noggin in a composition for topical administration described in this application is less than about 1%, less than about 2%, less than about 3%, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, of the composition, inclusive of all ranges and subranges therebetween. In certain embodiments, the concentration of Noggin in the composition for topical administration described in this application is less than about 3% of the composition. In some embodiments, the concentration of Noggin in the composition for topical administration described in this application can be any concentration suitable for the method.
In some embodiments, the concentration of one or more BMP peptides in a composition for topical administration described in this application is less than about 1%, less than about 2%, less than about 3%, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, of the composition, inclusive of all ranges and subranges therebetween. In some embodiments, the concentration of the BMP peptide in the composition for topical administration described in this application is less than about 10% of the composition. In some embodiments, the concentration of the BMP peptide in the composition for topical administration described in this application can be any concentration suitable for the method.
In some embodiments, the concentration of a protein associated with a BMP in a composition for topical administration described in this application is less than about 1%, less than about 2%, less than about 3%, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, of the composition, inclusive of all ranges and subranges therebetween. In some embodiments, the concentration of proteins associated with a BMP in the composition for topical administration described in this application is less than about 10% of the composition. In some embodiments, the concentration of a protein associated with a BMP in the composition for topical administration described in this application can be any concentration suitable for the method.
In some embodiments, the one or more BMPs described in this application are subdermally administered at a dose of about 50-1,500 ng/mL, about 100-1,000 ng/mL, about 150-950 ng/mL, about 200-900 ng/mL, about 250-850 ng/mL, about 300-800 ng/mL, about 350-400 ng/mL, about 100-950 ng/mL, about 50-400 ng/mL, about 200-1,500 ng/mL, about 500-600 ng/mL, about 550-850 ng/mL, or about 650-1,000 ng/mL per administration site, inclusive of all ranges and subranges therebetween, to a subject in need thereof. In some embodiments, the one or more BMPs described in this application are subdermally administered at a dose of about 100-1,000 ng/mL per administration site to a subject in need thereof. In some embodiments, the one or more BMPs described in this application are subdermally administered at any suitable dose per administration site to a subject in need thereof. In some embodiments, the one or more BMPs described in this application are subcutaneouslly or supraperiostially administered at any suitable dose per administration site to a subject in need thereof.
In some embodiments, Noggin is subdermally administered at a dose of about 50-1,500 ng/mL, about 100-1,000 ng/mL, about 150-950 ng/mL, about 200-900 ng/mL, about 250-850 ng/mL, about 300-800 ng/mL, about 350-400 ng/mL, about 100-950 ng/mL, about 50-400 ng/mL, about 200-1,500 ng/mL, about 500-600 ng/mL, about 550-850 ng/mL, or about 650-1,000 ng/mL per administration site, inclusive of all ranges and subranges therebetween, to a subject in need thereof. In some embodiments, Noggin is subdermally administered at a dose of about 100-1,000 ng/mL per administration site to a subject in need thereof. In some embodiments, Noggin is subdermally administered at any suitable dose per administration site to a subject in need thereof. In some embodiments, Noggin is subcutaneouslly or supraperiostially administered at any suitable dose per administration site to a subject in need thereof.
In some embodiments, other proteins associated with a BMP described in this application are subdermally administered at a dose of about 50-1,500 ng/mL, about 100-1,000 ng/mL, about 150-950 ng/mL, about 200-900 ng/mL, about 250-850 ng/mL, about 300-800 ng/mL, about 350-400 ng/mL, about 100-950 ng/mL, about 50-400 ng/mL, about 200-1,500 ng/mL, about 500-600 ng/mL, about 550-850 ng/mL, or about 650-1,000 ng/mL
per administration site, inclusive of all ranges and subranges therebetween, to a subject in need thereof. In some embodiments, other proteins associated with a BMP
described in this application are subdermally administered at a dose of about 100-1,000 ng/mL
per administration site to a subject in need thereof. In some embodiments, other proteins associated with a BMP described in this application are subdermally administered at any suitable dose per administration site to a subject in need thereof. In some embodiments, a protein associated with a BMP is subcutaneouslly or supraperiostially administered at any suitable dose per administration site to a subject in need thereof.
Dose ranges as described in this application provide guidance for the administration of provided pharmaceutical or cosmetic compositions to an adult. The amount to be administered to, for example, an elderly subject can be determined by a medical practitioner or person skilled in the art and can, in some embodiments, be lower or higher than the amount that is administered to a younger subject.
Compositions as described in this application can be administered in combination with one or more additional pharmaceutical or cosmetic agents (e.g., therapeutically and/or prophylactically active agents). The compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their activity, improve bioavailability, improve safety, reduce and/or modify metabolism, inhibit excretion, and/or modify distribution in a subject or cell. It will also be appreciated that the therapy employed may achieve a desired effect for the same purpose such as skin wrinkle reduction, and/or it may achieve different effects. In certain embodiments, a pharmaceutical or cosmetic composition described in this application, including a protein or peptide described in this application and an additional pharmaceutical agent, shows a synergistic effect that is absent in a pharmaceutical or cosmetic composition including one of the proteins or peptides described in this application and the additional pharmaceutical agent, but not both.
The composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical or cosmetic agents, which may be useful as, e.g., combination therapies. Pharmaceutical agents include therapeutically active agents.
Pharmaceutical agents also include prophylactically active agents.
Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In certain embodiments, the additional pharmaceutical or cosmetic agent is a pharmaceutical or cosmetic agent useful for preventing or alleviating signs of aging (e.g., preventing or reducing skin wrinkles, enhancing or preserving facial contours). Each additional pharmaceutical or cosmetic agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical or cosmetic agent. The additional pharmaceutical or cosmetic agents may also be administered together with each other and/or with the composition described in this application in a single dose or administered separately in different doses. The particular combination to employ in a regimen will take into account compatibility of the protein or peptide described in this application with the additional pharmaceutical or cosmetic agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical or cosmetic agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.
In some embodiments, one or more additional pharmaceutical or cosmetic agents can be any agents that are useful for anti-aging purposes. A non-limiting list of pharmaceutical or cosmetic agents can include botulinum toxin (Botox), fillers, sunscreens, skincare products, antioxidants such as polyphenols, flavonoids, Vitamins C, B3, E, and K, hormone replacement therapy, collagens, cell regulators, such as retinols and their derivates (retinyl palmitate, retinyl propionate, retinyl linoleate and other retinyl complex), peptides and growth factors, superficial peels (a-f3-, lipo-hydroxy acids (HA), trichloroacetic acid (TCA) 10-30%), medium-depth peels (TCA above 30 to 50%), deep peels (TCA > 50%, phenol), placental extract and polycyclic aromatic hydrocarbons (PAHs), natural plant such as tragacanth, natural oils extracts such as pomegranate oil, fennel oil, citrus oil, rosemary oil, chamomile oil, jojoba oil, rosehip oil, biological active ingredients such as acetyl hexapeptide-8, aspartic acid, minerals such as Zn or Mg Silicates, platelet-rich plasma (PRP), and other natural additives such as glutathione, hyaluronic acid. One or more aesthetic procedures (invasive or non-invasive) can be implemented in combination with compositions disclosed herein comprising one or more BMPs and/or proteins associated with a BMP. A
non-limiting list of aesthetic procedures can include visible light devices, intense pulsed light (HT), ablative and nonablative laser photo¨rejuvenation, radiofrequency (RF), injectable skin biostimulation and rejuvenation, prevention of dynamic wrinkles, correction of static, anatomical wrinkles, restoration (redistribution) of fat and volume loss, and skin augmentation and contouring.
In some embodiments, compositions can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical or cosmetic agents by using devices for enhanced penetration through the skin. For example, devices that rely on ultrasound, microcurrent, or microneedles can be used. In some embodiments, the devices are also used for delivering the one or more additional pharmaceutical or cosmetic agents.
Also encompassed by the disclosure are kits (e.g., pharmaceutical or cosmetic packs) comprising a composition comprising one or more BMPs and/or one or more proteins associated with a BMP for use in administering the composition to prevent or reduce skin wrinkles and/or to enhance or preserve facial contours. The kits provided may comprise a composition, such as a pharmaceutical or cosmetic composition, or a protein or peptide described in this application, and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical or cosmetic composition or a protein or peptide described in this application. In some embodiments, the pharmaceutical or cosmetic composition or a protein or peptide described in this application provided in a first container and a second container are combined to form one unit dosage form. Thus, in one aspect, provided are kits including a first container comprising a composition, protein, or peptide described in this application. In certain embodiments, the kits are useful for alleviating signs of aging in a subject in need thereof. In certain embodiments, the kits are useful for preventing or reducing the appearance of skin wrinkles in a subject in need thereof. In certain embodiments, the kits are useful for enhancing or preserving facial contours in a subject in need thereof.
In certain embodiments, a kit described in this application further includes instructions for using the kit. A kit described in this application may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information. In certain embodiments, the kits and instructions provide for alleviating signs of aging in a subject in need thereof. In certain embodiments, the kits and instructions provide for preventing or reducing the appearance of skin wrinkles in a subject in need thereof. In certain embodiments, the kits and instructions provide for improving skin texture or evenness of skin tone in a subject in need thereof. In certain embodiments, the kits and instructions provide for enhancing or preserving facial contours in a subject in need thereof. In certain embodiments, the kits and instructions provide for improving the attractiveness of facial contours in a subject in need thereof. A kit described in this application may include one or more additional pharmaceutical agents described in this application as a separate composition or one or more additional aesthetic procedures as a separate instruction (e.g., recommendation for receiving intense pulsed light).
Host cells The term "host cell" refers to a cell that can be used to express a polynucleotide, such as a polynucleotide that encodes a BMP or a protein associated with a BMP. The terms "genetically modified host cell," "recombinant host cell," and "recombinant strain" are used interchangeably and refer to host cells that have been genetically modified by, e.g., cloning and transformation methods, or by other methods known in the art (e.g., selective editing methods). Thus, the terms include a host cell (e.g., bacterial cell, yeast cell, fungal cell, insect cell, plant cell, mammalian cell, human cell, etc.) that has been genetically altered, modified, or engineered, so that it exhibits an altered, modified, or different genotype and/or phenotype, as compared to the naturally-occurring cell from which it was derived. It is understood that the term "cell," as used in this application, may refer to a single cell or a population of cells, such as a population of cells belonging to the same cell line or strain. Use of the singular term "cell" should not be construed to refer explicitly to a single cell rather than a population of cells.
The term "heterologous" with respect to a polynucleotide, such as a polynucleotide comprising a gene, is used interchangeably with the term "exogenous" and the term "recombinant" and refers to: a polynucleotide that has been artificially supplied to a biological system; a polynucleotide that has been modified within a biological system, or a polynucleotide whose expression or regulation has been manipulated within a biological system. A heterologous polynucleotide that is introduced into or expressed in a host cell may be a polynucleotide that comes from a different organism or species than the host cell, or may be a synthetic polynucleotide, or may be a polynucleotide that is also endogenously expressed in the same organism or species as the host cell. For example, a polynucleotide that is endogenously expressed in a host cell may be considered heterologous when it is situated non-naturally in the host cell; expressed recombinantly in the host cell, either stably or transiently; modified within the host cell; selectively edited within the host cell; expressed in a copy number that differs from the naturally occurring copy number within the host cell; or expressed in a non-natural way within the host cell, such as by manipulating regulatory regions that control expression of the polynucleotide. In some embodiments, a heterologous polynucleotide is a polynucleotide that is endogenously expressed in a host cell but whose expression is driven by a promoter that does not naturally regulate expression of the polynucleotide. In other embodiments, a heterologous polynucleotide is a polynucleotide that is endogenously expressed in a host cell and whose expression is driven by a promoter that does naturally regulate expression of the polynucleotide, but the promoter or another regulatory region is modified. In some embodiments, the promoter is recombinantly activated or repressed. For example, gene-editing based techniques may be used to regulate expression of a polynucleotide, including an endogenous polynucleotide, from a promoter, including an endogenous promoter. See, e.g., Chavez et al., Nat Methods. 2016 Jul; 13(7):
563-567. A heterologous polynucleotide may comprise a wild-type sequence or a mutant sequence as compared with a reference polynucleotide sequence.
Suitable host cells include, but are not limited to: yeast cells, bacterial cells, algal cells, plant cells, fungal cells, insect cells, and animal cells, including mammalian cells.
Suitable yeast host cells include, but are not limited to: Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, and Yarrowia. In some embodiments, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccaromyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Komagataella phaffii, formerly known as Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica.
In some embodiments, the yeast strain is an industrial polyploid yeast strain.
Other non-limiting examples of fungal cells include cells obtained from Aspergillus spp., Penicillium spp., Fusarium spp., Rhizopus spp., Acremonium spp., Neurospora spp., Sordaria spp., Magnaporthe spp., Allomyces spp., Ustilago spp., Botrytis spp., and Trichoderma spp.
In certain embodiments, the host cell is an algal cell such as, Chlamydomonas (e.g., C.
Reinhardtii) and Phormidium (P. sp. ATCC29409).
In other embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include gram positive, gram negative, and gram-variable bacterial cells. The host cell may be a species of, but not limited to: Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Saccharopolyspora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermos ynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia, and Zymomonas.
In some embodiments, the bacterial host strain is an industrial strain.
Numerous bacterial industrial strains are known and suitable for the methods and compositions described in this application. In some embodiments, the bacterial host cell is of the Agrobacterium species (e.g., A. radiobacter, A. rhizogenes, A. rubi), the Arthrobacterspecies (e.g., A. aurescens, A. citreus, A. globformis, A.
hydrocarboglutamicus, A. mysorens, A. nicotianae, A. paraffineus, A.
protophonniae, A.
roseoparaffinus, A. sulfureus, A. ureafaciens), the Bacillus species (e.g., B.
thuringiensis, B.
anthracis, B. megaterium, B. subtilis, B. lentus, B. circulars, B. pumilus, B.
lautus, B.
coagulans, B. brevis, B. firmus, B. alkaophius, B. lichernformis, B. clausii, B.
stearothermophilus, B. halodurans and B. amyloliquefaciens. In particular embodiments, the host cell will be an industrial Bacillus strain including but not limited to B. subtilis, B.
pumilus, B. lichernformis, B. megaterium, B. clausii, B. stearothermophilus and B.
amyloliquefaciens. In some embodiments, the host cell will be an industrial Clostridium species (e.g., C. acetobutylicum, C. tetani E88, C. lituseburense, C.
saccharobutylicum, C. perfringens, C. beijerinckii). In some embodiments, the host cell will be an industrial Corynebacterium species (e.g., C. glutamicum, C.
acetoacidophilum). In some embodiments, the host cell will be an industrial Escherichia species (e.g., E. coli). In some embodiments, the host cell will be an industrial Erwinia species (e.g., E. uredovora, E.
carotovora, E. ananas, E. herbicola, E. punctata, E. terreus). In some embodiments, the host cell will be an industrial Pantoea species (e.g., P. citrea, P. agglomerans).
In some embodiments, the host cell will be an industrial Pseudomonas species, (e.g., P. putida, P.
aeruginosa, P. mevalonii). In some embodiments, the host cell will be an industrial Streptococcus species (e.g., S. equisimiles, S. pyo genes, S.
uberis). In some embodiments, the host cell will be an industrial Streptomyces species (e.g., S. ambofaciens, S.
achromo genes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S.
fun gicidicus, S.
griseus, S. lividans). In some embodiments, the host cell will be an industrial Zymomonas species (e.g., Z. mobilis, Z. lipolytica), and the like.
The present disclosure is also suitable for use with a variety of animal cell types, including mammalian cells, for example, human (including HEK 293, HEK 293T, A549, HepG2, HeLa, WI38, PER.C6 and Bowes melanoma cells), non-human primate (including COS-1, COS-7) mouse (including 3T3, C2C12, ROS 17/2.8 (osteosarcoma cells), NSO, NS1, Sp2/0), hamster (CHO, BHK), monkey (COS, FRhL, Vero), insect cells, for example fall armyworm (including Sf9 and Sf21), silkmoth (including BmN), cabbage looper (including BTI-Tn-5B1-4) and common fruit fly (including Schneider 2), and hybridoma cell lines.
In some embodiments, the effect of BMPs and/or proteins associated with BMPs is tested in one or more cell types such as mesenchymal stem cells, preosteoblast cells, and/or osteoblast cells.
In various embodiments, strains that may be used in the practice of the disclosure including both prokaryotic and eukaryotic strains, and are readily accessible to the public from a number of culture collections such as American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
Culturing of Host Cells Any of the cells disclosed in this application can be cultured in media of any type (rich or minimal) and any composition prior to, during, and/or after contact and/or integration of a nucleic acid. The conditions of the culture or culturing process can be optimized through routine experimentation as would be understood by one of ordinary skill in the art. In some embodiments, the selected media is supplemented with various components. In some embodiments, the concentration and amount of a supplemental component is optimized. In some embodiments, other aspects of the media and growth conditions (e.g., pH, temperature, etc.) are optimized through routine experimentation. In some embodiments, the frequency that the media is supplemented with one or more supplemental components, and the amount of time that the cell is cultured, is optimized.
Culturing of the cells described in this application can be performed in culture vessels known and used in the art. In some embodiments, an aerated reaction vessel (e.g., a stirred tank reactor) is used to culture the cells. In some embodiments, a bioreactor or fermenter is used to culture the cell. Thus, in some embodiments, the cells are used in fermentation. As used in this application, the terms "bioreactor" and "fermenter" are interchangeably used and refer to an enclosure, or partial enclosure, in which a biological, biochemical and/or chemical reaction takes place that involves a living organism or part of a living organism. A "large-scale bioreactor" or "industrial-scale bioreactor" is a bioreactor that is used to generate a product on a commercial or quasi-commercial scale. Large scale bioreactors typically have volumes in the range of liters, hundreds of liters, thousands of liters, or more.
Non-limiting examples of bioreactors include: stirred tank fermenters, bioreactors agitated by rotating mixing devices, chemostats, bioreactors agitated by shaking devices, airlift fermenters, packed-bed reactors, fixed-bed reactors, fluidized bed bioreactors, bioreactors employing wave induced agitation, centrifugal bioreactors, roller bottles, and hollow fiber bioreactors, roller apparatuses (for example benchtop, cart-mounted, and/or automated varieties), vertically-stacked plates, spinner flasks, stirring or rocking flasks, shaken multi-well plates, MD bottles, T-flasks, Roux bottles, multiple-surface tissue culture propagators, modified fermenters, and coated beads (e.g., beads coated with serum proteins, nitrocellulose, or carboxymethyl cellulose to prevent cell attachment).
In some embodiments, the bioreactor includes a cell culture system where the cell (e.g., yeast cell) is in contact with moving liquids and/or gas bubbles. In some embodiments, the cell or cell culture is grown in suspension. In other embodiments, the cell or cell culture is attached to a solid phase carrier. Non-limiting examples of a carrier system includes microcarriers (e.g., polymer spheres, microbeads, and microdisks that can be porous or non-porous), cross-linked beads (e.g., dextran) charged with specific chemical groups (e.g., tertiary amine groups), 2D microcarriers including cells trapped in nonporous polymer fibers, 3D carriers (e.g., carrier fibers, hollow fibers, multicartridge reactors, and semi-permeable membranes that can comprising porous fibers), microcarriers having reduced ion exchange capacity, encapsulation cells, capillaries, and aggregates. In some embodiments, carriers are fabricated from materials such as dextran, gelatin, glass, or cellulose.
In some embodiments, industrial-scale processes are operated in continuous, semi-continuous or non-continuous modes. Non-limiting examples of operation modes are batch, fed batch, extended batch, repetitive batch, draw/fill, rotating-wall, spinning flask, and/or perfusion mode of operation. In some embodiments, a bioreactor allows continuous or semi-continuous replenishment of the substrate stock, for example a carbohydrate source and/or continuous or semi-continuous separation of the product, from the bioreactor.
In some embodiments, the bioreactor or fermenter includes a sensor and/or a control system to measure and/or adjust reaction parameters. Non-limiting examples of reaction parameters include biological parameters (e.g., growth rate, cell size, cell number, cell density, cell type, or cell state, etc.), chemical parameters (e.g., pH, redox-potential, concentration of reaction substrate and/or product, concentration of dissolved gases, such as oxygen concentration and CO2 concentration, nutrient concentrations, metabolite concentrations, concentration of an oligopeptide, concentration of an amino acid, concentration of a vitamin, concentration of a hormone, concentration of an additive, serum concentration, ionic strength, concentration of an ion, relative humidity, molarity, osmolarity, concentration of other chemicals, for example buffering agents, adjuvants, or reaction by-products), physical/mechanical parameters (e.g., density, conductivity, degree of agitation, pressure, and flow rate, shear stress, shear rate, viscosity, color, turbidity, light absorption, mixing rate, conversion rate, as well as thermodynamic parameters, such as temperature, light intensity/quality, etc.). Sensors to measure the parameters described in this application are well known to one of ordinary skill in the relevant mechanical and electronic arts. Control systems to adjust the parameters in a bioreactor based on the inputs from a sensor described in this application are well known to one of ordinary skill in the art in bioreactor engineering.
In some embodiments, the method involves batch fermentation (e.g., shake flask fermentation). General considerations for batch fermentation (e.g., shake flask fermentation) include the level of oxygen and glucose. For example, batch fermentation (e.g., shake flask fermentation) may be oxygen and glucose limited, so in some embodiments, the capability of a strain to perform in a well-designed fed-batch fermentation is underestimated.
In some embodiments, the cells of the present disclosure are adapted to produce BMPs or proteins associated with a BMP in vivo. In some embodiments, the cells are adapted to secrete one or more BMPs or proteins associated with a BMP. In some embodiments, the cells of the present disclosure are lysed, and the remaining lysates are recovered for subsequent use. In some embodiments, any of the methods described in this application may include isolation and/or purification of BMPs or proteins associated with a BMP. For example, the isolation and/or purification can involve one or more of cell lysis, centrifugation, extraction, column chromatography, distillation, crystallization, and lyophilization.
Expression of Nucleic Acids in Host Cells Aspects of the present disclosure relate to recombinant proteins, functional modifications and variants thereof, as well as their uses. For example, the methods described in this application may be used to produce BMPs or proteins associated with a BMP. The methods may comprise using a host cell comprising a protein or peptide disclosed in this application, cell lysate, isolated protein or peptide, or any combination thereof. Methods comprising recombinant expression of genes encoding a protein or peptide disclosed in this application in a host cell are encompassed by the present disclosure.
A nucleic acid encoding any of the recombinant polypeptides (e.g., BMP2, BMP9, Noggin) described in this application may be incorporated into any appropriate vector through any method known in the art. For example, the vector may be an expression vector, including but not limited to a viral vector (e.g., a lentiviral, retroviral, adenoviral, or adeno-associated viral vector), any vector suitable for transient expression, any vector suitable for constitutive expression, or any vector suitable for inducible expression (e.g., a galactose-inducible or doxycycline-inducible vector).
A vector encoding any of the recombinant polypeptides (e.g., BMP2, BMP9, Noggin) described in this application may be introduced into a suitable host cell using any method known in the art. Non-limiting examples of yeast transformation protocols are described in Gietz et al., Yeast transformation can be conducted by the LiAc/SS Carrier DNA/PEG
method. Methods Mol Biol. 2006;313:107-20, which is hereby incorporated by reference in its entirety. Host cells may be cultured under any conditions suitable as would be understood by one of ordinary skill in the art. For example, any media, temperature, and incubation conditions known in the art may be used. For host cells carrying an inducible vector, cells may be cultured with an appropriate inducible agent to promote expression.
In some embodiments, a vector replicates autonomously in the cell. In some embodiments, a vector integrates into a chromosome within a cell. A vector can contain one or more endonuclease restriction sites that are cut by a restriction endonuclease to insert and ligate a nucleic acid containing a gene described in this application to produce a recombinant vector that is able to replicate in a cell. Vectors are typically composed of DNA, although RNA vectors are also available. Cloning vectors include, but are not limited to: plasmids, fosmids, phagemids, virus genomes and artificial chromosomes. As used in this application, the terms "expression vector" or "expression construct" refer to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. In some embodiments, the nucleic acid sequence of a gene described in this application is inserted into a cloning vector so that it is operably joined to regulatory sequences and, in some embodiments, expressed as an RNA transcript. In some embodiments, the vector contains one or more markers, such as a selectable marker as described in this application, to identify cells transformed or transfected with the recombinant vector. In some embodiments, a host cell has already been transformed with one or more vectors. In some embodiments, a host cell that has been transformed with one or more vectors is subsequently transformed with one or more vectors.
In some embodiments, a host cell is transformed simultaneously with more than one vector.
In some embodiments, a cell that has been transformed with a vector or an expression cassette incorporates all or part of the vector or expression cassette into its genome. In some embodiments, the nucleic acid sequence of a gene described in this application is codon-optimized. Codon optimization may increase production of the gene product by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, including all values in between) relative to a reference sequence that is not codon-optimized.
In some embodiments, the nucleic acid encoding any of the proteins described in this application is under the control of regulatory sequences (e.g., enhancer sequences). In some embodiments, a nucleic acid is expressed under the control of a promoter. The promoter can be a native promoter, e.g., the promoter of the gene in its endogenous context, which provides normal regulation of expression of the gene. Alternatively, a promoter can be a promoter that is different from the native promoter of the gene, e.g., the promoter is different from the promoter of the gene in its endogenous context.
In some embodiments, the promoter is a eukaryotic promoter. Non-limiting examples of eukaryotic promoters include TDH3, PGK1, PKC1, PDC1, TEF1, TEF2, RPL18B, SSA1, TDH2, PYK1, TPI1, GAL1, GAL10, GAL7, GAL3, GAL2, MET3, MET25, HXT3, HXT7, ACT1, ADH1, ADH2, CUP1-1, EN02, and SOD1, as would be known to one of ordinary skill in the art (see, e.g., Addgene website: blog.addgene.org/plasmids-101-the-promoter-region). In some embodiments, the promoter is a prokaryotic promoter (e.g., bacteriophage or bacterial promoter). Non-limiting examples of bacteriophage promoters include Pls icon, T3, T7, SP6, and PL. Non-limiting examples of bacterial promoters include Pbad, PmgrB, Ptrc2, Plac/ara, Ptac, and Pm.
In some embodiments, the promoter is an inducible promoter. As used in this application, an "inducible promoter" is a promoter controlled by the presence or absence of a molecule. Non-limiting examples of inducible promoters include chemically regulated promoters and physically regulated promoters. For chemically regulated promoters, the transcriptional activity can be regulated by one or more compounds, such as methanol, alcohol, tetracycline, galactose, a steroid, a metal, an amino acid, or other compounds. For physically regulated promoters, transcriptional activity can be regulated by a phenomenon such as light or temperature. Non-limiting examples of tetracycline-regulated promoters include anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems (e.g., a tetracycline repressor protein (tetR), a tetracycline operator sequence (tet0) and a tetracycline transactivator fusion protein (tTA)). Non-limiting examples of steroid-regulated promoters include promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily. Non-limiting examples of metal-regulated promoters include promoters derived from metallothionein (proteins that bind and sequester metal ions) genes. Non-limiting examples of pathogenesis-regulated promoters include promoters induced by salicylic acid, ethylene or benzothiadiazole (BTH). Non-limiting examples of temperature/heat-inducible promoters include heat shock promoters.
Non-limiting examples of light-regulated promoters include light responsive promoters from plant cells. In certain embodiments, the inducible promoter is a galactose-inducible promoter. In some embodiments, the inducible promoter is induced by one or more physiological conditions (e.g., pH, temperature, radiation, osmotic pressure, saline gradients, cell surface binding, or concentration of one or more extrinsic or intrinsic inducing agents). Non-limiting examples of an extrinsic inducer or inducing agent include amino acids and amino acid analogs, saccharides and polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal containing compounds, salts, ions, enzyme substrate analogs, hormones or any combination.
In some embodiments, the promoter is a constitutive promoter. As used in this application, a "constitutive promoter" refers to an unregulated promoter that allows continuous transcription of a gene. Non-limiting examples of a constitutive promoter include TDH3, PGK1, PKC1, PDC1, TEF1, TEF2, RPL18B, SSA1, TDH2, PYK1, TPI1, HXT3, HXT7, ACT1, ADH1, ADH2, EN02, and SOD1. Other inducible promoters or constitutive promoters, including synthetic promoters, that may be known to one of ordinary skill in the art are also contemplated.
The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but generally include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. In particular, such 5' non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene.
Regulatory sequences may also include enhancer sequences or upstream activator sequences.
The vectors disclosed may include 5' leader or signal sequences. The regulatory sequence may also include a terminator sequence. In some embodiments, a terminator sequence marks the end of a gene in DNA during transcription. The choice and design of one or more appropriate vectors suitable for inducing expression of one or more genes described in this application in a heterologous organism is within the ability and discretion of one of ordinary skill in the art. Expression vectors containing the necessary elements for expression are commercially available and known to one of ordinary skill in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012).
Variants Aspects of the disclosure relate to nucleic acids encoding any of the polypeptides (e.g., BMP2, BMP7, BMP9, Noggin) described in this application. In some embodiments, a nucleic acid encompassed by the disclosure is a nucleic acid that hybridizes under high or medium stringency conditions to a nucleic acid encoding a protein or peptide (e.g., BMP2, BMP7, BMP9, Noggin) and is biologically active. For example, high stringency conditions of 0.2 to 1 x SSC at 65 C followed by a wash at 0.2 x SSC at 65 C can be used.
In some embodiments, a nucleic acid encompassed by the disclosure is a nucleic acid that hybridizes under low stringency conditions to a nucleic acid encoding a protein or peptide (e.g., BMP2, BMP7, BMP9, Noggin) and is biologically active. For example, low stringency conditions of 6 x SSC at room temperature followed by a wash at 2 x SSC at room temperature can be used. Other hybridization conditions include 3 x SSC at 40 or 50 C, followed by a wash in 1 or 2 x SSC at 20, 30, 40, 50, 60, or 65 'C.
Hybridizations can be conducted in the presence of formaldehyde, e.g., 10%, 20%, 30% 40% or 50%, which further increases the stringency of hybridization.
Theory and practice of nucleic acid hybridization is described, e.g., in S. Agrawal (ed.) Methods in Molecular Biology, volume 20; and Tijssen (1993) Laboratory Techniques in biochemistry and molecular biology-hybridization with nucleic acid probes, e.g., part I
chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays,"
Elsevier, New York provide a basic guide to nucleic acid hybridization.
Variants of proteins or peptides (or derivatives thereof) described in this application (e.g., BMP2, BMP7, BMP9, Noggin) are also encompassed by the present disclosure. A
variant may share at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a reference sequence, including all values in between.
Unless otherwise noted, the term "sequence identity," which is used interchangeably in this disclosure with the term "percent identity," as known in the art, refers to a relationship between the sequences of two polypeptides or polynucleotides, as determined by sequence comparison (alignment). In some embodiments, sequence identity is determined across the entire length of a sequence (e.g., BMP2, BMP7, BMP9, Noggin sequence). In some embodiments, sequence identity is determined over a region (e.g., a stretch of amino acids or nucleic acids, e.g., the sequence spanning an active site) of a sequence (e.g., BMP2, BMP7, BMP9, Noggin). For example, in some embodiments, sequence identity is determined over a region corresponding to at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or over 100% of the length of the reference sequence.
Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model, algorithm, or computer program. Identity of related polypeptides or nucleic acid sequences can be readily calculated by any of the methods known to one of ordinary skill in the art. The percent identity of two sequences (e.g., nucleic acid or amino acid sequences) may, for example, be determined using the algorithm of Karlin and Altschul Proc. Natl.
Acad. Sci.
USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad.
Sci. USA
90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST
programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990.
BLAST protein searches can be performed, for example, with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins described in this application. Where gaps exist between two sequences, Gapped BLAST can be utilized, for example, as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST ) can be used, or the parameters can be adjusted appropriately as would be understood by one of ordinary skill in the art.
Another local alignment technique which may be used, for example, is based on the Smith-Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981) "Identification of common molecular subsequences." J. Mol. Biol. 147:195-197). A general global alignment technique which may be used, for example, is the Needleman¨Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol.
48:443-453), which is based on dynamic programming. More recently, a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) was developed that purportedly produces global alignment of nucleic acid and amino acid sequences faster than other optimal global alignment methods, including the Needleman¨Wunsch algorithm. In some embodiments, the identity of two polypeptides is determined by aligning the two amino acid sequences, calculating the number of identical amino acids, and dividing by the length of one of the amino acid sequences. In some embodiments, the identity of two nucleic acids is determined by aligning the two nucleotide sequences and calculating the number of identical nucleotide and dividing by the length of one of the nucleic acids. For multiple sequence alignments, computer programs including Clustal Omega (Sievers et al., Mol Syst Biol. 2011 Oct 11;7:539) may be used.
In preferred embodiments, a sequence, including a nucleic acid or amino acid sequence, is found to have a specified percent identity to a reference sequence, when sequence identity is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad.
Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl.
Acad. Sci. USA
90:5873-77, 1993 (e.g., BLAST , NBLAST , XBLAST or Gapped BLAST programs, using default parameters of the respective programs).
In some embodiments, a sequence, including a nucleic acid or amino acid sequence, is found to have a specified percent identity to a reference sequence when sequence identity is determined using the Smith-Waterman algorithm (Smith, T.F. & Waterman, M.S.
(1981) "Identification of common molecular subsequences." J. Mol. Biol. 147:195-197) or the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol.
Biol. 48:443-453) using default parameters.
In some embodiments, a sequence, including a nucleic acid or amino acid sequence, is found to have a specified percent identity to a reference sequence when sequence identity is determined using a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) using default parameters.
In some embodiments, a sequence, including a nucleic acid or amino acid sequence, is found to have a specified percent identity to a reference sequence when sequence identity is determined using Clustal Omega (Sievers et al., Mol Syst Biol. 2011 Oct
11;7:539) using default parameters.
As used in this application, a residue (such as a nucleic acid residue or an amino acid residue) in sequence "X" is referred to as corresponding to a position or residue (such as a nucleic acid residue or an amino acid residue) "Z" in a different sequence "Y"
when the residue in sequence "X" is at the counterpart position of "Z" in sequence "Y"
when sequences X and Y are aligned using amino acid sequence alignment tools known in the art.
As used in this application, variant sequences may be homologous sequences. As used in this application, homologous sequences are sequences (e.g., nucleic acid or amino acid sequences) that share a certain percent identity (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%
percent identity, including all values in between). Homologous sequences include but are not limited to paralogous or orthologous sequences. Paralogous sequences arise from duplication of a gene within a genome of a species, while orthologous sequences diverge after a speciation event.
In some embodiments, a polypeptide variant (e.g., BMP2, BMP7, BMP9, Noggin variant) comprises a domain that shares a secondary structure (e.g., alpha helix, beta sheet) with a reference polypeptide (e.g., a reference BMP2, BMP7, BMP9, Noggin). In some embodiments, a polypeptide variant (e.g., BMP2, BMP7, BMP9, Noggin variant) shares a tertiary structure with a reference polypeptide (e.g., a reference BMP2, BMP7, BMP9, Noggin enzyme). As a non-limiting example, a polypeptide variant (e.g., BMP2, BMP7, BMP9, Noggin enzyme) may have low primary sequence identity (e.g., less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% sequence identity) compared to a reference polypeptide, but share one or more secondary structures (e.g., including but not limited to loops, alpha helices, or beta sheets), or have the same tertiary structure as a reference polypeptide. For example, a loop may be located between a beta sheet and an alpha helix, between two alpha helices, or between two beta sheets. Homology modeling may be used to compare two or more tertiary structures.
Functional variants of the recombinant proteins disclosed in this application are encompassed by the present disclosure. For example, functional variants may bind one or more of the same substrates or produce one or more of the same products. In other examples, functional variants may bind one or more of the same receptors associated with the BMP
signaling and/or regulatory pathways. Functional variants may be identified using any method known in the art. For example, the algorithm of Karlin and Altschul Proc. Natl.
Acad. Sci. USA 87:2264-68, 1990 described above may be used to identify homologous proteins with known functions.
Putative functional variants may also be identified by searching for polypeptides with functionally annotated domains. Databases including Pfam (Sonnhammer et al., Proteins.
1997 Jul;28(3):405-20) may be used to identify polypeptides with a particular domain.
Homology modeling may also be used to identify amino acid residues that are amenable to mutation (e.g., substitution, deletion, and/or insertion) without affecting function. A non-limiting example of such a method may include use of position-specific scoring matrix (PSSM) and an energy minimization protocol.
Position-specific scoring matrix (PSSM) uses a position weight matrix to identify consensus sequences (e.g., motifs). PSSM can be conducted on nucleic acid or amino acid sequences. Sequences are aligned and the method takes into account the observed frequency of a particular residue (e.g., an amino acid or a nucleotide) at a particular position and the number of sequences analyzed. See, e.g., Stormo et al., Nucleic Acids Res.
1982 May 11;10(9):2997-3011. The likelihood of observing a particular residue at a given position can be calculated. Without being bound by a particular theory, positions in sequences with high variability may be amenable to mutation (e.g., substitution, deletion, and/or insertion; e.g., PSSM score >0) to produce functional homologs. PSSM may be paired with calculation of a Rosetta energy function, which determines the difference between the wild-type and the single-point mutant. The Rosetta energy function calculates this difference as (AAGca/c).
With the Rosetta function, the bonding interactions between a mutated residue and the surrounding atoms are used to determine whether an amino acid substitution, deletion, or insertion increases or decreases protein stability. For example, an amino acid substitution, deletion, or insertion that is designated as favorable by the PSSM score (e.g.
PSSM score can then be analyzed using the Rosetta energy function to determine the potential impact of the mutation on protein stability. Without being bound by a particular theory, potentially stabilizing mutations are desirable for protein engineering (e.g., production of functional homologs). In some embodiments, a potentially stabilizing mutation has a AAGcaic value of less than -0.1 (e.g., less than -0.2, less than -0.3, less than -0.35, less than -0.4, less than -0.45, less than -0.5, less than -0.55, less than -0.6, less than -0.65, less than -0.7, less than -0.75, less than -0.8, less than -0.85, less than -0.9, less than -0.95, or less than -1.0) Rosetta energy units (R.e.u.). See, e.g., Goldenzweig et al., Mol Cell. 2016 Jul 21;63(2):337-346. Doi:
10.1016/j.molce1.2016.06.012.
In some embodiments, the coding sequence of a BMP or a protein associated with a BMP comprises a mutation at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more than 100 positions relative to a reference (e.g., a BMP or a protein associated with a BMP) coding sequence. In some embodiments, the coding sequence of a BMP or a protein associated with a BMP comprises a mutation in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100 or more codons of the coding sequence relative to a reference (e.g., a BMP or a protein associated with a BMP) coding sequence. As will be understood by one of ordinary skill in the art, a mutation within a codon may or may not change the amino acid that is encoded by the codon due to degeneracy of the genetic code. In some embodiments, the one or more mutation in the coding sequence do not alter the amino acid sequence of the coding sequence (e.g., a BMP or a protein associated with a BMP) relative to the amino acid sequence of a reference polypeptide (e.g., a BMP or a protein associated with a BMP).
It should be appreciated that sequences disclosed in the present application may or may not contain signal sequences.
In some embodiments, the one or more mutations in the coding sequence of a recombinant BMP or a protein associated with a BMP sequence alters the amino acid sequence of the polypeptide (e.g., a BMP or a protein associated with a BMP) relative to the amino acid sequence of a reference polypeptide (e.g., a BMP or a protein associated with a BMP). In some embodiments, the one or more mutations alters the amino acid sequence of the recombinant polypeptide (e.g., a BMP or a protein associated with a BMP) relative to the amino acid sequence of a reference polypeptide (e.g., a BMP or a protein associated with a BMP) and also alters (enhances or reduces) an activity of the polypeptide relative to the reference polypeptide.
The activity (e.g., specific activity) of any of the recombinant polypeptides described in this application (e.g., a BMP or a protein associated with a BMP) may be measured using routine methods.
The skilled artisan will also realize that mutations in a recombinant polypeptide (e.g., a BMP or a protein associated with a BMP) coding sequence may result in conservative amino acid substitutions to provide functionally equivalent variants of the foregoing polypeptides, e.g., variants that retain the activities of the polypeptides.
As used in this application, a "conservative amino acid substitution" refers to an amino acid substitution that does not alter the relative charge or size characteristics or functional activity of the protein in which the amino acid substitution is made.
In some instances, an amino acid is characterized by its R group. For example, an amino acid may comprise a nonpolar aliphatic R group, a positively charged R
group, a negatively charged R group, a nonpolar aromatic R group, or a polar uncharged R group.
Non-limiting examples of an amino acid comprising a nonpolar aliphatic R group include alanine, glycine, valine, leucine, methionine, and isoleucine. Non-limiting examples of an amino acid comprising a positively charged R group includes lysine, arginine, and histidine.
Non-limiting examples of an amino acid comprising a negatively charged R group include aspartate and glutamate. Non-limiting examples of an amino acid comprising a nonpolar, aromatic R group include phenylalanine, tyrosine, and tryptophan. Non-limiting examples of an amino acid comprising a polar uncharged R group include serine, threonine, cysteine, proline, asparagine, and glutamine.
Non-limiting examples of functionally equivalent variants of polypeptides may include conservative amino acid substitutions in the amino acid sequences of proteins disclosed in this application. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Additional non-limiting examples of conservative amino acid substitutions are provided in Table 1.
In some embodiments, 1,2, 3,4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 or more than 20 residues can be changed when preparing variant polypeptides.
In some embodiments, amino acids are replaced by conservative amino acid substitutions. In some embodiments, amino acids are replaced by non-conservative amino acid substitutions.
Table 1: Non-limiting examples of conservative amino acid substitutions Original Residue R Group Type Conservative Amino Acid Substitutions Ala (A) nonpolar aliphatic R group Cys, Gly, Ser Arg (R) positively charged R group His, Lys Mn (N) polar uncharged R group Asp, Gin, Glu Asp (D) negatively charged R group Asn, Gin, Giu Cs (C) polar uncharged R group Ala, Ser Gin (Q) polar uncharged R group A snõA.sp, Gin Giu (E) negatively charged R group Asn, Asp, Gin Gy (G) nonpolar aliphatic Rgroup Ma, Ser Uis (H) positively charged R group Arg, Tyr, Trp lie (I) nonpolar aliphatic R group Leu, Met, Val Leu (1_,) nonpolar aliphatic .R group Ile, Met, 'Val Lys (K) positively charged R group Arg, His Met (Ind) nonpolar aliphatic R group lie, Lou, Phe, Val .Pro (P) polar uncharged .R group Phc. (F) nonpolar aromatic R group Met, Trp, Tyr Ser (S) polar uncharged R group Ala, (ily, Thr Thr (T) polar uncharged R group Ala., Asn, Ser Trp (W) nonpolar aromatic R group His, Phe, Tyr, Met Tyr (Y) nonpolar aromatic R group His. Phe, Trp Val (V) nonpolar aliphatic R group Ile, Len, Met, Thr Amino acid substitutions in the amino acid sequence of a polypeptide to produce a recombinant polypeptide (e.g., a BMP or a protein associated with a BMP) variant having a desired property and/or activity can be made by alteration of the coding sequence of the polypeptide (e.g., a BMP or a protein associated with a BMP). Similarly, conservative amino acid substitutions in the amino acid sequence of a polypeptide to produce functionally equivalent variants of the polypeptide typically are made by alteration of the coding sequence of the recombinant polypeptide (e.g., a BMP or a protein associated with a BMP).
Mutations (e.g., substitutions, insertions, additions, or deletions) can be made in a nucleic acid sequence by a variety of methods known to one of ordinary skill in the art. For example, mutations (e.g., substitutions, insertions, additions, or deletions) can be made by PCR-directed mutation, site-directed mutagenesis according to the method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492, 1985), by chemical synthesis of a gene encoding a polypeptide, by gene editing methods, or by insertions, such as insertion of a tag (e.g., a HIS tag or a GFP tag). Mutations can include, for example, substitutions, insertions, additions, deletions, and translocations, generated by any method known in the art. Methods for producing mutations may be found in in references such as Molecular Cloning: A
Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York, 2010.
General Techniques The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the ordinary skill in the art (e.g., as disclosed in: Molecular Cloning: A Laboratory Manual, fourth edition (Green, et al., 2012 Cold Spring Harbor Press); Oligonucleotide Synthesis (M.
J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook, Vol. 3 (J. E. Cellis, ed., 2005) Academic Press; Animal Cell Culture (R. I.
Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E.
Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.
Griffiths, and D. G.
Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.);
Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.);
Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Cabs, eds., 1987); Short Protocols in Molecular Biology (F. M. Ausubel, et al., eds., 2002); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);
Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997);
Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989);
Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995). It is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited in the present application are incorporated by reference for the purposes or subject matter referenced herein.
EXAMPLES
In order that the invention described in the present application may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the systems and methods provided herein and are not to be construed in any way as limiting their scope.
Example 1: Screen to Identify Functional Expression of BMPs, BMP Peptides, Receptor Decoys, and Regulatory Proteins To identify BMPs, BMP peptides, receptor decoys, and/or regulatory proteins that could be functionally expressed in host cells, a library of BMPs (e.g., human mature BMP1, BMP 2, BMP 3, BMP 4, BMP 5, BMP 6, BMP 7, BMP 8a, BMP Sb, BMP 9, BMP10, BMPI 1, BMP12, BMP1.3, BMPI4, and/or BMP15) and derivatives of BMPs such as heterodimers and peptides derived from the above-listed BMPs, chim.eric BMPs, and rationally engineered BMPs is designed. The library may include naturally occurring BMPs and/or engineered versions of BM Ps. The library may also include one or more of proteins associated with BMPs including Activin. The library may include chimeric proteins that consist of domains from different BMPs or Activins, such. as a BMP2/7 chimeric protein or a BMP/Activin chimeric protein, for example. Proteins within the library, including chimeric proteins, may also include one or more amino acid modifications, such as amino acid substitutions, additions, deletions, or insertions. Without wishing to be bound by any theory, heterodimers, chimeric proteins, and amino acid modifications may increase the binding affinity of the ligands to BMP receptors, which include but are not limited to ALKI, AL,K2, ALIO, ALK6, ACVR2A, ACNR2B, BMPR2, and AMHR2, Engineered BMPs included in the library may exhibit increased potency compared to naturally occurring or wildtype 13 MPs.
Strategies for engineering BMPs may include -bioinformatics analysis of the BMP protein sequences. For example, bioinformatics analyses may be used to predict the fitness of the native amino acid at every position in the relevant wildtype protein sequences and to suggest favorable alternatives if the native amino acid is suboptimal. In some instances, BMPs within the library contain one or more point mutations relative to the corresponding wildtype B MP.
Receptor decoys such as BMP3, Activin 13, and Inhibin a are also designed, screened and tested. Without wishing to be bound by any theory, receptor decoys may bind to BMP
receptors without activating them. Receptor decoys may lower the activation of the SMAD
pathway induced by 131VIPs. Regulatory proteins, such as Activin, Gremlin, and/or Noggin, are also designed, screened and tested. Noggin and Gremlin are known to act as antagonists of BMP proteins, Receptor decoys and regulatory proteins may be naturally occurring proteins or may be engineered.
The screening and construction process may include DNA synthesis of DNA
sequences within the library that are cloned into a plasmid for expression in a host cell. For example, a host cell may be a yeast cell such as a P. pastoris cell, a bacterial cell such as an .Escherichia coli cell, and/or a mammalian cell, such as a 1-IEK 293 or CU() (Chinese hamster ovary) cell. The plasmids are transformed into the host cell and cultured in well plates. After growth at a defined temperature and time period, the library proteins produced by the host cells are assayed for activity and quantified using a method known the art such as ELBA
(enzyme-linked immunosorbent assay) or a -fluorescence detection assay.
Efficacy testing is performed for determining the presence and the level of alkaline phosphatase production, and by performing a Bone Gla Protein (BGP; osteocalcin) assay. Protein.s from the library are selected for further testing based on expression compatibility in the host cells as well as activity in the in vitro efficacy assay. Further testing may include testing the effects of proteins identified in the library on different cell types, including, for example, mesenchymal stem cells, preosteoblast cells, and/or osteoblast cells.
Example 2: Host Strain Engineering and Recombinant BMP Production Using P.
pastoris A P. pastoris strain is used that has been genetically altered for improved production of recombinant proteins. In general, strains will have a single amino acid auxotrophy (e.g., his4 or Ade2) to allow for selection of expression constructs following transformation. In addition, a limited number of protease knockouts (e.g., pep4 & prbl) or secretion pathway modifications (e.g., PB/1 overexpression) previously shown to be useful in general secretion screening strains may be introduced. BMP protein family members are known to retain biological activity even without sugar chains. However, since BMPs are relatively hydrophobic, recombinant BMP without sugar chains can cause protein aggregation. When using P. pastoris as a host cell, post-translational, modifications such. as glycosylation and folding can be performed. The host strain can be further modified to perform post-translational modifications (e.g., glycosylation) similar to the post-translational modifications (e.g., glycosylation) of human BMP 2 and BMP 7. The post-translational modifications will.
be evaluated by using proteoglycomics.
BMP2 and BMP7, and other candidate proteins identified in the screening are inserted into the genome of P pastoris. An episomal expression system is developed.
Various solubility and/or secretion tags (e.g., the S. cerevisiae mating factor) and codon optimization is tested to ensure optimal expression and secretion of the proteins. P
pastoris strains containing the candidate proteins are grown in 96-well plates. Cell lysates, supernatants, and/or purified proteins are assayed by quantification using ELISA or fluorescence assays.
Efficacy testing is then performed for determining the presence and/or the level of alkaline phosphatase production. The BGP assay is also conducted. The cell lysates, supernatants, and/or purified proteins are tested with mammalian cells to determine cell proliferation. A
fermentation process is developed for producing larger quantities of the candidate proteins.
For example, a bioreactor or fermenter can be used for culturing the host cells. Batch fermentation methods (e.g., shake flask or well plate fermentation) can be used.
Example 3: Host Strain Engineering and Recombinant BMP Production Using Mammalian Cells An advantage of the expression systems utilizing mammalian cells for generating recombinant proteins is the ability to introduce proper protein folding, post-translational modifications, and product assembly, which are important for complete and functional biological activities of mammalian proteins. A mammalian cell line capable of stably expressing BMPs and/or other candidate proteins is developed. The cell line is engineered for producing high protein titers. An expression system appropriate for mammalian cells and development of growth medium for optimal production is designed.
BMP2 and BMP7 and other potential candidate proteins are inserted into an episomal expression system for transient expression. Alternatively, BMP2 and BMP7 and other potential candidate proteins are inserted into a construct for stable expression. A strong promoter such as the CMV or CAG promoter is used to express the BMPs or other potential candidate proteins, which is optionally adjusted by adding enhancer elements.
Various solubility and/or secretion tags (e.g., the S. cerevisiae mating factor) are tested. Codon optimization is also tested to ensure optimal expression and secretion of the proteins, if needed. The episomal expression system is transfected into mammalian cells such as HEK-293 or CHO cells. Any mammalian cells suitable for producing recombinant proteins can be used. The mammalian cells are grown in liquid medium. Cell lysates, supernatants, or purified proteins are assayed by quantification using ELISA or fluorescence assays. Efficacy testing is performed for determining the presence and/or the level of alkaline phosphatase production. The BGP assay is also conducted. The cell lysates, supernatants, and/or purified proteins are also tested with mammalian cells to determine cell proliferation.
A fermentation process is developed for producing larger quantities of proteins. For example, a bioreactor or fermenter can be used for culturing the cell. Batch fermentation methods (e.g., shake flask fermentation) can be used.
Example 4: Administration of BMPs and/or BMP Associated Proteins in Human Subjects In order to examine the effects of BMPs and/or BMP associated proteins on anti-aging and aesthetic related characteristics, male and female subjects who are older than 18 years of age are recruited for the studies. Subjects who are at least 65 years of old (e.g., elderly) are a preferred group in some instances. Subject candidates are first screened for their health history and condition (e.g., allergies, pregnancies, drug and medications that may contraindicate the use of BMPs and BMP associated protein formulations, familial inheritance related to skin conditions), their routine skin care, and other aesthetic non-invasive procedures they have received according to various criteria that will be set forth.
Prior to any treatments, baseline profiles are developed for each recruited subject. The baseline profiles may include but are not limited to several headshots of the subjects, anthropometric facial skeleton measurements such as the skeleton features surrounding periorbital regions, midface regions, perinasal regions, and lower face regions, and wrinkles and fine lines assessments via dermatoscopy and digital fringe profilometry techniques, for example, and one or more questionnaires capturing the subjects self-assessment of attractiveness and desired improvements in facial contouring or skin texture or evenness of skin tone.
BMPs and/or BMP associated proteins are administered to the selected human subjects for testing the aesthetic and/or cosmetic effects of the proteins. As disclosed in the present application, any BMPs and BMP associated proteins suitable for the study designs may be chosen. For example, Activin, BMP2, BMP6, BMP7 or BMP9 can be administered to the subjects. In some instances, more than one BMP can be formulated for administration to the subjects. BMPs can be administered to the subjects with BMP associated proteins. For example, any of the BMPs can be administered to subjects in combination with Noggin for regulating the functions of BMPs in vivo. As disclosed in the present application, Noggin may function as a "off switch" to BMPs, so that the effects of BMPs can be attenuated or delayed. BMPs and BMP associated proteins can be formulated separately (i.e., in two different formulations) or prepared in the same formulation. When appropriate, subjects may receive administration of BMP associated proteins at the same time or after the administration of BMPs. Some subjects may be administered placebo treatments which will not contain any BMPs and/or BMP associated proteins.
BMPs and/or BMP associated proteins may be administered to the subject topically, such as in the form of a cream and/or serum, or via subdermal, subcutaneous or supraperiostial injections. BMPs and/or BMP associated proteins will optionally be encapsulated in a formulation for enhancing penetration of the proteins through the skin of the subjects.
For topical administrations, BMPs and BMP regulatory proteins such as Noggin may be formulated at a concentration of less than about 3%. BMP peptides may be formulated at a concentration of less than about 10% for topical administrations. Subjects who receive topical administration may optionally receive skin pre-treatments such as exfoliations or peels to minimize potential inconsistency of absorption of the cream or serum. For subdermal, subcutaneous or supraperiostial administration, BMPs and BMP regulatory proteins such as Noggin may be formulated at a concentration of about 100 to about 1,000 ng/mL
for each injection site. The doses of formulations for topical or subdermal, subcutaneous or supraperiostial administration can be adjusted based on the needs of a subject such as the estimated frequencies of the administrations and site of the administrations (e.g., face versus hand). For subdermal, subcutaneous or supraperiostial injections, BMPs and BMP
regulatory proteins can be either administered in a pre-formulated composition or in a formulation that will be prepared right before the injections. Alternatively, BMPs and BMP
regulatory proteins can be pre-mixed with dermal fillers prior to the injections. In some instances, a subject may receive both subdermal (or subcutaneous or supraperiostial) injection and topical administration.
Example 5: Effects of Administration of BMPs and BMP Associated Proteins on the Appearance of Skin of Subjects As described in Example 4, various BMPs and BMP regulatory proteins are administered to human subjects for determining anti-aging and aesthetic characteristics. After administration (e.g., topical or subdermal), the subjects are evaluated for any changes of the appearance of their skin and for changes in their perception of attractiveness. A list of skin characteristics and signs to be examined is developed. For example, characteristics for evaluation may include the appearance of skin wrinkles and fine lines around the face and neck (e.g., crinkle lines, permanent elastotic creases, dynamic expression lines, and/or gravitational folds), the appearance of facial contours, the appearance of facial bone structures, and/or the subjects' perception of improvement in attractiveness.
Depending on the type of administration, various time points post-administrations are assigned for conducting evaluations. For example, for subjects who receive subdermal (or subcutaneous or supraperiostial) injections, characteristics may be evaluated as soon as two days after the initial injection. In some instances, the subjects who receive subdermal (or subcutaneous or supraperiostial) injections are evaluated 10 or more days after the initial injection. For subjects who receive topical administration, characteristics may be evaluated, e.g., approximately 17 days after the initial application. In some instances, subjects who receive topical administration of cream and/or serum are evaluated 30 or more days after the initial application. Optionally, subjects are evaluated at multiple time points to monitor any longer-term effects. Subjects may be evaluated against their own baseline profiles and/or against skin conditions and changes of subjects who receive placebo treatments.
EQUIVALENTS
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described in the present application. Such equivalents are intended to be encompassed by the following claims. All references, including patent documents, disclosed in this application are incorporated by reference in their entirety, particularly for the disclosure referenced in this application.
As used in this application, a residue (such as a nucleic acid residue or an amino acid residue) in sequence "X" is referred to as corresponding to a position or residue (such as a nucleic acid residue or an amino acid residue) "Z" in a different sequence "Y"
when the residue in sequence "X" is at the counterpart position of "Z" in sequence "Y"
when sequences X and Y are aligned using amino acid sequence alignment tools known in the art.
As used in this application, variant sequences may be homologous sequences. As used in this application, homologous sequences are sequences (e.g., nucleic acid or amino acid sequences) that share a certain percent identity (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%
percent identity, including all values in between). Homologous sequences include but are not limited to paralogous or orthologous sequences. Paralogous sequences arise from duplication of a gene within a genome of a species, while orthologous sequences diverge after a speciation event.
In some embodiments, a polypeptide variant (e.g., BMP2, BMP7, BMP9, Noggin variant) comprises a domain that shares a secondary structure (e.g., alpha helix, beta sheet) with a reference polypeptide (e.g., a reference BMP2, BMP7, BMP9, Noggin). In some embodiments, a polypeptide variant (e.g., BMP2, BMP7, BMP9, Noggin variant) shares a tertiary structure with a reference polypeptide (e.g., a reference BMP2, BMP7, BMP9, Noggin enzyme). As a non-limiting example, a polypeptide variant (e.g., BMP2, BMP7, BMP9, Noggin enzyme) may have low primary sequence identity (e.g., less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% sequence identity) compared to a reference polypeptide, but share one or more secondary structures (e.g., including but not limited to loops, alpha helices, or beta sheets), or have the same tertiary structure as a reference polypeptide. For example, a loop may be located between a beta sheet and an alpha helix, between two alpha helices, or between two beta sheets. Homology modeling may be used to compare two or more tertiary structures.
Functional variants of the recombinant proteins disclosed in this application are encompassed by the present disclosure. For example, functional variants may bind one or more of the same substrates or produce one or more of the same products. In other examples, functional variants may bind one or more of the same receptors associated with the BMP
signaling and/or regulatory pathways. Functional variants may be identified using any method known in the art. For example, the algorithm of Karlin and Altschul Proc. Natl.
Acad. Sci. USA 87:2264-68, 1990 described above may be used to identify homologous proteins with known functions.
Putative functional variants may also be identified by searching for polypeptides with functionally annotated domains. Databases including Pfam (Sonnhammer et al., Proteins.
1997 Jul;28(3):405-20) may be used to identify polypeptides with a particular domain.
Homology modeling may also be used to identify amino acid residues that are amenable to mutation (e.g., substitution, deletion, and/or insertion) without affecting function. A non-limiting example of such a method may include use of position-specific scoring matrix (PSSM) and an energy minimization protocol.
Position-specific scoring matrix (PSSM) uses a position weight matrix to identify consensus sequences (e.g., motifs). PSSM can be conducted on nucleic acid or amino acid sequences. Sequences are aligned and the method takes into account the observed frequency of a particular residue (e.g., an amino acid or a nucleotide) at a particular position and the number of sequences analyzed. See, e.g., Stormo et al., Nucleic Acids Res.
1982 May 11;10(9):2997-3011. The likelihood of observing a particular residue at a given position can be calculated. Without being bound by a particular theory, positions in sequences with high variability may be amenable to mutation (e.g., substitution, deletion, and/or insertion; e.g., PSSM score >0) to produce functional homologs. PSSM may be paired with calculation of a Rosetta energy function, which determines the difference between the wild-type and the single-point mutant. The Rosetta energy function calculates this difference as (AAGca/c).
With the Rosetta function, the bonding interactions between a mutated residue and the surrounding atoms are used to determine whether an amino acid substitution, deletion, or insertion increases or decreases protein stability. For example, an amino acid substitution, deletion, or insertion that is designated as favorable by the PSSM score (e.g.
PSSM score can then be analyzed using the Rosetta energy function to determine the potential impact of the mutation on protein stability. Without being bound by a particular theory, potentially stabilizing mutations are desirable for protein engineering (e.g., production of functional homologs). In some embodiments, a potentially stabilizing mutation has a AAGcaic value of less than -0.1 (e.g., less than -0.2, less than -0.3, less than -0.35, less than -0.4, less than -0.45, less than -0.5, less than -0.55, less than -0.6, less than -0.65, less than -0.7, less than -0.75, less than -0.8, less than -0.85, less than -0.9, less than -0.95, or less than -1.0) Rosetta energy units (R.e.u.). See, e.g., Goldenzweig et al., Mol Cell. 2016 Jul 21;63(2):337-346. Doi:
10.1016/j.molce1.2016.06.012.
In some embodiments, the coding sequence of a BMP or a protein associated with a BMP comprises a mutation at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more than 100 positions relative to a reference (e.g., a BMP or a protein associated with a BMP) coding sequence. In some embodiments, the coding sequence of a BMP or a protein associated with a BMP comprises a mutation in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100 or more codons of the coding sequence relative to a reference (e.g., a BMP or a protein associated with a BMP) coding sequence. As will be understood by one of ordinary skill in the art, a mutation within a codon may or may not change the amino acid that is encoded by the codon due to degeneracy of the genetic code. In some embodiments, the one or more mutation in the coding sequence do not alter the amino acid sequence of the coding sequence (e.g., a BMP or a protein associated with a BMP) relative to the amino acid sequence of a reference polypeptide (e.g., a BMP or a protein associated with a BMP).
It should be appreciated that sequences disclosed in the present application may or may not contain signal sequences.
In some embodiments, the one or more mutations in the coding sequence of a recombinant BMP or a protein associated with a BMP sequence alters the amino acid sequence of the polypeptide (e.g., a BMP or a protein associated with a BMP) relative to the amino acid sequence of a reference polypeptide (e.g., a BMP or a protein associated with a BMP). In some embodiments, the one or more mutations alters the amino acid sequence of the recombinant polypeptide (e.g., a BMP or a protein associated with a BMP) relative to the amino acid sequence of a reference polypeptide (e.g., a BMP or a protein associated with a BMP) and also alters (enhances or reduces) an activity of the polypeptide relative to the reference polypeptide.
The activity (e.g., specific activity) of any of the recombinant polypeptides described in this application (e.g., a BMP or a protein associated with a BMP) may be measured using routine methods.
The skilled artisan will also realize that mutations in a recombinant polypeptide (e.g., a BMP or a protein associated with a BMP) coding sequence may result in conservative amino acid substitutions to provide functionally equivalent variants of the foregoing polypeptides, e.g., variants that retain the activities of the polypeptides.
As used in this application, a "conservative amino acid substitution" refers to an amino acid substitution that does not alter the relative charge or size characteristics or functional activity of the protein in which the amino acid substitution is made.
In some instances, an amino acid is characterized by its R group. For example, an amino acid may comprise a nonpolar aliphatic R group, a positively charged R
group, a negatively charged R group, a nonpolar aromatic R group, or a polar uncharged R group.
Non-limiting examples of an amino acid comprising a nonpolar aliphatic R group include alanine, glycine, valine, leucine, methionine, and isoleucine. Non-limiting examples of an amino acid comprising a positively charged R group includes lysine, arginine, and histidine.
Non-limiting examples of an amino acid comprising a negatively charged R group include aspartate and glutamate. Non-limiting examples of an amino acid comprising a nonpolar, aromatic R group include phenylalanine, tyrosine, and tryptophan. Non-limiting examples of an amino acid comprising a polar uncharged R group include serine, threonine, cysteine, proline, asparagine, and glutamine.
Non-limiting examples of functionally equivalent variants of polypeptides may include conservative amino acid substitutions in the amino acid sequences of proteins disclosed in this application. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Additional non-limiting examples of conservative amino acid substitutions are provided in Table 1.
In some embodiments, 1,2, 3,4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 or more than 20 residues can be changed when preparing variant polypeptides.
In some embodiments, amino acids are replaced by conservative amino acid substitutions. In some embodiments, amino acids are replaced by non-conservative amino acid substitutions.
Table 1: Non-limiting examples of conservative amino acid substitutions Original Residue R Group Type Conservative Amino Acid Substitutions Ala (A) nonpolar aliphatic R group Cys, Gly, Ser Arg (R) positively charged R group His, Lys Mn (N) polar uncharged R group Asp, Gin, Glu Asp (D) negatively charged R group Asn, Gin, Giu Cs (C) polar uncharged R group Ala, Ser Gin (Q) polar uncharged R group A snõA.sp, Gin Giu (E) negatively charged R group Asn, Asp, Gin Gy (G) nonpolar aliphatic Rgroup Ma, Ser Uis (H) positively charged R group Arg, Tyr, Trp lie (I) nonpolar aliphatic R group Leu, Met, Val Leu (1_,) nonpolar aliphatic .R group Ile, Met, 'Val Lys (K) positively charged R group Arg, His Met (Ind) nonpolar aliphatic R group lie, Lou, Phe, Val .Pro (P) polar uncharged .R group Phc. (F) nonpolar aromatic R group Met, Trp, Tyr Ser (S) polar uncharged R group Ala, (ily, Thr Thr (T) polar uncharged R group Ala., Asn, Ser Trp (W) nonpolar aromatic R group His, Phe, Tyr, Met Tyr (Y) nonpolar aromatic R group His. Phe, Trp Val (V) nonpolar aliphatic R group Ile, Len, Met, Thr Amino acid substitutions in the amino acid sequence of a polypeptide to produce a recombinant polypeptide (e.g., a BMP or a protein associated with a BMP) variant having a desired property and/or activity can be made by alteration of the coding sequence of the polypeptide (e.g., a BMP or a protein associated with a BMP). Similarly, conservative amino acid substitutions in the amino acid sequence of a polypeptide to produce functionally equivalent variants of the polypeptide typically are made by alteration of the coding sequence of the recombinant polypeptide (e.g., a BMP or a protein associated with a BMP).
Mutations (e.g., substitutions, insertions, additions, or deletions) can be made in a nucleic acid sequence by a variety of methods known to one of ordinary skill in the art. For example, mutations (e.g., substitutions, insertions, additions, or deletions) can be made by PCR-directed mutation, site-directed mutagenesis according to the method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492, 1985), by chemical synthesis of a gene encoding a polypeptide, by gene editing methods, or by insertions, such as insertion of a tag (e.g., a HIS tag or a GFP tag). Mutations can include, for example, substitutions, insertions, additions, deletions, and translocations, generated by any method known in the art. Methods for producing mutations may be found in in references such as Molecular Cloning: A
Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York, 2010.
General Techniques The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the ordinary skill in the art (e.g., as disclosed in: Molecular Cloning: A Laboratory Manual, fourth edition (Green, et al., 2012 Cold Spring Harbor Press); Oligonucleotide Synthesis (M.
J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook, Vol. 3 (J. E. Cellis, ed., 2005) Academic Press; Animal Cell Culture (R. I.
Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E.
Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.
Griffiths, and D. G.
Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.);
Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.);
Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Cabs, eds., 1987); Short Protocols in Molecular Biology (F. M. Ausubel, et al., eds., 2002); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);
Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997);
Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989);
Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995). It is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited in the present application are incorporated by reference for the purposes or subject matter referenced herein.
EXAMPLES
In order that the invention described in the present application may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the systems and methods provided herein and are not to be construed in any way as limiting their scope.
Example 1: Screen to Identify Functional Expression of BMPs, BMP Peptides, Receptor Decoys, and Regulatory Proteins To identify BMPs, BMP peptides, receptor decoys, and/or regulatory proteins that could be functionally expressed in host cells, a library of BMPs (e.g., human mature BMP1, BMP 2, BMP 3, BMP 4, BMP 5, BMP 6, BMP 7, BMP 8a, BMP Sb, BMP 9, BMP10, BMPI 1, BMP12, BMP1.3, BMPI4, and/or BMP15) and derivatives of BMPs such as heterodimers and peptides derived from the above-listed BMPs, chim.eric BMPs, and rationally engineered BMPs is designed. The library may include naturally occurring BMPs and/or engineered versions of BM Ps. The library may also include one or more of proteins associated with BMPs including Activin. The library may include chimeric proteins that consist of domains from different BMPs or Activins, such. as a BMP2/7 chimeric protein or a BMP/Activin chimeric protein, for example. Proteins within the library, including chimeric proteins, may also include one or more amino acid modifications, such as amino acid substitutions, additions, deletions, or insertions. Without wishing to be bound by any theory, heterodimers, chimeric proteins, and amino acid modifications may increase the binding affinity of the ligands to BMP receptors, which include but are not limited to ALKI, AL,K2, ALIO, ALK6, ACVR2A, ACNR2B, BMPR2, and AMHR2, Engineered BMPs included in the library may exhibit increased potency compared to naturally occurring or wildtype 13 MPs.
Strategies for engineering BMPs may include -bioinformatics analysis of the BMP protein sequences. For example, bioinformatics analyses may be used to predict the fitness of the native amino acid at every position in the relevant wildtype protein sequences and to suggest favorable alternatives if the native amino acid is suboptimal. In some instances, BMPs within the library contain one or more point mutations relative to the corresponding wildtype B MP.
Receptor decoys such as BMP3, Activin 13, and Inhibin a are also designed, screened and tested. Without wishing to be bound by any theory, receptor decoys may bind to BMP
receptors without activating them. Receptor decoys may lower the activation of the SMAD
pathway induced by 131VIPs. Regulatory proteins, such as Activin, Gremlin, and/or Noggin, are also designed, screened and tested. Noggin and Gremlin are known to act as antagonists of BMP proteins, Receptor decoys and regulatory proteins may be naturally occurring proteins or may be engineered.
The screening and construction process may include DNA synthesis of DNA
sequences within the library that are cloned into a plasmid for expression in a host cell. For example, a host cell may be a yeast cell such as a P. pastoris cell, a bacterial cell such as an .Escherichia coli cell, and/or a mammalian cell, such as a 1-IEK 293 or CU() (Chinese hamster ovary) cell. The plasmids are transformed into the host cell and cultured in well plates. After growth at a defined temperature and time period, the library proteins produced by the host cells are assayed for activity and quantified using a method known the art such as ELBA
(enzyme-linked immunosorbent assay) or a -fluorescence detection assay.
Efficacy testing is performed for determining the presence and the level of alkaline phosphatase production, and by performing a Bone Gla Protein (BGP; osteocalcin) assay. Protein.s from the library are selected for further testing based on expression compatibility in the host cells as well as activity in the in vitro efficacy assay. Further testing may include testing the effects of proteins identified in the library on different cell types, including, for example, mesenchymal stem cells, preosteoblast cells, and/or osteoblast cells.
Example 2: Host Strain Engineering and Recombinant BMP Production Using P.
pastoris A P. pastoris strain is used that has been genetically altered for improved production of recombinant proteins. In general, strains will have a single amino acid auxotrophy (e.g., his4 or Ade2) to allow for selection of expression constructs following transformation. In addition, a limited number of protease knockouts (e.g., pep4 & prbl) or secretion pathway modifications (e.g., PB/1 overexpression) previously shown to be useful in general secretion screening strains may be introduced. BMP protein family members are known to retain biological activity even without sugar chains. However, since BMPs are relatively hydrophobic, recombinant BMP without sugar chains can cause protein aggregation. When using P. pastoris as a host cell, post-translational, modifications such. as glycosylation and folding can be performed. The host strain can be further modified to perform post-translational modifications (e.g., glycosylation) similar to the post-translational modifications (e.g., glycosylation) of human BMP 2 and BMP 7. The post-translational modifications will.
be evaluated by using proteoglycomics.
BMP2 and BMP7, and other candidate proteins identified in the screening are inserted into the genome of P pastoris. An episomal expression system is developed.
Various solubility and/or secretion tags (e.g., the S. cerevisiae mating factor) and codon optimization is tested to ensure optimal expression and secretion of the proteins. P
pastoris strains containing the candidate proteins are grown in 96-well plates. Cell lysates, supernatants, and/or purified proteins are assayed by quantification using ELISA or fluorescence assays.
Efficacy testing is then performed for determining the presence and/or the level of alkaline phosphatase production. The BGP assay is also conducted. The cell lysates, supernatants, and/or purified proteins are tested with mammalian cells to determine cell proliferation. A
fermentation process is developed for producing larger quantities of the candidate proteins.
For example, a bioreactor or fermenter can be used for culturing the host cells. Batch fermentation methods (e.g., shake flask or well plate fermentation) can be used.
Example 3: Host Strain Engineering and Recombinant BMP Production Using Mammalian Cells An advantage of the expression systems utilizing mammalian cells for generating recombinant proteins is the ability to introduce proper protein folding, post-translational modifications, and product assembly, which are important for complete and functional biological activities of mammalian proteins. A mammalian cell line capable of stably expressing BMPs and/or other candidate proteins is developed. The cell line is engineered for producing high protein titers. An expression system appropriate for mammalian cells and development of growth medium for optimal production is designed.
BMP2 and BMP7 and other potential candidate proteins are inserted into an episomal expression system for transient expression. Alternatively, BMP2 and BMP7 and other potential candidate proteins are inserted into a construct for stable expression. A strong promoter such as the CMV or CAG promoter is used to express the BMPs or other potential candidate proteins, which is optionally adjusted by adding enhancer elements.
Various solubility and/or secretion tags (e.g., the S. cerevisiae mating factor) are tested. Codon optimization is also tested to ensure optimal expression and secretion of the proteins, if needed. The episomal expression system is transfected into mammalian cells such as HEK-293 or CHO cells. Any mammalian cells suitable for producing recombinant proteins can be used. The mammalian cells are grown in liquid medium. Cell lysates, supernatants, or purified proteins are assayed by quantification using ELISA or fluorescence assays. Efficacy testing is performed for determining the presence and/or the level of alkaline phosphatase production. The BGP assay is also conducted. The cell lysates, supernatants, and/or purified proteins are also tested with mammalian cells to determine cell proliferation.
A fermentation process is developed for producing larger quantities of proteins. For example, a bioreactor or fermenter can be used for culturing the cell. Batch fermentation methods (e.g., shake flask fermentation) can be used.
Example 4: Administration of BMPs and/or BMP Associated Proteins in Human Subjects In order to examine the effects of BMPs and/or BMP associated proteins on anti-aging and aesthetic related characteristics, male and female subjects who are older than 18 years of age are recruited for the studies. Subjects who are at least 65 years of old (e.g., elderly) are a preferred group in some instances. Subject candidates are first screened for their health history and condition (e.g., allergies, pregnancies, drug and medications that may contraindicate the use of BMPs and BMP associated protein formulations, familial inheritance related to skin conditions), their routine skin care, and other aesthetic non-invasive procedures they have received according to various criteria that will be set forth.
Prior to any treatments, baseline profiles are developed for each recruited subject. The baseline profiles may include but are not limited to several headshots of the subjects, anthropometric facial skeleton measurements such as the skeleton features surrounding periorbital regions, midface regions, perinasal regions, and lower face regions, and wrinkles and fine lines assessments via dermatoscopy and digital fringe profilometry techniques, for example, and one or more questionnaires capturing the subjects self-assessment of attractiveness and desired improvements in facial contouring or skin texture or evenness of skin tone.
BMPs and/or BMP associated proteins are administered to the selected human subjects for testing the aesthetic and/or cosmetic effects of the proteins. As disclosed in the present application, any BMPs and BMP associated proteins suitable for the study designs may be chosen. For example, Activin, BMP2, BMP6, BMP7 or BMP9 can be administered to the subjects. In some instances, more than one BMP can be formulated for administration to the subjects. BMPs can be administered to the subjects with BMP associated proteins. For example, any of the BMPs can be administered to subjects in combination with Noggin for regulating the functions of BMPs in vivo. As disclosed in the present application, Noggin may function as a "off switch" to BMPs, so that the effects of BMPs can be attenuated or delayed. BMPs and BMP associated proteins can be formulated separately (i.e., in two different formulations) or prepared in the same formulation. When appropriate, subjects may receive administration of BMP associated proteins at the same time or after the administration of BMPs. Some subjects may be administered placebo treatments which will not contain any BMPs and/or BMP associated proteins.
BMPs and/or BMP associated proteins may be administered to the subject topically, such as in the form of a cream and/or serum, or via subdermal, subcutaneous or supraperiostial injections. BMPs and/or BMP associated proteins will optionally be encapsulated in a formulation for enhancing penetration of the proteins through the skin of the subjects.
For topical administrations, BMPs and BMP regulatory proteins such as Noggin may be formulated at a concentration of less than about 3%. BMP peptides may be formulated at a concentration of less than about 10% for topical administrations. Subjects who receive topical administration may optionally receive skin pre-treatments such as exfoliations or peels to minimize potential inconsistency of absorption of the cream or serum. For subdermal, subcutaneous or supraperiostial administration, BMPs and BMP regulatory proteins such as Noggin may be formulated at a concentration of about 100 to about 1,000 ng/mL
for each injection site. The doses of formulations for topical or subdermal, subcutaneous or supraperiostial administration can be adjusted based on the needs of a subject such as the estimated frequencies of the administrations and site of the administrations (e.g., face versus hand). For subdermal, subcutaneous or supraperiostial injections, BMPs and BMP
regulatory proteins can be either administered in a pre-formulated composition or in a formulation that will be prepared right before the injections. Alternatively, BMPs and BMP
regulatory proteins can be pre-mixed with dermal fillers prior to the injections. In some instances, a subject may receive both subdermal (or subcutaneous or supraperiostial) injection and topical administration.
Example 5: Effects of Administration of BMPs and BMP Associated Proteins on the Appearance of Skin of Subjects As described in Example 4, various BMPs and BMP regulatory proteins are administered to human subjects for determining anti-aging and aesthetic characteristics. After administration (e.g., topical or subdermal), the subjects are evaluated for any changes of the appearance of their skin and for changes in their perception of attractiveness. A list of skin characteristics and signs to be examined is developed. For example, characteristics for evaluation may include the appearance of skin wrinkles and fine lines around the face and neck (e.g., crinkle lines, permanent elastotic creases, dynamic expression lines, and/or gravitational folds), the appearance of facial contours, the appearance of facial bone structures, and/or the subjects' perception of improvement in attractiveness.
Depending on the type of administration, various time points post-administrations are assigned for conducting evaluations. For example, for subjects who receive subdermal (or subcutaneous or supraperiostial) injections, characteristics may be evaluated as soon as two days after the initial injection. In some instances, the subjects who receive subdermal (or subcutaneous or supraperiostial) injections are evaluated 10 or more days after the initial injection. For subjects who receive topical administration, characteristics may be evaluated, e.g., approximately 17 days after the initial application. In some instances, subjects who receive topical administration of cream and/or serum are evaluated 30 or more days after the initial application. Optionally, subjects are evaluated at multiple time points to monitor any longer-term effects. Subjects may be evaluated against their own baseline profiles and/or against skin conditions and changes of subjects who receive placebo treatments.
EQUIVALENTS
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described in the present application. Such equivalents are intended to be encompassed by the following claims. All references, including patent documents, disclosed in this application are incorporated by reference in their entirety, particularly for the disclosure referenced in this application.
Claims (65)
1. A method of preventing or reducing skin wrinkles or improving skin texture or evenness of skin tone, the method comprising administering to a subject in need thereof an effective amount of a composition comprising one or more bone morphogenetic proteins (BMPs), or variants or derivatives thereof.
2. A method of enhancing or preserving facial contours or improving the attractiveness of facial contours, the method comprising administering to a subject in need thereof an effective amount of a composition comprising one or more bone morphogenetic proteins (BMPs), or variants or derivatives thereof.
3. The method of claim 1 or 2, wherein the BMP, or variant or derivative thereof, is selected from the group consisting of BMP2, BMP6, BMP7, and BMP9, or variants or derivatives thereof.
4. The method of any one of claims 1-3, wherein the BMP, or variant or derivative thereof, is BMP2, or a variant or derivative thereof.
5. The method of any one of claims 1-3, wherein the BMP, or variant or derivative thereof, is BMP6, or a variant or derivative thereof.
6. The method of any one of claims 1-3, wherein the BMP, or variant or derivative thereof, is BMP7, or a variant or derivative thereof.
7. The method of any one of claims 1-3, wherein the BMP, or variant or derivative thereof, is BMP9, or a variant or derivative thereof.
8. The method of claim 1 or 2, wherein the composition comprises BMP2, BMP7, BMP6, and BMP9, or variants or derivatives thereof.
9. The method of any one of claims 1-7, wherein the composition further comprises one or more proteins associated with a BMP, or a variant or derivative thereof.
10. The method of claim 8, wherein the protein associated with a BMP is Noggin, or a variant or derivative thereof.
11. The method of claim 8, wherein the protein associated with a BMP is a BMP peptide, or a variant or derivative thereof.
12. The method of claim 8, wherein the protein associated with a BMP is Activin, or a variant or derivative thereof.
13. The method of any one of claims 1-12, wherein the subject is a human subject having, suspected of having, or at risk of developing, skin wrinkles.
14. The method of any one of claims 1-13, wherein the subject is a human subject who desires improving skin texture or evenness of skin tone.
15. The method of claim 13, wherein the skin wrinkles are facial and/or neck wrinkles.
16. The method of any one of claims 1-15, wherein the subject is a human subject having, suspected of having, or at risk of having, loss of natural facial contours.
17. The method of any one of claims 1-16, wherein the subject is a human subject who desires a change in facial contours to improve attractiveness.
18. The method of any one of claims 1-17, wherein the subject is elderly.
19. The method of any one of claims 1-17, wherein the subject is not elderly.
20. The method of any one of claims 2-19, wherein enhancing or preserving facial contours comprises inhibiting or reversing bone resorption.
21. The method of any one of claims 1-20, wherein the one or more BMPs, or variants or derivatives thereof, stimulates the formation of adipocytes in the skin.
22. The method of any one of claims 1-20, wherein the one or more BMPs, or variants or derivatives thereof, stimulates dedifferentiation of keratinocytes.
23. The method of any one of claims 1-20, wherein the one or more BMPs, or variants or derivatives thereof, reduces or modulates melanin formation by melanocytes.
24. The method of claim 10, wherein the presence of Noggin, or a variant or derivative thereof, reduces potential side effects of the one or more BMPs, or variants or derivatives thereof.
25. The method of any one of claims 1-24, wherein the composition is administered topically.
26. The method of claim 25, wherein the composition is administered in a cream or serum.
27. The method of any one of claims 1-24, wherein the composition is administered through subdermal injection.
28. The method of claim 27, wherein the subdermal injection is near periosteum.
29. The method of any one of claims 1-28, wherein the concentration of the one or more BMPs, or variants or derivatives thereof, in the composition is less than about 3%.
30. The method of claim 10 or 24, wherein the concentration of Noggin, or a variant or derivative thereof, in the composition is less than about 3%.
31. The method of claim 11, wherein the concentration of the BMP peptide, or a variant or derivative thereof, in the composition is less than about 10%.
32. The method of claim 24, wherein the one or more BMPs, or variants or derivatives thereof, are administered at a dose of about 100-1,000 ng/mL per administration site.
33. The method of claim 24, wherein the method further comprises administering Noggin, or a variant or derivative thereof, at a dose of about 100-1,000 ng/mL per administration site.
34. A composition comprising one or more bone morphogenetic proteins (BMPs), or variants or derivatives thereof, in a pharmaceutically acceptable carrier for use in preventing or reducing skin wrinkles or improving skin texture or evenness of skin tone.
35. A composition comprising one or more bone morphogenetic proteins (BMPs), or variants or derivatives thereof, in a pharmaceutically acceptable carrier for use in enhancing or preserving facial contours or improving the attractiveness of facial contours.
36. The composition of claim 34 or 35, wherein the BMP, or variant or derivative thereof, is selected from the group consisting of BMP2, BMP7, and BMP9, or variants or derivatives thereof.
37. The composition of any one of claims 34-36, wherein the BMP, or variant or derivative thereof, is BMP2, or a variant or derivative thereof.
38. The composition of any one of claims 34-36, wherein the BMP, or variant or derivative thereof, is BMP6, or a variant or derivative thereof.
39. The composition of any one of claims 34-36, wherein the BMP, or variant or derivative thereof, is BMP7, or a variant or derivative thereof.
40. The composition of any one of claims 34-36, wherein the BMP, or variant or derivative thereof, is BMP9, or a variant or derivative thereof.
41. The composition of claim 34 or 35, wherein the composition comprises BMP2, BMP6, BMP7 and BMP9, or variants or derivatives thereof.
42. The composition of any one of claims 34-41, wherein the composition further comprises one or more proteins associated with a BMP, or a variant or derivative thereof.
43. The composition of claim 42, wherein the protein associated with a BMP
is Noggin, or a variant or derivative thereof.
is Noggin, or a variant or derivative thereof.
44. The composition of claim 42, wherein the protein associated with BMP is a BMP
peptide, or a variant or derivative thereof.
peptide, or a variant or derivative thereof.
45. The composition of claim 42, wherein the protein associated with a BMP
is Activin, or a variant or derivative thereof.
is Activin, or a variant or derivative thereof.
46. The composition of any one of claims 34-45, wherein the composition is a cream or serum.
47. The composition of any one of claims 34-46, wherein the concentration of the one or more BMPs, or variants or derivatives thereof, in the composition is less than about 3%.
48. The composition of claim 43, wherein the concentration of Noggin, or a variant or derivative thereof, in the composition is less than about 3%.
49. The composition of claim 44, wherein the concentration of the BMP
peptide, or a variant or derivative thereof, in the composition is less than about 10%.
peptide, or a variant or derivative thereof, in the composition is less than about 10%.
50. The composition of any one of claims 34-45, wherein the composition is formulated for subdermal injection.
51. The composition of claim 50, wherein the composition further comprises a dermal filler.
52. The composition of any one of claims 46-49, wherein the composition further comprises one or more agents that enhance penetration.
53. The method of any one of claims 1-33, wherein the one or more BMPs, or variants or derivatives thereof, are recombinantly produced.
54. The method of claim 53, wherein the one or more BMPs, or variants or derivatives thereof, are recombinantly produced in a mammalian cell.
55. The method of claim 54, wherein the mammalian cell is a HEK 293 cell or a CHO
cell.
cell.
56. The method of claim 53, wherein the one or more BMPs, or variants or derivatives thereof, are recombinantly produced in a yeast cell.
57. The method of claim 56, wherein the yeast cell is a Pichia pastoris cell.
58. The composition of any one of claims 34-52, wherein the one or more BMPs, or variants or derivatives thereof, are recombinantly produced.
59. The composition of claim 58, wherein the one or more BMPs, or variants or derivatives thereof, are recombinantly produced in a mammalian cell.
60. The composition of claim 59, wherein the mammalian cell is a HEK 293 cell or a CHO cell.
61. The composition of claim 58, wherein the one or more BMPs, or variants or derivatives thereof, are recombinantly produced in a yeast cell.
62. The composition of claim 61, wherein the yeast cell is a Pichia pastoris cell.
63. The composition of claim 58, wherein the one or more BMPs, or variants or derivatives thereof, are recombinantly produced in an E. coli cell.
64. A kit comprising a composition comprising one or more bone morphogenetic proteins (BMPs), or variants or derivatives thereof, and instructions for use in administering the composition to prevent or reduce skin wrinkles or to improve skin texture or evenness of skin tone.
65. A kit comprising a composition comprising one or more bone morphogenetic proteins (BMPs), or variants or derivatives thereof, and instructions for use in administering the composition to enhance or preserve facial contours or to improve the attractiveness of facial contours.
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