WO2020163771A1 - Structure, manufacturing and uses of hoxd12-pde8a cell-penetrating peptides - Google Patents
Structure, manufacturing and uses of hoxd12-pde8a cell-penetrating peptides Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Definitions
- the embodiments disclosed herein relate generally to HoxD12-PDE8A cell- penetrating peptides, compositions thereof and methods for treating and preventing disease, such as cancer through, administration of the HoxD12-PDE8A cell-penetrating peptides. These peptides facilitate entry into tissues, cells and the nucleus of cells thus allowing the PDE8A fusion protein to reach its site of action for therapeutic purposes.
- genes may be regulated through the direct delivery of biologically active molecules, such as nucleic acids, peptides and proteins, to their intracellular and intranuclear sites of action to influence gene expression either directly or indirectly through interference with transcription, translation or transcription factor production and action and also missing or defective protein products may be replaced to provide these types of molecules in individuals with germ-line or somatic mutations.
- biologically active molecules such as nucleic acids, peptides and proteins
- the use of biologically important proteins is hampered by the inability of these proteins to reach intracellular sites and tissue sites where they normally function.
- patients having certain genotypes, including genetic mutations are cannot be treated with conventional therapeutics and/or develop a resistance to conventional therapeutics over time.
- conjugates comprising: a first region comprising: (i) a homeodomain structure and having a polypeptide sequence set forth in SEQ ID No. 1 or a variant thereof or (ii) at least one of a HOX D12 homeodomain; and a second region comprising a cAMP-degrading phosphodiesterase-8A (PDE8A) - C-Raf complex disruptor.
- a first region comprising: (i) a homeodomain structure and having a polypeptide sequence set forth in SEQ ID No. 1 or a variant thereof or (ii) at least one of a HOX D12 homeodomain
- PDE8A cAMP-degrading phosphodiesterase-8A
- compositions comprising a conjugate according to any embodiment disclosed and described herein and a pharmaceutically acceptable carrier.
- kits for treating cancer in a subject comprising administering a conjugate for a period of time sufficient to increase C-Raf- S259 phosphorylation and/or reduce ERK signaling.
- the PDE8A - C-Raf complex disruptor is PDE8A or a fragment thereof.
- the PDE8A has a polypeptide sequence set forth in SEQ. ID No. 2 or a variant thereof.
- the second region is conjugated to the C-terminus of the first region. In other embodiments, the second region is conjugated to the N- terminus of the first region.
- the conjugate is in the form of a fusion protein.
- the conjugate has a polypeptide sequence set forth in SEQ. ID No. 3.
- the conjugate further comprises a linker sequence between the first and second regions.
- the first region is linked to the second region with a peptidic bond or a non-peptidic bond.
- the linker sequence is a thioester linker.
- the composition is in the form of an inhalable composition, an enema, a topical composition, or an injectable composition including injectable implants for sustained release.
- the composition further comprises a B-Raf inhibitor.
- the formulation is administered as an intravenous solution.
- the conjugate elicits slow cancer cell growth and/or stops cancer cell growth compared to a control.
- the control is a peptide comprising the second region when not conjugated to the first region.
- the cancer is melanoma, malignant melanoma, colon cancer, colorectal cancer, lung cancer, urinary bladder carcinoma, and/or thyroid carcinoma.
- the cancer is B-Raf resistant melanoma.
- the cancer is metastatic cancer.
- the subject has previously been administered a B- Raf inhibitor. In some embodiments, the subject is resistant to a B-Raf inhibitor.
- the conjugate interacts with and/or prevents endogenous PDE8A binding with endogenous C-Raf. In some embodiments, the conjugate potentiates PKA-mediated inhibitory phosphorylation. In some embodiments, at least a portion of total C-Raf remains complexed with PDE8A.
- the subject is administered a B-Raf inhibitor, for example, the B-Raf inhibitor is administering prior to, simultaneously with, and/or following administration of a conjugate or composition disclosed and described herein.
- the subject has wildtype BRAF, NRAS gain-of- function mutations, KRAS gain-of-function mutations, and/or HRAS gain-of-function mutations.
- the autoimmune disease is multiple sclerosis or rheumatoid arthritis.
- FIG. 1 shows exemplary conjugates of HoxD12 (SEQ. ID Nos, 23, 25, 27, and 29) and PDE8A (residues 454-465) (SEQ. ID Nos 24, 26, 28, and 30) formed by thioester or disulfide linkages.
- FIGs. 2A-2C show the effects of HoxD12-PDE8A cell-penetrating conjugates (also referred to herein as“PPL008”) on pERK levels and rate of cell proliferation.
- Normalized phospho-ERK (mean ⁇ SEM) following treatment with DMSO (lane 1 ), PLX4032 B-Raf inhibitor (lane 2), PPL008 conjugates (lanes 1 1 -14) or PLX co-treatments with PDE8A - C-Raf peptide disrupters (original stearylated‘disrupter’ lane 3, or scrambled control , lane 4) or PPL-008 conjugates (lanes 7-10) in: (A) MM415 (NRAS Q61 L) and (B) A375 (BRAF V600E) human malignant melanoma cell lines.
- FIGs. 4A-4D show PPL008-C/N dose response in MM415 (NRAS Q61 L) human malignant melanoma cell line.
- FIGs. 4C(ii) and 4D(ii) show representative traces of normalized cell index of each treatment shown below. N, PPL-008N; C, PPL-008C.
- FIGs. 5A-5C demonstrate in vivo suppression of phospho-ERK signaling in a MM415 murine xenograft model. In vivo suppression of phospho-ERK signaling in an MM415 murine xenograft model.
- FIG. 5A shows normalized total pERK1/2
- FIG. 5B shows pERK1 (T202, 44kDa)
- FIG. 5A shows normalized total pERK1/2
- FIG. 5B shows pERK1 (T202, 44kDa)
- FIGs. 6A-6B are schematics showing the dual inhibition of B-Raf and C-Raf that inhibits melanoma tumor progression.
- FIG. 6A shows B-Raf inhibition leads to the Ras negative feedback mechanism switching to C-Raf driven tumorigenesis via potentiation of the Raf/MEK/ERK signalling axis.
- FIG. 6B shows PPL-008 cell- penetrating peptide (PDE8A - C-Raf disrupter peptide) binds to C-Raf, preventing PDE8A localization within the C-Raf cAMP microdomain and exposing serine 259 - C- Raf to inhibitory phosphorylation by PKA.
- Co-treatment with B-Raf inhibitor and PPL- 008 blocks onco-Ras driven tumor progression via inhibition of the Raf/MEK/ERK axis.
- Melanoma is the most aggressive form of skin cancer, with a wide range of treatments currently available and many more at pre-clinical and clinical phases 8 ⁇ 32 ⁇ 33 .
- First line B-Raf inhibitors are capable of managing the majority of melanoma patients that express the BRAF V600E mutation 33 .
- B-Raf inhibitors become ineffective and tumors persist - warranting the development of novel effective treatments 10 18 .
- conjugates comprising: a first region comprising: (i) a homeodomain structure and having a polypeptide sequence set forth in SEQ ID No. 1 or a variant thereof or (ii) at least one of a HOX D12 homeodomain; and a second region comprising a cAMP-degrading phosphodiesterase-8A (PDE8A) - C-Raf complex disruptor.
- PDE8A cAMP-degrading phosphodiesterase-8A
- homeodomain structure refers to HOX-derived homeodomains, such as the human HOX D12 and HOX C12 sequences shown in Table 1 as SEQ ID No. 1 and SEQ ID No. 4, respectively, or variants or portions thereof.
- homology refers to identity or near identity of nucleotide or amino acid sequences.
- nucleotide mismatches can occur at the third or wobble base in the codon without causing amino acid substitutions in the translated polypeptide sequence.
- minor nucleotide modifications e.g., substitutions, insertions or deletions
- substitutions, insertions or deletions in certain regions of the gene sequence can be tolerated whenever such modifications result in changes in amino acid sequence that do not alter functionality of the final gene product.
- Homologs of specific DNA sequences may be identified by those skilled in the art using the test of cross hybridization of nucleic acids under conditions of stringency as is well understood m the art (as described in Hames et al. , Nucleic Acid Hybridisation, (1985) IRL Press, Oxford, UK). Extent of homology is often measured in terms of percentage of identity between the sequences compared.
- variant refers to a polypeptide or protein that differs from a reference polypeptide or protein, but retains essential properties.
- a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall (homologous) and, in many regions, identical.
- a variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions).
- a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
- a variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
- Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
- Exemplary substitutions that take one or more of the foregoing characteristics into consideration are well known to those of skill in the art and include, but are not limited to (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (lie: Leu, Val), (Leu: lie, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: lie, Leu).
- Embodiments of this disclosure therefore, consider functional or biological equivalents of a polypeptide or protein as set forth above.
- and proteins can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide and protein of interest.
- D-amino acids can be used for the first region and/or the second region. It is contemplated that using D-amino acids for at least the second region will reduce intracellular degradation and/or increase the pharmacological half- life.
- Identity is a relationship between two or more polypeptide or protein sequences, as determined by comparing the sequences.
- identity also refers to the degree of sequence relatedness between polypeptides or proteins, as determined by the match between strings of such sequences. “Identity” can be readily calculated by known bioinformational methods.
- the first region of the conjugate or fusion protein embodiments disclosed may comprise a natural or synthetic 60-amino acid peptide or variant or portion thereof derived from the HOX D12 or HOX C12 gene or variants or portions thereof.
- the HOX D12 (SEQ ID No. 1 ) amino acid sequence is:
- the HOX C12 (SEQ ID No. 4) amino acid sequence is:
- synthetic variants may be used provided that they retain the ability to translocate the membrane. Synthetic variants will generally differ from the naturally-occurring proteins by substitution, particularly conservative substitution.
- the phrase“conservative amino acid changes” herein means replacing an amino acid from one of the amino acid groups, namely hydrophobic, polar, acidic or basic, with an amino acid from within the same group. An example of such a change is the replacement of valine by methionine and vice versa. Other examples of conservative substitutions may be seen by reference to Table 2 below: [0045] Table 2: Conservative Amino Acid Substitutions.
- Such variants may be synthesized directly or prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions corresponding to the naturally-occurring sequence either side of the insertion site. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed to make the encoded protein.
- These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.
- Variants that retain at least 50% sequence identity with the claimed 60-amino acid sequences or variants or portions thereof derived from HOX-D12 will likely maintain their cell permeability characteristics and retain their human characteristics resulting in low immunogenicity potential.
- the ability of a naturally occurring or synthetic HOX sequence to translocate the membrane may be tested by routine methods known in the art.
- Second Region Peptides or Proteins may comprise a cAMP-degrading phosphodiesterase-8A (PDE8A) - C-Raf complex disruptor.
- PDE8A cAMP-degrading phosphodiesterase-8A
- the second region also may or may not be from the same species as the first region, but the first and second regions will be present in the conjugate or fusion protein embodiments in a manner different from the natural situation.
- the second region of the fusion protein or conjugate embodiments may be a cAMP-degrading phosphodiesterase-8A (PDE8A) - C-Raf complex disruptor peptide or protein of any length as long as it is biologically active on its target when included in the fusion protein or conjugate.
- PDE8A - C-Raf complex disruptor is PDE8A or a fragment thereof, for example, in certain embodiments, the PDE8A has a polypeptide sequence set forth in SEQ. ID No. 2 or a variant thereof.
- the conjugate comprises polypeptide sequence set forth in SEQ. ID No. 3.
- conjugates comprises a category of structures, including fusion proteins, in which the first region, a homeodomain sequence or variant or portion thereof, is conjugated to the PDE8A - C-Raf complex disruptor directly via a peptide bond or other type of bond including both covalent and non- covalent bonds.
- Conjugates may include a linker region that connects the homeodomain sequence to the PDE8A - C-Raf complex disruptor that is not naturally associated with the first region.
- phrase“not naturally associated with” means that entire sequence of the conjugate or fusion protein is not found in nature, and that the entire sequence is not encoded for by a single gene found in nature.
- linkers short, connecting sequences
- fusion protein is used to refer to a particular subcategory of conjugate that exists when no such linkers are used to form the conjugate and the domains are linked entirely by peptide bonds.
- the first region and the second regions may be linked by a cleavable linker region this may be any region suitable for this purpose provided the function of the conjugate is not compromised by its addition.
- the second region is conjugated to the C-terminus of the first region. In other embodiments, the second region is conjugated to the N-terminus of the first region.
- the cleavable linker region is a protease cleavable linker, although other linkers, cleavable for example by small molecules, may be used. These include Met-X sites, cleavable by cyanogen bromide, Asn-Gly, cleavable by hydroxylamine, Asp-Pro, cleavable by weak acid and Trp-X cleavable by, inter alia, NBS-skatole. Protease cleavage sites require milder cleavage conditions and are found in, for example, factor Xa, thrombin and collagenase. Any of these may be used.
- the cleavable linker region may be one that is targeted by endocellular proteases. Linkers may not be required for function but linkers may be included between first and second regions to allow targeted release of the second region without compromising function or to enhance biological activity of the second region with linker cleavage.
- the conjugates of the present disclosure may comprise a linker sequence between the first and second regions.
- the first region is linked to the second region with a peptidic bond or a non-peptidic bond.
- the linker sequence is a thioester linker.
- composition embodiments disclosed herein may comprise one or more of the cell-penetrating conjugates disclosed and described herein and a pharmaceutically acceptable carrier, diluent or excipient.
- pharmaceutically acceptable carrier diluent or excipient refers to any substance, not itself a therapeutic agent, used as a carrier or vehicle for delivery of a therapeutic agent to a subject or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a unit dose of the composition, and that does not produce unacceptable toxicity or interaction with other components in the composition.
- compositions may comprise any agents that may aid, regulate, release or increase entry into the body compartment, tissue, intracellular or intranuclear target site, such as binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilizing agent(s), or other agents.
- An injectable implant for the sustained release of the protein may also be used to obtain prolonged exposure and action.
- sustained release refers to formulations from which the conjugate is released at a slow rate allowing for a longer period of exposure at active concentrations.
- compositions comprising one or more cell-penetrating conjugates disclosed herein can be administered, depending on condition to be treated or other considerations, in any number of ways, for example without limitation, by any one or more of the following: (1 ) inhalation; (2) in the form of a suppository or pessary; (3) in the form of a topical lotion, solution, cream, ointment or dusting powder; (4) by use of a skin patch; (5) orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavoring or coloring agents; (6) injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously; (7) for ophthalmic diseases, they may be formulated as eye drops or for intraocular injection; (8) for parenteral administration, they may be
- the composition is in the form of an inhalable composition, an enema, a topical composition, or an injectable composition including injectable implants for sustained release.
- the cell-penetrating conjugates further comprise a B- Raf inhibitor.
- Non-limiting examples of suitable B-Raf inhibitors include vemurafenib (aslso known as PLX4032 and Zelboraf, Daiichi-Sankyo), encorafenib, XL281 (also known as BMS-908662, Bristol-Myers Squibb), LGX818 (Novartis), PLX3603 (Hofmann-LaRoche), RAF265 (Novartis), R05185426 (Hofmann-LaRoche), and GSK21 18436 (also known as dabrafenib and Tafinlar, GlaxoSmithKline).
- vemurafenib as PLX4032 and Zelboraf, Daiichi-Sankyo
- encorafenib XL281
- LGX818 Novartis
- PLX3603 Hofmann-LaRoche
- RAF265 Novartis
- R05185426 Hof
- the active concentration of cell-penetrating conjugates in cell culture is less than about 1 15 mM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 65 pM, less than about 60 pM, less than about 55 pM, less than about 50 pM, less than about 45 pM, less than about 40 pM, less than about 35 pM, less than about 30 pM, less than about 25 pM, less than about 20 pM, less than about 15 pM, less than about 10 pM, less than about 5 pM, or less than about 1 pM for example, less than about 1 pM to about 3 pM, less than about 1 pM to about 6 pM, less than about 1 pM to about 8 pM, less than about 1 pM to about 15 pM, less than about 1 pM to about 25 pM, less than about 1 pM
- the dosage delivered (daily or as required) in mouse models (per 20 g mouse) through (1 ) an intravenous (i.v.) or intraperitoneal (i.p.) injection, (2) a topical formulation, or (3) an inhaled formulation is at least 500 pg, at least 450 pg, at least 400 pg, at least 350 pg, at least 300 pg, at least 250 pg, at least 200 pg, at least 150 pg, at least 100 pg, at least 80 pg, at least 70 pg, at least 60 pg, at least 50 pg, at least 40 pg, at least 30 pg, at least 20 pg, at least 10 pg, or at least 1 pg, for example, about 1 pg to about 500 pg, about 10 pg to about 450 pg, about 20 pg to about 400 pg about 30 pg to about 350 pg, about 30 p
- the dosage delivered (daily or as required) through an intravenous (i.v.) or intraperitoneal (i.p.) injection in a mouse model is at least 100 mg/kg, less than about 80 mg/kg, less than about 45 mg/kg, less than about 40 mg/kg, less than about 30 mg/kg, less than about 25 mg/kg, less than about 20 mg/kg, less than about 15 mg/kg, less than about 12 mg/kg, less than about 10 mg/kg, less than about 8 mg/kg, less than about 4 mg/kg, less than about 2 mg/kg, or less than about 1 mg/kg, for example, less than about 1 mg/kg to about 50 mg/kg, about 5 mg/kg to about 40 mg/kg, about 8 mg/kg to about 30 mg/kg, about 10 mg/kg to about 20 mg/kg, or about 12 mg/kg to about 15 mg/kg, about 8 mg/kg to about 12 mg/kg, about 5 mg/kg to about 9 mg/kg, about 3 mg/kg, less than about 1 mg/
- the dosage delivered (daily or as required) through an inhaled formulation in humans is at least about 600 mg, at least about 500 mg, at least about 450 mg, at least about 400 mg, at least about 350 mg, at least about 300 mg, at least about 250 mg, at least about 200 mg, at least about 150 mg, at least about 125 mg, at least about 100 mg, at least about 75 mg, at least about 50 mg, at least about 25 mg, at least about 20 mg, at least about 15 mg, at least about 10 mg, at least about 5 mg, at least about 1 mg, at least about 500 pg, at least about 450 pg, at least about 400 pg, at least about 350 pg, at least about 300 pg, at least about 250 pg, at least about 200 pg, at least about 150 pg, at least about 100 pg, at least about 80 pg, at least about 70 pg, at least about 60 pg, at least about 50 pg,
- the dosage delivered (daily or as required) through topical formulation in humans and in mouse models is less than about 5% wt/vol, less than about 4.5% wt/vol, less than about 3.5% wt/vol, less than about 2.5% wt/vol, less than about 1 .5% wt/vol, less than about 0.5% wt/vol, less than about 0.4% wt/vol, less than about 0.3% wt/vol, less than about 0.2%, less than about 0.1 % wt/vol, less than about 0.09% wt/vol, less than about 0.08% wt/vol, less than about 0.07% wt/vol, less than about 0.06% wt/vol, less than about 0.05% wt/vol, less than about 0.04% wt/vol, less than about 0.03% wt/vol, less than about 0.02% wt/vol, less than about 0.01 % wt/vol, less than about 0.008%
- the dosage delivered (daily or as required) through a topical formulation in humans is less than about 70 pg, less than about 50 pg, less than about 45 pg, less than about 40 pg, less than about 30 pg, less than about 25 pg, less than about 20 pg, less than about 15 pg, less than about 12 pg, less than about 10 pg, less than about 8 pg, less than about 4 pg, less than about 2 pg, or less than about 1 pg, for example, about 1 pg to about 50 pg, about 5 pg to about 40 pg, about 8 pg to about 30 pg, about 10 pg to about 20 pg, or about 12 pg to about 15 pg, about 8 pg to about 12 pg, about 5 pg to about 9, about 3 pg to about 6 pg, about 2 pg to about 5 pg, or
- the systemic dosage delivered (daily or as required) through an intravenous, subcutaneous or intramuscular injection or an injectable implant for sustained release formulations in humans is less than about 100 mg/kg, less than about 80 mg/kg, less than about 45 mg/kg, less than about 40 mg/kg, less than about 30 mg/kg, less than about 25 mg/kg, less than about 20 mg/kg, less than about 15 mg/kg, less than about 12 mg/kg, less than about 10 mg/kg, less than about 8 mg/kg, less than about 4 mg/kg, less than about 2 mg/kg, less than about 1 mg/kg, less than about 0.1 mg/kg, or less than about 0.01 mg/kg, for example, less than about 0.01 mg/kg to about 50 mg/kg, less than about 5 mg/kg to about 40 mg/kg, less than about 8 mg/kg to about 30 mg/kg, less than about 10 mg/kg to about 20 mg/kg, or less than about 12 mg/kg to about 15 mg/kg, less than
- the dosage delivered (daily or as required) to humans (based on 70 kg weight) through any formulation other than an intravenous, subcutaneous, or intramuscular injection or injectable implant for the sustained release, inhaled or topical formulation is at least about 600 mg, at least about 500 mg, at least about 450 mg, at least about 400 mg, at least about 350 mg, at least about 300 mg, at least about 250 mg, at least about 200 mg, at least about 150 mg, at least about 125 mg, at least about 100 mg, at least about 75 mg, at least about 50 mg, at least about 25 mg, at least about 20 mg, at least about 15 mg, at least about 10 mg, at least about 5 mg, at least about 1 mg, at least about 500 pg, at least about 450 pg, at least about 400 pg, at least about 350 pg, at least about 300 pg, at least about 250 pg, at least about 200 pg, at least about 150 pg, at least about 100 pg, at least about 600 mg, at least about 500
- novel cell-penetrating peptides and compositions thereof for use in treating and/or preventing diseases and disorders.
- kits for treating cancer or autoimmune diseases in a subject comprising administering a formulation comprising the conjugates or compositions disclosed herein.
- the cell-penetrating conjugates, compositions, formulations, and methods can be used to treat any type of cancer.
- cancer include skin cancer (e.g., melanoma, malignant melanoma), lung cancer (e.g., non-small lung cell carcinoma), urinary bladder carcinoma, thyroid cancer, prostate cancer, endometrial cancer, lung cancer, breast cancer, colon cancer, and colorectal cancer.
- the cancer is melanoma.
- the cancer is a gain-of- function onco-ras mutation cancer.
- the cancer is a B-Raf resistant cancer, for example, B-Raf resistant melanoma.
- the cancer is metastatic cancer.
- the cell-penetrating conjugates, compositions, formulations, and methods can be used to treat any type of autoimmune disease.
- autoimmune diseases include multiple sclerosis and rheumatoid arthritis.
- cell-penetrating peptides can be administered orally or via injection (e.g., subcutaneous injection, intramuscular injection, intravenous injection, or intrathecal injection).
- cell-penetrating peptides can be administered by different routes.
- one cell-penetrating peptide can be administered orally and a second cell-penetrating peptide can be administered via injection.
- cell-penetrating peptides can be administered following resection of a tumor.
- Cell- penetrating peptides can be administered to a mammal in any amount, at any frequency, and for any duration effective to achieve a desired outcome (e.g., to increase progression-free survival or to increase the time to progression).
- cell-penetrating peptides can be administered to a mammal having skin cancer to reduce the progression rate of melanoma by 5, 10, 25, 50, 75, 100, or more percent.
- the progression rate can be reduced such that no additional cancer progression is detected.
- Any method can be used to determine whether or not the progression rate of skin cancer is reduced.
- the progression rate of skin cancer can be assessed by imaging tissue at different time points and determining the amount of cancer cells present. The amounts of cancer cells determined within tissue at different times can be compared to determine the progression rate. After treatment as described herein, the progression rate can be determined again over another time interval.
- the stage of skin cancer after treatment can be determined and compared to the stage before treatment to determine whether or not the progression rate was reduced.
- the subject has previously been administered a B- Raf inhibitor.
- Subjects previously administered a B-Raf inhibit may develop a resistance.
- the subject has or is developing resistance to a B- Raf inhibitor.
- cell-penetrating peptides, composition, or formulation is administered systemically or locally (e.g. intraocular injection, enema formulation, inhalation, intratumor injection) at intervals of 6 hours, 12 hours, daily or every other day or on a weekly or monthly basis to elicit the desired benefit or otherwise provide a therapeutic effect.
- the cell-penetrating peptides, composition, or formulation is administered as required to elicit the desired benefit or otherwise provide a therapeutic effect.
- the subject(s) upon treatment of one or more human or animal subjects with any of the cell-penetrating peptides, compositions, or formulations thereof the subject(s) will exhibit one or more of the following outcomes:
- the subject is treated over a period, for example, of about 1 day through the lifetime of the subject, over a period of about 1 day to about 200 weeks, about 1 day to about 100 weeks, about 1 day to about 80 weeks, about 1 day to about 50 weeks, about 1 day to about 40 weeks, about 1 day to about 20 weeks, about 1 day to about 15 weeks, about 1 day to about 12 weeks, about 1 day to about 10 weeks, about 1 day to about 5 weeks, about 1 week to about 4 weeks, about 2 weeks to about 3 weeks, about 1 day to about 2 weeks, about 1 week, about 1 to 5 days, about 1 to 3 days, or about 1 to 2 days.
- the treatment group members, or the treatment group(s) will exhibit one or more of the following outcomes, each compared to baseline or control, unless otherwise indicated:
- the (1 ) subject(s) or (2) treatment group(s) as disclosed in the studies in the examples, including experimental animals such as mice in animal models exhibit one or more of the following outcomes compared to controls: (a) an increase in progression-free survival of at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, 6 about months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, or more;
- a reduction in progression rate of cancer of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);
- an increase in PKA-mediated inhibitory phosphorylation of C-Raf (e.g., serine 259 phosphorylation) of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control); and/or
- At least a portion of total C-Raf remains complexed with PDE8A. In some embodiments at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% of total C-Raf remains complexed with PDE8A.
- the subject is administered a B-Raf inhibitor.
- the B- Raf inhibitor may be administered prior to, simultaneously with, and/or following administration of the cell-penetrating conjugates, compositions, or formulations disclosed herein.
- B-Raf is a serine/threonine protein kinase that is part of the RAS - RAF - MEK - ERK signalling axis, involved in regulating many cellular processes including: differentiation, proliferation, survival and apoptosis 5 .
- This signalling pathway is believed to be crucial to melanoma progression, with the V600E mutation resulting in B-Raf protein conformational changes that constitutively activate B-Raf and downstream MEK - ERK signalling 5 .
- B-Raf-specific small molecule inhibitors (and eventually MEK inhibitors) were developed and found to dramatically improve patient prognosis, survival rate and lead to tumor regression through suppression of downstream ERK signalling 6- 9 .
- B-Raf inhibitor resistance was developed in many patients through paradoxical activation of ERK; allowing the cancer to persist 10-14 .
- Pathway reactivation is believed to occur as a result of oncogenic mutations in a number of genes, including NRAS (20% of cases; Q61 K/R/L most frequent) and KRAS (2% of cases) gain-of- function mutations 15-18 .
- B-Raf preferentially heterodimerises to C-Raf (vs.
- B-Raf inhibition results in a negative feedback mechanism that switches from B-Raf to C-Raf activation by Ras and subsequent tumor invasion and metastasis 19 20 .
- C-Raf has become a key therapeutic target for the development of new treatments able to suppress RAS-mediated tumor progression in B-Raf inhibitor resistant melanoma.
- HoxD12-PDE8A cell-penetrating peptides also referred to herein as“PPL-008 conjugates”. Briefly, HoxD12 (also referred to herein as“Cell Porter®”, Portage Pharmaceuticals) was conjugated onto the C or N-terminal of the disrupter (PDE8A) via either thioester (C or N giving PPL-008C (conjugate of SEQ. ID Nos. 25 and 26) or PPL-008N (conjugate of SEQ.
- Linkers were intended to provide more molecular flexibility for the cargo (i.e. , PDE8A) to bind to its target without interference by the larger HoxD12 structure.
- PPL-008-CSS and PPL- 008-NSS were designed to include a disulfide bond between the disruptor peptide and the HoxD12 cell penetrating peptide sequence.
- Intracellular release of the cargo by cleavage of the disulfide bond occurs because of the reducing environment within the cell and not any enzymatic cleavage process. It was contemplated that CCS and NSS cell-penetrating conjugates would allow the PDE8A peptide to function inside the cell without being attached to the larger HoxD12 peptide.
- mice were subcutaneously injected at the site of tumor with PPL-008 peptide drug dissolved in a 5% dextrose - water solution at either 25mg/kg or 100mg/kg. Mice were euthanized and the tumors were harvested at varying time points post-treatment: 30 min, 1 h, 2 h, 4 h, 8 h, 12 h. Tumors were frozen down at -80°C prior to preparation into lysates for follow-up western blot analyses.
- A375 (V600E) and MM415 (BRAF wt, KRAS wt, NR AS Q61 L) human malignant melanoma epithelial skin cell lines were cultured with RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS, v/v), 1 % L-glutamine (v/v), 1 % penicillin-streptomycin (v/v) (all Sigma-Aldrich) and incubated at 37°C, 5% C02 and 95% humidity. Cells were split at -80% confluency, using 0.05% trypsin-EDTA, 1 :5.
- MM415 cell adhesion Following MM415 cell adhesion, cells were treated with one of the peptide disrupters for 2 h, followed by PLX (1 mM). The slope (i.e. rate of cell proliferation/growth) was measured based on the normalized cell index from the point in which treatments were administered, until the response had plateaued appropriately.
- Results from western blot analyses are represented as mean ⁇ SEM (n > 3).
- Results from xCELLigence cell proliferation assay are represented as mean ⁇ STDEV (n > 3).
- P ⁇ 0.05 indicates data are significant, with significance determined via unpaired t-test using GraphPad Prism software.
- NRAS Q61 L NRAS Q61 L cell line.
- PPL-008 conjugates (10mM) could suppress phospho-ERK signalling in MM415 cells
- pERK levels were determined via western blot
- pERK was significantly reduced in the human A375 malignant melanoma cell line (BRAF V600E) following PLX treatment (1 mM) ( Figure 2B, lanes 2,3,4) with PPL-008 analogues providing no ERK inhibition as a mono-treatment
- MM415 cells were co-treated with PLX (1 mM), following pre-treatment with a dose range of PPL-008C or PPL-008N (1 nM - 10mM).
- the levels of pERK and cell proliferation rates determined as previously described ( Figure 4).
- the levels of pERK triggered by PLX ( Figure 4A and 4B, lane 1 vs lane 2) were reduced at all concentrations following PPL-008N treatment, with the higher [10mM] dose causing the most significant reduction ( *** P ⁇ 0.001 , Figure 4A).
- PPL-008C was chosen as the lead peptide disrupter due to its consistency in attenuating pERK signalling and cell proliferation as both a single treatment and co-treatment with PLX. Briefly, PPL-008C was administered subcutaneously at the site of the tumor as a single treatment. Tumors were removed at varying time points post-treatment and pERK expression was assessed via western blot (N > 3, Figure 5).
- PPL-008C significantly suppressed pERK1 , pERK2 and total pERK levels over all time-points in the time course and at both doses (25mg/kg and 100mg/kg), excluding the 25mg/kg - 12Hr treatment ( * P ⁇ 0.05, Figure 5). This shows that a single PPL-008C treatment can attenuate Raf - MEK- ERK signalling relatively quickly (within 30 minutes) and can maintain this inhibition for at least 12 hours at higher concentrations (100mg/kg, Figure 5). Maximal pERK inhibition occurred 2 hours post treatment with 100mg/kg PPL-008C.
- a FAK scaffold inhibitor disrupts FAK and VEGFR-3 signaling and blocks melanoma growth by targeting both tumor and endothelial cells. Cell Cycle. 13, 2542-2553
- the embodiments disclosed herein relate generally to HoxD12-PDE8A cell- penetrating peptides, compositions thereof and methods for treating and preventing disease, such as cancer through, administration of the HoxD12-PDE8A cell-penetrating peptides. These peptides facilitate entry into tissues, cells and the nucleus of cells thus allowing the PDE8A fusion protein to reach its site of action for therapeutic purposes.
- genes may be regulated through the direct delivery of biologically active molecules, such as nucleic acids, peptides and proteins, to their intracellular and intranuclear sites of action to influence gene expression either directly or indirectly through interference with transcription, translation or transcription factor production and action and also missing or defective protein products may be replaced to provide these types of molecules in individuals with germ-line or somatic mutations.
- biologically active molecules such as nucleic acids, peptides and proteins
- the use of biologically important proteins is hampered by the inability of these proteins to reach intracellular sites and tissue sites where they normally function.
- patients having certain genotypes, including genetic mutations are cannot be treated with conventional therapeutics and/or develop a resistance to conventional therapeutics over time.
- conjugates comprising: a first region comprising: (i) a homeodomain structure and having a polypeptide sequence set forth in SEQ ID No. 1 or a variant thereof or (ii) at least one of a HOX D12 homeodomain; and a second region comprising a cAMP-degrading phosphodiesterase-8A (PDE8A) - C-Raf complex disruptor.
- a first region comprising: (i) a homeodomain structure and having a polypeptide sequence set forth in SEQ ID No. 1 or a variant thereof or (ii) at least one of a HOX D12 homeodomain
- PDE8A cAMP-degrading phosphodiesterase-8A
- compositions comprising a conjugate according to any embodiment disclosed and described herein and a pharmaceutically acceptable carrier.
- kits for treating cancer in a subject comprising administering a conjugate for a period of time sufficient to increase C-Raf- S259 phosphorylation and/or reduce ERK signaling.
- the PDE8A - C-Raf complex disruptor is PDE8A or a fragment thereof.
- the PDE8A has a polypeptide sequence set forth in SEQ. ID No. 2 or a variant thereof.
- the second region is conjugated to the C-terminus of the first region. In other embodiments, the second region is conjugated to the N- terminus of the first region.
- the conjugate is in the form of a fusion protein.
- the conjugate has a polypeptide sequence set forth in SEQ. ID No. 3.
- the conjugate further comprises a linker sequence between the first and second regions.
- the first region is linked to the second region with a peptidic bond or a non-peptidic bond.
- the linker sequence is a thioester linker.
- the composition is in the form of an inhalable composition, an enema, a topical composition, or an injectable composition including injectable implants for sustained release.
- the composition further comprises a B-Raf inhibitor.
- the formulation is administered as an intravenous solution.
- the conjugate elicits slow cancer cell growth and/or stops cancer cell growth compared to a control.
- the control is a peptide comprising the second region when not conjugated to the first region.
- the cancer is melanoma, malignant melanoma, colon cancer, colorectal cancer, lung cancer, urinary bladder carcinoma, and/or thyroid carcinoma.
- the cancer is B-Raf resistant melanoma.
- the cancer is metastatic cancer.
- the subject has previously been administered a B- Raf inhibitor. In some embodiments, the subject is resistant to a B-Raf inhibitor.
- the conjugate interacts with and/or prevents endogenous PDE8A binding with endogenous C-Raf. In some embodiments, the conjugate potentiates PKA-mediated inhibitory phosphorylation. In some embodiments, at least a portion of total C-Raf remains complexed with PDE8A.
- the subject is administered a B-Raf inhibitor, for example, the B-Raf inhibitor is administering prior to, simultaneously with, and/or following administration of a conjugate or composition disclosed and described herein.
- the subject has wildtype BRAF, NRAS gain-of- function mutations, KRAS gain-of-function mutations, and/or HRAS gain-of-function mutations.
- the autoimmune disease is multiple sclerosis or rheumatoid arthritis.
- FIG. 1 shows exemplary conjugates of HoxD12 (SEQ. ID Nos, 23, 25, 27, and 29) and PDE8A (residues 454-465) (SEQ. ID Nos 24, 26, 28, and 30) formed by thioester or disulfide linkages.
- FIGs. 2A-2C show the effects of HoxD12-PDE8A cell-penetrating conjugates (also referred to herein as“PPL008”) on pERK levels and rate of cell proliferation.
- Normalized phospho-ERK (mean ⁇ SEM) following treatment with DMSO (lane 1 ), PLX4032 B-Raf inhibitor (lane 2), PPL008 conjugates (lanes 1 1 -14) or PLX co-treatments with PDE8A - C-Raf peptide disrupters (original stearylated‘disrupter’ lane 3, or scrambled control , lane 4) or PPL-008 conjugates (lanes 7-10) in: (A) MM415 (NRAS Q61 L) and (B) A375 (BRAF V600E) human malignant melanoma cell lines.
- FIGs. 4A-4D show PPL008-C/N dose response in MM415 (NRAS Q61 L) human malignant melanoma cell line.
- FIGs. 4C(ii) and 4D(ii) show representative traces of normalized cell index of each treatment shown below. N, PPL-008N; C, PPL-008C.
- FIGs. 5A-5C demonstrate in vivo suppression of phospho-ERK signaling in a MM415 murine xenograft model. In vivo suppression of phospho-ERK signaling in an MM415 murine xenograft model.
- FIG. 5A shows normalized total pERK1/2
- FIG. 5B shows pERK1 (T202, 44kDa)
- FIG. 5A shows normalized total pERK1/2
- FIG. 5B shows pERK1 (T202, 44kDa)
- FIGs. 6A-6B are schematics showing the dual inhibition of B-Raf and C-Raf that inhibits melanoma tumor progression.
- FIG. 6A shows B-Raf inhibition leads to the Ras negative feedback mechanism switching to C-Raf driven tumorigenesis via potentiation of the Raf/MEK/ERK signalling axis.
- FIG. 6B shows PPL-008 cell- penetrating peptide (PDE8A - C-Raf disrupter peptide) binds to C-Raf, preventing PDE8A localization within the C-Raf cAMP microdomain and exposing serine 259 - C- Raf to inhibitory phosphorylation by PKA.
- Co-treatment with B-Raf inhibitor and PPL- 008 blocks onco-Ras driven tumor progression via inhibition of the Raf/MEK/ERK axis.
- Melanoma is the most aggressive form of skin cancer, with a wide range of treatments currently available and many more at pre-clinical and clinical phases 8 ⁇ 32 ⁇ 33 .
- First line B-Raf inhibitors are capable of managing the majority of melanoma patients that express the BRAF V600E mutation 33 .
- B-Raf inhibitors become ineffective and tumors persist - warranting the development of novel effective treatments 10 18 .
- conjugates comprising: a first region comprising: (i) a homeodomain structure and having a polypeptide sequence set forth in SEQ ID No. 1 or a variant thereof or (ii) at least one of a HOX D12 homeodomain; and a second region comprising a cAMP-degrading phosphodiesterase-8A (PDE8A) - C-Raf complex disruptor.
- PDE8A cAMP-degrading phosphodiesterase-8A
- homeodomain structure refers to HOX-derived homeodomains, such as the human HOX D12 and HOX C12 sequences shown in Table 1 as SEQ ID No. 1 and SEQ ID No. 4, respectively, or variants or portions thereof.
- homology refers to identity or near identity of nucleotide or amino acid sequences.
- nucleotide mismatches can occur at the third or wobble base in the codon without causing amino acid substitutions in the translated polypeptide sequence.
- minor nucleotide modifications e.g., substitutions, insertions or deletions
- substitutions, insertions or deletions in certain regions of the gene sequence can be tolerated whenever such modifications result in changes in amino acid sequence that do not alter functionality of the final gene product.
- Homologs of specific DNA sequences may be identified by those skilled in the art using the test of cross hybridization of nucleic acids under conditions of stringency as is well understood m the art (as described in Hames et al. , Nucleic Acid Hybridisation, (1985) IRL Press, Oxford, UK). Extent of homology is often measured in terms of percentage of identity between the sequences compared.
- variant refers to a polypeptide or protein that differs from a reference polypeptide or protein, but retains essential properties.
- a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall (homologous) and, in many regions, identical.
- a variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions).
- a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
- a variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
- Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
- Exemplary substitutions that take one or more of the foregoing characteristics into consideration are well known to those of skill in the art and include, but are not limited to (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (lie: Leu, Val), (Leu: lie, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: lie, Leu).
- Embodiments of this disclosure therefore, consider functional or biological equivalents of a polypeptide or protein as set forth above.
- and proteins can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide and protein of interest.
- D-amino acids can be used for the first region and/or the second region. It is contemplated that using D-amino acids for at least the second region will reduce intracellular degradation and/or increase the pharmacological half- life.
- Identity is a relationship between two or more polypeptide or protein sequences, as determined by comparing the sequences.
- identity also refers to the degree of sequence relatedness between polypeptides or proteins, as determined by the match between strings of such sequences. “Identity” can be readily calculated by known bioinformational methods.
- the first region of the conjugate or fusion protein embodiments disclosed may comprise a natural or synthetic 60-amino acid peptide or variant or portion thereof derived from the HOX D12 or HOX C12 gene or variants or portions thereof.
- the HOX D12 (SEQ ID No. 1 ) amino acid sequence is:
- the HOX C12 (SEQ ID No. 4) amino acid sequence is:
- synthetic variants may be used provided that they retain the ability to translocate the membrane. Synthetic variants will generally differ from the naturally-occurring proteins by substitution, particularly conservative substitution.
- the phrase“conservative amino acid changes” herein means replacing an amino acid from one of the amino acid groups, namely hydrophobic, polar, acidic or basic, with an amino acid from within the same group. An example of such a change is the replacement of valine by methionine and vice versa. Other examples of conservative substitutions may be seen by reference to Table 2 below: [0045] Table 2: Conservative Amino Acid Substitutions.
- Such variants may be synthesized directly or prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions corresponding to the naturally-occurring sequence either side of the insertion site. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed to make the encoded protein.
- These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.
- Variants that retain at least 50% sequence identity with the claimed 60-amino acid sequences or variants or portions thereof derived from HOX-D12 will likely maintain their cell permeability characteristics and retain their human characteristics resulting in low immunogenicity potential.
- the ability of a naturally occurring or synthetic HOX sequence to translocate the membrane may be tested by routine methods known in the art.
- Second Region Peptides or Proteins may comprise a cAMP-degrading phosphodiesterase-8A (PDE8A) - C-Raf complex disruptor.
- PDE8A cAMP-degrading phosphodiesterase-8A
- the second region also may or may not be from the same species as the first region, but the first and second regions will be present in the conjugate or fusion protein embodiments in a manner different from the natural situation.
- the second region of the fusion protein or conjugate embodiments may be a cAMP-degrading phosphodiesterase-8A (PDE8A) - C-Raf complex disruptor peptide or protein of any length as long as it is biologically active on its target when included in the fusion protein or conjugate.
- PDE8A - C-Raf complex disruptor is PDE8A or a fragment thereof, for example, in certain embodiments, the PDE8A has a polypeptide sequence set forth in SEQ. ID No. 2 or a variant thereof.
- the conjugate comprises polypeptide sequence set forth in SEQ. ID No. 3.
- conjugates comprises a category of structures, including fusion proteins, in which the first region, a homeodomain sequence or variant or portion thereof, is conjugated to the PDE8A - C-Raf complex disruptor directly via a peptide bond or other type of bond including both covalent and non- covalent bonds.
- Conjugates may include a linker region that connects the homeodomain sequence to the PDE8A - C-Raf complex disruptor that is not naturally associated with the first region.
- phrase“not naturally associated with” means that entire sequence of the conjugate or fusion protein is not found in nature, and that the entire sequence is not encoded for by a single gene found in nature.
- linkers short, connecting sequences
- fusion protein is used to refer to a particular subcategory of conjugate that exists when no such linkers are used to form the conjugate and the domains are linked entirely by peptide bonds.
- the first region and the second regions may be linked by a cleavable linker region this may be any region suitable for this purpose provided the function of the conjugate is not compromised by its addition.
- the second region is conjugated to the C-terminus of the first region. In other embodiments, the second region is conjugated to the N-terminus of the first region.
- the cleavable linker region is a protease cleavable linker, although other linkers, cleavable for example by small molecules, may be used. These include Met-X sites, cleavable by cyanogen bromide, Asn-Gly, cleavable by hydroxylamine, Asp-Pro, cleavable by weak acid and Trp-X cleavable by, inter alia, NBS-skatole. Protease cleavage sites require milder cleavage conditions and are found in, for example, factor Xa, thrombin and collagenase. Any of these may be used.
- the cleavable linker region may be one that is targeted by endocellular proteases. Linkers may not be required for function but linkers may be included between first and second regions to allow targeted release of the second region without compromising function or to enhance biological activity of the second region with linker cleavage.
- the conjugates of the present disclosure may comprise a linker sequence between the first and second regions.
- the first region is linked to the second region with a peptidic bond or a non-peptidic bond.
- the linker sequence is a thioester linker.
- composition embodiments disclosed herein may comprise one or more of the cell-penetrating conjugates disclosed and described herein and a pharmaceutically acceptable carrier, diluent or excipient.
- pharmaceutically acceptable carrier diluent or excipient refers to any substance, not itself a therapeutic agent, used as a carrier or vehicle for delivery of a therapeutic agent to a subject or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a unit dose of the composition, and that does not produce unacceptable toxicity or interaction with other components in the composition.
- compositions may comprise any agents that may aid, regulate, release or increase entry into the body compartment, tissue, intracellular or intranuclear target site, such as binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilizing agent(s), or other agents.
- An injectable implant for the sustained release of the protein may also be used to obtain prolonged exposure and action.
- sustained release refers to formulations from which the conjugate is released at a slow rate allowing for a longer period of exposure at active concentrations.
- compositions comprising one or more cell-penetrating conjugates disclosed herein can be administered, depending on condition to be treated or other considerations, in any number of ways, for example without limitation, by any one or more of the following: (1 ) inhalation; (2) in the form of a suppository or pessary; (3) in the form of a topical lotion, solution, cream, ointment or dusting powder; (4) by use of a skin patch; (5) orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavoring or coloring agents; (6) injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously; (7) for ophthalmic diseases, they may be formulated as eye drops or for intraocular injection; (8) for parenteral administration, they may be
- the composition is in the form of an inhalable composition, an enema, a topical composition, or an injectable composition including injectable implants for sustained release.
- the cell-penetrating conjugates further comprise a B- Raf inhibitor.
- Non-limiting examples of suitable B-Raf inhibitors include vemurafenib (aslso known as PLX4032 and Zelboraf, Daiichi-Sankyo), encorafenib, XL281 (also known as BMS-908662, Bristol-Myers Squibb), LGX818 (Novartis), PLX3603 (Hofmann-LaRoche), RAF265 (Novartis), R05185426 (Hofmann-LaRoche), and GSK21 18436 (also known as dabrafenib and Tafinlar, GlaxoSmithKline).
- vemurafenib as PLX4032 and Zelboraf, Daiichi-Sankyo
- encorafenib XL281
- LGX818 Novartis
- PLX3603 Hofmann-LaRoche
- RAF265 Novartis
- R05185426 Hof
- the active concentration of cell-penetrating conjugates in cell culture is less than about 1 15 mM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 65 pM, less than about 60 pM, less than about 55 pM, less than about 50 pM, less than about 45 pM, less than about 40 pM, less than about 35 pM, less than about 30 pM, less than about 25 pM, less than about 20 pM, less than about 15 pM, less than about 10 pM, less than about 5 pM, or less than about 1 pM for example, less than about 1 pM to about 3 pM, less than about 1 pM to about 6 pM, less than about 1 pM to about 8 pM, less than about 1 pM to about 15 pM, less than about 1 pM to about 25 pM, less than about 1 pM
- the dosage delivered (daily or as required) in mouse models (per 20 g mouse) through (1 ) an intravenous (i.v.) or intraperitoneal (i.p.) injection, (2) a topical formulation, or (3) an inhaled formulation is at least 500 pg, at least 450 pg, at least 400 pg, at least 350 pg, at least 300 pg, at least 250 pg, at least 200 pg, at least 150 pg, at least 100 pg, at least 80 pg, at least 70 pg, at least 60 pg, at least 50 pg, at least 40 pg, at least 30 pg, at least 20 pg, at least 10 pg, or at least 1 pg, for example, about 1 pg to about 500 pg, about 10 pg to about 450 pg, about 20 pg to about 400 pg about 30 pg to about 350 pg, about 30 p
- the dosage delivered (daily or as required) through an intravenous (i.v.) or intraperitoneal (i.p.) injection in a mouse model is at least 100 mg/kg, less than about 80 mg/kg, less than about 45 mg/kg, less than about 40 mg/kg, less than about 30 mg/kg, less than about 25 mg/kg, less than about 20 mg/kg, less than about 15 mg/kg, less than about 12 mg/kg, less than about 10 mg/kg, less than about 8 mg/kg, less than about 4 mg/kg, less than about 2 mg/kg, or less than about 1 mg/kg, for example, less than about 1 mg/kg to about 50 mg/kg, about 5 mg/kg to about 40 mg/kg, about 8 mg/kg to about 30 mg/kg, about 10 mg/kg to about 20 mg/kg, or about 12 mg/kg to about 15 mg/kg, about 8 mg/kg to about 12 mg/kg, about 5 mg/kg to about 9 mg/kg, about 3 mg/kg, less than about 1 mg/
- the dosage delivered (daily or as required) through an inhaled formulation in humans is at least about 600 mg, at least about 500 mg, at least about 450 mg, at least about 400 mg, at least about 350 mg, at least about 300 mg, at least about 250 mg, at least about 200 mg, at least about 150 mg, at least about 125 mg, at least about 100 mg, at least about 75 mg, at least about 50 mg, at least about 25 mg, at least about 20 mg, at least about 15 mg, at least about 10 mg, at least about 5 mg, at least about 1 mg, at least about 500 pg, at least about 450 pg, at least about 400 pg, at least about 350 pg, at least about 300 pg, at least about 250 pg, at least about 200 pg, at least about 150 pg, at least about 100 pg, at least about 80 pg, at least about 70 pg, at least about 60 pg, at least about 50 pg,
- the dosage delivered (daily or as required) through topical formulation in humans and in mouse models is less than about 5% wt/vol, less than about 4.5% wt/vol, less than about 3.5% wt/vol, less than about 2.5% wt/vol, less than about 1 .5% wt/vol, less than about 0.5% wt/vol, less than about 0.4% wt/vol, less than about 0.3% wt/vol, less than about 0.2%, less than about 0.1 % wt/vol, less than about 0.09% wt/vol, less than about 0.08% wt/vol, less than about 0.07% wt/vol, less than about 0.06% wt/vol, less than about 0.05% wt/vol, less than about 0.04% wt/vol, less than about 0.03% wt/vol, less than about 0.02% wt/vol, less than about 0.01 % wt/vol, less than about 0.008%
- the dosage delivered (daily or as required) through a topical formulation in humans is less than about 70 pg, less than about 50 pg, less than about 45 pg, less than about 40 pg, less than about 30 pg, less than about 25 pg, less than about 20 pg, less than about 15 pg, less than about 12 pg, less than about 10 pg, less than about 8 pg, less than about 4 pg, less than about 2 pg, or less than about 1 pg, for example, about 1 pg to about 50 pg, about 5 pg to about 40 pg, about 8 pg to about 30 pg, about 10 pg to about 20 pg, or about 12 pg to about 15 pg, about 8 pg to about 12 pg, about 5 pg to about 9, about 3 pg to about 6 pg, about 2 pg to about 5 pg, or
- the systemic dosage delivered (daily or as required) through an intravenous, subcutaneous or intramuscular injection or an injectable implant for sustained release formulations in humans is less than about 100 mg/kg, less than about 80 mg/kg, less than about 45 mg/kg, less than about 40 mg/kg, less than about 30 mg/kg, less than about 25 mg/kg, less than about 20 mg/kg, less than about 15 mg/kg, less than about 12 mg/kg, less than about 10 mg/kg, less than about 8 mg/kg, less than about 4 mg/kg, less than about 2 mg/kg, less than about 1 mg/kg, less than about 0.1 mg/kg, or less than about 0.01 mg/kg, for example, less than about 0.01 mg/kg to about 50 mg/kg, less than about 5 mg/kg to about 40 mg/kg, less than about 8 mg/kg to about 30 mg/kg, less than about 10 mg/kg to about 20 mg/kg, or less than about 12 mg/kg to about 15 mg/kg, less than
- the dosage delivered (daily or as required) to humans (based on 70 kg weight) through any formulation other than an intravenous, subcutaneous, or intramuscular injection or injectable implant for the sustained release, inhaled or topical formulation is at least about 600 mg, at least about 500 mg, at least about 450 mg, at least about 400 mg, at least about 350 mg, at least about 300 mg, at least about 250 mg, at least about 200 mg, at least about 150 mg, at least about 125 mg, at least about 100 mg, at least about 75 mg, at least about 50 mg, at least about 25 mg, at least about 20 mg, at least about 15 mg, at least about 10 mg, at least about 5 mg, at least about 1 mg, at least about 500 pg, at least about 450 pg, at least about 400 pg, at least about 350 pg, at least about 300 pg, at least about 250 pg, at least about 200 pg, at least about 150 pg, at least about 100 pg, at least about 600 mg, at least about 500
- novel cell-penetrating peptides and compositions thereof for use in treating and/or preventing diseases and disorders.
- kits for treating cancer or autoimmune diseases in a subject comprising administering a formulation comprising the conjugates or compositions disclosed herein.
- the cell-penetrating conjugates, compositions, formulations, and methods can be used to treat any type of cancer.
- cancer include skin cancer (e.g., melanoma, malignant melanoma), lung cancer (e.g., non-small lung cell carcinoma), urinary bladder carcinoma, thyroid cancer, prostate cancer, endometrial cancer, lung cancer, breast cancer, colon cancer, and colorectal cancer.
- the cancer is melanoma.
- the cancer is a gain-of- function onco-ras mutation cancer.
- the cancer is a B-Raf resistant cancer, for example, B-Raf resistant melanoma.
- the cancer is metastatic cancer.
- the cell-penetrating conjugates, compositions, formulations, and methods can be used to treat any type of autoimmune disease.
- autoimmune diseases include multiple sclerosis and rheumatoid arthritis.
- cell-penetrating peptides can be administered orally or via injection (e.g., subcutaneous injection, intramuscular injection, intravenous injection, or intrathecal injection).
- cell-penetrating peptides can be administered by different routes.
- one cell-penetrating peptide can be administered orally and a second cell-penetrating peptide can be administered via injection.
- cell-penetrating peptides can be administered following resection of a tumor.
- Cell- penetrating peptides can be administered to a mammal in any amount, at any frequency, and for any duration effective to achieve a desired outcome (e.g., to increase progression-free survival or to increase the time to progression).
- cell-penetrating peptides can be administered to a mammal having skin cancer to reduce the progression rate of melanoma by 5, 10, 25, 50, 75, 100, or more percent.
- the progression rate can be reduced such that no additional cancer progression is detected.
- Any method can be used to determine whether or not the progression rate of skin cancer is reduced.
- the progression rate of skin cancer can be assessed by imaging tissue at different time points and determining the amount of cancer cells present. The amounts of cancer cells determined within tissue at different times can be compared to determine the progression rate. After treatment as described herein, the progression rate can be determined again over another time interval.
- the stage of skin cancer after treatment can be determined and compared to the stage before treatment to determine whether or not the progression rate was reduced.
- the subject has previously been administered a B- Raf inhibitor.
- Subjects previously administered a B-Raf inhibit may develop a resistance.
- the subject has or is developing resistance to a B- Raf inhibitor.
- cell-penetrating peptides, composition, or formulation is administered systemically or locally (e.g. intraocular injection, enema formulation, inhalation, intratumor injection) at intervals of 6 hours, 12 hours, daily or every other day or on a weekly or monthly basis to elicit the desired benefit or otherwise provide a therapeutic effect.
- the cell-penetrating peptides, composition, or formulation is administered as required to elicit the desired benefit or otherwise provide a therapeutic effect.
- the subject(s) upon treatment of one or more human or animal subjects with any of the cell-penetrating peptides, compositions, or formulations thereof the subject(s) will exhibit one or more of the following outcomes:
- the subject is treated over a period, for example, of about 1 day through the lifetime of the subject, over a period of about 1 day to about 200 weeks, about 1 day to about 100 weeks, about 1 day to about 80 weeks, about 1 day to about 50 weeks, about 1 day to about 40 weeks, about 1 day to about 20 weeks, about 1 day to about 15 weeks, about 1 day to about 12 weeks, about 1 day to about 10 weeks, about 1 day to about 5 weeks, about 1 week to about 4 weeks, about 2 weeks to about 3 weeks, about 1 day to about 2 weeks, about 1 week, about 1 to 5 days, about 1 to 3 days, or about 1 to 2 days.
- the treatment group members, or the treatment group(s) will exhibit one or more of the following outcomes, each compared to baseline or control, unless otherwise indicated:
- the (1 ) subject(s) or (2) treatment group(s) as disclosed in the studies in the examples, including experimental animals such as mice in animal models exhibit one or more of the following outcomes compared to controls: (a) an increase in progression-free survival of at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, 6 about months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, or more;
- a reduction in progression rate of cancer of at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control);
- an increase in PKA-mediated inhibitory phosphorylation of C-Raf (e.g., serine 259 phosphorylation) of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% (actual % change or median % change compared to baseline or control); and/or
- At least a portion of total C-Raf remains complexed with PDE8A. In some embodiments at least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 35%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%, for example, about 30% to about 99%, about 80% to about 90%, about 70% to about 90%, about 60% to about 90%, about 50% to about 90%, about 40% to about 90%, about 35% to about 90%, about 30% to about 90%, about 25% to about 90%, about 5% to about 85%, or about 10% to about 80% of total C-Raf remains complexed with PDE8A.
- the subject is administered a B-Raf inhibitor.
- the B- Raf inhibitor may be administered prior to, simultaneously with, and/or following administration of the cell-penetrating conjugates, compositions, or formulations disclosed herein.
- B-Raf is a serine/threonine protein kinase that is part of the RAS - RAF - MEK - ERK signalling axis, involved in regulating many cellular processes including: differentiation, proliferation, survival and apoptosis 5 .
- This signalling pathway is believed to be crucial to melanoma progression, with the V600E mutation resulting in B-Raf protein conformational changes that constitutively activate B-Raf and downstream MEK - ERK signalling 5 .
- B-Raf-specific small molecule inhibitors (and eventually MEK inhibitors) were developed and found to dramatically improve patient prognosis, survival rate and lead to tumor regression through suppression of downstream ERK signalling 6- 9 .
- B-Raf inhibitor resistance was developed in many patients through paradoxical activation of ERK; allowing the cancer to persist 10-14 .
- Pathway reactivation is believed to occur as a result of oncogenic mutations in a number of genes, including NRAS (20% of cases; Q61 K/R/L most frequent) and KRAS (2% of cases) gain-of- function mutations 15-18 .
- B-Raf preferentially heterodimerises to C-Raf (vs.
- B-Raf inhibition results in a negative feedback mechanism that switches from B-Raf to C-Raf activation by Ras and subsequent tumor invasion and metastasis 19 20 .
- C-Raf has become a key therapeutic target for the development of new treatments able to suppress RAS-mediated tumor progression in B-Raf inhibitor resistant melanoma.
- HoxD12-PDE8A cell-penetrating peptides also referred to herein as“PPL-008 conjugates”. Briefly, HoxD12 (also referred to herein as“Cell Porter®”, Portage Pharmaceuticals) was conjugated onto the C or N-terminal of the disrupter (PDE8A) via either thioester (C or N giving PPL-008C (conjugate of SEQ. ID Nos. 25 and 26) or PPL-008N (conjugate of SEQ.
- Linkers were intended to provide more molecular flexibility for the cargo (i.e. , PDE8A) to bind to its target without interference by the larger HoxD12 structure.
- PPL-008-CSS and PPL- 008-NSS were designed to include a disulfide bond between the disruptor peptide and the HoxD12 cell penetrating peptide sequence.
- Intracellular release of the cargo by cleavage of the disulfide bond occurs because of the reducing environment within the cell and not any enzymatic cleavage process. It was contemplated that CCS and NSS cell-penetrating conjugates would allow the PDE8A peptide to function inside the cell without being attached to the larger HoxD12 peptide.
- mice were subcutaneously injected at the site of tumor with PPL-008 peptide drug dissolved in a 5% dextrose - water solution at either 25mg/kg or 100mg/kg. Mice were euthanized and the tumors were harvested at varying time points post-treatment: 30 min, 1 h, 2 h, 4 h, 8 h, 12 h. Tumors were frozen down at -80°C prior to preparation into lysates for follow-up western blot analyses.
- A375 (V600E) and MM415 (BRAF wt, KRAS wt, NR AS Q61 L) human malignant melanoma epithelial skin cell lines were cultured with RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS, v/v), 1 % L-glutamine (v/v), 1 % penicillin-streptomycin (v/v) (all Sigma-Aldrich) and incubated at 37°C, 5% C02 and 95% humidity. Cells were split at -80% confluency, using 0.05% trypsin-EDTA, 1 :5.
- MM415 cell adhesion Following MM415 cell adhesion, cells were treated with one of the peptide disrupters for 2 h, followed by PLX (1 mM). The slope (i.e. rate of cell proliferation/growth) was measured based on the normalized cell index from the point in which treatments were administered, until the response had plateaued appropriately.
- Results from western blot analyses are represented as mean ⁇ SEM (n > 3).
- Results from xCELLigence cell proliferation assay are represented as mean ⁇ STDEV (n > 3).
- P ⁇ 0.05 indicates data are significant, with significance determined via unpaired t-test using GraphPad Prism software.
- NRAS Q61 L NRAS Q61 L cell line.
- PPL-008 conjugates (10mM) could suppress phospho-ERK signalling in MM415 cells
- pERK levels were determined via western blot
- pERK was significantly reduced in the human A375 malignant melanoma cell line (BRAF V600E) following PLX treatment (1 mM) ( Figure 2B, lanes 2,3,4) with PPL-008 analogues providing no ERK inhibition as a mono-treatment
- MM415 cells were co-treated with PLX (1 mM), following pre-treatment with a dose range of PPL-008C or PPL-008N (1 nM - 10mM).
- the levels of pERK and cell proliferation rates determined as previously described ( Figure 4).
- the levels of pERK triggered by PLX ( Figure 4A and 4B, lane 1 vs lane 2) were reduced at all concentrations following PPL-008N treatment, with the higher [10mM] dose causing the most significant reduction ( *** P ⁇ 0.001 , Figure 4A).
- PPL-008C was chosen as the lead peptide disrupter due to its consistency in attenuating pERK signalling and cell proliferation as both a single treatment and co-treatment with PLX. Briefly, PPL-008C was administered subcutaneously at the site of the tumor as a single treatment. Tumors were removed at varying time points post-treatment and pERK expression was assessed via western blot (N > 3, Figure 5).
- PPL-008C significantly suppressed pERK1 , pERK2 and total pERK levels over all time-points in the time course and at both doses (25mg/kg and 100mg/kg), excluding the 25mg/kg - 12Hr treatment ( * P ⁇ 0.05, Figure 5). This shows that a single PPL-008C treatment can attenuate Raf - MEK- ERK signalling relatively quickly (within 30 minutes) and can maintain this inhibition for at least 12 hours at higher concentrations (100mg/kg, Figure 5). Maximal pERK inhibition occurred 2 hours post treatment with 100mg/kg PPL-008C.
- a FAK scaffold inhibitor disrupts FAK and VEGFR-3 signaling and blocks melanoma growth by targeting both tumor and endothelial cells. Cell Cycle. 13, 2542-2553
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US20040018605A1 (en) * | 1996-03-25 | 2004-01-29 | Incyte Corporation | Cyclic nucleotide phosphodiesterase |
US20170204383A1 (en) * | 2012-03-28 | 2017-07-20 | Caroline Emery | C-RAF Mutants that Confer Resistance to RAF Inhibitors |
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