WO2016073435A1 - Conjugated polypeptides and uses thereof - Google Patents
Conjugated polypeptides and uses thereof Download PDFInfo
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- WO2016073435A1 WO2016073435A1 PCT/US2015/058764 US2015058764W WO2016073435A1 WO 2016073435 A1 WO2016073435 A1 WO 2016073435A1 US 2015058764 W US2015058764 W US 2015058764W WO 2016073435 A1 WO2016073435 A1 WO 2016073435A1
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- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—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
- 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
- A61K47/54—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 an organic compound
- A61K47/55—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 an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—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
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/585—Calcitonins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention is in the field of medicine. More particularly, the present invention relates to conjugated polypeptide compounds, or pharmaceutically acceptable salts thereof, which are agonists of both the human parathyroid and calcitonin receptors, as well as pharmaceutical compositions and methods of use comprising the same.
- Spinal fusion is a surgical procedure in which adjacent vertebrae are grafted together to limit or eliminate the range of motion in the joint space between the affected bones.
- Spinal fusions are performed to address pain and morbidity associated with degenerative conditions such as degenerative disc disease (DDD), spondylosis, and spondylolisthesis; congenital deformities, including kyphosis and scoliosis; as well as some vertebral fractures.
- DDD degenerative disc disease
- spondylosis spondylosis
- spondylolisthesis congenital deformities, including kyphosis and scoliosis
- Posterolateral fusion involves placing bone grafting substance between the transverse processes of adjacent vertebrae in the posterior of the spine.
- the vertebrae may then be fixed in place using metal rods on each side of the vertebrae, attached to the pedicles of each vertebra with screws and/or wire.
- Interbody fusion involves removing an intervertebral disc and placing the bone graft substance into the intervertebral space between adjacent vertebrae.
- An interbody device or spacer may be placed between the affected vertebral bodies to maintain alignment and disc-space height. Fusion occurs between the endplates of the adjacent vertebral bodies.
- the interbody fusion may be stabilized with the placement of rods, plates, screws or wire to facilitate the fusion process.
- Types of interbody fusion procedures include anterior lumbar interbody fusion (ALIF), posterior lumbar interbody fusion (PLIF), and transforaminal lumbar interbody fusion which differ according to location and angle of approach to the spine.
- PTH Parathyroid hormone
- PTH binding to the PTH receptor 1 directly induces bone formation through activation of cyclic AMP and canonical wnt-signaling pathways, while indirectly inducing osteoclast differentiation and bone resorption through induced expression of RANK ligand on the osteoblast cell surface.
- Teriparatide (rhPTH(l -34)) marketed as Forteo®, is a thirty four amino acid N-terminal fragment of PTH that has been shown to increase bone formation activity in osteoporotic patients and is the only bone anabolic agent approved in the United States to treat osteoporosis.
- Calcitonin (CT) is a thirty two amino acid peptide which mediates calcium homeostasis.
- sCT salmon calcitonin
- sCT has been used in the treatment of post-menopausal osteoporosis, Paget's disease and in the treatment of hypercalcemia.
- sCT has also been reported to decrease spinal pain associated with osteoporotic vertebral compression fractures (Knopp et al, Osteoporosis Int., 16(10); 1281-1290 (Oct. 2005)).
- Combining the bone anabolic properties of PTH with the anti-bone resorptive properties of sCT provides an alternative therapeutic approach for improving outcomes for spinal fusion patients, as well as inducing bone healing or treating conditions associated with bone loss or degeneration.
- Currently, to achieve such therapeutic effect would require co-administration of separate agents, either through administration of separate formulations (each containing a separate active agent), or administration of a single, co-formulation containing each of the individual agents. While co-administration of separate formulations would permit flexibility in dose and timing of administration, the inconvenience and possible discomfort associated with multiple injections may reduce patient compliance.
- the present invention provides conjugated polypeptide compounds, or pharmaceutically acceptable salts thereof, useful as adjunct therapies for spinal fusion surgery, or as agents for bone healing or treating conditions associated with bone loss or degeneration.
- the present invention provides a compound consisting of:
- Xaa26 of the sequence given by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 is a cysteine residue modified by attachment to the maleimide-PEG linker through the thioether bond;
- Xaal8 of the sequence given by SEQ ID NO: 10 is a lysine residue modified by attachment to the maleimide-PEG linker through the amide bond
- n 40 to 50
- the present invention provides a compound consisting of:
- Xaa26 of the sequence given by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 is a cysteine residue modified by attachment to the maleimide-PEG linker through the thioether bond;
- Xaal 8 of the sequence given by SEQ ID NO: 10 is a lysine residue modified by attachment to the maleimide-PEG linker through the amide bond
- n 40 to 50
- the present invention provides a compound consisting of:
- Xaa26 of the sequence given by SEQ ID NO: 7 is a cysteine residue modified by attachment to the maleimide-PEG linker through the thioether bond;
- Xaal 8 of the sequence given by SEQ ID NO: 10 is a lysine residue modified by attachment to the maleimide-PEG linker through the amide bond
- n 40 to 50
- the present invention provides a compound consisting of: (a) a first polypeptide which is given by a sequence which is Ser-Val-Ser-Glu-Ile-Gln-
- Xaa26 of the sequence given by SEQ ID NO: 8 is a cysteine residue modified by attachment to the maleimide-PEG linker through the thioether bond;
- Xaal 8 of the sequence given by SEQ ID NO: 10 is a lysine residue modified by attachment to the maleimide-PEG linker through the amide bond
- n 40 to 50
- the present invention provides a compound consisting of: (a) a first polypeptide which is given by a sequence which is Ser-Val-Ser-Glu-Ile-Gln-
- Xaa26 of the sequence given by SEQ ID NO: 9 is a cysteine residue modified by attachment to the maleimide-PEG linker through the thioether bond;
- Xaal 8 of the sequence given by SEQ ID NO: 10 is a lysine residue modified by attachment to the maleimide-PEG linker through the amide bond
- n 40 to 50
- the present invention provides any of the afore-mentioned compounds wherein n represents 43 to 47. As yet another particular embodiment, the present invention provides any of the afore-mentioned compounds wherein n represents 45.
- the present invention also provides methods of use comprising administering to a spinal fusion patient an effective amount of a conjugated polypeptide compound of the present invention, or a pharmaceutically acceptable salt thereof.
- the present invention provides methods of use comprising administering to a spinal fusion patient an effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, wherein said compound is administered prior to spinal fusion surgery.
- the present invention provides methods of use comprising administering to a spinal fusion patient an effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, wherein said compound is administered during spinal fusion surgery.
- the present invention provides methods of use comprising administering to a spinal fusion patient an effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, wherein said compound is administered after spinal fusion surgery.
- the compound of the present invention, or pharmaceutically acceptable salt thereof is administered to a spinal fusion patient prior to, during or after posteriolateral fusion surgery (PLF) or
- TLIF transforaminal lumbar interbody
- the present invention further provides methods for bone healing, for example in patients with bone fractures such as vertebral fractures, as well as methods for treating conditions associated with bone loss or degeneration such as osteoporosis, osteopenia, or osteogenesis imperfecta, said methods comprising administering to a patient in need thereof a compound of the present invention, or a pharmaceutically acceptable salt thereof.
- the present invention also provides a compound of the present invention, or a pharmaceutically acceptable salt thereof, for use in therapy. Additionally, the present invention provides a compound of the present invention, or a pharmaceutically acceptable salt thereof, for use as an adjunct to spinal fusion surgery. More particularly, the present invention provides a compound of the present invention, or a pharmaceutically acceptable salt thereof, for use as an adjunct to spinal fusion surgery, wherein said compound, or a pharmaceutically acceptable salt thereof, is administered to a spinal fusion patient prior to, during or after spinal fusion surgery. The present invention also provides a compound of the present invention, or a pharmaceutically acceptable salt thereof, for use in bone healing or in the treatment of osteoporosis, osteopenia, or osteogenesis imperfecta.
- the present invention also provides the use of a compound of the present invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for spinal fusion surgery. More particularly, the present invention also provides the use of a compound of the present invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for spinal fusion surgery, wherein said medicament is administered to a spinal fusion patient prior to, during or after spinal fusion surgery. Further, the present invention provides the use of a compound of the present invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for bone healing or for the treatment of osteoporosis, osteopenia, or osteogenesis imperfecta.
- the present invention further provides pharmaceutical compositions comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.
- the present invention provides a pharmaceutical composition comprising a plurality of any one of the compounds of the present invention, or a pharmaceutically acceptable salt thereof, wherein the number average molecular weight of the PEG polymer of the formula "-(O- CH2-CH2) n -" in said plurality of compounds or salts is 1800 to 2200 daltons, and a pharmaceutically acceptable carrier, diluent or excipient.
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising a plurality of any one of the compounds of the present invention, or a pharmaceutically acceptable salt thereof, wherein the number average molecular weight of the PEG polymer of the formula "-(O- CH2-CH2) n -" in said plurality of compounds or salts is 1900 to 2100 daltons, and a pharmaceutically acceptable carrier, diluent or excipient.
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising a plurality of any one of the compounds of the present invention, or a pharmaceutically acceptable salt thereof, wherein the number average molecular weight of the PEG polymer of the formula "-(0-CH2-CH2) n -" in said plurality of compounds or salts is about 2000 daltons, and a pharmaceutically acceptable carrier, diluent or excipient.
- the present invention also provides a process for preparing a conjugated polypeptide compound comprising:
- n 40 to 50 and the number average molecular weight of the PEG polymers of the formula "-(0-CH2-CH2) n -" in said reagent is 2000 daltons, under suitable conditions such that an amide bond is formed between Lysl8 of the polypeptide given by SEQ ID NO:4 and the ester moiety of said compound;
- step (ii) reacting the product of step (i) with a polypeptide given by the sequence which is SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3 under suitable conditions such that a thioether bond is formed between Cys26 of the polypeptide given by SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3 and the maleimide moiety of the product of step (i).
- the present invention also provides a conjugated polypeptide compound prepared according to afore-mentioned process.
- the conjugated polypeptide compounds of the present invention are bi-functional, meaning they are capable of interacting with, and modulating the activity of, two distinct targets.
- the conjugated polypeptides of the present invention are agonists of both the PTH receptor and the CT receptor and, more particularly, the human PTH and human CT receptor.
- the conjugated polypeptide compounds of the present invention may also counter tolerability limitations seen with individual administration of PTH(l-34) (e.g., hypercalcemia, hypotension) and sCT (e.g., hypocalcemia, hypertension), thus potentially allowing for administration at doses higher than currently, clinically-approved for one or both of PTH(l-34) and sCT.
- PTH(l-34) e.g., hypercalcemia, hypotension
- sCT e.g., hypocalcemia, hypertension
- the conjugated polypeptide compounds of the present invention may be useful, for example, as adjuncts to spinal fusion surgery.
- bone graft substances are employed to provide proteins and other substances to stimulate new bone growth in the affected space.
- Autograft (autologous bone graft) procedures employ the patient's own bone material as the grafting substance.
- the bone material which must be separately harvested from areas such as the iliac crest of the pelvis, ribs or the spine, provides a source of viable osteocytes, bone morphogenic proteins (e.g. BMP-2) and a calcified matrix for new bone growth.
- BMP-2 bone morphogenic proteins
- BGS procedures include allograft procedures (e.g., bone material previously harvested from sources such as cadavers and obtained through tissue banks or the like); demineralized bone matrix(DBM) procedures which employ allograft bone material that has been demineralized to expose bone-forming proteins; ceramic-based bone graft extender procedures which provide a calcium matrix for bone growth; and procedures employing bone graft substances containing bone forming proteins (e.g., bone morphogenic proteins) or other growth factors (e.g. TGF-beta, PDGF, FGF), such as platelet rich plasma.
- allograft procedures e.g., bone material previously harvested from sources such as cadavers and obtained through tissue banks or the like
- demineralized bone matrix(DBM) procedures which employ allograft bone material that has been demineralized to expose bone-forming proteins
- ceramic-based bone graft extender procedures which provide a calcium matrix for bone growth
- conjugated polypeptides of the present invention may be administered to the patient systemically. Such systemic administration may provide flexibility in dosing, including dosing prior to, during or after bone fusion surgery as well as repeat dosing.
- Systemic administration refers to administration by parenteral routes, including intravenous, intramuscular, subcutaneous, intraperitoneal or intranasal routes of administration. Preferred routes of administration of the conjugated polypeptide compounds of the present invention include intravenous, subcutaneous or intraperitoneal routes.
- the conjugated polypeptide compounds of the present invention are agonists of both the PTH and CT receptors, they may also be useful in situations or conditions in which PTH or sCT is indicated.
- the conjugated peptide compounds of the present invention may build bone mass, increase bone biomechanical strength, restore bone architecture, and/or reduce the risk of vertebral and non- vertebral bone fractures in osteoporotic patients, particularly those who are at high risk of fracture.
- the conjugated polypeptide compounds of the invention may also be useful as bone- building or bone-healing agents, for example in fracture repair, or to treat or prevent disorders associated with bone loss or degeneration, such as osteoporosis, osteopenia and osteogenesis imperfecta.
- the term "patient in need thereof refers to a human or non-human mammal, and more preferably a human, which has been diagnosed as having a condition or disorder for which treatment or administration with a compound of the present invention is indicated.
- a “spinal fusion patient”, as used herein, refers to a human or non-human mammal, which either has been diagnosed as needing spinal fusion surgery, is undergoing spinal fusion surgery or has previously undergone spinal fusion surgery. More particularly, a “spinal fusion patient” refers to a human who has been diagnosed as needing posteriolateral or transforaminal lumbar interbody fusion surgery, or is undergoing posteriolateral or transforaminal lumbar interbody fusion surgery, or has previously undergone posteriolateral or transforaminal lumbar interbody fusion surgery.
- the term "effective amount” refers to the amount or dose of a conjugated polypeptide compound of the present invention, or a pharmaceutically acceptable salt thereof, which upon single or multiple dose administration to the patient, provides the desired pharmacological effect in the patient.
- An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by considering a number of factors such as the species of mammal; its size, age, and general health; the specific disease or surgical procedure involved; the degree or severity of the disease or malady; the response of the individual patient; the particular compound or composition administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; and the use of any concomitant medications.
- Maleimide-PEG linker refers to a chemical moiety comprising a polyethylene glycol (PEG) polymer and a derivatized maleimide functional group.
- PEG polyethylene glycol
- the maleimide-PEG linker of the compounds of the present invention has the following structure, wherein the dashed lines represent the locations of covalent attachments to the first and second polypeptides: lypeptide
- the linker is covalently attached to the second polypeptide through an amide bond to residue 18 of the second polypeptide, and covalently attached to the first polypeptide through a thioether bond to residue 26 of the first polypeptide.
- the PEG polymer portion of the linker is represented by the chemical moiety of the formula "-(0-CH2-CH2) n -" wherein "n” represents the number of repeating ethylene glycol monomer units that are present in the polymer of a given molecular weight.
- Pegylation reagents are typically described by reference to the molecular weight (in daltons or kilodaltons) of the PEG polymer portion of the PEG-containing compounds in the reagent.
- the molecular weight in daltons or kilodaltons
- commercially available PEG-containing reagents generally have some degree of polydisperity, meaning that the number of repeating ethylene glycol monomer units contained within the reagent (the "n") varies over a range, typically over a narrow range.
- the reference to the PEG polymer molecular weight is typically a reference to the average molecular weight of the PEG polymers contained within the reagent.
- the pegylation reagent used to introduce the maleimide-PEG linker to the conjugated polypeptides of the present invention MAL-PEG2000-NHS ester (JenKem Technologies, A5001-1), has a molecular weight of 2000 ⁇ 200 daltons, reported as the number average molecular weight (Mn).
- Mn is calculated according the formula ( ⁇ NjMj)/( ⁇ Nj), where ⁇ NjMj represents the total mass of all molecules in the sample and ⁇ Nj represents to the total number of molecules in the sample.
- Suitable techniques are known in the art for determining number average molecular weight including gel permeation chromatography (GPC) and matrix-assisted laser desporption/ionization (MALDI) mass spectrometry.
- the ethylene glycol monomer (O-CH2-CH2) of the reagent used to prepare the compounds of the present invention has a molecular weight of 44 g/mol or 44 daltons.
- the reported number average molecular weight of 2000 ⁇ 200 daltons of the reagent correlates to an "n" of between 40.9 and 50 ethylene glycol monomer units, or more generally between 40 and 50 ethylene glycol monomer units, in the PEG-containing compounds of the reagent.
- using the same reagent will produce compounds of the present invention which contain maleimide-PEG linkers having between 40 and 50 ethylene glycol monomer (O-CH2-CH2) units.
- the conjugated polypeptide compounds of the present invention contain an intra- chain disulfide bond formed between sulfhydral side chains of cysteine residues within the second polypeptide.
- bonds may be readily generated by one of skill in the art using conventional chemical methods and may be depicted by a line connecting affected cysteine residues, or by a "-S-S-" designation between affected cysteine residues.
- the presence of the disulfide bond may be represented by brackets occurring before and after the affected cysteine residues in the polypeptide chain.
- polypeptide chains of the present invention are depicted by their sequence of amino acids from N-terminus to C-terminus, when read from left to right, with each amino acid represented by either their single letter or three-letter amino acid abbreviation. Unless otherwise stated herein, all amino acids used in the preparation of the polypeptides of the present invention are L-amino acids.
- the "N-terminus” (or amino terminus) of an amino acid, or a polypeptide chain refers to the free amine group on the amino acid, or the free amine group on the first amino acid residue of the polypeptide chain.
- C-terminus or carboxy terminus of an amino acid, or a polypeptide chain, refers to the free carboxy group on the amino acid, or the free carboxy group on the final amino acid residue of the polypeptide chain.
- amidated refers to an optional replacement of the hydroxyl moiety of the carboxy terminus of an amino acid, or the final amino acid residue of a polypeptide chain, with an amino (-NH2) group.
- aminoated refers to a required replacement of the hydroxyl moiety of the carboxy terminus of an amino acid, or the final amino acid residue of a polypeptide chain, with an amino (-NH2) group.
- Amidation of a carboxy terminus of an amino acid, or the final amino acid of a polypeptide chain may be depicted by an "-NH2" designation following the amino acid single letter or three-letter abbreviation.
- acetylated when used herein, refers to a required replacement of a hydrogen atom of the amino terminus of an amino acid, or the first amino acid residue of a polypeptide chain, with an acetyl group (-C(0)CH3). Acetylation of the amino terminus of an amino acid, or the first amino acid residue of a polypeptide chain, may be depicted by an "Ac-" designation preceding the amino acid single letter or three-letter abbreviation.
- the conjugated polypeptides of the present invention can react with any of a number of inorganic and organic acids or bases to form pharmaceutically acceptable salts.
- Preferred pharmaceutically acceptable salts of the conjugated polypeptides of the present invention include acetate and chloride salts. More preferred are the acetate salts of the compounds of Examples 1-3 herein. Especially preferred is the acetate salt exemplified in Example 1.
- Methods for preparation of the pharmaceutically acceptable salts of the present invention are well known to the skilled artisan (See: Stahl et al., "Handbook of Pharmaceutical Salts: Properties, Selection and Use," VCHA/Wiley-VCH (2002); and Berge et al., Journal of Pharmaceutical Sciences, 66: 1-19, 1977) Compound Synthesis
- the individual polypeptide chains of the conjugated polypeptide compounds of the present invention can be synthesized using manual or automated solid-phase synthesis procedures.
- Automated peptide synthesizers are commercially available from, for example, Applied Biosystems and Protein Technologies, Inc. Reagents for solid-phase synthesis are readily available from commercial sources.
- an N-protected amino acid is coupled to an N-terminal-deprotected amino acid attached at its C-terminus to a solid phase support resin through a suitable linker.
- the coupling reaction is typically carried out at room temperature and in an inert solvent such as dimethylformamide, N-methylpyrrolidone or methylene chloride in the presence of coupling agents such as diisopropyl-carbodiimide (DIC) and 1- hydroxybenzotriazole (HOBt).
- DIC diisopropyl-carbodiimide
- HOBt 1- hydroxybenzotriazole
- Typical side chain protecting groups include tBoc (a tertiary butyloxycarbonyl) for lysine and tryptophan residues; Pbf (a 2, 2, 4, 6, 7-pentamethyldihydrobenzofuran-5-sulfonyl group) for arginine residues; tBu (a tertiary butyl group) for serine, aspartic acid, and glutamic acid residues; and Trt (a triphenylmethyl group) for glutamine, histidine, and asparagine residues.
- tBoc a tertiary butyloxycarbonyl
- Pbf a 2, 2, 4, 6, 7-pentamethyldihydrobenzofuran-5-sulfonyl group
- tBu a tertiary butyl group
- Trt a triphenylmethyl group for glutamine, histidine, and asparagine residues.
- N- protecting group is removed from the resulting peptide under basic conditions, such as 20% piperidine in DMF, and the coupling reaction is repeated with the next desired N-protected amino acid to be added to the peptide chain.
- Suitable amine protecting groups are well known in the art and are described, for example, in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, 1991. Commonly used examples include tBoc and Fmoc
- polypeptide chains of the conjugated compounds of the invention can be synthesized with either a C-terminal free acid or carboxamide.
- the type of derivatized polystyrene resin solid phase support used for the synthesis will determine the C-terminal moiety after cleavage.
- a number of linkers are well known and routinely used in the art.
- resins incorporating Rink amide MB HA or Rink amide AM linkers are typically used with Fmoc synthesis, while MBHA resin is generally used with tBoc synthesis.
- 2-chlorotrityl or Wang resin is typically used for Fmoc synthesis, while tBoc synthesis generally employs PAM resin.
- Methods for loading the first amino acid to the resin are also well known in the art.
- the conjugated polypeptide compounds of the present invention contain an intra- chain disulfide bond formed between the sulfhydral side chains of cysteine residues 1 and 7 of the second polypeptide.
- Intra-chain disulfide bonds can be readily introduced using typical chemical methods and may be depicted by a line connecting affected cysteine residues, or by a "-S-S-" designation between affected cysteine residues.
- the presence of the disulfide bond may be represented by brackets occurring before and after the affected cysteine residues in the polypeptide chain.
- Crude peptides are typically purified using reverse-phase High Performance Liquid Chromatography (rp-HPLC) on C 8 or CI 8 columns using water-acetonitrile gradients containing 0.05 to 0.1% TFA. Purity can be verified by analytical rp-HPLC. Identity of peptides can be verified by mass spectrometry. Peptides can be solubilized in aqueous buffers over a wide pH range.
- rp-HPLC reverse-phase High Performance Liquid Chromatography
- Conjugation of the polypeptides to the PEG-containing linker may be carried out using well-characterized chemical synthetic reactions. It is preferable that a molar excess of the PEG polymer relative to the peptide is used to drive the reaction to completion. Excess PEG reagent may be separated from the conjugated polypeptide products by conventional separation methods such as rp-HPLC.
- the PEG-reagent used to conjugate the PEG-containing linker to each of the first and second polypeptides comprises the maleimide-PEG N-hydroxysuccininimide ester of the following structure:
- n is between 40 and 50.
- the conjugation chemistry used in preparing the compounds of the present invention involves (1) reaction of the N-hydroxysuccinimide ester moiety with the amine side chain of Lys 18 of the polypeptide given by SEQ ID NO :4 to form an amide bond, and (2) reaction of the double bond of the maleimide moiety with the sulfhydral side chain of Cys26 of the polypeptide given by SEQ ID NO:l, SEQ ID NO:2 or SEQ ID
- polypeptide given by the sequence which is SEQ ID NO: 4 has been engineered to contain only a single lysine residue in its sequence (supplying a primary amine side chain) and the polypeptides given by the sequences of SEQ ID NOs: 1 - 3 have been engineered to contain only a single cysteine residue (supplying a sulfhydral group.)
- the conjugated polypeptide compounds of the present invention are intended for administration via parenteral routes, including intravenous, intramuscular, subcutaneous or intraperitoneal routes of administration.
- the present invention also provides pharmaceutical compositions suitable for parenteral administration comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.
- Such carriers, diluents and/or excipients may include dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents and the like.
- An exemplary composition includes 35 to 400 ⁇ g/ml of a conjugated polypeptide of the present invention in a solution of 20 mM NaH 2 PC>4 in 0.9% NaCl, 3 mg/ml mannitol, at pH ⁇ 4.5.
- the conjugated polypeptide compounds of the invention may also be lyophilized for storage and reconstituted in a suitable vehicle prior to use or administration. Remington. The Science and Practice of Pharmacy, 19 th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA 1995 provides a compendium of formulation techniques for those of skill in art.
- SEQ ID NP Polypeptide sequence a ' b ' 0
- the protecting groups used include: tBoc (a tertiary butyloxycarbonyl) for Lys and Trp residues; Pbf (a 2, 2, 4, 6, 7- pentamethyldihydrobenzofuran-5-sulfonyl group) for Arg residues; tBu (a tertiary butyl group) for Ser, Asp, and Glu residues; and Trt (a triphenylmethyl group) for Gin, His, and Asn residues.
- Amino acid side chain derivatives used are: Arg(Pbf), Asn(Trt),
- hydroxybenzotriazole (1 :1: 1 molar ratio) in dimethylformamide (DMF).
- N- terminal acetylation of the polypeptide amino acid sequence as given in SEQ ID NO:4 is carried out by treating the resin bound peptide with about 20 equivalents of acetic anhydride in DMF.
- SEQ ID NO:2 and SEQ ID NO:3 are also prepared:
- the polypeptide given by the sequence SEQ ID NO:4 is mixed with MAL- PEG2000-NHS (JenKem Technology USA, cat#: A5001-1, 1.2 eq.) in anhydrous DMF with N,N-Diisopropylethylamine (5 eq.).
- the reaction is monitored by LC-MS and typically complete within 4 hr.
- the reaction mixture is then quenched with 10% AcOH in water, and purified on a reversed-phase HPLC column (Waters SymmetryPrep 7 ⁇ , 19 x 300mm, cat.
- the maleimide-PEG - SEQ ID NO: 10 fragment and the polypeptide given by the sequence SEQ ID NO: 1 are mixed in a mixture of buffer (200 mM NH 4 OAc, 10 mM EDTA, pH 6.8) and acetonitirle at room temperature. The reaction is monitored by LC- MS and typically complete within 5 min. The mixture is purified on a reversed-phase HPLC column (Waters SymmetryPrep 7 ⁇ , 19 x 300mm, cat. No: WAT066245) as described above to provide the conjugated polypeptide compound comprising the polypeptides given by SEQ ID NO: 7 and SEQ ID NO: 10 as the trifluoroacetic acid salt, with a yield of -87%. (SEQ ID NO: 7 representing the modification of Cys26 of the sequence given by SEQ ID NO:l following conjugation to the maleimide-PEG- SEQ ID NO: 10 fragment.)
- the conjugated polypeptide-TFA salt is dissolved in acetonitrile/HiO and treated with AG1-X8 Resin (BIO-RAD, cat. #140-1443 acetate form, 50 eq. per basic reside on the construct), and shaken at rt. for 30 min. The filtrate and washes (3 times) are combined and lyophilized to provide the conjugated polypeptide as the acetate salt with a mass recovery of -88%.
- AG1-X8 Resin BIO-RAD, cat. #140-1443 acetate form, 50 eq. per basic reside on the construct
- the conjugated polypeptide compounds of Examples 2 and 3 are prepared as the acetate salts.
- Underlined amino acid residues represent the points of attachment of the maleimide- PEG linker to each of the first and second polypeptide sequences.
- Conjugated peptides of the present invention are evaluated for their ability to stimulate cAMP production in HEK-293 cells overexpressing either human PTHRl or human CT receptors. Briefly, various concentrations of conjugated peptide compound are incubated for 30 minutes at room temperature, with cells expressing either the human PTHRl or human CT receptor. Following incubation with test compound, cAMP levels are quantitated by cAMP HTRF assay kit (Cisbio 62AM4PEC). Reaction plates are read on an EnVision plate reader using HTRF optimized protocol (PerkinElmer). EC50s are calculated using Graphpad Prism nonlinear regression curve fitting.
- hPTH(l-34) may be included as a positive control in the human PTHR1 receptor assays, while sCT may be included as a positive control in the human CT receptor assays.
- the conjugated peptides of the present invention may be evaluated in a mature osteopenic, ovariectomized rat cortical defect model to ascertain systemic effects on bone healing and vertebrae.
- This model may mimic the impaired bone healing observed in post-menopausal women with osteoporosis ( Komatsu, et al., Endocrinology, 150:1570- 1579, 2009)
- BMD bone mass density
- qCT quantitative computed tomography
- BMD GE Locus Ultra CT scanner
- Micro ViewABA Very ViewABA (Version 2.3a4 GE Healthcare, London, Ontario, Canada) and entails converting the mean intensity results (in Hounsfield units) generated for each region of interest to BMD by performing a linear transformation of data using intensity values from a hydroxyapatite phantom as a reference.
- L5 vertebrae may be scanned post-necropsy using an Aloka LaTheta LTC-100 CT scanner (Aloka Co., Ltd., Tokyo, Japan). Briefly, vertebrae are wrapped in parafilm, positioned in 2D and scanned using landmark structures. Total BMD is determined using the manufacture's software package (SYS-C320-V 1.5).
- Biomarker analyses Serum samples may also be taken from test animals during the course of the study for biomarker analysis. For example, the serum levels of rat osteocalcin (OCN) may be determined by an enzyme immunoassay (EIA) (Biomedical Technologies Inc.
- the serum levels of N-terminal propeptide of type I procollagen may be determined by EIA (rat/mouse PINP, Immunodiagnostic Systems Inc. Scottsdale, AZ) following the manufacturer's protocol.
- the serum levels of fragments of type I collagen may be determined by EIA (RatLaps Immunodiagnostic Systems Inc. Scottsdale, AZ) following the manufacturer's protocol.
- the data demonstrates that the conjugated polypeptide compound of Example 1 is efficacious in building bone, as measured by BMD, in a rat model of cortical bone injury.
- the data demonstrates that the compound of Example 1, following subcutaneous administration at all doses tested, significantly promotes bone formation at the site of cortical bone injury (i.e. femur, Table 2) at both day 21 and day 35 post- surgery.
- the data demonstrates that the compound of Example 1 significantly promotes bone formation at a site remote from the location of injury (i.e. L5 vertebra, Table 3) at all doses tested at day 35 post-surgery.
- Example 1 Treatments with Example 1 produced no effects on the bone formation marker PINP, however, at the highest dose administered (420 ⁇ g/kg) (3X/wk), the compound of Example 1 significantly increased intact osteocalcin at day 11 post-surgery, with a trend toward significance at day 34. In addition, this dose of Example 1 produced a significant decrease in the bone resorption marker CTX- 1 , at both day 11 - and day 34 post-surgery.
- the rat screw implant model has been used to test the pharmacological effect of osseous integration into implants and implant fixation.
- PTH was shown to enhance osseous integration into implants and implant fixation in the rat screw implant model (Skripitz, et ah, Journal of Orthopaedic Research, 23: 1266-1270, 2005).
- a similar rat model may be used to evaluate the effects of the conjugated peptide compounds of the present invention on osseous integration into implants and also on implant fixation.
- Rats Female Sprague Dawley rats are maintained on a 12hr light/dark cycle at 22 °C with ad lib access to food (TD 89222 with 0.5% Ca and 0.4%P, Teklad, Madison, WI) and water. Rats are then ovariectomized and allowed to lose bone for about 7 months. At 13 months, animals are randomized into treatment groups and surgical procedures are performed as follows. Rats are surgically implanted with titanium screws (2x4mm) into both tibiae of the medial lateral side, at 5mm below the growth plate. Test compound or vehicle (20 mM NaH2P04 in 0.9% NaCl, 3 mg/ml mannitol) is administered
- a biomechanical pull to failure force test is performed on both tibiae ex vivo using an industrial digital force gauge (Mark-10, model M3-50, ESM301, IN, USA). Pull to failure force is tested at a speed of lOmm/minute. After the mechanical test, screws are carefully removed. Tibiae and L5 vertebrae are then scanned using quantitative computed tomography with a 60- ⁇ voxel size (Aloka LaTheta LTC-100 model CT scanner). Group differences are assessed with JMP version 5.1 software, Dunnett's T test.
- Biomarker Analysis Collected sera may be analyzed for biomarkers as described above. Briefly, the serum levels of rat osteocalcin (OCN) may be determined by EIA (Biomedical Technologies Inc. Stoughton, MA) following the manufacturer's protocol using a 1:10 dilution. The serum levels of N-terminal propeptide of type I procollagen (P1NP) may be determined by EIA (rat/mouse PINP, Immunodiagnostic Systems Inc. Scottsdale, AZ) following the manufacturer's protocol. The serum levels of fragments of type I collagen (CTX-I) may be determined by EIA (RatLaps Immunodiagnostic Systems Inc. Scottsdale, AZ) following the manufacturer's protocol.
- OCN rat osteocalcin
- P1NP N-terminal propeptide of type I procollagen
- CX-I fragments of type I collagen
- the compound of Example 1 also dose-dependently increased the bone formation markers PINP and osteocalcin at days 14 and 28, while suppressing the bone resorption marker CTX-1 at day 28 at the dose of 140 ⁇ g/kg.
- PTH has previously been shown to stimulate de novo bone formation to restore bone loss and promote bone healing in clinical and pre-clinical studies. (Compston, Bone, 40: 1447-1452, 2007; Datta, World J. Orthop., 18; 2(8): 67-74, 2011) Previously, PTH was also shown to enhance spinal fusion in rats and patients (Ohtori et ah, Spine (Phila Pa. 1976), 2012 Nov. 1; 37(23): E1464-1468; Ohtori et al, Spine (Phila Pa. 1976), 2013 Apr. 15; 38(8): E487-492).
- a rat posterior lateral spinal fusion model with autografts harvested from the iliac crest may be employed as follows. Methods
- An iliac crest surgery to harvest autograft material (0.5x0.5cm) from the left iliac crest is conducted on male, 17 week old Sprague-Dawley rats weighing 450-530g.
- the bone graft material is then immediately transplanted to the decorticated L5 and L6 transverse processes of the lumbar vertebrae of the same animal.
- Digital radiographs are taken at the day of surgery to ensure graft positioning.
- Test compound dosed for example at 140ug/kg (vehicle: 20 mM NaH2P04 in 0.9% NaCl, 3 mg/ml mannitol, at pH 4.5), is given subcutaneously once a week, twice a week or three times a week to different groups starting on day 3 post surgery. Animals are sacrificed after 4 weeks.
- Micro-CT images showed full fusion rates in rats treated with the compound of Example 1 (140ug/kg) of 61.5% (8 of 13), 38.5% (5 of 13) and 53.3% (8 of 15) for three times, twice or once per week treatment regimens, respectively, compared to 26.7% (4 of 13) in the vehicle control.
- Rats were measured via incapacitance testing on day 4 post osteotomy for baseline pain. On day 7 post osteotomy, the rats were randomized using BRAT (the Block Randomization Allocation Tool) and baseline pain measurements into five groups of 6 animals and dosed with either vehicle (20 mM NaH 2 P0 4 in 0.9% NaCl, 3 mg/mL mannitol, pH 4.5), 70, 140 or 420 ⁇ g/kg of the compound of Example 1. Rats were dosed again on days 10 and 14 post osteotomy. Dose volume was 1 ml/kg. Pain was measured via incapacitance testing on days 8, 9, 11, 15 and 16 post-osteotomy with animals from each dose group measured randomly throughout the day of measurement. The following table provides the profile of activity obtained in this study with the compound of Example 1.
- BRAT Block Randomization Allocation Tool
- Example 1 3.81 5.44 14.42 30.69 36.11 ⁇ /kg ( ⁇ 1.52) ( ⁇ 1.53) ( ⁇ 1.16 ( ⁇ 2.45)* ( ⁇ 3.72)*
- Example 1 5.30 11.87 22.67 32.94 40.92 ⁇ g ⁇ kg ( ⁇ 2.40) ( ⁇ 2.52) ( ⁇ 3.89)* ( ⁇ 4.58)* ( ⁇ 4.59)*
- Example 1 6.24 16.76 23.32 38.15 47.44 420Mg/kg ( ⁇ 0.96) ( ⁇ 1.97)* ( ⁇ 2.66)* ( ⁇ 2.85)* ( ⁇ 2.02)*
- the compound of Example 1 significantly reduced pain compared to vehicle on days 9, 11, 15 and 16 post-osteotomy at 42C ⁇ g/kg; on days 11, 15 and 16 post-osteotomy at 14C ⁇ g/kg; and on days 15 and 16 at 7C ⁇ g/kg post-osteotomy (*p ⁇ 0.05).
- Rats were measured via incapacitance testing on day 4 post-osteotomy for baseline pain. On day 7 post-osteotomy, rats were randomized using BRAT and baseline pain measurements into five groups of 6 animals and dosed with either vehicle (20 mM NaH 2 P0 4 in 0.9% NaCl, 3 mg/mL mannitol, pH 4.5), 10, 35, 70 or 140 ⁇ g/kg of the compound of Example 1. In this study, rats were dosed daily for 5 days. Dose volume was lml/kg with pain measured via incapacitance testing on days 8, 9, 11, 14 and 16 post- osteotomy. Note that if pain was measured on the same day as dosing, pain was measured after dosing with rats randomized for pain measurement throughout the day of measurement. The following table provides the profile of activity obtained in this study with the compound of Example 1.
- Example 1 1.69 4.47 9.76 0.59 9.77 lOMg/kg ( ⁇ 1.95) ( ⁇ 1.03) ( ⁇ 2.40) ( ⁇ 3.26) ( ⁇ 2.08)
- Example 1 2.78 7.28 18.70 8.79 13.99 35 ⁇ g/kg ( ⁇ 2.71) ( ⁇ 1.92) ( ⁇ 2.16)* ( ⁇ 4.51) ( ⁇ 3.22)
- Example 1 4.55 11.39 23.60 9.63 16.79 ⁇ /kg ( ⁇ 1.19) ( ⁇ 0.70) ( ⁇ 2.45)* ( ⁇ 4.72) ( ⁇ 3.90)
- Example 1 3.00 12.73 29.13 18.80 21.76 140Mg/kg ( ⁇ 2.83) ( ⁇ 1.03) ( ⁇ 2.08)* ( ⁇ 4.37) ( ⁇ 3.71)* Pain efficacy was not seen until day 11 post osteotomy at which time point all doses above and including 35 ⁇ g/kg of Example 1 were significantly different from vehicle, but not different from each other (*p ⁇ 0.05).
- the lC ⁇ g/kg dose of Example 1 did not significantly reduce pain compared to vehicle on any day measured.
- the 14C ⁇ g/kg dose showed pain efficacy on the same day post- osteotomy, whereas the 7C ⁇ g/kg dose showed earlier efficacy and the 35ug/kg dose also showed efficacy at this time point.
- Example 1 The compound of Example 1 was compared to clinically relevant doses of PTH and salmon calcitonin (sCT). As in the previous studies, rats were measured via
- the rats were randomized using BRAT and baseline pain measurements into five groups of 6 animals and dosed with either vehicle (20 mM NaH 2 P0 4 in 0.9% NaCl, 3 mg/mL mannitol, pH 4.5) (dosed every 3-4 days), 140 ⁇ g/kg of the compound of Example 1 (dosed every 3-4 days), 3 ⁇ g/kg hPTH(l-34) (SEQ ID NO: 6, synthesized in-house, Eli Lilly and Company) (dosed every day), 3 ⁇ g/kg sCT (BACHEM) (dosed every day) or 3 ⁇ g/kg PTH(l-34) + 3 ⁇ g/kg sCT (dosed every 3-4 days).
- vehicle 20 mM NaH 2 P0 4 in 0.9% NaCl, 3 mg/mL mannitol, pH 4.5
- 140 ⁇ g/kg of the compound of Example 1 dosed every 3-4 days
- 3 ⁇ g/kg hPTH(l-34) SEQ ID NO: 6, synthesized in-house, Eli Lilly
- test compounds were administered using the same vehicle (20 mM NaH 2 P0 4 in 0.9% NaCl, 3 mg/mL mannitol, pH 4.5), dosed at a volume of lml/kg, with pain measured via incapacitance testing on days 7, 8, 10, 11, 14 and 15 post-osteotomy. Note that if pain was measured on the same day as dosing, pain was measured after dosing with rats randomized for pain
- sCT purchased from BACHEM
- 3 ⁇ g/kg sCT dosed daily provided significant pain efficacy compared to vehicle on all days when pain was measured, with the exception of the first day (day 7) (*p ⁇ 0.05).
- 3ug/kg PTH daily did not show pain efficacy on any day in comparison to vehicle.
- the combination dose of sCT plus PTH showed significant pain efficacy compared to vehicle on days 11, 14 and 15 post-osteotomy and the compound of Example 1 showed efficacy compared to vehicle on days 10 and 11 post-osteotomy (*p ⁇ 0.05).
- the single dose of sCT and sCT in combination with PTH were significantly different from PTH alone on days 14 and 15 post-osteotomy.
- SEQ ID NO:l (Example 1, first polypeptide sequence prior to modification)
- SEQ ID NO:2 (Example 2, first polypeptide sequence prior to modification)
- SEQ ID NO:3 (Example 3, first polypeptide sequence prior to modification)
- SEQ ID NO:4 (Examples 1-3, second polypeptide sequence prior to modification)
- SEQ ID NO: 6 (hPTH(l-34)) Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg- Val-Glu-T ⁇ -Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe
- SEQ ID NO: 7 (Example 1, first polypeptide sequence after modification)
- Xaa at position 26 is cysteine residue modified by thioether bond formation to maleimide-PEG linker
- SEQ ID NO: 8 (Example 2, first polypeptide sequence after modification)
- Xaa at position 26 is cysteine residue modified by thioether bond formation to maleimide-PEG linker
- SEQ ID NO: 9 (Example 3, first polypeptide sequence after modification)
- Xaa at position 26 is cysteine residue modified by thioether bond formation to maleimide-PEG linker
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Abstract
The present invention relates to conjugated polypeptide compounds, or pharmaceutically acceptable salts thereof, which are agonists of the human parathyroid and calcitonin receptors and which are useful as adjuncts to spinal fusion surgery, or as agents for bone healing or treating conditions associated with bone loss or degeneration, as well as pharmaceutical compositions and methods of use comprising the same.
Description
CONJUGATED POLYPEPTIDES AND USES THEREOF
The present invention is in the field of medicine. More particularly, the present invention relates to conjugated polypeptide compounds, or pharmaceutically acceptable salts thereof, which are agonists of both the human parathyroid and calcitonin receptors, as well as pharmaceutical compositions and methods of use comprising the same.
Spinal fusion is a surgical procedure in which adjacent vertebrae are grafted together to limit or eliminate the range of motion in the joint space between the affected bones. Spinal fusions are performed to address pain and morbidity associated with degenerative conditions such as degenerative disc disease (DDD), spondylosis, and spondylolisthesis; congenital deformities, including kyphosis and scoliosis; as well as some vertebral fractures.
Posterolateral fusion (PLF) involves placing bone grafting substance between the transverse processes of adjacent vertebrae in the posterior of the spine. The vertebrae may then be fixed in place using metal rods on each side of the vertebrae, attached to the pedicles of each vertebra with screws and/or wire. As the bone graft substance ossifies, it forms continuous bone, fusing the vertebrae above and below the site of the graft.
Interbody fusion involves removing an intervertebral disc and placing the bone graft substance into the intervertebral space between adjacent vertebrae. An interbody device or spacer may be placed between the affected vertebral bodies to maintain alignment and disc-space height. Fusion occurs between the endplates of the adjacent vertebral bodies. As with PLF, the interbody fusion may be stabilized with the placement of rods, plates, screws or wire to facilitate the fusion process. Types of interbody fusion procedures include anterior lumbar interbody fusion (ALIF), posterior lumbar interbody fusion (PLIF), and transforaminal lumbar interbody fusion which differ according to location and angle of approach to the spine.
Risks or complications associated with current spinal fusion procedures include the risk of fusion failure (pseudoarthrosis) and the need for revision surgery, postoperative pain and morbidity and potentially long recovery times. Thus, there remains a need for alternative therapies which could lead to better outcomes for patients. In particular, there remains a need for a systemically-administered pharmaceutical agent which could be used as an adjunct therapy to spinal fusion procedures.
Parathyroid hormone (PTH) is an eighty four amino acid peptide which plays a central role in bone remodeling. PTH binding to the PTH receptor 1 directly induces bone formation through activation of cyclic AMP and canonical wnt-signaling pathways, while indirectly inducing osteoclast differentiation and bone resorption through induced expression of RANK ligand on the osteoblast cell surface. Teriparatide (rhPTH(l -34)), marketed as Forteo®, is a thirty four amino acid N-terminal fragment of PTH that has been shown to increase bone formation activity in osteoporotic patients and is the only bone anabolic agent approved in the United States to treat osteoporosis. Calcitonin (CT) is a thirty two amino acid peptide which mediates calcium homeostasis. The binding of CT to receptors in the kidney results in inhibition of calcium reabsorption, while binding to receptors on osteoclasts results in inhibition of bone resorption. In therapeutic applications, salmon calcitonin (sCT) is typically employed due to its superior bioactivity relative to human CT. Clinically, sCT has been used in the treatment of post-menopausal osteoporosis, Paget's disease and in the treatment of hypercalcemia. sCT has also been reported to decrease spinal pain associated with osteoporotic vertebral compression fractures (Knopp et al, Osteoporosis Int., 16(10); 1281-1290 (Oct. 2005)).
Combining the bone anabolic properties of PTH with the anti-bone resorptive properties of sCT, provides an alternative therapeutic approach for improving outcomes for spinal fusion patients, as well as inducing bone healing or treating conditions associated with bone loss or degeneration. Currently, to achieve such therapeutic effect would require co-administration of separate agents, either through administration of separate formulations (each containing a separate active agent), or administration of a single, co-formulation containing each of the individual agents. While co-administration of separate formulations would permit flexibility in dose and timing of administration, the inconvenience and possible discomfort associated with multiple injections may reduce patient compliance. On the other hand, while single administration of a co-formulation of multiple agents would provide convenience, the difficulty and/or expense associated with designing a suitable pharmaceutical formulation that provides the necessary stability and bioavailability for each individual agent may be prohibitive. Furthermore, any treatment regimen which entails administration of separate agents will incur added manufacturing and regulatory costs associated with the development of each individual agent.
The present invention provides conjugated polypeptide compounds, or pharmaceutically acceptable salts thereof, useful as adjunct therapies for spinal fusion surgery, or as agents for bone healing or treating conditions associated with bone loss or degeneration. Particularly, the present invention provides a compound consisting of:
(a) a first polypeptide which is given by a sequence selected from the group consisting of:
i. Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Arg-His-Leu- Ala-Ser-Met-Glu-Arg-Val-Glu-Tφ-Leu-Arg-Xaa26-Leu-Leu-Gln- Asp-Val-His-Asn-Phe (SEQ ID NO: 7),
ii. Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Arg-His-Leu- Ala-Ser-Met-Glu-Arg -Val-Glu-Trp-Leu-Arg- Xaa26-Leu-Leu- Gln-Glu-Val-His-Asn-Phe (SEQ ID NO: 8), and iii. Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu- Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg- Xaa26-Lys-Leu- Gln-Asp-Val-His-Asn-Phe (SEQ ID NO: 9);
(b) a second polypeptide which is given by the sequence which is Ac-[Cys- Ser-Asn-Leu-Ser-Thr-Cys]-Val-Leu-Gly-Arg-Leu-Ser-Gln-Glu-Leu-His- Xaal8-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 (SEQ ID NO: 10); and
(c) a maleimide- PEG linker of the formula
Xaa26 of the sequence given by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 is a cysteine residue modified by attachment to the maleimide-PEG linker through the thioether bond;
the C-terminus of Phe34 of the sequence given by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 is optionally amidated;
Xaal8 of the sequence given by SEQ ID NO: 10 is a lysine residue modified by attachment to the maleimide-PEG linker through the amide bond, and
n represents 40 to 50,
or a pharmaceutically acceptable salt thereof.
More particularly, the present invention provides a compound consisting of:
(a) a first polypeptide which is given by a sequence selected from the group consisting of:
i. Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Arg-His-Leu-
Ala-Ser-Met-Glu-Arg-Val-Glu-Tφ-Leu-Arg-Xaa26-Leu-Leu-Gln- Asp-Val-His-Asn-Phe (SEQ ID NO: 7),
ii. Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Arg-His-Leu- Ala-Ser-Met-Glu-Arg -Val-Glu-Trp-Leu-Arg- Xaa26-Leu-Leu- Gln-Glu-Val-His-Asn-Phe (SEQ ID NO : 8), and iii. Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu- Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg- Xaa26-Lys-Leu- Gln-Asp-Val-His-Asn-Phe (SEQ ID NO: 9);
(b) a second polypeptide which is given by a sequence which is Ac-[Cys-Ser- Asn-Leu-Ser-Thr-Cys]-Val-Leu-Gly-Arg-Leu-Ser-Gln-Glu-Leu-His-
Xaal8-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 (SEQ ID NO: 10); and
(c) a maleimide-PEG linker of the formula
wherein,
Xaa26 of the sequence given by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 is a cysteine residue modified by attachment to the maleimide-PEG linker through the thioether bond;
the C-terminus of Phe34 of the sequence given by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 is amidated;
Xaal 8 of the sequence given by SEQ ID NO: 10 is a lysine residue modified by attachment to the maleimide-PEG linker through the amide bond, and
n represents 40 to 50,
or a pharmaceutically acceptable salt thereof.
Even more particularly, the present invention provides a compound consisting of:
(a) a first polypeptide which is given by a sequence which is Ser-Val-Ser-Glu-Ile-Gln- Leu-Met-His-Asn-Leu-Gly-Arg-His-Leu-Ala-Ser-Met-Glu-Arg-Val-Glu-Tφ-Leu- Arg-Xaa26-Leu-Leu-Gln-Asp-Val-His-Asn-Phe (SEQ ID NO:7);
(b) a second polypeptide which is given by a sequence which is Ac-[Cys-Ser-Asn- Leu-Ser-Thr-Cys]-Val-Leu-Gly-Arg-Leu-Ser-Gln-Glu-Leu-His-Xaal8-Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 (SEQ ID NO: 10); and
(c) a maleimide-PEG linker of the formula
Xaa26 of the sequence given by SEQ ID NO: 7 is a cysteine residue modified by attachment to the maleimide-PEG linker through the thioether bond;
the C-terminus of Phe34 of the sequence given by SEQ ID NO: 7 is amidated;
Xaal 8 of the sequence given by SEQ ID NO: 10 is a lysine residue modified by attachment to the maleimide-PEG linker through the amide bond, and
n represent 40 to 50,
or a pharmaceutically acceptable salt thereof.
Even more particularly, the present invention provides a compound consisting of: (a) a first polypeptide which is given by a sequence which is Ser-Val-Ser-Glu-Ile-Gln-
Leu-Met-His-Asn-Leu-Gly-Arg-His-Leu-Ala-Ser-Met-Glu-Arg -Val-Glu-Trp- Leu-Arg- Xaa26-Leu-Leu-Gln-Glu-Val-His-Asn-Phe (SEQ ID NO: 8);
(b) a second polypeptide which is given by a sequence which is Ac-[Cys-Ser-Asn- Leu-Ser-Thr-Cys]-Val-Leu-Gly-Arg-Leu-Ser-Gln-Glu-Leu-His-Xaal8-Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 (SEQ ID NO: 10); and
(c) a maleimide-PEG linker of the formula
Xaa26 of the sequence given by SEQ ID NO: 8 is a cysteine residue modified by attachment to the maleimide-PEG linker through the thioether bond;
the C-terminus of Phe34 of the sequence given by SEQ ID NO: 8 is amidated;
Xaal 8 of the sequence given by SEQ ID NO: 10 is a lysine residue modified by attachment to the maleimide-PEG linker through the amide bond, and
n represents 40 to 50,
or a pharmaceutically acceptable salt thereof.
Even more particularly, the present invention provides a compound consisting of: (a) a first polypeptide which is given by a sequence which is Ser-Val-Ser-Glu-Ile-Gln-
Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Τφ- Leu-Arg- Xaa26-Lys-Leu-Gln-Asp-Val-His-Asn-Phe (SEQ ID NO: 9);
(b) a second polypeptide which is given by a sequence which is Ac-[Cys-Ser-Asn- Leu-Ser-Thr-Cys]-Val-Leu-Gly-Arg-Leu-Ser-Gln-Glu-Leu-His-Xaal8-Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 (SEQ ID NO: 10); and
(c) a maleimide-PEG linker of the formula
wherein,
Xaa26 of the sequence given by SEQ ID NO: 9 is a cysteine residue modified by attachment to the maleimide-PEG linker through the thioether bond;
the C-terminus of Phe34 of the sequence given by SEQ ID NO: 9 is amidated;
Xaal 8 of the sequence given by SEQ ID NO: 10 is a lysine residue modified by attachment to the maleimide-PEG linker through the amide bond, and
n represents 40 to 50,
or a pharmaceutically acceptable salt thereof.
As another particular embodiment, the present invention provides any of the afore-mentioned compounds wherein n represents 43 to 47. As yet another particular embodiment, the present invention provides any of the afore-mentioned compounds wherein n represents 45.
The present invention also provides methods of use comprising administering to a spinal fusion patient an effective amount of a conjugated polypeptide compound of the present invention, or a pharmaceutically acceptable salt thereof. As a particular embodiment, the present invention provides methods of use comprising administering to a spinal fusion patient an effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, wherein said compound is administered prior to spinal fusion surgery. As another particular embodiment, the present invention provides methods of use comprising administering to a spinal fusion patient an effective amount of
a compound of the present invention, or a pharmaceutically acceptable salt thereof, wherein said compound is administered during spinal fusion surgery. As yet another particular embodiment, the present invention provides methods of use comprising administering to a spinal fusion patient an effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, wherein said compound is administered after spinal fusion surgery. Even more particular, the compound of the present invention, or pharmaceutically acceptable salt thereof, is administered to a spinal fusion patient prior to, during or after posteriolateral fusion surgery (PLF) or
transforaminal lumbar interbody (TLIF) fusion surgery.
The present invention further provides methods for bone healing, for example in patients with bone fractures such as vertebral fractures, as well as methods for treating conditions associated with bone loss or degeneration such as osteoporosis, osteopenia, or osteogenesis imperfecta, said methods comprising administering to a patient in need thereof a compound of the present invention, or a pharmaceutically acceptable salt thereof.
The present invention also provides a compound of the present invention, or a pharmaceutically acceptable salt thereof, for use in therapy. Additionally, the present invention provides a compound of the present invention, or a pharmaceutically acceptable salt thereof, for use as an adjunct to spinal fusion surgery. More particularly, the present invention provides a compound of the present invention, or a pharmaceutically acceptable salt thereof, for use as an adjunct to spinal fusion surgery, wherein said compound, or a pharmaceutically acceptable salt thereof, is administered to a spinal fusion patient prior to, during or after spinal fusion surgery. The present invention also provides a compound of the present invention, or a pharmaceutically acceptable salt thereof, for use in bone healing or in the treatment of osteoporosis, osteopenia, or osteogenesis imperfecta.
The present invention also provides the use of a compound of the present invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for spinal fusion surgery. More particularly, the present invention also provides the use of a compound of the present invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for spinal fusion surgery, wherein said medicament is administered to a spinal fusion patient prior to, during or after spinal
fusion surgery. Further, the present invention provides the use of a compound of the present invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for bone healing or for the treatment of osteoporosis, osteopenia, or osteogenesis imperfecta.
The present invention further provides pharmaceutical compositions comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient. In addition, the present invention provides a pharmaceutical composition comprising a plurality of any one of the compounds of the present invention, or a pharmaceutically acceptable salt thereof, wherein the number average molecular weight of the PEG polymer of the formula "-(O- CH2-CH2)n-" in said plurality of compounds or salts is 1800 to 2200 daltons, and a pharmaceutically acceptable carrier, diluent or excipient. More particularly, the present invention provides a pharmaceutical composition comprising a plurality of any one of the compounds of the present invention, or a pharmaceutically acceptable salt thereof, wherein the number average molecular weight of the PEG polymer of the formula "-(O- CH2-CH2)n-" in said plurality of compounds or salts is 1900 to 2100 daltons, and a pharmaceutically acceptable carrier, diluent or excipient. Even more particularly, the present invention provides a pharmaceutical composition comprising a plurality of any one of the compounds of the present invention, or a pharmaceutically acceptable salt thereof, wherein the number average molecular weight of the PEG polymer of the formula "-(0-CH2-CH2)n-" in said plurality of compounds or salts is about 2000 daltons, and a pharmaceutically acceptable carrier, diluent or excipient.
The present invention also provides a process for preparing a conjugated polypeptide compound comprising:
(i) reacting a polypeptide given by the sequence which is SEQ ID NO:4 with a reagent comprising a compound of the formula
(ii) reacting the product of step (i) with a polypeptide given by the sequence which is SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3 under suitable conditions such that a thioether bond is formed between Cys26 of the polypeptide given by SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3 and the maleimide moiety of the product of step (i). Further, the present invention also provides a conjugated polypeptide compound prepared according to afore-mentioned process.
The conjugated polypeptide compounds of the present invention are bi-functional, meaning they are capable of interacting with, and modulating the activity of, two distinct targets. Specifically, the conjugated polypeptides of the present invention are agonists of both the PTH receptor and the CT receptor and, more particularly, the human PTH and human CT receptor. By combining agonist properties of PTH and sCT into a single compound, it is believed that the compounds of the present invention will demonstrate bone anabolic activity similar to PTH, and anti-resorptive and/or analgesic activity similar to sCT. Furthermore, by combining activities of PTH and sCT into a single molecule, the conjugated polypeptide compounds of the present invention may also counter tolerability limitations seen with individual administration of PTH(l-34) (e.g., hypercalcemia,
hypotension) and sCT (e.g., hypocalcemia, hypertension), thus potentially allowing for administration at doses higher than currently, clinically-approved for one or both of PTH(l-34) and sCT.
The conjugated polypeptide compounds of the present invention may be useful, for example, as adjuncts to spinal fusion surgery. During spinal fusion procedures, bone graft substances are employed to provide proteins and other substances to stimulate new bone growth in the affected space. Autograft (autologous bone graft) procedures employ the patient's own bone material as the grafting substance. The bone material, which must be separately harvested from areas such as the iliac crest of the pelvis, ribs or the spine, provides a source of viable osteocytes, bone morphogenic proteins (e.g. BMP-2) and a calcified matrix for new bone growth. Autografts were long-considered the gold standard in spinal fusion procedures due to their proven efficacy, low cost and reduced risk of graft-tissue rejection, however, due to the added pain and morbidity associated with the bone-harvesting procedures, bone graft substitute (BGS) procedures are increasing in use. BGS procedures include allograft procedures (e.g., bone material previously harvested from sources such as cadavers and obtained through tissue banks or the like); demineralized bone matrix(DBM) procedures which employ allograft bone material that has been demineralized to expose bone-forming proteins; ceramic-based bone graft extender procedures which provide a calcium matrix for bone growth; and procedures employing bone graft substances containing bone forming proteins (e.g., bone morphogenic proteins) or other growth factors (e.g. TGF-beta, PDGF, FGF), such as platelet rich plasma.
While bone graft substances provide a means for supplying proteins and other factors necessary for stimulating new bone formation, the bone graft substance must be applied locally during the surgical procedure. The conjugated polypeptides of the present invention, however, may be administered to the patient systemically. Such systemic administration may provide flexibility in dosing, including dosing prior to, during or after bone fusion surgery as well as repeat dosing. "Systemic administration", as used herein, refers to administration by parenteral routes, including intravenous, intramuscular, subcutaneous, intraperitoneal or intranasal routes of administration. Preferred routes of
administration of the conjugated polypeptide compounds of the present invention include intravenous, subcutaneous or intraperitoneal routes.
Because the conjugated polypeptide compounds of the present invention are agonists of both the PTH and CT receptors, they may also be useful in situations or conditions in which PTH or sCT is indicated. For example, the conjugated peptide compounds of the present invention may build bone mass, increase bone biomechanical strength, restore bone architecture, and/or reduce the risk of vertebral and non- vertebral bone fractures in osteoporotic patients, particularly those who are at high risk of fracture. Thus, the conjugated polypeptide compounds of the invention may also be useful as bone- building or bone-healing agents, for example in fracture repair, or to treat or prevent disorders associated with bone loss or degeneration, such as osteoporosis, osteopenia and osteogenesis imperfecta.
As used herein, the term "patient in need thereof refers to a human or non-human mammal, and more preferably a human, which has been diagnosed as having a condition or disorder for which treatment or administration with a compound of the present invention is indicated.
A "spinal fusion patient", as used herein, refers to a human or non-human mammal, which either has been diagnosed as needing spinal fusion surgery, is undergoing spinal fusion surgery or has previously undergone spinal fusion surgery. More particularly, a "spinal fusion patient" refers to a human who has been diagnosed as needing posteriolateral or transforaminal lumbar interbody fusion surgery, or is undergoing posteriolateral or transforaminal lumbar interbody fusion surgery, or has previously undergone posteriolateral or transforaminal lumbar interbody fusion surgery.
As used herein the term "effective amount" refers to the amount or dose of a conjugated polypeptide compound of the present invention, or a pharmaceutically acceptable salt thereof, which upon single or multiple dose administration to the patient, provides the desired pharmacological effect in the patient. An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by considering a number of factors such as the species of mammal; its size, age, and general health; the specific disease or surgical procedure involved; the degree or severity of the disease or malady; the response of the individual patient; the particular compound or composition
administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; and the use of any concomitant medications.
"Maleimide-PEG linker", as used herein, refers to a chemical moiety comprising a polyethylene glycol (PEG) polymer and a derivatized maleimide functional group. The maleimide-PEG linker of the compounds of the present invention has the following structure, wherein the dashed lines represent the locations of covalent attachments to the first and second polypeptides: lypeptide
Second polypeptide
The linker is covalently attached to the second polypeptide through an amide bond to residue 18 of the second polypeptide, and covalently attached to the first polypeptide through a thioether bond to residue 26 of the first polypeptide. The PEG polymer portion of the linker is represented by the chemical moiety of the formula "-(0-CH2-CH2)n-" wherein "n" represents the number of repeating ethylene glycol monomer units that are present in the polymer of a given molecular weight.
Pegylation reagents are typically described by reference to the molecular weight (in daltons or kilodaltons) of the PEG polymer portion of the PEG-containing compounds in the reagent. However, as one of skill in the art will appreciate, commercially available PEG-containing reagents generally have some degree of polydisperity, meaning that the number of repeating ethylene glycol monomer units contained within the reagent (the
"n") varies over a range, typically over a narrow range. Thus, the reference to the PEG polymer molecular weight is typically a reference to the average molecular weight of the PEG polymers contained within the reagent. For example, the pegylation reagent used to introduce the maleimide-PEG linker to the conjugated polypeptides of the present invention, MAL-PEG2000-NHS ester (JenKem Technologies, A5001-1), has a molecular weight of 2000 ± 200 daltons, reported as the number average molecular weight (Mn). The Mn is calculated according the formula (∑NjMj)/(∑Nj), where∑NjMj represents the total mass of all molecules in the sample and∑Nj represents to the total number of molecules in the sample. Suitable techniques are known in the art for determining number average molecular weight including gel permeation chromatography (GPC) and matrix-assisted laser desporption/ionization (MALDI) mass spectrometry.
In the present case, the ethylene glycol monomer (O-CH2-CH2) of the reagent used to prepare the compounds of the present invention has a molecular weight of 44 g/mol or 44 daltons. Thus, the reported number average molecular weight of 2000 ± 200 daltons of the reagent correlates to an "n" of between 40.9 and 50 ethylene glycol monomer units, or more generally between 40 and 50 ethylene glycol monomer units, in the PEG-containing compounds of the reagent. Likewise, using the same reagent will produce compounds of the present invention which contain maleimide-PEG linkers having between 40 and 50 ethylene glycol monomer (O-CH2-CH2) units.
The conjugated polypeptide compounds of the present invention contain an intra- chain disulfide bond formed between sulfhydral side chains of cysteine residues within the second polypeptide. Such bonds may be readily generated by one of skill in the art using conventional chemical methods and may be depicted by a line connecting affected cysteine residues, or by a "-S-S-" designation between affected cysteine residues.
Alternatively, where the disulfide bond exists between cysteine residues in a polypeptide chain, the presence of the disulfide bond may be represented by brackets occurring before and after the affected cysteine residues in the polypeptide chain.
The polypeptide chains of the present invention are depicted by their sequence of amino acids from N-terminus to C-terminus, when read from left to right, with each amino acid represented by either their single letter or three-letter amino acid abbreviation. Unless otherwise stated herein, all amino acids used in the preparation of the polypeptides
of the present invention are L-amino acids. The "N-terminus" (or amino terminus) of an amino acid, or a polypeptide chain, refers to the free amine group on the amino acid, or the free amine group on the first amino acid residue of the polypeptide chain. Likewise, the "C-terminus" (or carboxy terminus) of an amino acid, or a polypeptide chain, refers to the free carboxy group on the amino acid, or the free carboxy group on the final amino acid residue of the polypeptide chain.
The term "optionally amidated", when used herein, refers to an optional replacement of the hydroxyl moiety of the carboxy terminus of an amino acid, or the final amino acid residue of a polypeptide chain, with an amino (-NH2) group. Likewise, the term "amidated", when used herein, refers to a required replacement of the hydroxyl moiety of the carboxy terminus of an amino acid, or the final amino acid residue of a polypeptide chain, with an amino (-NH2) group. Amidation of a carboxy terminus of an amino acid, or the final amino acid of a polypeptide chain, may be depicted by an "-NH2" designation following the amino acid single letter or three-letter abbreviation.
The term "acetylated", when used herein, refers to a required replacement of a hydrogen atom of the amino terminus of an amino acid, or the first amino acid residue of a polypeptide chain, with an acetyl group (-C(0)CH3). Acetylation of the amino terminus of an amino acid, or the first amino acid residue of a polypeptide chain, may be depicted by an "Ac-" designation preceding the amino acid single letter or three-letter abbreviation.
The conjugated polypeptides of the present invention can react with any of a number of inorganic and organic acids or bases to form pharmaceutically acceptable salts. Preferred pharmaceutically acceptable salts of the conjugated polypeptides of the present invention include acetate and chloride salts. More preferred are the acetate salts of the compounds of Examples 1-3 herein. Especially preferred is the acetate salt exemplified in Example 1. Methods for preparation of the pharmaceutically acceptable salts of the present invention are well known to the skilled artisan (See: Stahl et al., "Handbook of Pharmaceutical Salts: Properties, Selection and Use," VCHA/Wiley-VCH (2002); and Berge et al., Journal of Pharmaceutical Sciences, 66: 1-19, 1977)
Compound Synthesis
General Chemistry
The individual polypeptide chains of the conjugated polypeptide compounds of the present invention can be synthesized using manual or automated solid-phase synthesis procedures. Automated peptide synthesizers are commercially available from, for example, Applied Biosystems and Protein Technologies, Inc. Reagents for solid-phase synthesis are readily available from commercial sources.
Typically, an N-protected amino acid is coupled to an N-terminal-deprotected amino acid attached at its C-terminus to a solid phase support resin through a suitable linker. The coupling reaction is typically carried out at room temperature and in an inert solvent such as dimethylformamide, N-methylpyrrolidone or methylene chloride in the presence of coupling agents such as diisopropyl-carbodiimide (DIC) and 1- hydroxybenzotriazole (HOBt). In addition, one of skill in the art will appreciate that amino acid side chains that are acidic, basic or highly polar are likely to be reactive and must also be protected to prevent unwanted reactions from occurring on the side chains. Typical side chain protecting groups include tBoc (a tertiary butyloxycarbonyl) for lysine and tryptophan residues; Pbf (a 2, 2, 4, 6, 7-pentamethyldihydrobenzofuran-5-sulfonyl group) for arginine residues; tBu (a tertiary butyl group) for serine, aspartic acid, and glutamic acid residues; and Trt (a triphenylmethyl group) for glutamine, histidine, and asparagine residues. After coupling, the N- protecting group is removed from the resulting peptide under basic conditions, such as 20% piperidine in DMF, and the coupling reaction is repeated with the next desired N-protected amino acid to be added to the peptide chain. Suitable amine protecting groups are well known in the art and are described, for example, in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, 1991. Commonly used examples include tBoc and Fmoc
(fluorenylmethoxycarbonyl). After completion of synthesis, peptides are cleaved from the solid-phase support with simultaneous side-chain deprotection using standard treatment methods under acidic conditions such as TFA.
The skilled artisan will appreciate that the polypeptide chains of the conjugated compounds of the invention can be synthesized with either a C-terminal free acid or carboxamide. The type of derivatized polystyrene resin solid phase support used for the
synthesis will determine the C-terminal moiety after cleavage. A number of linkers are well known and routinely used in the art. For the synthesis of C-terminal amide peptides, resins incorporating Rink amide MB HA or Rink amide AM linkers are typically used with Fmoc synthesis, while MBHA resin is generally used with tBoc synthesis. For the generation of C-terminal acid peptides, 2-chlorotrityl or Wang resin is typically used for Fmoc synthesis, while tBoc synthesis generally employs PAM resin. Methods for loading the first amino acid to the resin are also well known in the art.
The conjugated polypeptide compounds of the present invention contain an intra- chain disulfide bond formed between the sulfhydral side chains of cysteine residues 1 and 7 of the second polypeptide. Intra-chain disulfide bonds can be readily introduced using typical chemical methods and may be depicted by a line connecting affected cysteine residues, or by a "-S-S-" designation between affected cysteine residues. Alternatively, where the disulfide bond exists between cysteine residues in a polypeptide chain, the presence of the disulfide bond may be represented by brackets occurring before and after the affected cysteine residues in the polypeptide chain.
Crude peptides are typically purified using reverse-phase High Performance Liquid Chromatography (rp-HPLC) on C 8 or CI 8 columns using water-acetonitrile gradients containing 0.05 to 0.1% TFA. Purity can be verified by analytical rp-HPLC. Identity of peptides can be verified by mass spectrometry. Peptides can be solubilized in aqueous buffers over a wide pH range.
Conjugation of the polypeptides to the PEG-containing linker may be carried out using well-characterized chemical synthetic reactions. It is preferable that a molar excess of the PEG polymer relative to the peptide is used to drive the reaction to completion. Excess PEG reagent may be separated from the conjugated polypeptide products by conventional separation methods such as rp-HPLC. The PEG-reagent used to conjugate the PEG-containing linker to each of the first and second polypeptides comprises the maleimide-PEG N-hydroxysuccininimide ester of the following structure:
The conjugation chemistry used in preparing the compounds of the present invention involves (1) reaction of the N-hydroxysuccinimide ester moiety with the amine side chain of Lys 18 of the polypeptide given by SEQ ID NO :4 to form an amide bond, and (2) reaction of the double bond of the maleimide moiety with the sulfhydral side chain of Cys26 of the polypeptide given by SEQ ID NO:l, SEQ ID NO:2 or SEQ ID
NO: 3 to form a thioether bond. For example, the polypeptide given by the sequence which is SEQ ID NO: 4 has been engineered to contain only a single lysine residue in its sequence (supplying a primary amine side chain) and the polypeptides given by the sequences of SEQ ID NOs: 1 - 3 have been engineered to contain only a single cysteine residue (supplying a sulfhydral group.)
Pharmaceutical Compositions
The conjugated polypeptide compounds of the present invention are intended for administration via parenteral routes, including intravenous, intramuscular, subcutaneous or intraperitoneal routes of administration. Thus, the present invention also provides pharmaceutical compositions suitable for parenteral administration comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient. Such carriers, diluents and/or
excipients may include dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents and the like. An exemplary composition includes 35 to 400 μg/ml of a conjugated polypeptide of the present invention in a solution of 20 mM NaH2PC>4 in 0.9% NaCl, 3 mg/ml mannitol, at pH ~ 4.5. The conjugated polypeptide compounds of the invention may also be lyophilized for storage and reconstituted in a suitable vehicle prior to use or administration. Remington. The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA 1995 provides a compendium of formulation techniques for those of skill in art.
The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
Example 1
(n = 40-50)
Synthesis of polypeptides given by SEP ID NO: l and SEP ID NO:4
SEQ ID NP: Polypeptide sequence a' b' 0
1 Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Arg-
Ηϊβ-Εε^Α^ΒεΓ-Μεί-Ο^-Α^^Ι-Ο^-Τφ-Ι^Ηΐ-Α^^β-
Leu-Leu-Gln-Asp-Val-His-Asn-Phe-NH2
4 Ac-[Cys-Ser-Asn-Leu-Ser-Thr-Cys]-Val-Leu-Gly-Arg-Leu-
Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-
Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2
a The designation "-NH2" following the carboxy-terminus amino acids represents amidation of the amino acid.
b The designation "Ac-" preceding the amino-terminus amino acid of SEQ ID NO:4 represents acetylation of the amino acid.
0 Brackets indicate presence of disulfide bond between cysteine residues
Synthesis of each polypeptide is performed on Rapp AM RAM Fmoc-amide polystyrene resin (Rapp Polymere Tubingen, Germany) (substitution 0.6 to 0.7 mmol/g). The synthesis is performed using the Fmoc main-chain protecting group strategy. Amino acid side chains that are acidic, basic or highly polar are protected to prevent unwanted reactions from occurring on the side chains. The protecting groups used include: tBoc (a tertiary butyloxycarbonyl) for Lys and Trp residues; Pbf (a 2, 2, 4, 6, 7- pentamethyldihydrobenzofuran-5-sulfonyl group) for Arg residues; tBu (a tertiary butyl group) for Ser, Asp, and Glu residues; and Trt (a triphenylmethyl group) for Gin, His, and Asn residues. Amino acid side chain derivatives used are: Arg(Pbf), Asn(Trt),
Asp(OtBu), Cys(Trt), Gln(Trt), Glu(OtBu), His(Trt), Lys(Boc), Ser(OtBu), Trp(Boc). Coupling of amino acids to the polypeptide chain is carried out with approximately 10 equivalents of amino acid activated with diisopropylcarbodiimide (DIC) and
hydroxybenzotriazole (HOBt) (1 :1: 1 molar ratio) in dimethylformamide (DMF). N- terminal acetylation of the polypeptide amino acid sequence as given in SEQ ID NO:4 is carried out by treating the resin bound peptide with about 20 equivalents of acetic anhydride in DMF.
Concomitant cleavage from the resin and side chain protecting group removal is carried out in a solution containing trifluoroacetic acid: triisopropylsilane: H20
(95%:2.5%:2.5%) for 2 hours at room temperature. Cleaveage of the polypeptides from the Rink amide resin generates the C-terminal carboxamide form of each polypeptide. Polypeptides are precipitated with diethyl ether. The linear polypeptide amino acid sequence as given in SEQ ID NO:4 was dissolved in a mixture of 0.1M NH4HCO3 Buffer and acetonitrile with 10% DMSO by volume at 37°C overnight to form the intramolecular disulfide bond. Both polypeptides are purified on a reversed-phase HPLC column (Waters SymmetryPrep 7μιη, 19 x 300mm, cat. No: WAT066245) at a flow rate of 16
mL/min with a linear AB gradient where A = 0. 05% TF A/water and B = 0. 05%
TFA/acetonitrile. Polypeptide purity and molecular weight is confirmed on an Agilent 1100 15 Series LC-MS system with a single quadrupole MS detector. Analytical HPLC separation is done on a Waters SymmetryShield RP18 Column (cat. no. 186000179, 3.5 μιη, 4.6 x 100 mm.) with a linear AB gradient of 6 to 60% B over 14 minutes in which A = 0. 05% TFA/HiO and B = 0. 05%TFA acetonitrile and the flow rate is 1 ml/min. Each polypeptide is purified to > 95% purity and confirmed to have molecular weight corresponding to the calculated value within 1 amu. Yield: -55% for polypeptide of SEQ ID NO: 1; -37% for polypeptide of SEQ ID NO:4.
Following procedures essentially as described above, the polypeptides as given by
SEQ ID NO:2 and SEQ ID NO:3 are also prepared:
a The designation "-NH2" following the carboxy-terminus amino acids represents amidation of the amino acid.
Polypeptide Conjugation
The polypeptide given by the sequence SEQ ID NO:4 is mixed with MAL- PEG2000-NHS (JenKem Technology USA, cat#: A5001-1, 1.2 eq.) in anhydrous DMF with N,N-Diisopropylethylamine (5 eq.). The reaction is monitored by LC-MS and typically complete within 4 hr. The reaction mixture is then quenched with 10% AcOH in water, and purified on a reversed-phase HPLC column (Waters SymmetryPrep 7μιη, 19 x 300mm, cat. No: WAT066245) at a flow rate of 16 mL/min with a linear AB gradient where A = 0.05% TF A/water and B = 0.05% TFA/acetonitrile to provide the maleimide-PEG-SEQ ID NO: 10 fragment with a yield of -67%. (SEQ ID NO: 10
representing the modification of Lysig. of the sequence given by SEQ ID NO:4 following pegylation)
The maleimide-PEG - SEQ ID NO: 10 fragment and the polypeptide given by the sequence SEQ ID NO: 1 are mixed in a mixture of buffer (200 mM NH4OAc, 10 mM EDTA, pH 6.8) and acetonitirle at room temperature. The reaction is monitored by LC- MS and typically complete within 5 min. The mixture is purified on a reversed-phase HPLC column (Waters SymmetryPrep 7μιη, 19 x 300mm, cat. No: WAT066245) as described above to provide the conjugated polypeptide compound comprising the polypeptides given by SEQ ID NO: 7 and SEQ ID NO: 10 as the trifluoroacetic acid salt, with a yield of -87%. (SEQ ID NO: 7 representing the modification of Cys26 of the sequence given by SEQ ID NO:l following conjugation to the maleimide-PEG- SEQ ID NO: 10 fragment.)
The conjugated polypeptide-TFA salt is dissolved in acetonitrile/HiO and treated with AG1-X8 Resin (BIO-RAD, cat. #140-1443 acetate form, 50 eq. per basic reside on the construct), and shaken at rt. for 30 min. The filtrate and washes (3 times) are combined and lyophilized to provide the conjugated polypeptide as the acetate salt with a mass recovery of -88%.
Following procedures essentially as described above for Example 1, but using polypeptides given by the sequences of SEQ ID NO:2 or SEQ ID NO:3 in place of the polypeptide given by SEQ ID NO : 1 , the conjugated polypeptide compounds of Examples 2 and 3 are prepared as the acetate salts.
Example 2
J— C-S-
(SEQ ID O: 10)
Example 3
H— S-V-S-E-I-Q^M-H-N^G-K-H^N-S-M-E-R-V-E-W^R N K^Q-D-V-H-N-F— N H 2
(SEQ ID NO: 9)
V- C 1 -S-N-L-S-T-C 1 -V^G-R^S-Q-E^H— ^ X I^-Q-T-Y-P-R-T-N-T-G-S-G-T-P— N H■■
(SEQ IDNO: 10)
The complete sequences for the first and second polypeptides for each of Examples 1-3, are as indicated below.
Example First polypeptide sequence Second polypeptide sequence
No. a.d a,b,c,d
1 Ser-Val-Ser-Glu-Ile-Gln- Ac-[Cys-Ser-Asn-Leu-Ser-Thr- Leu-Met-His-Asn-Leu-Gly- Cys] - Val-Leu-Gly- Arg-Leu- Ser- Arg-His-Leu-Ala-Ser-Met- Gln-Glu-Leu-His- Xaa^g-Leu- Glu-Arg-Val-Glu-Trp-Leu- Gln-Thr-Tyr-Pro-Arg-Thr-Asn-
a The designation "-NH2" following the carboxy-terminus amino acids of each of the first and second polypeptide sequences represents amidation of the amino acid.
b The designation "Ac-" preceding the amino-terminus amino acid of the second polypeptide sequence represents acetylation of the amino acid.
0 Brackets indicate presence of disulfide bond between cysteine residues
d Underlined amino acid residues represent the points of attachment of the maleimide- PEG linker to each of the first and second polypeptide sequences.
Biological Activity
In vitro modulation of PTH Receptor or CT receptor:
Conjugated peptides of the present invention are evaluated for their ability to stimulate cAMP production in HEK-293 cells overexpressing either human PTHRl or human CT receptors. Briefly, various concentrations of conjugated peptide compound are incubated for 30 minutes at room temperature, with cells expressing either the human PTHRl or human CT receptor. Following incubation with test compound, cAMP levels are quantitated by cAMP HTRF assay kit (Cisbio 62AM4PEC). Reaction plates are read
on an EnVision plate reader using HTRF optimized protocol (PerkinElmer). EC50s are calculated using Graphpad Prism nonlinear regression curve fitting. hPTH(l-34) may be included as a positive control in the human PTHR1 receptor assays, while sCT may be included as a positive control in the human CT receptor assays. Following procedures essentially as described above, the exemplified conjugated peptides of the present invention demonstrate the following profile of activity:
Table 1
* hPTH(l-34) obtained from BACHEM.
** sCT prepared in-house at Eli Lilly and Company (SEQ ID NO: 5)
The data in Table 1 demonstrates that the exemplified conjugated polypeptide compounds of the present invention are potent agonists of both human PTHR1 and human CTR.
In vivo Potency in Rat Cortical Defect Model:
The conjugated peptides of the present invention may be evaluated in a mature osteopenic, ovariectomized rat cortical defect model to ascertain systemic effects on bone healing and vertebrae. This model may mimic the impaired bone healing observed in
post-menopausal women with osteoporosis (Komatsu, et al., Endocrinology, 150:1570- 1579, 2009)
Methods
Six-month old, virgin, virus antibody free, female, Sprague Dawley rats (Harlan,
Indianapolis) are maintained on a 12hr light/dark cycle at 22°C with ad lib access to food (TD 89222 with 0.5% Ca and 0.4%P, Teklad, Madison, WI) and water. Animals are ovariectomized by the vendor and allowed to lose bone for 2 months, weighed and randomized into treatment groups. Cortical defect surgery is performed essentially as previously described in Komatsu, et al., Endocrinology, 150:1570-1579, 2009. Briefly, 2 mm diameter holes are drilled through the diaphysis of the left and right femurs with an electric drill and a 2 mm bit. This hole extends through both the anterior and posterior cortices. Subcutaneous treatment with vehicle (20 mM NaH2P04 in 0.9% NaCl, 3 mg/ml mannitol) or test compound is initiated 1 day-post surgery and continues for 5 weeks (35 days.)
Healing of bone is monitored longitudinally in vivo by assessing bone mass density ("BMD") through the use of quantitative computed tomography ("qCT") using a GE Locus Ultra CT scanner (GE Healthcare, London, Ontario, Canada). Cylindrical regions of interest (ROI) passing through each cortical defect or the whole bone (2mm cross-section including site of cortical defects) may be generated and analyzed using ImageJ to determine average intensity profiles. Region-specific bone mineral density (BMD) may then be quantified for each ROI positioned in the anterior or posterior cortex, and for the whole bone, by analyzing the femoral diaphysis at the location of the cortical defect. Analysis are conducted using Micro ViewABA (Version 2.3a4 GE Healthcare, London, Ontario, Canada) and entails converting the mean intensity results (in Hounsfield units) generated for each region of interest to BMD by performing a linear transformation of data using intensity values from a hydroxyapatite phantom as a reference.
Analyses post-necropsy: In addition to in vivo qCT, L5 vertebrae may be scanned post-necropsy using an Aloka LaTheta LTC-100 CT scanner (Aloka Co., Ltd., Tokyo, Japan). Briefly, vertebrae are wrapped in parafilm, positioned in 2D and scanned using landmark structures. Total BMD is determined using the manufacture's software package (SYS-C320-V 1.5).
Biomarker analyses: Serum samples may also be taken from test animals during the course of the study for biomarker analysis. For example, the serum levels of rat osteocalcin (OCN) may be determined by an enzyme immunoassay (EIA) (Biomedical Technologies Inc. Stoughton, MA) following the manufacturer's protocol using a 1: 10 dilution. The serum levels of N-terminal propeptide of type I procollagen (PINP) may be determined by EIA (rat/mouse PINP, Immunodiagnostic Systems Inc. Scottsdale, AZ) following the manufacturer's protocol. The serum levels of fragments of type I collagen (CTX-I) may be determined by EIA (RatLaps Immunodiagnostic Systems Inc. Scottsdale, AZ) following the manufacturer's protocol.
Results
Following procedures essentially as described above, the compound of Example 1, when administered subcutaneously at doses of 47, 140 and 420 μg/ g, 3X/wk (n = 8 for each treatment group), displayed the following profile of activity on whole bone (femur) BMD, L5 vertebrae BMD and the biomarkers PINP, OCN and CTX-1.
Table 2
Significant relative to vehicle (Dunnett's Test, p < 0.05)
Table 3
Significant relative to vehicle (Dunnett's Test, p < 0.05)
Table 4
Significant relative to vehicle (Dunnett's Test, p < 0.05)
The data demonstrates that the conjugated polypeptide compound of Example 1 is efficacious in building bone, as measured by BMD, in a rat model of cortical bone injury. Specifically, the data demonstrates that the compound of Example 1, following subcutaneous administration at all doses tested, significantly promotes bone formation at the site of cortical bone injury (i.e. femur, Table 2) at both day 21 and day 35 post- surgery. In addition, the data demonstrates that the compound of Example 1 significantly promotes bone formation at a site remote from the location of injury (i.e. L5 vertebra, Table 3) at all doses tested at day 35 post-surgery. Treatments with Example 1 produced
no effects on the bone formation marker PINP, however, at the highest dose administered (420 μg/kg) (3X/wk), the compound of Example 1 significantly increased intact osteocalcin at day 11 post-surgery, with a trend toward significance at day 34. In addition, this dose of Example 1 produced a significant decrease in the bone resorption marker CTX- 1 , at both day 11 - and day 34 post-surgery.
Notably, in a separate study, performed essentially as described above, but using doses of sCT or hPTH (1-38) believed to be clinically relevant, administration of sCT (BACHEM) ^g/kg 2X/wk), PTH (1-38) (BACHEM) ^g/kg/d) or the combination of sCT ^g/kg 2X/wk) and PTH ^g/kg/d) failed to significantly increase whole bone (femur) BMD at day 35 relative to vehicle controls.
In vivo Potency in Rat Osseous Integration Model:
Because many orthopaedic surgical procedures utilize metal implants, including spinal fusion procedures (Ohtori et ah, Spine (Phila Pa. 1976), 2012 Nov. 1 ; 37(23): E1464-1468; Ohtori et al, Spine (Phila Pa. 1976), 2013 Apr. 15; 38(8): E487-492), the rat screw implant model has been used to test the pharmacological effect of osseous integration into implants and implant fixation. Previously, PTH was shown to enhance osseous integration into implants and implant fixation in the rat screw implant model (Skripitz, et ah, Journal of Orthopaedic Research, 23: 1266-1270, 2005). A similar rat model may be used to evaluate the effects of the conjugated peptide compounds of the present invention on osseous integration into implants and also on implant fixation.
Methods
Female Sprague Dawley rats are maintained on a 12hr light/dark cycle at 22 °C with ad lib access to food (TD 89222 with 0.5% Ca and 0.4%P, Teklad, Madison, WI) and water. Rats are then ovariectomized and allowed to lose bone for about 7 months. At 13 months, animals are randomized into treatment groups and surgical procedures are performed as follows. Rats are surgically implanted with titanium screws (2x4mm) into both tibiae of the medial lateral side, at 5mm below the growth plate. Test compound or vehicle (20 mM NaH2P04 in 0.9% NaCl, 3 mg/ml mannitol) is administered
subcutaneously, for example 20, 60 or 140 μg/kg 3 times per week, starting from the
same day as surgery for 28 days. The animals are injected with alizarin complexone 30mg/kg on the day of first dose, and calcein (lOmg/kg) at day 13 and 3 days prior to sacrifice. Sera are collected by tail bleed at baseline (right before the first dose) and at day 14, and by cardiac puncture at day 28 during the sacrifice for biomarker analysis. At sacrifice, both tibia and the LI -6 vertebra are removed and stored in 50% ethanol/saline and stored at 4°C for histological, computed tomography and/or biomechanical analyses. A biomechanical pull to failure force test is performed on both tibiae ex vivo using an industrial digital force gauge (Mark-10, model M3-50, ESM301, IN, USA). Pull to failure force is tested at a speed of lOmm/minute. After the mechanical test, screws are carefully removed. Tibiae and L5 vertebrae are then scanned using quantitative computed tomography with a 60-μιη voxel size (Aloka LaTheta LTC-100 model CT scanner). Group differences are assessed with JMP version 5.1 software, Dunnett's T test.
Biomarker Analysis: Collected sera may be analyzed for biomarkers as described above. Briefly, the serum levels of rat osteocalcin (OCN) may be determined by EIA (Biomedical Technologies Inc. Stoughton, MA) following the manufacturer's protocol using a 1:10 dilution. The serum levels of N-terminal propeptide of type I procollagen (P1NP) may be determined by EIA (rat/mouse PINP, Immunodiagnostic Systems Inc. Scottsdale, AZ) following the manufacturer's protocol. The serum levels of fragments of type I collagen (CTX-I) may be determined by EIA (RatLaps Immunodiagnostic Systems Inc. Scottsdale, AZ) following the manufacturer's protocol.
Results
Following procedures essentially as described above, the compound of Example 1, at doses of 60 and 140 μg/kg 3X/wk (n = 10 for each treatment group) increased screw implant pull-out force by -95% and -86%, respectively, relative to vehicle treated controls. In addition, all doses of Example 1 tested (20, 60 and 140 μg/kg, 3X/wk) increased lumbar vertebra L5 BMD (mg/cm ) by -15%, -18% and -23%, respectively, relative to vehicle treated controls. The compound of Example 1 also dose-dependently increased the bone formation markers PINP and osteocalcin at days 14 and 28, while suppressing the bone resorption marker CTX-1 at day 28 at the dose of 140 μg/kg. Taken
together, these data suggest that the conjugated polypeptide compound of Example 1 has beneficial effects in enhancing osseous integration at the site of implant, as well as in sites remote from bone injury. In vivo Efficacy in Rat Posteriolateral Fusion Model:
PTH has previously been shown to stimulate de novo bone formation to restore bone loss and promote bone healing in clinical and pre-clinical studies. (Compston, Bone, 40: 1447-1452, 2007; Datta, World J. Orthop., 18; 2(8): 67-74, 2011) Previously, PTH was also shown to enhance spinal fusion in rats and patients (Ohtori et ah, Spine (Phila Pa. 1976), 2012 Nov. 1; 37(23): E1464-1468; Ohtori et al, Spine (Phila Pa. 1976), 2013 Apr. 15; 38(8): E487-492). To evaluate the effects of the conjugated peptide compounds of the present invention, a rat posterior lateral spinal fusion model with autografts harvested from the iliac crest may be employed as follows. Methods
An iliac crest surgery to harvest autograft material (0.5x0.5cm) from the left iliac crest is conducted on male, 17 week old Sprague-Dawley rats weighing 450-530g. The bone graft material is then immediately transplanted to the decorticated L5 and L6 transverse processes of the lumbar vertebrae of the same animal. Digital radiographs are taken at the day of surgery to ensure graft positioning. Test compound, dosed for example at 140ug/kg (vehicle: 20 mM NaH2P04 in 0.9% NaCl, 3 mg/ml mannitol, at pH 4.5), is given subcutaneously once a week, twice a week or three times a week to different groups starting on day 3 post surgery. Animals are sacrificed after 4 weeks.
Spinal fusion rate and quality are evaluated with three-dimensional micro- computed tomography images (μΟΎ40, 9 Scanco Medical, Bassersdorf, Switzerland). Whole lumbar vertebral columns are scanned by μCT using a resolution of 36 μιη per voxel. The three-dimensional images are used to assess osseous tissue fusion using a scoring system where no fusion scores zero, partial fusion scores 3 and full fusion scores 5. In addition, L3 lumbar vertebrae are removed at time of sacrifice and are scanned with quantitative CT (qCT) (Aloka and Skycan 1174) for systemic bone effects.
Significance of mean group fusion rate and fusion scores are evaluated by Fisher exact test with significance defined as a p value of less than 0.05 vs. vehicle controls.
Results
Following procedures substantially as described above, Micro-CT images showed full fusion rates in rats treated with the compound of Example 1 (140ug/kg) of 61.5% (8 of 13), 38.5% (5 of 13) and 53.3% (8 of 15) for three times, twice or once per week treatment regimens, respectively, compared to 26.7% (4 of 13) in the vehicle control. Mean fusion scores were 4.08 ± 1.32 (n = 13), 3.62 ± 1.26 (n = 13), and 3.92 ± 1.28 (n = 15) for three times, twice or once per week treatment regimens, respectively, compared to 3.53 ± 0.92 (n = 15) in the vehicle control. While these results did not reach significance, they suggest that the conjugated peptide of Example 1 is efficacious in promoting spinal fusion in a clinically relevant model. No significant differences in lumbar vertebra L3 BMC and BMD were observed between groups evaluated by ex vivo qCT.
In vivo Efficacy in Rat Osteotomy Model of Bone Pain
Spinal fusion is a painful procedure and it is believed that the conjugated peptide compounds of the present invention may provide pain relief for spinal fusion patients. This is supported by various reports in the literature detailing the analgesic properties of sCT. To assess the analgesic properties of compounds of the present invention, a surrogate rat model may be used where aged ovariectomized rats undergo a unilateral osteotomy of the femur and pain is measured via incapacitance testing, as described below.
Methods
Aged (33-36 weeks) ovariectomized female Sprague Dawley rats (Harlan, Indianapolis, IN) that are osteopenic (10 weeks post ovariectomy) undergo a unilateral osteotomy of the femur of the right hind leg where the bone is cut and stabilized with a fixator. Pain assessment and dosing is begun four days post-osteotomy. Pain is assessed in the animals using incapacitance testing, which measures the difference in hind paw
weight bearing between the leg with the osteotomy and the contra-lateral un-operated leg. Pain is then calculated as a percent decrease in pain compared to a baseline pain reading taken four days post-osteotomy. Rats are single-housed and maintained in a constant temperature, and on a 12 hour light/ 12 hour dark cycle. Animals have free access to food and water at all times except during data collection.
Following procedures substantively as described above, the following results were obtained for various dosing regimens of the compound of Example 1
Results
(a) Dose response study with test compound dosed every 3-4 days
Rats were measured via incapacitance testing on day 4 post osteotomy for baseline pain. On day 7 post osteotomy, the rats were randomized using BRAT (the Block Randomization Allocation Tool) and baseline pain measurements into five groups of 6 animals and dosed with either vehicle (20 mM NaH2P04 in 0.9% NaCl, 3 mg/mL mannitol, pH 4.5), 70, 140 or 420μg/kg of the compound of Example 1. Rats were dosed again on days 10 and 14 post osteotomy. Dose volume was 1 ml/kg. Pain was measured via incapacitance testing on days 8, 9, 11, 15 and 16 post-osteotomy with animals from each dose group measured randomly throughout the day of measurement. The following table provides the profile of activity obtained in this study with the compound of Example 1.
Table 5
% Reduction in Pain (± SEM)
Treatment Day 8 Day 9 Day 11 Day 15 Day 16
Vehicle -1.54 4.01 11.44 18.16 21.47
(± 1.65) (± 0.86) (± 3.81) (± 3.67) (± 4.53)
Example 1 3.81 5.44 14.42 30.69 36.11 ΙΟμ /kg (± 1.52) (± 1.53) (± 1.16 (± 2.45)* (± 3.72)*
Example 1 5.30 11.87 22.67 32.94 40.92 ^g^kg (± 2.40) (± 2.52) (± 3.89)* (± 4.58)* (± 4.59)*
Example 1 6.24 16.76 23.32 38.15 47.44 420Mg/kg (± 0.96) (± 1.97)* (± 2.66)* (± 2.85)* (± 2.02)*
The compound of Example 1 significantly reduced pain compared to vehicle on days 9, 11, 15 and 16 post-osteotomy at 42C^g/kg; on days 11, 15 and 16 post-osteotomy at 14C^g/kg; and on days 15 and 16 at 7C^g/kg post-osteotomy (*p<0.05). These results demonstrate that the conjugated polypeptide compound of Example 1 is efficacious in reducing pain in an osteotomy model of bone pain.
(b) Dose response study with test compound dosed every day
Rats were measured via incapacitance testing on day 4 post-osteotomy for baseline pain. On day 7 post-osteotomy, rats were randomized using BRAT and baseline pain measurements into five groups of 6 animals and dosed with either vehicle (20 mM NaH2P04 in 0.9% NaCl, 3 mg/mL mannitol, pH 4.5), 10, 35, 70 or 140μg/kg of the compound of Example 1. In this study, rats were dosed daily for 5 days. Dose volume was lml/kg with pain measured via incapacitance testing on days 8, 9, 11, 14 and 16 post- osteotomy. Note that if pain was measured on the same day as dosing, pain was measured after dosing with rats randomized for pain measurement throughout the day of measurement. The following table provides the profile of activity obtained in this study with the compound of Example 1.
Table 6
% Reduction in Pain (± SEM )
Treatment Day 8 Day 9 Day 11 Day 14 Day 16
Vehicle -0.16 4.38 8.87 9.52 11.98
(± 1.90) (± 1.42) (± 2.69) (± 1.05) (± 2.27)
Example 1 1.69 4.47 9.76 0.59 9.77 lOMg/kg (± 1.95) (± 1.03) (± 2.40) (± 3.26) (± 2.08)
Example 1 2.78 7.28 18.70 8.79 13.99 35μg/kg (± 2.71) (± 1.92) (± 2.16)* (± 4.51) (± 3.22)
Example 1 4.55 11.39 23.60 9.63 16.79 ΙΟμ /kg (± 1.19) (± 0.70) (± 2.45)* (± 4.72) (± 3.90)
Example 1 3.00 12.73 29.13 18.80 21.76 140Mg/kg (± 2.83) (± 1.03) (± 2.08)* (± 4.37) (± 3.71)*
Pain efficacy was not seen until day 11 post osteotomy at which time point all doses above and including 35μg/kg of Example 1 were significantly different from vehicle, but not different from each other (*p<0.05). The lC^g/kg dose of Example 1 did not significantly reduce pain compared to vehicle on any day measured. In comparison to the previous study, the 14C^g/kg dose showed pain efficacy on the same day post- osteotomy, whereas the 7C^g/kg dose showed earlier efficacy and the 35ug/kg dose also showed efficacy at this time point. These data suggest that daily dosing may allow for earlier pain efficacy as seen with the 7C^g/kg dose in this study compared to the previous dose response study. It was also seen that once dosing was stopped, pain efficacy diminished and trended towards that seen in the vehicle group.
(c) Comparison of test compound to relevant doses of PTH and sCT
The compound of Example 1 was compared to clinically relevant doses of PTH and salmon calcitonin (sCT). As in the previous studies, rats were measured via
incapacitance testing on day 4 post-osteotomy for baseline pain. On day 7 post osteotomy, the rats were randomized using BRAT and baseline pain measurements into five groups of 6 animals and dosed with either vehicle (20 mM NaH2P04 in 0.9% NaCl, 3 mg/mL mannitol, pH 4.5) (dosed every 3-4 days), 140μg/kg of the compound of Example 1 (dosed every 3-4 days), 3μg/kg hPTH(l-34) (SEQ ID NO: 6, synthesized in-house, Eli Lilly and Company) (dosed every day), 3μg/kg sCT (BACHEM) (dosed every day) or 3μg/kg PTH(l-34) + 3μg/kg sCT (dosed every 3-4 days). All test compounds were administered using the same vehicle (20 mM NaH2P04 in 0.9% NaCl, 3 mg/mL mannitol, pH 4.5), dosed at a volume of lml/kg, with pain measured via incapacitance testing on days 7, 8, 10, 11, 14 and 15 post-osteotomy. Note that if pain was measured on the same day as dosing, pain was measured after dosing with rats randomized for pain
measurement throughout the day of measurement.
Table 7
% Reduction in Pain (± SEM)
Treatment Day 7 Day 8 Day 10 Day 11 Day 14 Day 15
Vehicle 0.59 2.29 7.68 9.27 18.46 23.43
a (PTH prepared in house: SEQ ID NO:6)
b (sCT purchased from BACHEM) 3μg/kg sCT dosed daily provided significant pain efficacy compared to vehicle on all days when pain was measured, with the exception of the first day (day 7) (*p<0.05). 3ug/kg PTH daily did not show pain efficacy on any day in comparison to vehicle. The combination dose of sCT plus PTH showed significant pain efficacy compared to vehicle on days 11, 14 and 15 post-osteotomy and the compound of Example 1 showed efficacy compared to vehicle on days 10 and 11 post-osteotomy (*p<0.05). In addition the single dose of sCT and sCT in combination with PTH, were significantly different from PTH alone on days 14 and 15 post-osteotomy. These results indicate that the pain efficacy seen with the compound of Example 1 is likely due to the second polypeptide, the sequence of which is given by SEQ ID NO: 10.
Data for the results in Tables 5-7 were calculated as means (n = 6) with standard error of the means (±SEM). Statistical tests of hypothesis were based on a two way repeated measurement analysis of variance (ANOVA) model with day and treatment group as factors and percentage change in weight difference from baseline used as the response variable. In all studies, reported p-values for comparisons between each treatment and vehicle groups were adjusted for multiplicity within each day (similar to Dunnett's test, but adapted for longitudinal data). In the dose response, reported p-values
for comparisons between test compound at the same dose were not adjusted for multiplicity. In the daily dosing study, reported p-values for comparisons between doses of test compound were adjusted for multiplicity within each day. Differences were considered to be significant if the reported p-value was less than 0.05. Statistical analysis was conducted using the R statistical package (R Core Team (2013), i^ilwwwM^
Polypeptide Sequences
SEQ ID NO:l (Example 1, first polypeptide sequence prior to modification)
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Arg-His-Leu-Ala-Ser-Met-Glu-Arg- Val-Glu-Tφ-Leu-Arg-Cys-Leu-Leu-Gln-Asp-Val-His-Asn-Phe
(Phe at position 34 may be amidated)
SEQ ID NO:2 (Example 2, first polypeptide sequence prior to modification)
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Arg-His-Leu-Ala-Ser-Met-Glu-Arg -Val-Glu-Tφ-Leu-Arg-Cys-Leu-Leu-Gln-Glu-Val-His-Asn-Phe
(Phe at position 34 may be amidated)
SEQ ID NO:3 (Example 3, first polypeptide sequence prior to modification)
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg- Val-Glu-Tφ-Leu-Arg-Cys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe
(Phe at position 34 may be amidated)
SEQ ID NO:4 (Examples 1-3, second polypeptide sequence prior to modification)
Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Arg-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu- Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro
(Cys at position 1 is acetylated)
(Disulfide bond between cysteine residues (1) and (7))
(Pro at position 32 is amidated)
SEQ ID NO: 5 (sCT)
Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu- Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro
(Disulfide bond between cysteine residues (1) and (7))
(Pro at position 32 is amidated)
SEQ ID NO: 6 (hPTH(l-34))
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg- Val-Glu-Tφ-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe
(Phe at position 34 is amidated)
SEQ ID NO: 7 (Example 1, first polypeptide sequence after modification)
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Arg-His-Leu-Ala-Ser-Met-Glu-Arg- Val-Glu-Tφ-Leu-Arg-Xaa26-Leu-Leu-Gln-Asp-Val-His-Asn-Phe
(Xaa at position 26 is cysteine residue modified by thioether bond formation to maleimide-PEG linker)
(Phe at position 34 may be amidated)
SEQ ID NO: 8 (Example 2, first polypeptide sequence after modification)
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Arg-His-Leu-Ala-Ser-Met-Glu-Arg -Val-Glu-Tφ-Leu-Arg- Xaa26-Leu-Leu-Gln-Glu-Val-His-Asn-Plle
(Xaa at position 26 is cysteine residue modified by thioether bond formation to maleimide-PEG linker)
(Phe at position 34 may be amidated)
SEQ ID NO: 9 (Example 3, first polypeptide sequence after modification)
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-
Xaa26-Lys-Leu-Gln-Asp-Val-His-Asn-Phe
(Xaa at position 26 is cysteine residue modified by thioether bond formation to maleimide-PEG linker)
(Phe at position 34 may be amidated)
SEQ ID NO: 10 (Examples 1-3, second polypeptide sequence after modification)
Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Arg-Leu-Ser-Gln-Glu-Leu-His-Xaal8-Leu- Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro
(Cys at position 1 is acetylated)
(Disulfide bond between cysteine residues (1) and (7))
(Xaa at position 18 is lysine residue modified by amide bond formation to maleimide-PEG linker)
(Pro at position 32 is amidated)
Claims
1. A compound consisting of:
(a) a first polypeptide which is given by a sequence selected from the group consisting of:
i. Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Arg-His-Leu- Ala-Ser-Met-Glu-Arg-Val-Glu-Tφ-Leu-Arg-Xaa26-Leu-Leu-Gln- Asp-Val-His-Asn-Phe (SEQ ID NO: 7),
ii. Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Arg-His-Leu- Ala-Ser-Met-Glu-Arg -Val-Glu-Trp-Leu-Arg- Xaa26-Leu-Leu-
Gln-Glu-Val-His-Asn-Phe (SEQ ID NO: 8), and iii. Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu- Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg- Xaa26-Lys-Leu- Gln-Asp-Val-His-Asn-Phe (SEQ ID NO: 9);
(b) a second polypeptide which is given by the sequence which is Ac-[Cys-
Ser-Asn-Leu-Ser-Thr-Cys]-Val-Leu-Gly-Arg-Leu-Ser-Gln-Glu-Leu-His- Xaal8-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 (SEQ ID NO: 10); and
(c) a maleimide-PEG linker of the formula
wherein,
Xaa26 of the sequence given by SEQ ID NO:7, SEQ ID NO: 8 or SEQ ID NO: 9 is a cysteine residue modified by attachment to the maleimide-PEG linker through the thioether bond;
the C-terminus of Phe34 of the sequence given by SEQ ID NO:7, SEQ ID
NO: 8 or SEQ ID NO: 9 is optionally amidated;
Xaal8 of the sequence given by SEQ ID NO: 10 is a lysine residue modified by attachment to the maleimide-PEG linker through the amide bond, and
n represents 40 to 50,
or a pharmaceutically acceptable salt thereof.
2. A compound or salt according to Claim 1 wherein the C-terminus of Phe34 of the sequence given by SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 is amidated.
3. A compound or salt according to any one of Claims 1-2 wherein said first polypeptide is given by SEQ ID NO:7.
4. A compound or salt according to any one of Claims 1-2 wherein said first polypeptide is given by SEQ ID NO: 8.
5. A compound or salt according to any one of Claims 1-2 wherein said first polypeptide is given by SEQ ID NO:9.
6. A salt according to any one of Claims 3-5 wherein said salt is the acetate salt.
7. A salt according to Claim 1 which is given by the formula
H— S-V-S-E-I-Q^M-H-N-L-G-R-H-L-A-S-M-E-R-V-E-W-L-R N — L-L-Q-D-V-H-N-F— N H
(SEQ ID NO: 7)
V- C-S-N-L-S-T-C-V-L-G-R-L-S-Q-E-L-H— "
(SEQ ID NO: 10)
8. A salt according to Claim 1 which is given by the formula
H— S-V-S-E-I-Q^M-H-N-L-G-R-H-L-A-S-M-E-R-V-E-W-L-R— N — L-L-Q-E-V-H-N-F— (SEQ ID NO: 8)
(n = 40-50) H
•acetate salt ~ C-S-N-L-S-T-C-V-L-G-R-L-S-Q-E-L-H— [j L-Q-T-Y-P-R-T-N-T-G-S-G-T-P— N H
(SEQ ID NO: 10)
9. A salt according to Claim 1 which is given by the formula
H— S-V-S-E-I-Q^M-H-N^G-K-H^N-S-M-E-R-V-E-W^R κ ^ — K-I^Q-D-V-H-N-F— N H
10. A pharmaceutical composition comprising a compound or salt according to any one of Claims 1-9 and a pharmaceutically acceptable carrier, diluent, or excipient.
11. A pharmaceutical composition comprising a plurality of any one of the compounds or salts according to any one of Claims 1-9, wherein the number average molecular weight of the PEG polymer of the formula "-(0-Ο¾-Ο¾)η-" in said plurality of compounds or salts is 1800 to 2200 daltons, and a pharmaceutically acceptable carrier, diluent or excipient.
12. The composition according to Claim 11 , wherein the number average molecular weight of the PEG polymer of the formula "-(0-Ο¾-Ο¾)η-" in said plurality of compounds or salts is 1900 to 2100 daltons.
13. The composition according to Claim 12, wherein the number average molecular weight of the PEG polymer of the formula "-(0-Ο¾-Ο¾)η-" in said plurality of compounds or salts is about 2000 daltons.
14. A method of using a compound, salt or composition according to any one of Claims 1-13 comprising administering to a spinal fusion patient an effective amount of said compound, salt or composition.
15. The method according to Claim 14 wherein said compound, salt or composition is administered prior to spinal fusion surgery.
16. The method of Claim 14 wherein said compound, salt or composition is administered during spinal fusion surgery.
17. The method of Claim 14 wherein said compound, salt or composition is administered after spinal fusion surgery.
18. A method according to any one of Claims 14-17 wherein the spinal fusion surgery is posteriolateral fusion surgery or transforaminal lumbar interbody fusion surgery.
19. A method for bone healing or treating osteoporosis, osteopenia or osteogenesis imperfecta comprising administering to a patient in need thereof an effective amount of a compound, salt or composition according to any one of Claims 1-13.
20. A compound, salt or composition according to any one of Claims 1 -13 for use in therapy.
21. A compound, salt or composition according to any one of Claims 1 - 13 for use as an adjunct to spinal fusion surgery.
22. A compound, salt or composition according to any one of Claims 1 -13 for use in bone healing or in the treatment of osteoporosis, osteopenia or osteogenesis imperfecta.
23. A process for preparing a conjugated polypeptide compound comprising:
(i) reacting a polypeptide given the sequence which is SEQ ID NO:4 with a reagent comprising a compound of the formula
(ii) reacting the product of step (i) with a polypeptide given by the sequence which is SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3 under suitable conditions such that a thioether bond is formed between Cys26 of the polypeptide given by SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3 and the maleimide moiety of the product of step (i).
24. A conjugated polypeptide compound prepared according to the process of Claim 23.
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| US201462074849P | 2014-11-04 | 2014-11-04 | |
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| CN107722259A (en) * | 2017-11-02 | 2018-02-23 | 湖南华腾制药有限公司 | A kind of polyethylene glycol maleimation Top profit derivative and preparation method thereof |
| CN107793563A (en) * | 2017-10-27 | 2018-03-13 | 湖南华腾制药有限公司 | A kind of polyethylene glycol omega-amino acid maleimide derivatives and preparation method thereof |
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Cited By (4)
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| CN107793563A (en) * | 2017-10-27 | 2018-03-13 | 湖南华腾制药有限公司 | A kind of polyethylene glycol omega-amino acid maleimide derivatives and preparation method thereof |
| CN107793563B (en) * | 2017-10-27 | 2019-09-20 | 湖南华腾制药有限公司 | A kind of polyethylene glycol omega-amino acid maleimide derivatives and preparation method thereof |
| CN107722259A (en) * | 2017-11-02 | 2018-02-23 | 湖南华腾制药有限公司 | A kind of polyethylene glycol maleimation Top profit derivative and preparation method thereof |
| CN107722259B (en) * | 2017-11-02 | 2019-12-17 | 湖南华腾医药有限公司 | Polyethylene glycol maleimido topril derivative and preparation method thereof |
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