WO2002098232A1 - Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same - Google Patents

Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same Download PDF

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Publication number
WO2002098232A1
WO2002098232A1 PCT/US2002/017574 US0217574W WO02098232A1 WO 2002098232 A1 WO2002098232 A1 WO 2002098232A1 US 0217574 W US0217574 W US 0217574W WO 02098232 A1 WO02098232 A1 WO 02098232A1
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WIPO (PCT)
Prior art keywords
mixture
insulin
oligomer
moiety
polyethylene glycol
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PCT/US2002/017574
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English (en)
French (fr)
Inventor
Nnochiri N. Ekwuribe
Christopher H. Price
Aslam M. Ansari
Amy L. Odenbaugh
Balasingam Radhakrishnan
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Nobex Corporation
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Priority to DK02737359.6T priority Critical patent/DK1404178T3/en
Priority to ES02737359.6T priority patent/ES2564820T3/es
Priority to KR10-2003-7015910A priority patent/KR20040004692A/ko
Priority to AU2002310291A priority patent/AU2002310291B2/en
Priority to MXPA03011284A priority patent/MXPA03011284A/es
Priority to EP02737359.6A priority patent/EP1404178B1/en
Priority to CA2449426A priority patent/CA2449426C/en
Publication of WO2002098232A1 publication Critical patent/WO2002098232A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin

Definitions

  • the present invention relates to drug-oligomer conjugates, and, more particularly, to insulin drug-oligomer conjugates.
  • the insulin molecule consists of two chains of amino acids linked by disulf ⁇ de bonds (mw 6,000).
  • the ⁇ - cells of the pancreatic islets secrete a single chain precursor of insulin, known as proinsulin.
  • Proteolysis of proinsulin results in removal of four basic amino acids (numbers 31, 32, 64 and 65 in the proinsulin chain: Arg, Arg, Lys, Arg respectively) and the connecting ("C") polypeptide.
  • the A chain has glycine at the amino terminus
  • the B chain has phenylalanine at the amino terminus.
  • Insulin may exist as a monomer, dimer or a hexamer formed from three of the dimers. The hexamer is coordinated with two Zn 2+ atoms. Biological activity resides in the monomer. Although until recently bovine and porcine insulin were used almost exclusively to treat diabetes in humans, numerous variations in insulin between species are known. Porcine insulin is most similar to human insulin, from which it differs only in having an alanine rather than threonine residue at the B-chain C-terminus. Despite these differences most mammalian insulin has comparable specific activity. Until recently animal extracts provided all insulin used for treatment of the disease.
  • Insulin is necessary for normal utilization of glucose by most cells in the body.
  • human insulin e.g., HumulinTM insulin, commercially available from Eli Lilly and Company, Indianapolis, IN.
  • Insulin is necessary for normal utilization of glucose by most cells in the body.
  • the normal ability to use glucose is inhibited, thereby increasing blood sugar levels (hyperglycemia).
  • hypoglycemia As glucose accumulates in the blood, excess levels of sugar are excreted in the urine (glycosuria).
  • Other symptoms of diabetes include increased urinary volume and frequency, thirst, itching, hunger, weight loss, and weakness.
  • Type I diabetes insulin-dependent diabetes mellitus
  • IDDM insulin-dependent diabetes mellitus
  • IDDM insulin-dependent diabetes mellitus
  • IDDM juvenile onset diabetes
  • IDDM insulin is not secreted by the pancreas and must be provided from an external source.
  • Type II adult-onset diabetes can ordinarily be controlled by diet although in some advanced cases insulin is required.
  • Treatment of diabetes typically requires regular injections of insulin.
  • the use of insulin as a treatment for diabetes dates to 1922, when Banting et al. ("Pancreatic Extracts in the Treatment of Diabetes Mellitus," Can. Med. Assoc. J.. 12: 141-146 (1922)) showed that the active extract from the pancreas had therapeutic effects in diabetic dogs.
  • Treatment of a diabetic patient in that same year with pancreatic extracts resulted in a dramatic, life-saving clinical improvement. Due to the inconvenience of insulin injections, massive efforts to improve insulin administration and bioassimilation have been undertaken.
  • insulin Even if insulin survives this enzymatic attack, the biological barriers that must be traversed before insulin can reach its receptors in vivo may limit oral administration of insulin. For example, insulin may possess low membrane permeability, limiting its ability to pass from the lumen into the bloodstream.
  • compositions such as insulin have been conjugated with polydispersed mixtures of polyethylene glycol or polydispersed mixtures of polyethylene glycol containing polymers to provide polydispersed mixtures of drug-oligomer conjugates.
  • polydispersed mixtures of polyethylene glycol or polydispersed mixtures of polyethylene glycol containing polymers to provide polydispersed mixtures of drug-oligomer conjugates.
  • U.S. Patent No. 4,179,337 to Davis et al. proposes conjugating polypeptides such as insulin with various polyethylene glycols such as MPEG- 1900 and MPEG-5000 supplied by Union Carbide.
  • U.S. Patent No. 5,567,422 to Greenwald proposes the conjugation of biologically active nucleophiles with polyethylene glycols such as m-PEG-OH (Union Carbide), which has a number average molecular weight of 5,000 Daltons.
  • U.S. Patent No. 5,359,030 to Ekwuribe proposes conjugating polypeptides such as insulin with polyethylene glycol modified glycolipid polymers and polyethylene glycol modified fatty acid polymers.
  • the number average molecular weight of polymer resulting from each combination is preferred to be in the range of from about 500 to about 10,000 Daltons.
  • Polyethylene glycol is typically produced by base-catalyzed ring-opening polymerization of ethylene oxide. The reaction is initiated by adding ethylene oxide to ethylene glycol, with potassium hydroxide as catalyst. This process results in a polydispersed mixture of polyethylene glycol polymers having a number average molecular weight within a given range of molecular weights.
  • PEG products offered by Sigma- Aldrich of Milwaukee, Wisconsin are provided in polydispersed mixtures such as PEG 400 (M n 380- 420); PEG 1,000 (M span 950-1,050); PEG 1,500 (M n 1,400-1,600); and PEG 2,000 (M n 1,900- 2,200).
  • a non-polydispersed mixture of insulin- oligomer conjugates comprising polyethylene glycol according to embodiments of the present invention exhibits higher in vivo activity than a polydispersed mixture of similar conjugates having the same number average molecular weight. This heightened activity may result in lower dosage requirements.
  • a non-polydispersed mixture of insulin-oligomer conjugates comprising polyethylene glycol according to embodiments of the present invention is typically more effective at surviving an in vitro model of intestinal digestion than polydispersed mixtures of similar conjugates.
  • non-polydispersed mixtures of insulin-oligomer conjugates comprising polyethylene glycol according to embodiments of the present invention also typically result in less inter-subject variability than polydispersed mixtures of similar conjugates.
  • a substantially monodispersed mixture of conjugates each comprising an insulin drug coupled to an oligomer that comprises a polyethylene glycol moiety is provided.
  • the polyethylene glycol moiety preferably has at least two, three, or four polyethylene glycol subunits and, most preferably, has at least seven polyethylene glycol subunits.
  • the oligomer preferably further comprises a lipophilic moiety.
  • the insulin drug is preferably human insulin.
  • the oligomer is preferably covalently coupled to Lys B29 of the human insulin.
  • the conjugate is preferably amphiphilically balanced such that the conjugate is aqueously soluble and able to penetrate biological membranes.
  • the mixture is preferably a monodispersed mixture and is most preferably a purely monodispersed mixture.
  • the oligomer comprises a first polyethylene glycol moiety covalently coupled to the insulin by a non-hydrolyzable bond and a second polyethylene glycol moiety covalently coupled to the first polyethylene glycol moiety by a hydrolyzable bond.
  • each conjugate includes human insulin covalently coupled at Lys B29 of the human insulin to a carboxyhc acid moiety of an oligomer that comprises hexanoic acid covalently coupled at the end distal to the carboxyhc acid moiety to a methyl terminated polyethylene glycol moiety having at least 7 polyethylene glycol subunits.
  • Substantially monodispersed mixtures of conjugates according to these embodiments of the present invention preferably have improved properties when compared with those of polydispersed mixtures.
  • a substantially monodispersed mixture of conjugates is provided where each conjugate comprises an insulin drug coupled to an oligomer including a polyethylene glycol moiety, and the mixture has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of insulin drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture.
  • a substantially monodispersed mixture of conjugates where each conjugate comprises an insulin drug coupled to an oligomer including a polyethylene glycol moiety, and the mixture has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of insulin drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture.
  • a substantially monodispersed mixture of conjugates where each conjugate comprises an insulin drug coupled to an oligomer including a polyethylene glycol moiety, and the mixture has an increased resistance to degradation by chymotrypsin when compared to the resistance to degradation by chymotrypsin of a polydispersed mixture of insulin drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture.
  • a substantially monodispersed mixture of conjugates where each conjugate comprises an insulin drug coupled to an oligomer including a polyethylene glycol moiety, and the mixture has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of insulin drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture.
  • Substantially monodispersed mixtures of conjugates according to embodiments of the present invention preferably have two or more of the above-described properties. More preferably, substantially monodispersed mixtures of conjugates according to embodiments of the present invention have three or more of the above-described properties. Most preferably, substantially monodispersed mixtures of conjugates according to embodiments of the present invention have all four of the above-described properties.
  • a mixture of conjugates where each conjugate includes an insulin drug coupled to an oligomer that comprises a polyethylene glycol moiety, and the mixture has a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • a mixture of conjugates is provided where each conjugate includes an insulin drug coupled to an oligomer that comprises a polyethylene glycol moiety, and the mixture has a dispersity coefficient (DC) greater than 10,000 where
  • n is the number of different molecules in the sample
  • Nj is the number of i- molecules in the sample.
  • Mj is the mass of the molecule.
  • a mixture of conjugates is provided in which each conjugate includes an insulin drug coupled to an oligomer and has the same number of polyethylene glycol subunits.
  • each conjugate is the same and has the formula:
  • L is a linker moiety
  • G, G' and G" are individually selected spacer moieties
  • R is a lipophilic moiety and R' is a polyalkylene glycol moiety, or R' is the lipophilic moiety and R is the polyalkylene glycol moiety; T is a terminating moiety; j, k, m and n are individually 0 or 1 ; and p is an integer between 1 and the number of nucleophilic residues on the insulin drug.
  • Insulin-oligomer conjugate mixtures according to embodiments of the present invention may provide increased in vivo activity and/or lowered inter-subject variability and/or decreased degradation by chymotrypsin when compared to conventional polydispersed insulin-oligomer conjugate mixtures.
  • Figure 1 illustrates a generic scheme for synthesizing a mixture of activated polymers comprising a polyethylene glycol moiety and a fatty acid moiety according to embodiments of the present invention
  • Figure 2 illustrates a scheme for synthesizing a mixture of mPEG according to embodiments of the present invention
  • Figure 3 illustrates a scheme for synthesizing a mixture of activated mPEG7-hexyl oligomers according to embodiments of the present invention
  • Figure 4 illustrates a scheme for synthesizing a mixture of activated mPEG7-octyl oligomers according to embodiments of the present invention
  • Figure 5 illustrates a scheme for synthesizing a mixture of activated mPEG-decyl oligomers according to embodiments of the present invention
  • Figure 6 illustrates a scheme for synthesizing a mixture of activated stearate-PEG6 oligomers according to embodiments of the present invention
  • Figure 7 illustrates a scheme for synthesizing a mixture of activated stearate-PEG8 oligomers according to embodiments of the present invention
  • Figure 8 illustrates a scheme for synthesizing a mixture of activated PEG3 oligomers according to embodiments of the present invention
  • Figure 9 illustrates a scheme for synthesizing a mixture of activated palmitate-PEG3 oligomers according to embodiments of the present invention.
  • Figure 10 illustrates a scheme for synthesizing a mixture of activated PEG6 oligomers according to embodiments of the present invention
  • Figure 11 illustrates a scheme for synthesizing various propylene glycol monomers according to embodiments of the present invention
  • Figure 12 illustrates a scheme for synthesizing various propylene glycol polymers according to embodiments of the present invention
  • Figure 13 illustrates a scheme for synthesizing various propylene glycol polymers according to embodiments of the present invention
  • Figure 14 illustrates a comparison of results obtained with a Cytosensor ® Microphysiometer, which provides an indication of the activity of a compound, for mixtures of insulin-oligomer conjugates according to embodiments of the present invention compared with polydispersed conjugate mixtures and insulin, which are provided for comparison purposes only and do not form part of the invention;
  • Figure 15 illustrates a comparison of chymotrypsin degradation of insulin-oligomer conjugates according to embodiments of the present invention with a conventional polydispersed mixture of insulin-oligomer conjugates, which is provided for comparison purposes only and does not form part of the invention
  • Figure 16 illustrates the effect of a mixture of mPEG7-hexyl-insulin, monoconjugate, according to embodiments of the present invention on plasma glucose in fasted beagles;
  • Figure 17 illustrates, for comparison purposes, the effect of a polydispersed mixture of mPEG7 aV g-hexyl-insulin, monoconjugate, which is not part of the present invention, on plasma glucose in fasted beagles;
  • Figure 18 illustrates the inter-subj ect variability of a mixture of mPEG4-hexyl-insulin monoconjugates according to embodiments of the present invention administered to fasted beagles;
  • Figure 19 illustrates the inter-subject variability of a mixture of mPEG7-hexyl-insulin monoconjugates according to embodiments of the present invention administered to fasted beagles;
  • Figure 20 illustrates the inter-subject variability for a mixture of mPEGlO-hexyl- insulin monoconjugates according to embodiments of the present invention administered to fasted beagles;
  • Figure 21 illustrates, for comparison purposes, the inter-subject variability of a polydispersed mixture of mPEG7 aV g-hexyl-insulin monoconjugates, which is not part of the present invention, administered to fasted beagles.
  • non-polydispersed is used to describe a mixture of compounds having a dispersity that is in contrast to the polydispersed mixtures described in U.S. Patent No. 4,179,337 to Davis et al.; U.S. Patent No. 5,567,422 to Greenwald; U.S. Patent No. 5,405,877 to Greenwald et al.; and U.S. Patent No. 5,359,030 to Ekwuribe.
  • substantially monodispersed is used to describe a mixture of compounds wherein at least about 95 percent of the compounds in the mixture have the same molecular weight.
  • the term "monodispersed” is used to describe a mixture of compounds wherein about 100 percent of the compounds in the mixture have the same molecular weight.
  • the term “substantially purely monodispersed” is used to describe a mixture of compounds wherein at least about 95 percent of the compounds in the mixture have the same molecular weight and have the same molecular structure.
  • a substantially purely monodispersed mixture is a substantially monodispersed mixture, but a substantially monodispersed mixture is not necessarily a substantially purely monodispersed mixture.
  • the term "purely monodispersed” is used to describe a mixture of compounds wherein about 100 percent of the compounds in the mixture have the same molecular weight and have the same molecular structure. Thus, a purely monodispersed mixture is a monodispersed mixture, but a monodispersed mixture is not necessarily a purely monodispersed mixture.
  • the term "weight average molecular weight” is defined as the sum of the products of the weight fraction for a given molecule in the mixture times the mass of the molecule for each molecule in the mixture. The "weight average molecular weight” is represented by the symbol M w .
  • number average molecular weight is defined as the total weight of a mixture divided by the number of molecules in the mixture and is represented by the symbol M n .
  • DC disersity coefficient
  • n is the number of different molecules in the sample
  • Nj is the number of i ⁇ molecules in the sample
  • fh is the number of i ⁇ molecules in the sample
  • intra-subject variability means the variability in activity occurring within the same subject when the subject is administered the same dose of a drug or pharmaceutical composition at different times.
  • inter-subject variability means the variability in activity between two or more subjects when each subject is administered the same dose of a given drug or pharmaceutical formulation.
  • insulin drug means a drug possessing all or some of the biological activity of insulin.
  • insulin means human insulin, bovine insulin, porcine insulin or whale insulin, provided by natural, synthetic, or genetically engineered sources.
  • insulin analog means insulin wherein one or more of the amino acids have been replaced while retaining some or all of the activity of the insulin. The analog is described by noting the replacement amino acids with the position of the replacement as a superscript followed by a description of the insulin. For example, "Pro B29 insulin, human” means that the lysine typically found at the B29 position of a human insulin molecule has been replaced with proline. Insulin analogs may be obtained by various means, as will be understood by those skilled in the art.
  • amino acids may be substituted for other amino acids in the insulin structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules.
  • structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant polypeptide, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity, i.e., still obtain a biological functionally equivalent polypeptide.
  • substitution of amino acids whose hydropathic indices are within ⁇ 2 of each other is preferred, those which are within ⁇ 1 of each other are particularly preferred, and those within ⁇ 0.5 of each other are even more particularly preferred.
  • Patent 4,554,101 the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine ( ⁇ 3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); seine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 of each other is preferred, those which are within ⁇ 1 of each other are particularly preferred, and those within ⁇ 0.5 of each other are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions i.e., amino acids that may be interchanged without significantly altering the biological activity of the polypeptide
  • amino acids that may be interchanged without significantly altering the biological activity of the polypeptide include, for example: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • insulin fragment means a segment of the amino acid sequence found in the insulin that retains some or all of the activity of the insulin. Insulin fragments are denoted by stating the position(s) in an amino acid sequence followed by a description of the amino acid. For example, a "B25-B30 human insulin” fragment would be the six amino acid sequence corresponding to the B25, B26, B27, B28, B29 and B30 positions in the human insulin amino acid sequence.
  • insulin fragment analog means a segment of the amino acid sequence found in the insulin molecule wherein one or more of the amino acids in the segment have been replace while retaining some or all of the activity of the insulin.
  • PEG refers to straight or branched polyethylene glycol polymers, and includes the monomethylether of polyethylene glycol (mPEG).
  • PEG subunit and polyethylene glycol subunit refer to a single polyethylene glycol unit, i.e., _(CH 2 CH 2 O)— .
  • lipophilic means the ability to dissolve in lipids and/or the ability to penetrate, interact with and/or traverse biological membranes
  • lipophilic moiety or “lipophile” means a moiety which is lipophilic and/or which, when attached to another chemical entity, increases the lipophilicity of such chemical entity.
  • lipophilic moieties include, but are not limited to, alkyls, fatty acids, esters of fatty acids, cholesteryl, adamantyl and the like.
  • lower alkyl refers to substituted or unsubstituted alkyl moieties having from one to five carbon atoms.
  • higher alkyl refers to substituted or unsubstituted alkyl moieties having six or more carbon atoms.
  • a substantially monodispersed mixture of insulin-oligomer conjugates is provided.
  • at least about 96, 97, 98 or 99 percent of the conjugates in the mixture have the same molecular weight.
  • the mixture is a monodispersed mixture.
  • the mixture is a substantially purely monodispersed mixture.
  • at least about 96, 97, 98 or 99 percent of the conjugates in the mixture have the same molecular weight and have the same molecular structure.
  • the mixture is a purely monodispersed mixture.
  • the insulin drug is preferably insulin. More preferably, the insulin drug is human insulin.
  • insulin drug may be selected from various insulin drugs known to those skilled in the art including, for example, proinsulin, insulin analogs, insulin fragments, and insulin fragment analogs.
  • Insulin analogs include, but are not limited to, Asp B28 human insulin, Lys B28 human insulin, Leu B28 human insulin, Val B28 human
  • Insulin fragments include, but are not limited to, B22-B30 human insulin, B23-B30 human insulin, B25-B30 human insulin, B26-B30 human insulin, B27-B30 human insulin, B29-B30 human insulin, the A chain of human insulin, and the B chain of human insulin.
  • Insulin fragment analogs may be provided by substituting one or more amino acids as described above in an insulin fragment.
  • the oligomer may be various oligomers comprising a polyethylene glycol moiety as will be understood by those skilled in the art.
  • the polyethylene glycol moiety of the oligomer has at least 2, 3 or 4 polyethylene glycol subunits. More preferably, the polyethylene glycol moiety has at least 5 or 6 polyethylene glycol subunits and, most preferably, the polyethylene glycol moiety has at least 7 polyethylene glycol subunits.
  • the oligomer may comprise one or more other moieties as will be understood by those skilled in the art including, but not limited to, additional hydrophilic moieties, lipophilic moieties, spacer moieties, linker moieties, and terminating moieties.
  • the various moieties in the oligomer are covalently coupled to one another by either hydrolyzable or non- hydrolyzable bonds.
  • the oligomer may further comprise one or more additional hydrophilic moieties (i.e., moieties in addition to the polyethylene glycol moiety) including, but not limited to, sugars, polyalkylene oxides, and polyamine/PEG copolymers.
  • additional hydrophilic moieties i.e., moieties in addition to the polyethylene glycol moiety
  • polyethylene glycol is a polyalkylene oxide
  • the additional hydrophilic moiety may be a polyethylene glycol moiety. Adjacent polyethylene glycol moieties will be considered to be the same moiety if they are coupled by an ether bond.
  • the moiety i.e., moieties in addition to the polyethylene glycol moiety
  • O-C 2 H 4 — O-C 2 H 4 — O-C 2 H 4 — O-C 2 H 4 -O-C 2 H 4 — O-C 2 H 4 -O-C 2 H 4 — is a single polyethylene glycol moiety having six polyethylene glycol subunits. If this moiety were the only hydrophilic moiety in the oligomer, the oligomer would not contain an additional hydrophilic moiety. Adjacent polyethylene glycol moieties will be considered to be different moieties if they are coupled by a bond other than an ether bond. For example, the moiety
  • oligomers according to embodiments of the present invention comprise a polyethylene glycol moiety and no additional hydrophilic moieties.
  • the oligomer may further comprise one or more lipophilic moieties as will be understood by those skilled in the art.
  • the lipophilic moiety is preferably a saturated or unsaturated, linear or branched alkyl moiety or a saturated or unsaturated, linear or branched fatty acid moiety.
  • the lipophilic moiety is an alkyl moiety, it is preferably a linear, saturated or unsaturated alkyl moiety having 1 to 28 carbon atoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms.
  • the lipophilic moiety is a fatty acid moiety, it is preferably a natural fatty acid moiety that is linear, saturated or unsaturated, having 2 to 18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14 carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5 or 6 carbon atoms.
  • the oligomer may further comprise one or more spacer moieties as will be understood by those skilled in the art.
  • Spacer moieties may, for example, be used to separate a hydrophilic moiety from a lipophilic moiety, to separate a lipophilic moiety or hydrophilic moiety from the insulin drug, to separate a first hydrophilic or lipophilic moiety from a second hydrophilic or lipophilic moiety, or to separate a hydrophilic moiety or lipophilic moiety from a linker moiety.
  • Spacer moieties are preferably selected from the group consisting of sugar, cholesterol and glycerine moieties.
  • the oligomer may further comprise one or more linker moieties that are used to couple the oligomer with the insulin drug as will be understood by those skilled in the art.
  • Linker moieties are preferably selected from the group consisting of alkyl and fatty acid moieties.
  • the oligomer may further comprise one or more terminating moieties at the one or more ends of the oligomer which are not coupled to the insulin drug.
  • the terminating moiety is preferably an alkyl or alkoxy moiety, and is more preferably a lower alkyl or lower alkoxy moiety. Most preferably, the terminating moiety is methyl or methoxy. While the terminating moiety is preferably an alkyl or alkoxy moiety, it is to be understood that the terminating moiety may be various moieties as will be understood by those skilled in the art including, but not limited to, sugars, cholesterol, alcohols, and fatty acids.
  • the oligomer is preferably covalently coupled to the insulin drug.
  • the insulin drug is coupled to the oligomer utilizing a hydrolyzable bond (e.g., an ester or carbonate bond).
  • a hydrolyzable coupling may provide an insulin drug-oligomer conjugate that acts as a prodrug.
  • a hydrolyzable coupling may provide for a time-release or controlled-release effect, administering the insulin drag over a given time period as one or more oligomers are cleaved from their respective insulin drug-oligomer conjugates to provide the active drug.
  • the insulin drug is coupled to the oligomer utilizing a non-hydrolyzable bond (e.g., a carbamate, amide, or ether bond).
  • a non-hydrolyzable bond may be preferable when it is desirable to allow the insulin drug-oligomer conjugate to circulate in the bloodstream for an extended period of time, preferably at least 2 hours.
  • the oligomer further comprises one or more bonding moieties that are used to covalently couple the oligomer with the insulin drug as will be understood by those skilled in the art.
  • Bonding moieties are preferably selected from the group consisting of covalent bond(s), ester moieties, carbonate moieties, carbamate moieties, amide moieties and secondary amine moieties. More than one moiety on the oligomer may be covalently coupled to the insulin drug.
  • oligomer is preferably covalently coupled to the insulin drug
  • the oligomer may be non-covalently coupled to the insulin drug to form a non-covalently conjugated insulin drug-oligomer complex.
  • non-covalent couplings include, but are not limited to, hydrogen bonding, ionic bonding, Van der Waals bonding, and micellular or liposomal encapsulation.
  • oligomers may be suitably constructed, modified and/or appropriately functionalized to impart the ability for non-covalent conjugation in a selected manner (e.g., to impart hydrogen bonding capability), as will be understood by those skilled in the art.
  • oligomers may be derivatized with various compounds including, but not limited to, amino acids, oligopeptides, peptides, bile acids, bile acid derivatives, fatty acids, fatty acid derivatives, salicylic acids, salicylic acid derivatives, aminosalicylic acids, and aminosalicylic acid derivatives.
  • the resulting oligomers can non-covalently couple (complex) with drug molecules, pharmaceutical products, and/or pharmaceutical excipients.
  • the resulting complexes preferably have balanced lipophilic and hydrophilic properties.
  • oligomers may be derivatized with amine and/or alkyl amines. Under suitable acidic conditions, the resulting oligomers can form non- covalently conjugated complexes with drug molecules, pharmaceutical products and/or pharmaceutical excipients.
  • the products resulting from such complexation preferably have balanced lipophilic and hydrophilic properties.
  • More than one oligomer may be coupled to the insulin drug.
  • the oligomers in the plurality are preferably the same. However, it is to be understood that the oligomers in the plurality may be different from one another, or, alternatively, some of the oligomers in the plurality may be the same and some may be different.
  • all of the bonds coupling the plurality of oligomers to the insulin drag may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more of the oligomers is rapidly removed from the insulin drug by hydrolysis in the body and one or more of the oligomers is slowly removed from the insulin drag by hydrolysis in the body.
  • the oligomer may be coupled to the insulin drug at various nucleophilic residues of the insulin drug including, but not limited to, nucleophilic hydroxyl functions and/or amino functions.
  • nucleophilic hydroxyl function may be found, for example, at serine and/or tyrosine residues
  • a nucleophilic amino function may be found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini of the polypeptide.
  • the coupling preferably forms a secondary amine.
  • the oligomer may be coupled to an amino functionality of the insulin, including the amino functionality of Grj ⁇ 1 , the amino functionality of Phe B1 , and the amino functionality of Lys 329 .
  • the oligomer is preferably coupled to the amino functionality of Lys B29 .
  • the oligomers are preferably coupled to the amino functionality of Phe B1 and the amino functionality of Lys B29 . While more than one oligomer may be coupled to the human insulin, a higher activity (improved glucose lowering ability) is observed for the mono-conjugated human insulin.
  • Substantially monodispersed mixtures of insulin drag-oligomer conjugates of the present invention may be synthesized by various methods. For example, a substantially monodispersed mixture of oligomers consisting of carboxyhc acid and polyethylene glycol is synthesized by contacting a substantially monodispersed mixture of carboxylic acid with a substantially monodispersed mixture of polyethylene glycol under conditions sufficient to provide a substantially monodispersed mixture of oligomers. The oligomers of the substantially monodispersed mixture are then activated so that they are capable of reacting with an insulin drug to provide an insulin drug-oligomer conjugate.
  • a synthesis route for providing a substantially monodispersed mixture of activated oligomers is illustrated in Figure 3 and described in Examples 11-18 hereinbelow.
  • Another embodiment of a synthesis route for providing a substantially monodispersed mixture of activated oligomers is illustrated in Figure 4 and described in Examples 19-24 hereinbelow.
  • Still another embodiment of a synthesis route for providing a substantially monodispersed mixture of activated oligomers is illustrated in Figure 5 and described in Examples 25-29 hereinbelow.
  • Yet another embodiment of a synthesis route for providing a substantially monodispersed mixture of activated oligomers is illustrated in Figure 6 and described in Examples 30-31 hereinbelow.
  • FIG. 7 Another embodiment of a synthesis route for providing a substantially monodispersed mixture of activated oligomers is illustrated in Figure 7 and described in Examples 32-37 hereinbelow. Still another embodiment of a synthesis route for providing a substantially monodispersed mixture of activated oligomers is illustrated in Figure 8 and described in Example 38 hereinbelow. Yet another embodiment of a synthesis route for providing a substantially monodispersed mixture of activated oligomers is illustrated in Figure 9 and described in Example 39 hereinbelow. Another embodiment of a synthesis route for providing a substantially monodispersed mixture of activated oligomers is illustrated in Figure 10 and described in Example 40 hereinbelow.
  • the substantially monodispersed mixture of activated oligomers may be reacted with a substantially monodispersed mixture of insulin drugs under conditions sufficient to provide a mixture of insulin drag-oligomer conjugates.
  • a preferred synthesis is described in Example 41 hereinbelow.
  • the reaction conditions e.g., selected molar ratios, solvent mixtures and/or pH
  • the reaction conditions may be controlled such that the mixture of insulin drag-oligomer conjugates resulting from the reaction of the substantially monodispersed mixture of activated oligomers and the substantially monodispersed mixture of insulin drugs is a substantially monodispersed mixture.
  • conjugation at the amino functionality of lysine may be suppressed by maintaining the pH of the reaction solution below the pK a of lysine.
  • the mixture of insulin drag-oligomer conjugates may be separated and isolated utilizing, for example, HPLC as described below in Example 50 to provide a substantially monodispersed mixture of insulin drug-oligomer conjugates, for example mono-, di-, or tri-conjugates.
  • the degree of conjugation (e.g., whether the isolated molecule is a mono-, di-, or tri-conjugate) of a particular isolated conjugate may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, mass spectroscopy.
  • the particular conjugate structure (e.g., whether the oligomer is at Gi ⁇ 1 , Phe B1 or Lys B29 a human insulin monoconjugate) may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, sequence analysis, peptide mapping, selective enzymatic cleavage, and/or endopeptidase cleavage.
  • one or more of the reaction sites on the insulin drug may be blocked by, for example, reacting the insulin drug with a suitable reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9-fluorenylmethoxycarbonyl) (N- FMOC).
  • a suitable reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9-fluorenylmethoxycarbonyl) (N- FMOC).
  • t-BOC N-tert-butoxycarbonyl
  • N- FMOC N-(9-fluorenylmethoxycarbonyl)
  • the substantially monodispersed mixture of blocked insulin drags may be reacted with the substantially monodispersed mixture of activated oligomers to provide a mixture of insulin drag-oligomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues.
  • the insulin drug- oligomer conjugates may be de-blocked as will be understood by those skilled in the art. If necessary, the mixture of insulin drag-oligomer conjugates may then be separated as described above to provide a substantially monodispersed mixture of insulin drug-oligomer conjugates. Alternatively, the mixture of insulin drug-oligomer conjugates may be separated prior to de-blocking.
  • each conjugate includes human insulin covalently coupled at Lys 29 of the insulin to a carboxyhc acid moiety of an oligomer that comprises hexanoic acid covalently coupled at the end distal to the carboxyhc acid moiety to a methyl terminated polyethylene glycol moiety having at least 7 polyethylene glycol subunits.
  • each conjugate in the substantially monodispersed mixture consists of human insulin covalently coupled at Lys B29 of the insulin to a carboxyhc acid moiety of an oligomer that consists of hexanoic acid covalently coupled at the end distal to the carboxyhc acid moiety to a methyl terminated polyethylene glycol moiety having 7 polyethylene glycol subunits.
  • Substantially monodispersed mixtures of conjugates according to these embodiments of the present invention preferably have improved properties when compared with those of polydispersed mixtures.
  • a substantially monodispersed mixture of insulin drug- oligomer conjugates preferably has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of insulin drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture.
  • the number average molecular weight of the substantially monodispersed mixture and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography such as gel permeation chromatography as described, for example, in H.R. Allcock & F.W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991).
  • a substantially monodispersed mixture of insulin drug-oligomer conjugates preferably has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of insulin drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture.
  • the number average molecular weight of the substantially monodispersed mixture and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • the in vitro activity of a particular mixture may be measured by various methods, as will be understood by those skilled in the art.
  • the in vitro activity is measured using a Cytosensor® Microphysiometer commercially available from Molecular Devices Corporation of Sunnyvale, California.
  • the microphysiometer monitors small changes in the rates of extracellular acidification in response to a drag being added to cultured cells in a transwell. This response is proportional to the activity of the molecule under study.
  • the in vitro activity of the substantially monodispersed mixture is at least about 5 percent greater than the in vitro activity of the polydispersed mixture. More preferably, the in vitro activity of the substantially monodispersed mixture is at least about 10 percent greater than the in vitro activity of the polydispersed mixture.
  • a substantially monodispersed mixture of insulin drag- oligomer conjugates preferably has an increased resistance to degradation by chymotrypsin when compared to the resistance to degradation by chymotrypsin of a polydispersed mixture of insulin drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture.
  • Resistance to chymotrypsin corresponds to the percent remaining when the molecule to be tested is digested in chymotrypsin using a procedure similar to the one outlined in Example 52 below.
  • the number average molecular weight of the substantially monodispersed mixture and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • the resistance to degradation by chymotrypsin of the substantially monodispersed mixture is at least about 10 percent greater than the resistance to degradation by chymotrypsin of the polydispersed mixture. More preferably, the resistance to degradation by chymotrypsin of the substantially monodispersed mixture is at least about 20 percent greater than the resistance to degradation by chymotrypsin of the polydispersed mixture.
  • a substantially monodispersed mixture of insulin drug- oligomer conjugates preferably has an inter-subject variability that is less than the inter- subject variability of a polydispersed mixture of insulin drag-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture.
  • the number average molecular weight of the substantially monodispersed mixture and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • the inter-subject variability may be measured by various methods as will be understood by those skilled in the art.
  • the inter-subject variability is preferably calculated as follows.
  • the area under a dose response curve (i.e., the area between the dose- response curve and a baseline value) is determined for each subject.
  • the average AUC for all subjects is determined by summing the AUCs of each subject and dividing the sum by the number of subjects.
  • the absolute value of the difference between the subject's AUC and the average AUC is then determined for each subject.
  • the absolute values of the differences obtained are then summed to give a value that represents the inter-subject variability.
  • Lower values represent lower inter-subject variabilities and higher values represent higher inter- subject variabilities.
  • the inter-subject variability of the substantially monodispersed mixture is at least about 10 percent less than the inter-subject variability of the polydispersed mixture. More preferably, the inter-subject variability of the substantially monodispersed mixture is at least about 25 percent less than the inter-subject variability of the polydispersed mixture.
  • Substantially monodispersed mixtures of conjugates according to embodiments of the present invention preferably have two or more of the above-described properties. More preferably, substantially monodispersed mixtures of conjugates according to embodiments of the present invention have three or more of the above-described properties. Most preferably, substantially monodispersed mixtures of conjugates according to embodiments of the present invention have all four of the above-described properties.
  • Each conjugate in the mixture includes an insulin drug coupled to an oligomer that comprises a polyethylene glycol moiety.
  • the standard deviation is preferably less than about 14 Daltons and is more preferably less than about 11 Daltons.
  • the molecular weight distribution may be determined by methods known to those skilled in the art including, but not limited to, size exclusion chromatography such as gel permeation chromatography as described, for example, in H.R. Allcock & F.W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991). The standard deviation of the molecular weight distribution may then be determined by statistical methods as will be understood by those skilled in the art.
  • the insulin drug is preferably insulin. More preferably, the insulin drug is human insulin.
  • the insulin drag may be selected from various insulin drags known to those skilled in the art including, for example, proinsulin, insulin analogs, insulin fragments, and insulin fragment analogs.
  • Insulin analogs include, but are not limited to, Asp B28 human insulin, Lys B28 human insulin, Leu B28 human insulin, Val 328 human insulin, Ala B28 human insulin, Asp B28 Pro 329 human insulin, Lys B28 Pro B29 human insulin, Leu 328 Pro B29 human insulin, Val B28 Pro B29 human insulin, Ala B28 Pro B29 human insulin, as well as analogs provided using the substitution guidelines described above.
  • Insulin fragments include, but are not limited to, B22-B30 human insulin, B23-B30 human insulin, B25-B30 human insulin, B26-B30 human insulin, B27-B30 human insulin, B29-B30 human insulin, the A chain of human insulin, and the B chain of human insulin.
  • Insulin fragment analogs may be provided by substituting one or more amino acids as described above in an insulin fragment.
  • the oligomer may be various oligomers comprising a polyethylene glycol moiety as will be understood by those skilled in the art.
  • the polyethylene glycol moiety of the oligomer has at least 2, 3 or 4 polyethylene glycol subunits. More preferably, the polyethylene glycol moiety has at least 5 or 6 polyethylene glycol subunits and, most preferably, the polyethylene glycol moiety has at least 7 polyethylene glycol subunits.
  • the oligomer may comprise one or more other moieties as will be understood by those skilled in the art including, but not limited to, additional hydrophilic moieties, lipophilic moieties, spacer moieties, linker moieties, and terminating moieties.
  • the various moieties in the oligomer are covalently coupled to one another by either hydrolyzable or non- hydrolyzable bonds.
  • the oligomer may further comprise one or more additional hydrophilic moieties (i.e., moieties in addition to the polyethylene glycol moiety) including, but not limited to, sugars, polyalkylene oxides, and polyamine/PEG copolymers.
  • additional hydrophilic moieties i.e., moieties in addition to the polyethylene glycol moiety
  • polyethylene glycol is a polyalkylene oxide
  • the additional hydrophilic moiety may be a polyethylene glycol moiety. Adjacent polyethylene glycol moieties will be considered to be the same moiety if they are coupled by an ether bond.
  • the moiety i.e., moieties in addition to the polyethylene glycol moiety
  • oligomers according to embodiments of the present invention comprise a polyethylene glycol moiety and no additional hydrophilic moieties.
  • the oligomer may further comprise one or more lipophilic moieties as will be understood by those skilled in the art.
  • the lipophilic moiety is preferably a saturated or unsaturated, linear or branched alkyl moiety or a saturated or unsaturated, linear or branched fatty acid moiety.
  • the lipophilic moiety is an alkyl moiety, it is preferably a linear, saturated or unsaturated alkyl moiety having 1 to 28 carbon atoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms.
  • the lipophilic moiety is a fatty acid moiety, it is preferably a natural fatty acid moiety that is linear, saturated or unsaturated, having 2 to 18 carbon atoms.
  • the fatty acid moiety has 3 to 14 carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5 or 6 carbon atoms.
  • the oligomer may further comprise one or more spacer moieties as will be understood by those skilled in the art. Spacer moieties may, for example, be used to separate a hydrophilic moiety from a lipophilic moiety, to separate a lipophilic moiety or hydrophilic moiety from the insulin drag, to separate a first hydrophilic or lipophilic moiety from a second hydrophilic or lipophilic moiety, or to separate a hydrophilic moiety or lipophilic moiety from a linker moiety. Spacer moieties are preferably selected from the group consisting of sugar, cholesterol and glycerine moieties.
  • the oligomer may further comprise one or more linker moieties that are used to couple the oligomer with the insulin drag as will be understood by those skilled in the art.
  • Linker moieties are preferably selected from the group consisting of alkyl and fatty acid moieties.
  • the oligomer may further comprise one or more terminating moieties at the one or more ends of the oligomer which are not coupled to the insulin drug.
  • the terminating moiety is preferably an alkyl or alkoxy moiety, and is more preferably a lower alkyl or lower alkoxy moiety. Most preferably, the terminating moiety is methyl or methoxy. While the terminating moiety is preferably an alkyl or alkoxy moiety, it is to be understood that the terminating moiety may be various moieties as will be understood by those skilled in the art including, but not limited to, sugars, cholesterol, alcohols, and fatty acids.
  • the oligomer is preferably covalently coupled to the insulin drag.
  • the insulin drug is coupled to the oligomer utilizing a hydrolyzable bond (e.g., an ester or carbonate bond).
  • a hydrolyzable coupling may provide an insulin drag-oligomer conjugate that acts as a prodrag.
  • a hydrolyzable coupling may provide for a time-release or controlled-release effect, administering the insulin drug over a given time period as one or more oligomers are cleaved from their respective insulin drug-oligomer conjugates to provide the active drag.
  • the insulin drug is coupled to the oligomer utilizing a non-hydrolyzable bond (e.g., a carbamate, amide, or ether bond).
  • the oligomer When the oligomer is covalently coupled to the insulin drug, the oligomer further comprises one or more bonding moieties that are used to covalently couple the oligomer with the insulin drug as will be understood by those skilled in the art. Bonding moieties are preferably selected from the group consisting of covalent bond(s), ester moieties, carbonate moieties, carbamate moieties, amide moieties and secondary amine moieties. More than one moiety on the oligomer may be covalently coupled to the insulin drag.
  • the oligomer is preferably covalently coupled to the insulin drag, it is to be understood that the oligomer may be non-covalently coupled to the insulin drug to form a non-covalently conjugated insulin drug-oligomer complex.
  • non-covalent couplings include, but are not limited to, hydrogen bonding, ionic bonding, Van der Waals bonding, and micellular or liposomal encapsulation.
  • oligomers may be suitably constructed, modified and/or appropriately functionalized to impart the ability for non-covalent conjugation in a selected manner (e.g., to impart hydrogen bonding capability), as will be understood by those skilled in the art.
  • oligomers may be derivatized with various compounds including, but not limited to, amino acids, oligopeptides, peptides, bile acids, bile acid derivatives, fatty acids, fatty acid derivatives, salicylic acids, salicylic acid derivatives, aminosalicylic acids, and aminosalicylic acid derivatives.
  • the resulting oligomers can non-covalently couple (complex) with drug molecules, pharmaceutical products, and/or pharmaceutical excipients.
  • the resulting complexes preferably have balanced lipophilic and hydrophilic properties.
  • oligomers may be derivatized with amine and/or alkyl amines. Under suitable acidic conditions, the resulting oligomers can form non- covalently conjugated complexes with drug molecules, pharmaceutical products and/or pharmaceutical excipients.
  • the products resulting from such complexation preferably have balanced lipophilic and hydrophilic properties.
  • More than one oligomer may be coupled to the insulin drug.
  • the oligomers in the plurality are preferably the same. However, it is to be understood that the oligomers in the plurality may be different from one another, or, alternatively, some of the oligomers in the plurality may be the same and some may be different.
  • all of the bonds coupling the plurality of oligomers to the insulin drug may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more of the oligomers is rapidly removed from the insulin drag by hydrolysis in the body and one or more of the oligomers is slowly removed from the insulin drag by hydrolysis in the body.
  • the oligomer may be coupled to the insulin drug at various nucleophilic residues of the insulin drag including, but not limited to, nucleophilic hydroxyl functions and/or amino functions.
  • the insulin drug is a polypeptide
  • a nucleophilic hydroxyl function may be found, for example, at serine and/or tyrosine residues
  • a nucleophilic amino function may be found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini of the polypeptide.
  • the coupling preferably forms a secondary amine.
  • the oligomer may be coupled to an amino functionality of the insulin, including the amino functionality of Gi ⁇ 1 , the amino functionality of Phe B1 , and the amino functionality of Lys 329 .
  • the oligomer When one oligomer is coupled to the human insulin, the oligomer is preferably coupled to the amino functionality of Lys 329 . When two oligomers are coupled to the human insulin, the oligomers are preferably coupled to the amino functionality of Phe 31 and the amino functionality of Lys 329 . While more than one oligomer may be coupled to the human insulin, a higher activity (improved glucose lowering ability) is observed for the mono-conjugated human insulin.
  • Mixtures of insulin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons may be synthesized by various methods.
  • a mixture of oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons consisting of carboxyhc acid and polyethylene glycol is synthesized by contacting a mixture of carboxyhc acid having a molecular weight distribution with a standard deviation of less than about 22 Daltons with a mixture of polyethylene glycol having a molecular weight distribution with a standard deviation of less than about 22 Daltons under conditions sufficient to provide a mixture of oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • the oligomers of the mixture having a molecular weight distribution with a standard deviation of less than about 22 Daltons are then activated so that they are capable of reacting with an insulin drag to provide an insulin drag-oligomer conjugate.
  • a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 3 and described in Examples 11-18 hereinbelow.
  • Another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 4 and described in Examples 19-24 hereinbelow.
  • Still another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 5 and described in Examples 25-29 hereinbelow.
  • Yet another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 6 and described in Examples 30-31 hereinbelow.
  • Another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 7 and described in Examples 32-37 hereinbelow.
  • Still another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 8 and described in Example 38 hereinbelow.
  • Yet another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 9 and described in Example 39 hereinbelow.
  • Another embodiment of a synthesis route for providing a mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is illustrated in Figure 10 and described in Example 40 hereinbelow.
  • the mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons is reacted with a mixture of insulin drugs having a molecular weight distribution with a standard deviation of less than about 22 Daltons under conditions sufficient to provide a mixture of insulin dragroligomer conjugates.
  • a preferred synthesis is described in Example 41 hereinbelow.
  • the reaction conditions e.g., selected molar ratios, solvent mixtures and/or pH
  • the mixture of insulin drug-oligomer conjugates resulting from the reaction of the mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons and the mixture of insulin drugs having a molecular weight distribution with a standard deviation of less than about 22 Daltons is a mixture having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • conjugation at the amino functionality of lysine may be suppressed by maintaining the pH of the reaction solution below the pK a of lysine.
  • the mixture of insulin drag-oligomer conjugates may be separated and isolated utilizing, for example, HPLC as described below in Example 50 to provide a mixture of insulin drag-oligomer conjugates, for example mono-, di-, or tri-conjugates, having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • the degree of conjugation e.g., whether the isolated molecule is a mono-, di-, or tri-conjugate
  • a particular isolated conjugate may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, mass spectroscopy.
  • the particular conjugate structure (e.g., whether the oligomer is at Gly ⁇ 1 , Phe 31 or Lys 329 of a human insulin monoconjugate) may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, sequence analysis, peptide mapping, selective enzymatic cleavage, and/or endopeptidase cleavage.
  • one or more of the reaction sites on the insulin drag may be blocked by, for example, reacting the insulin drug with a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9-fluorenylmethoxycarbonyl) (N-FMOC).
  • a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9-fluorenylmethoxycarbonyl) (N-FMOC).
  • t-BOC N-tert-butoxycarbonyl
  • N-FMOC N-(9-fluorenylmethoxycarbonyl)
  • the mixture of blocked insulin drugs having a molecular weight distribution with a standard deviation of less than about 22 Daltons may be reacted with the mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons to provide a mixture of insulin drug- oligomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues.
  • the insulin drug-oligomer conjugates may be de-blocked as will be understood by those skilled in the art.
  • the mixture of insulin drag-oligomer conjugates may then be separated as described above to provide a mixture of insulin drag-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • the mixture of insulin drug-oligomer conjugates may be separated prior to de-blocking.
  • Mixtures of insulin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably have improved properties when compared with those of polydispersed mixtures.
  • a mixture of insulin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of insulin drug-oligomer conjugates having the same number average molecular weight as the mixture of insulin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • the number average molecular weight of the mixture of insulin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography such as gel permeation chromatography as described, for example, in H.R. Allcock & F.W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991).
  • a mixture of insulin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of insulin drag-oligomer conjugates having the same number average molecular weight as the mixture of insulin drag-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • the number average molecular weight of the mixture of insulin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • the in vitro activity of a particular mixture may be measured by various methods, as will be understood by those skilled in the art.
  • the in vitro activity is measured using a Cytosensor® Microphysiometer commercially available from Molecular Devices Corporation of Sunnyvale, California.
  • the microphysiometer monitors small changes in the rates of extracellular acidification in response to a drag being added to cultured cells in a transwell. This response is proportional to the activity of the molecule under study.
  • the in vitro activity of the mixture of insulin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons is at least about 5 percent greater than the in vitro activity of the polydispersed mixture. More preferably, the in vitro activity of the mixture of insulin drag-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons is at least about 10 percent greater than the in vitro activity of the polydispersed mixture.
  • a mixture of insulin drag-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably has an increased resistance to degradation by chymotrypsin when compared to the resistance to degradation by chymotrypsin of a polydispersed mixture of insulin drag- oligomer conjugates having the same number average molecular weight as the mixture of insulin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • Resistance to chymotrypsin corresponds to the percent remaining when the molecule to be tested is digested in chymotrypsin using a procedure similar to the one outlined in Example 52 below.
  • the number average molecular weight of the mixture of insulin drug- oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • the resistance to degradation by chymotrypsin of the mixture of insulin drug- oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons is at least about 10 percent greater than the resistance to degradation by chymotrypsin of the polydispersed mixture.
  • the resistance to degradation by chymotrypsin of the mixture of insulin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons is at least about 20 percent greater than the resistance to degradation by chymotrypsin of the polydispersed mixture.
  • a mixture of insulin drag-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons preferably has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of insulin drug-oligomer conjugates having the same number average molecular weight as the mixture of insulin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • the number average molecular weight of the mixture of insulin drag-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • the inter-subject variability may be measured by various methods as will be understood by those skilled in the art.
  • the inter-subject variability is preferably calculated- as follows.
  • the area under a dose response curve (AUC) i.e., the area between the dose- response curve and a baseline value) is determined for each subject.
  • the average AUC for all subjects is determined by summing the AUCs of each subject and dividing the sum by the number of subjects.
  • the absolute value of the difference between the subject's AUC and the average AUC is then determined for each subject.
  • the absolute values of the differences obtained are then summed to give a value that represents the inter-subject variability.
  • Lower values represent lower inter-subject variabilities and higher values represent higher inter- subject variabilities.
  • the inter-subject variability of the mixture of insulin drug- oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons is at least about 10 percent less than the inter-subject variability of the polydispersed mixture.
  • the inter-subject variability of the mixture of insulin drag-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons is at least about 25 percent less than the inter-subject variability of the polydispersed mixture.
  • Mixtures of insulin drag-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons according to embodiments of the present invention preferably have two or more of the above-described properties. More preferably, mixtures of insulin drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons according to embodiments of the present invention have three or more of the above-described properties. Most preferably, mixtures of insulin drag-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons according to embodiments of the present invention have all four of the above-described properties.
  • each conjugate includes an insulin drag coupled to an oligomer that comprises a polyethylene glycol moiety, and the mixture has a dispersity coefficient (DC) greater than 10,000 where
  • n is the number of different molecules in the sample
  • Nj is the number of i- molecules in the sample
  • Mj is the mass of the i ⁇ molecule.
  • the mixture of conjugates preferably has a dispersity coefficient greater than 100,000. More preferably, the dispersity coefficient of the conjugate mixture is greater than 500,000 and, most preferably, the dispersity coefficient is greater than 10,000,000.
  • the variables n, Nj, and Mi may be determined by various methods as will be understood by those skilled in the art, including, but not limited to, methods described below in Example 49.
  • the insulin drag is preferably insulin. More preferably, the insulin drug is human insulin.
  • the insulin drag may be selected from various insulin drags known to those skilled in the art including, for example, proinsulin, insulin analogs, insulin fragments, and insulin fragment analogs.
  • Insulin analogs include, but are not limited to, Asp human insulin, Lys human insulin, Leu human insulin, Val human R R R 0 R R R Q insulin, Ala human insulin, Asp Pro human insulin, Lys Pro human insulin, Leu 328 Pro 329 human insulin, Val B28 Pro B29 human insulin, Ala B28 Pro B29 human insulin, as well as analogs provided using the substitution guidelines described above.
  • Insulin fragments include, but are not limited to, B22-B30 human insulin, B23-B30 human insulin, B25-B30 human insulin, B26-B30 human insulin, B27-B30 human insulin, B29-B30 human insulin, the A chain of human insulin, and the B chain of human insulin.
  • Insulin fragment analogs may be provided by substituting one or more amino acids as described above in an insulin fragment.
  • the oligomer may be various oligomers comprising a polyethylene glycol moiety as will be understood by those skilled in the art.
  • the polyethylene glycol moiety of the oligomer has at least 2, 3 or 4 polyethylene glycol subunits. More preferably, the polyethylene glycol moiety has at least 5 or 6 polyethylene glycol subunits and, most preferably, the polyethylene glycol moiety has at least 7 polyethylene glycol subunits.
  • the oligomer may comprise one or more other moieties as will be understood by those skilled in the art including, but not limited to, additional hydrophilic moieties, lipophilic moieties, spacer moieties, linker moieties, and terminating moieties.
  • the various moieties in the oligomer are covalently coupled to one another by either hydrolyzable or non- hydrolyzable bonds.
  • the oligomer may further comprise one or more additional hydrophilic moieties (i.e., moieties in addition to the polyethylene glycol moiety) including, but not limited to, sugars, polyalkylene oxides, and polyamine/PEG copolymers.
  • additional hydrophilic moieties i.e., moieties in addition to the polyethylene glycol moiety
  • polyethylene glycol is a polyalkylene oxide
  • the additional hydrophilic moiety may be a polyethylene glycol moiety. Adjacent polyethylene glycol moieties will be considered to be the same moiety if they are coupled by an ether bond.
  • the moiety i.e., moieties in addition to the polyethylene glycol moiety
  • oligomers according to embodiments of the present invention comprise a polyethylene glycol moiety and no additional hydrophilic moieties.
  • the oligomer may further comprise one or more lipophilic moieties as will be understood by those skilled in the art.
  • the lipophilic moiety is preferably a saturated or unsaturated, linear or branched alkyl moiety or a saturated or unsaturated, linear or branched fatty acid moiety.
  • the lipophilic moiety is an alkyl moiety, it is preferably a linear, saturated or unsaturated alkyl moiety having 1 to 28 carbon atoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms.
  • the lipophilic moiety is a fatty acid moiety, it is preferably a natural fatty acid moiety that is linear, saturated or unsaturated, having 2 to 18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14 carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5 or 6 carbon atoms.
  • the oligomer may further comprise one or more spacer moieties as will be understood by those skilled in the art.
  • Spacer moieties may, for example, be used to separate a hydrophilic moiety from a lipophilic moiety, to separate a lipophilic moiety or hydrophilic moiety from the insulin drag, to separate a first hydrophilic or lipophilic moiety from a second hydrophilic or lipophilic moiety, or to separate a hydrophilic moiety or lipophilic moiety from a linker moiety.
  • Spacer moieties are preferably selected from the group consisting of sugar, cholesterol and glycerine moieties.
  • the oligomer may further comprise one or more linker moieties that are used to couple the oligomer with the insulin drag as will be understood by those skilled in the art.
  • Linker moieties are preferably selected from the group consisting of alkyl and fatty acid moieties.
  • the oligomer may further comprise one or more terminating moieties at the one or more ends of the oligomer which are not coupled to the insulin drag.
  • the terminating moiety is preferably an alkyl or alkoxy moiety, and is more preferably a lower alkyl or lower alkoxy moiety. Most preferably, the terminating moiety is methyl or methoxy. While the terminating moiety is preferably an alkyl or alkoxy moiety, it is to be understood that the terminating moiety may be various moieties as will be understood by those skilled in the art including, but not limited to, sugars, cholesterol, alcohols, and fatty acids.
  • the oligomer is preferably covalently coupled to the insulin drug.
  • the insulin drag is coupled to the oligomer utilizing a hydrolyzable bond (e.g., an ester or carbonate bond).
  • a hydrolyzable coupling may provide an insulin drag-oligomer conjugate that acts as a prodrag.
  • a hydrolyzable coupling may provide for a time-release or controlled-release effect, administering the insulin drag over a given time period as one or more oligomers are cleaved from their respective insulin drag-oligomer conjugates to provide the active drug.
  • the insulin drug is coupled to the oligomer utilizing a non-hydrolyzable bond (e.g., a carbamate, amide, or ether bond).
  • a non-hydrolyzable bond may be preferable when it is desirable to allow the insulin drug-oligomer conjugate to circulate in the bloodstream for an extended period of time, preferably at least 2 hours.
  • the oligomer further comprises one or more bonding moieties that are used to covalently couple the oligomer with the insulin drag as will be understood by those skilled in the art.
  • Bonding moieties are preferably selected from the group consisting of covalent bond(s), ester moieties, carbonate moieties, carbamate moieties, amide moieties and secondary amine moieties. More than one moiety on the oligomer may be covalently coupled to the insulin drug.
  • oligomer is preferably covalently coupled to the insulin drag
  • the oligomer may be non-covalently coupled to the insulin drug to form a non-covalently conjugated insulin drag-oligomer complex.
  • non-covalent couplings include, but are not limited to, hydrogen bonding, ionic bonding, Van der Waals bonding, and micellular or liposomal encapsulation.
  • oligomers may be suitably constructed, modified and/or appropriately functionalized to impart the ability for non-covalent conjugation in a selected manner (e.g., to impart hydrogen bonding capability), as will be understood by those skilled in the art.
  • oligomers may be derivatized with various compounds including, but not limited to, amino acids, oligopeptides, peptides, bile acids, bile acid derivatives, fatty acids, fatty acid derivatives, salicylic acids, salicylic acid derivatives, aminosalicylic acids, and aminosalicylic acid derivatives.
  • the resulting oligomers can non-covalently couple (complex) with drug molecules, pharmaceutical products, and/or pharmaceutical excipients.
  • the resulting complexes preferably have balanced lipophilic and hydrophilic properties.
  • oligomers may be derivatized with amine and/or alkyl amines. Under suitable acidic conditions, the resulting oligomers can form non- covalently conjugated complexes with drag molecules, pharmaceutical products and/or pharmaceutical excipients.
  • the products resulting from such complexation preferably have balanced lipophilic and hydrophilic properties.
  • More than one oligomer may be coupled to the insulin drug.
  • the oligomers in the plurality are preferably the same. However, it is to be understood that the oligomers in the plurality may be different from one another, or, alternatively, some of the oligomers in the plurality may be the same and some may be different.
  • all of the bonds coupling the plurality of oligomers to the insulin drug may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more of the oligomers is rapidly removed from the insulin drug by hydrolysis in the body and one or more of the oligomers is slowly removed from the insulin drug by hydrolysis in the body.
  • the oligomer may be coupled to the insulin drug at various nucleophilic residues of the insulin drag including, but not limited to, nucleophilic hydroxyl functions and/or amino functions.
  • nucleophilic hydroxyl function may be found, for example, at serine and/or tyrosine residues
  • a nucleophilic amino function may be found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini of the polypeptide.
  • the coupling preferably forms a secondary amine.
  • the oligomer may be coupled to an amino functionality of the insulin, including the amino functionality of Gly ⁇ , the amino functionality of Phe 31 , and the amino functionality of Lys 329 .
  • the oligomer is preferably coupled to the amino functionality of Lys 329 .
  • the oligomers are preferably coupled to the amino functionality of Phe 31 and the amino functionality of Lys 329 . While more than one oligomer may be coupled to the human insulin, a higher activity (improved glucose lowering ability) is observed for the mono-conjugated human insulin.
  • Mixtures of insulin drag-oligomer conjugates having a dispersity coefficient greater than 10,000 may be synthesized by various methods. For example, a mixture of oligomers having a dispersity coefficient greater than 10,000 consisting of carboxyhc acid and polyethylene glycol is synthesized by contacting a mixture of carboxyhc acid having a dispersity coefficient greater than 10,000 with a mixture of polyethylene glycol having a dispersity coefficient greater than 10,000 under conditions sufficient to provide a mixture of oligomers having a dispersity coefficient greater than 10,000. The oligomers of the mixture having a dispersity coefficient greater than 10,000 are then activated so that they are capable of reacting with an insulin drug to provide an insulin drag-oligomer conjugate.
  • a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 3 and described in Examples 11-18 hereinbelow.
  • Another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 4 and described in Examples 19-24 hereinbelow.
  • Still another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 5 and described in Examples 25-29 hereinbelow.
  • Yet another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 6 and described in Examples 30-31 hereinbelow.
  • FIG. 7 Another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 7 and described in Examples 32-37 hereinbelow. Still another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 8 and described in Example 38 hereinbelow. Yet another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 9 and described in Example 39 hereinbelow. Another embodiment of a synthesis route for providing a mixture of activated oligomers having a dispersity coefficient greater than 10,000 is illustrated in Figure 10 and described in Example 40 hereinbelow.
  • the mixture of activated oligomers having a dispersity coefficient greater than 10,000 is reacted with a mixture of insulin drugs having a dispersity coefficient greater than 10,000 under conditions sufficient to provide a mixture of insulin drug-oligomer conjugates.
  • a preferred synthesis is described in Example 41 hereinbelow.
  • the reaction conditions e.g., selected molar ratios, solvent mixtures and/or pH
  • the reaction conditions may be controlled such that the mixture of insulin drug-oligomer conjugates resulting from the reaction of the mixture of activated oligomers having a dispersity coefficient greater than 10,000 and the mixture of insulin drugs having a dispersity coefficient greater than 10,000 is a mixture having a dispersity coefficient greater than 10,000.
  • conjugation at the amino functionality of lysine may be suppressed by maintaining the pH of the reaction solution below the pK a of lysine.
  • the mixture of insulin drug- oligomer conjugates may be separated and isolated utilizing, for example, HPLC as described below in Example 50 to provide a mixture of insulin drug-oligomer conjugates, for example mono-, di-, or tri-conjugates, having a dispersity coefficient greater than 10,000.
  • the degree of conjugation (e.g., whether the isolated molecule is a mono-, di-, or tri-conjugate) of a particular isolated conjugate may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, mass spectroscopy.
  • the particular conjugate structure (e.g., whether the oligomer is at Gly Phe 31 or Lys 329 of a human insulin monoconjugate) may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, sequence analysis, peptide mapping, selective enzymatic cleavage, and/or endopeptidase cleavage.
  • one or more of the reaction sites on the insulin drag may be blocked by, for example, reacting the insulin drag with a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9-fluorenylmethoxycarbony ⁇ ) (N-FMOC).
  • a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9-fluorenylmethoxycarbony ⁇ ) (N-FMOC).
  • t-BOC N-tert-butoxycarbonyl
  • N-FMOC N-(9-fluorenylmethoxycarbony ⁇
  • the mixture of blocked insulin drugs having a dispersity coefficient greater than 10,000 may be reacted with the mixture of activated oligomers having a dispersity coefficient greater than 10,000 to provide a mixture of insulin drag-oligomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues.
  • the insulin drag-oligomer conjugates may be de-blocked as will be understood by those skilled in the art. If necessary, the mixture of insulin drag-oligomer conjugates may then be separated as described above to provide a mixture of insulin drag-oligomer conjugates having a dispersity coefficient greater than 10,000. Alternatively, the mixture of insulin drug-oligomer conjugates may be separated prior to de-blocking.
  • Mixtures of insulin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably have improved properties when compared with those of polydispersed mixtures.
  • a mixture of insulin drag-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of insulin drug-oligomer conjugates having the same number average molecular weight as the mixture of insulin drug-oligomer conjugates having a dispersity coefficient greater than 10,000.
  • the number average molecular weight of the mixture of insulin drag-oligomer conjugates having a dispersity coefficient greater than 10,000 and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography such as gel permeation chromatography as described, for example, in H.R. Allcock & F.W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991).
  • a mixture of insulin drag-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of insulin drag-oligomer conjugates having the same number average molecular weight as the mixture of insulin drug-oligomer conjugates having a dispersity coefficient greater than 10,000.
  • the number average molecular weight of the mixture of insulin drug- oligomer conjugates having a dispersity coefficient greater than 10,000 and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • the in vitro activity of a particular mixture may be measured by various methods, as will be understood by those skilled in the art.
  • the in vitro activity is measured using a Cytosensor® Microphysiometer commercially available from Molecular Devices Corporation of Sunnyvale, California.
  • the microphysiometer monitors small changes in the rates of extracellular acidification in response to a drag being added to cultured cells in a transwell. This response is proportional to the activity of the molecule under study.
  • the in vitro activity of the mixture of insulin drag-oligomer conjugates having a dispersity coefficient greater than 10,000 is at least about 5 percent greater than the in vitro activity of the polydispersed mixture. More preferably, the in vitro activity of the mixture of insulin drag-oligomer conjugates having a dispersity coefficient greater than 10,000 is at least about 10 percent greater than the in vitro activity of the polydispersed mixture.
  • a mixture of insulin drag-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably has an increased resistance to degradation by chymotrypsin when compared to the resistance to degradation by chymotrypsin of a polydispersed mixture of insulin drag-oligomer conjugates having the same number average molecular weight as the mixture of insulin drag-oligomer conjugates having a dispersity coefficient greater than 10,000.
  • Resistance to chymotrypsin corresponds to the percent remaining when the molecule to be tested is digested in chymotrypsin using a procedure similar to the one outlined in Example 52 below.
  • the number average molecular weight of the mixture of insulin drug- oligomer conjugates having a dispersity coefficient greater than 10,000 and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • the resistance to degradation by chymotrypsin of the mixture of insulin drag-oligomer conjugates having a dispersity coefficient greater than 10,000 is at least about 10 percent greater than the resistance to degradation by chymotrypsin of the polydispersed mixture.
  • the resistance to degradation by chymotrypsin of the mixture of insulin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 is at least about 20 percent greater than the resistance to degradation by chymotrypsin of the polydispersed mixture.
  • a mixture of insulin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 preferably has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of insulin drug-oligomer conjugates having the same number average molecular weight as the mixture of insulin drag- oligomer conjugates having a dispersity coefficient greater than 10,000.
  • the number average molecular weight of the mixture of insulin drag-oligomer conjugates having a dispersity coefficient greater than 10,000 and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • the inter-subject variability may be measured by various methods as will be understood by those skilled in the art.
  • the inter-subject variability is preferably calculated as follows.
  • the area under a dose response ' curve (AUC) i.e., the area between the dose-response curve and a baseline value
  • AUC dose response ' curve
  • the average AUC for all subjects is determined by summing the AUCs of each subject and dividing the sum by the number of subjects.
  • the absolute value of the difference between the subject's AUC and the average AUC is then determined for each subject.
  • the absolute values of the differences obtained are then summed to give a value that represents the inter-subject variability.
  • Lower values represent lower inter-subject variabilities and higher values represent higher inter-subject variabilities.
  • the inter-subject variability of the mixture of insulin drag-oligomer conjugates having a dispersity coefficient greater than 10,000 is at least about 10 percent less than the inter- subject variability of the polydispersed mixture. More preferably, the inter-subject variability of the mixture of insulin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 is at least about 25 percent less than the inter-subject variability of the polydispersed mixture.
  • Mixtures of insulin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 according to embodiments of the present invention preferably have two or more of the above-described properties. More preferably, mixtures of insulin drag-oligomer conjugates having a dispersity coefficient greater than 10,000 according to embodiments of the present invention have three or more of the above-described properties. Most preferably, mixtures of insulin drug-oligomer conjugates having a dispersity coefficient greater than 10,000 according to embodiments of the present invention have all four of the above- described properties.
  • each conjugate includes an insulin drag coupled to an oligomer, and has the same number of polyethylene glycol subunits is provided.
  • the insulin drug is preferably insulin. More preferably, the insulin drag is human insulin.
  • the insulin drug may be selected from various insulin drugs known to those skilled in the art including, for example, proinsulin, insulin analogs, insulin fragments, and insulin fragment analogs.
  • Insulin analogs include, but are not limited to, Asp 328 human insulin, Lys 328 human insulin, Leu 328 human insulin, Val 328 human insulin, Ala 328 human insulin, Asp 328 Pro B29 human insulin, Lys B28 Pro 329 human insulin, Leu B28 Pro B29 human insulin, Val B28 Pro B29 human insulin, Ala 328 Pro B29 human insulin, as well as analogs provided using the substitution guidelines described above.
  • Insulin fragments include, but are not limited to, B22-B30 human insulin, B23-B30 human insulin, B25-B30 human insulin, B26-B30 human insulin, B27-B30 human insulin, B29-B30 human insulin, the A chain of human insulin, and the B chain of human insulin.
  • Insulin fragment analogs may be provided by substituting one or more amino acids as described above in an insulin fragment.
  • the oligomer may be various oligomers comprising a polyethylene glycol moiety as will be understood by those skilled in the art.
  • the polyethylene glycol moiety of the oligomer has at least 2, 3 or 4 polyethylene glycol subunits.
  • the polyethylene glycol moiety has at least 5 or 6 polyethylene glycol subunits and, most preferably, the polyethylene glycol moiety has at least 7 polyethylene glycol subunits.
  • the oligomer may comprise one or more other moieties as will be understood by those skilled in the art including, but not limited to, additional hydrophilic moieties, lipophilic moieties, spacer moieties, linker moieties, and terminating moieties.
  • the various moieties in the oligomer are covalently coupled to one another by either hydrolyzable or non- hydrolyzable bonds.
  • the oligomer may further comprise one or more additional hydrophilic moieties (i.e., moieties in addition to the polyethylene glycol moiety) including, but not limited to, sugars, polyalkylene oxides, and polyamine/PEG copolymers.
  • additional hydrophilic moieties i.e., moieties in addition to the polyethylene glycol moiety
  • polyethylene glycol is a polyalkylene oxide
  • the additional hydrophilic moiety may be a polyethylene glycol moiety. Adjacent polyethylene glycol moieties will be considered to be the same moiety if they are coupled by an ether bond.
  • the moiety i.e., moieties in addition to the polyethylene glycol moiety
  • oligomers according to embodiments of the present invention comprise a polyethylene glycol moiety and no additional hydrophilic moieties.
  • the oligomer may further comprise one or more lipophilic moieties as will be understood by those skilled in the art.
  • the lipophilic moiety is preferably a saturated or unsaturated, linear or branched alkyl moiety or a saturated or unsaturated, linear or branched fatty acid moiety.
  • the lipophilic moiety is an alkyl moiety, it is preferably a linear, saturated or unsaturated alkyl moiety having 1 to 28 carbon atoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms.
  • the lipophilic moiety is a fatty acid moiety, it is preferably a natural fatty acid moiety that is linear, saturated or unsaturated, having 2 to 18 carbon atoms.
  • the fatty acid moiety has 3 to 14 carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5 or 6 carbon atoms.
  • the oligomer may further comprise one or more spacer moieties as will be understood by those skilled in the art. Spacer moieties may, for example, be used to separate a hydrophilic moiety from a lipophilic moiety, to separate a lipophilic moiety or hydrophilic moiety from the insulin drug, to separate a first hydrophilic or lipophilic moiety from a second hydrophilic or lipophilic moiety, or to separate a hydrophilic moiety or lipophilic moiety from a linker moiety. Spacer moieties are preferably selected from the group consisting of sugar, cholesterol and glycerine moieties.
  • the oligomer may further comprise one or more linker moieties that are used to couple the oligomer with the insulin drug as will be understood by those skilled in the art.
  • Linker moieties are preferably selected from the group consisting of alkyl and fatty acid moieties.
  • the oligomer may further comprise one or more terminating moieties at the one or more ends of the oligomer which are not coupled to the insulin drug.
  • the terminating moiety is preferably an alkyl or alkoxy moiety, and is more preferably a lower alkyl or lower alkoxy moiety. Most preferably, the terminating moiety is methyl or methoxy. While the terminating moiety is preferably an alkyl or alkoxy moiety, it is to be understood that the terminating moiety may be various moieties as will be understood by those skilled in the art including, but not limited to, sugars, cholesterol, alcohols, and fatty acids.
  • the oligomer is preferably covalently coupled to the insulin drug.
  • the insulin drug is coupled to the oligomer utilizing a hydrolyzable bond (e.g., an ester or carbonate bond).
  • a hydrolyzable coupling may provide an insulin drug-oligomer conjugate that acts as a prodrag.
  • a hydrolyzable coupling may provide for a time-release or controlled-release effect, administering the insulin drag over a given time period as one or more oligomers are cleaved from their respective insulin drug-oligomer conjugates to provide the active drag.
  • the insulin drag is coupled to the oligomer utilizing a non-hydrolyzable bond (e.g., a carbamate, amide, or ether bond).
  • the oligomer When the oligomer is covalently coupled to the insulin drug, the oligomer further comprises one or more bonding moieties that are used to covalently couple the oligomer with the insulin drag as will be understood by those skilled in the art. Bonding moieties are preferably selected from the group consisting of covalent bond(s), ester moieties, carbonate moieties, carbamate moieties, amide moieties and secondary amine moieties. More than one moiety on the oligomer may be covalently coupled to the insulin drag.
  • the oligomer is preferably covalently coupled to the insulin drag, it is to be understood that the oligomer may be non-covalently coupled to the insulin drug to form a non-covalently conjugated insulin drag-oligomer complex.
  • non-covalent couplings include, but are not limited to, hydrogen bonding, ionic bonding, Van der Waals bonding, and micellular or liposomal encapsulation.
  • oligomers may be suitably constructed, modified and/or appropriately functionalized to impart the ability for non-covalent conjugation in a selected manner (e.g., to impart hydrogen bonding capability), as will be understood by those skilled in the art.
  • oligomers may be derivatized with various compounds including, but not limited to, amino acids, oligopeptides, peptides, bile acids, bile acid derivatives, fatty acids, fatty acid derivatives, salicylic acids, salicylic acid derivatives, aminosalicylic acids, and aminosalicylic acid derivatives.
  • the resulting oligomers can non-covalently couple (complex) with drag molecules, pharmaceutical products, and/or pharmaceutical excipients.
  • the resulting complexes preferably have balanced lipophilic and hydrophilic properties.
  • oligomers may be derivatized with amine and/or alkyl amines. Under suitable acidic conditions, the resulting oligomers can form non- covalently conjugated complexes with drug molecules, pharmaceutical products and/or pharmaceutical excipients.
  • the products resulting from such complexation preferably have balanced lipophilic and hydrophilic properties.
  • More than one oligomer may be coupled to the insulin drag.
  • the oligomers in the plurality are preferably the same. However, it is to be understood that the oligomers in the plurality may be different from one another, or, alternatively, some of the oligomers in the plurality may be the same and some may be different.
  • all of the bonds coupling the plurality of oligomers to the insulin drug may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more of the oligomers is rapidly removed from the insulin drug by hydrolysis in the body and one or more of the oligomers is slowly removed from the insulin drug by hydrolysis in the body.
  • the oligomer may be coupled to the insulin drag at various nucleophilic residues of the insulin drug including, but not limited to, nucleophilic hydroxyl functions and/or amino functions.
  • a nucleophilic hydroxyl function may be found, for example, at serine and/or tyrosine residues, and a nucleophilic amino function may be found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini of the polypeptide.
  • the coupling preferably forms a secondary amine.
  • the oligomer may be coupled to an amino functionality of the insulin, including the amino functionality of Gly A1 , the amino functionality of Phe 31 , and the amino functionality of Lys 329 .
  • the oligomer When one oligomer is coupled to the human insulin, the oligomer is preferably coupled to the amino functionality of Lys 329 . When two oligomers are coupled to the human insulin, the oligomers are preferably coupled to the amino functionality of Phe 31 and the amino functionality of Lys 329 . While more than one oligomer may be coupled to the human insulin, a higher activity (improved glucose lowering ability) is observed for the mono-conjugated human insulin.
  • Mixtures of insulin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits may be synthesized by various methods. For example, a mixture of oligomers consisting of carboxyhc acid and polyethylene glycol where each oligomer in the mixture has the same number of polyethylene glycol subunits is synthesized by contacting a mixture of carboxyhc acid with a mixture of polyethylene glycol where each polyethylene glycol molecule in the mixture has the same number of polyethylene glycol subunits under conditions sufficient to provide a mixture of oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits.
  • the oligomers of the mixture where each oligomer in the mixture has the same number of polyethylene glycol subunits are then activated so that they are capable of reacting with an insulin drug to provide an insulin drug-oligomer conjugate.
  • a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 3 and described in Examples 11-18 hereinbelow.
  • Another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 4 and described in Examples 19-24 hereinbelow.
  • Still another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 5 and described in Examples 25-29 hereinbelow.
  • Yet another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 6 and described in Examples 30-31 hereinbelow.
  • Another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 7 and described in Examples 32-37 hereinbelow.
  • Still another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 8 and described in Example 38 hereinbelow.
  • Yet another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 9 and described in Example 39 hereinbelow.
  • Another embodiment of a synthesis route for providing a mixture of activated oligomers having a mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is illustrated in Figure 10 and described in Example 40 hereinbelow.
  • reaction conditions e.g., selected molar ratios, solvent mixtures and/or pH
  • the mixture of insulin drugs is a mixture of conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits.
  • conjugation at the amino functionality of lysine may be suppressed by maintaining the pH of the reaction solution below the pK a of lysine.
  • the mixture of insulin drug-oligomer conjugates may be separated and isolated utilizing, for example, HPLC as described in Example 50 hereinbelow to provide a mixture of insulin drug-oligomer conjugates, for example mono-, di-, or tri-conjugates, where each conjugate in the mixture has the same number of polyethylene glycol subunits.
  • the degree of conjugation e.g., whether the isolated molecule is a mono-, di-, or tri-conjugate
  • a particular isolated conjugate may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, mass spectroscopy.
  • the particular conjugate structure (e.g., whether the oligomer is at Gly ⁇ , Phe 31 or Lys B29 of a human insulin monoconjugate) may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, sequence analysis, peptide mapping, selective enzymatic cleavage, and/or endopeptidase cleavage.
  • one or more of the reaction sites on the insulin drag may be blocked by, for example, reacting the insulin drag with a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9-fluorenylmethoxycarbonyl) (N-FMOC).
  • a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9-fluorenylmethoxycarbonyl) (N-FMOC).
  • t-BOC N-tert-butoxycarbonyl
  • N-FMOC N-(9-fluorenylmethoxycarbonyl)
  • the mixture of blocked insulin drags may be reacted with the mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits to provide a mixture of insulin drug-oligomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues.
  • the insulin drug-oligomer conjugates may be de-blocked as will be understood by those skilled in the art. If necessary, the mixture of insulin drag-oligomer conjugates may then be separated as described above to provide a mixture of insulin drag-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits. Alternatively, the mixture of insulin drug-oligomer conjugates may be separated prior to de-blocking.
  • Mixtures of insulin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits according to these embodiments of the present invention preferably have improved properties when compared with those of polydispersed mixtures.
  • a mixture of insulin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits preferably has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of insulin drug-oligomer conjugates having the same number average molecular weight as the mixture of insulin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits.
  • the number average molecular weight of the mixture of insulin drag- oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography such as gel permeation chromatography as described, for example, in H.R. Allcock & F.W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991).
  • a mixture of insulin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits preferably has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of insulin drag-oligomer conjugates having the same number average molecular weight as the mixture of insulin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits.
  • the number average molecular weight of the mixture of insulin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion cliromatography.
  • the in vitro activity of a particular mixture may be measured by various methods, as will be understood by those skilled in the art.
  • the in vitro activity is measured using a Cytosensor® Microphysiometer commercially available from Molecular Devices Corporation of Sunnyvale, California. The microphysiometer monitors small changes in the rates of extracellular acidification in response to a drug being added to cultured cells in a transwell. This response is proportional to the activity of the molecule under study.
  • the in vitro activity of the mixture of insulin drag-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits is at least about 5 percent greater than the in vitro activity of the polydispersed mixture. More preferably, the in vitro activity of the mixture of insulin drag-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits is at least about 10 percent greater than the in vitro activity of the polydispersed mixture.
  • a mixture of insulin drag-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits preferably has an increased resistance to degradation by chymotrypsin when compared to the resistance to degradation by chymotrypsin of a polydispersed mixture of insulin drug-oligomer conjugates having the same number average molecular weight as the mixture of insulin drag-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits. Resistance to chymotrypsin corresponds to the percent remaining when the molecule to be tested is digested in chymotrypsin using a procedure similar to the one outlined in Example 52 below.
  • the number average molecular weight of the mixture of insulin drag-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion cliromatography.
  • the resistance to degradation by chymotrypsin of the mixture of insulin drag-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits is at least about 10 percent greater than the resistance to degradation by chymotrypsin of the polydispersed mixture.
  • the resistance to degradation by chymotrypsin of the mixture of insulin drag-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits is at least about 20 percent greater than the resistance to degradation by chymotrypsin of the polydispersed mixture.
  • a mixture of insulin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits preferably has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of insulin drug-oligomer conjugates having the same number average molecular weight as the mixture of insulin drag-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits.
  • the number average molecular weight of the mixture of insulin drug- oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • the inter-subject variability may be measured by various methods as will be understood by those skilled in the art.
  • the inter-subject variability is preferably calculated as follows.
  • the area under a dose response curve (AUC) i.e., the area between the dose- response curve and a baseline value) is determined for each subject.
  • the average AUC for all subjects is determined by summing the AUCs of each subject and dividing the sum by the number of subjects.
  • the inter-subject variability of the mixture of insulin drag- oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits is at least about 10 percent less than the inter-subject variability of the polydispersed mixture. More preferably, the inter-subject variability of the mixture of insulin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits is at least about 25 percent less than the inter-subject variability of the polydispersed mixture.
  • Mixtures of insulin drag-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits according to embodiments of the present invention preferably have two or more of the above-described properties. More preferably, mixtures of insulin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits according to embodiments of the present invention have three or more of the above-described properties. Most preferably, mixtures of insulin drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits according to embodiments of the present invention have all four of the above-described properties.
  • each conjugate has the same molecular weight and has the structure of Formula A:
  • B is a bonding moiety
  • L is a linker moiety
  • G, G' and G" are individually selected spacer moieties
  • R is a lipophilic moiety and R is a polyalkylene glycol moiety, or R' is the lipophilic moiety and R is the polyalkylene glycol moiety; T is a terminating moiety; j, k, m and n are individually 0 or 1; and p is an integer between 1 and the number of nucleophilic residues on the insulin drag.
  • the insulin drug is preferably insulin. More preferably, the insulin drag is human insulin. However, it is to be understood that the insulin drag may be selected from various insulin drugs known to those skilled in the art including, for example, proinsulin, insulin analogs, insulin fragments, and insulin fragment analogs. Insulin analogs include, but are not limited to, Asp 328 human insulin, Lys 328 human insulin, Leu 328 human insulin, Val 328 human insulin, Ala 328 human insulin, Asp 328 Pro B29 human insulin, Lys B28 Pro B29 human insulin, Leu 328 Pro 329 human insulin, Val B28 Pro B29 human insulin, Ala B28 Pro 329 human insulin, as well as analogs provided using the substitution guidelines described above.
  • Insulin fragments include, but are not limited to, B22-B30 human insulin, B23-B30 human insulin, B25-B30 human insulin, B26-B30 human insulin, B27-B30 human insulin, B29-B30 human insulin, the A chain of human insulin, and the B chain of human insulin.
  • Insulin fragment analogs may be provided by substituting one or more amino acids as described above in an insulin fragment.
  • the polyalkylene glycol moiety of the oligomer preferably has at least 2, 3 or 4 polyalkylene glycol subunits. More preferably, the polyalkylene glycol moiety has at least 5 or 6 polyalkylene glycol subunits and, most preferably, the polyethylene glycol moiety has at least 7 polyalkylene glycol subunits.
  • the polyalkylene glycol moiety is preferably a lower polyalkylene glycol moiety such as a polyethylene glycol moiety, a polypropylene glycol moiety, or a polybutylene glycol moiety. More preferably, the polyalkylene glycol moiety is a polyethylene glycol moiety or a polypropylene glycol moiety.
  • the polyalkylene glycol moiety is a polyethylene glycol moiety.
  • the polyalkylene glycol moiety is a polypropylene glycol moiety, the moiety preferably has a uniform (i.e., not random) structure.
  • An exemplary polypropylene glycol moiety having a uniform structure is as follows:
  • This uniform polypropylene glycol structure may be described as having only one methyl substituted carbon atom adjacent each oxygen atom in the polypropylene glycol chain.
  • Such uniform polypropylene glycol moieties may exhibit both lipophilic and hydrophilic characteristics and thus be useful in providing amphiphilic insulin drag-oligomer conjugates without the use of lipophilic polymer moieties.
  • coupling the secondary alcohol moiety of the polypropylene glycol moiety with an drug may provide the insulin drug (e.g., human insulin) with improved resistance to degradation caused by enzymes such as trypsin and chymotrypsin found, for example, in the gut.
  • insulin drug e.g., human insulin
  • enzymes such as trypsin and chymotrypsin found, for example, in the gut.
  • Uniform polypropylene glycol according to embodiments of the present invention is preferably synthesized as illustrated in Figures 11 through 13, which will now be described.
  • 1,2-propanediol 53 is reacted with a primary alcohol blocking reagent to provide a secondary alcohol extension monomer 54.
  • the primary alcohol blocking reagent may be various primary alcohol blocking reagents as will be understood by those skilled in the art including, but not limited to, silylchloride compounds such as t- butyldiphenylsilylchloride and t-butyldimethylsilylchloride, and esterification reagents such as Ac 2 O.
  • the primary alcohol blocking reagent is a primary alcohol blocking reagent that is substantially non-reactive with secondary alcohols, such as t- butyldiphenylsilylchloride or t-butyldimethylsilylchloride.
  • the secondary alcohol extension monomer (54) may be reacted with methanesulfonyl chloride (MeSO 2 Cl) to provide a primary extension alcohol monomer mesylate 55.
  • the secondary alcohol extension monomer 54 may be reacted with a secondary alcohol blocking reagent to provide compound 56.
  • the secondary alcohol blocking reagent may be various secondary alcohol blocking reagents as will be understood by those skilled in the art including, but not limited to, benzyl chloride.
  • the compound 56 may be reacted with a Bi de-blocking reagent to remove the blocking moiety B 1 and provide a primary alcohol extension monomer 57.
  • the Bi de-blocking reagent may be selected from various de-blocking reagents as will be understood by one skilled in the art.
  • the Bi de-blocking reagent is a de- esterification reagent, such as a base (e.g., potassium carbonate).
  • the Bi de-blocking reagent is preferably tetrabutylammonium fluoride (TBAF).
  • TBAF tetrabutylammonium fluoride
  • the primary alcohol extension monomer 57 may be reacted with methane sulfonyl chloride to provide a secondary alcohol extension monomer mesylate 58.
  • the primary alcohol extension monomer 54 and the secondary alcohol extension monomer 57 may be capped as follows.
  • the secondary alcohol extension monomer 54 may be reacted with a capping reagent to provide a compound 59.
  • the capping reagent may be various capping reagents as will be understood by those skilled in the art including, but not limited to, alkyl halides such as methyl chloride.
  • the compound 59 may be reacted with a Bi de-blocking agent as described above to provide a primary alcohol capping monomer 60.
  • the primary alcohol capping monomer 60 may be reacted with methane sulfonyl chloride to provide the secondary alcohol capping monomer mesylate 61.
  • the primary alcohol extension monomer 57 may be reacted with a capping reagent to provide a compound 62.
  • the capping reagent may be various capping reagents as described above.
  • the compound 62 may be reacted with a B 2 de-blocking reagent to remove the blocking moiety B 2 and provide a secondary alcohol capping monomer 63.
  • the B de-blocking reagent may be various deblocking agents as will be understood by those skilled in the art including, but not limited to, H 2 in the presence of a palladium/activated carbon catalyst.
  • the secondary alcohol capping monomer may be reacted with methanesulfonyl chloride to provide a primary alcohol capping monomer mesylate 64. While the embodiments illustrated in Figure 11 show the synthesis of capping monomers, it is to be understood that similar reactions may be performed to provide capping polymers.
  • chain extensions may be effected by reacting a primary alcohol extension mono- or poly-mer such as the primary alcohol extension monomer 57 with a primary alcohol extension mono- or poly-mer mesylate such as the primary alcohol extension monomer mesylate 55 to provide various uniform polypropylene chains or by reacting a secondary alcohol extension mono- or poly-mer such as the secondary alcohol extension monomer 54 with a secondary alcohol extension mono-or poly-mer mesylate such as the secondary alcohol extension monomer mesylate 58.
  • the primary alcohol extension monomer mesylate 55 is reacted with the primary alcohol extension monomer 57 to provide a dimer compound 65.
  • the secondary alcohol extension monomer mesylate 58 may be reacted with the secondary alcohol extension monomer 54 to provide the dimer compound 65.
  • the Bi blocking moiety on the dimer compound 65 may be removed using a B] de-blocking reagent as described above to provide a primary alcohol extension dimer 66.
  • the primary alcohol extension dimer 66 may be reacted with methane sulfonyl chloride to provide a secondary alcohol extension dimer mesylate 67.
  • the B blocking moiety on the dimer compound 65 may be removed using the B 2 de-blocking reagent as described above to provide a secondary alcohol extension dimer 69.
  • the secondary alcohol extension dimer 69 may be reacted with methane sulfonyl chloride to provide a primary alcohol extension dimer mesylate 70.
  • the chain extension process may be repeated to achieve various other chain lengths.
  • the primary alcohol extension dimer 66 may be reacted with the primary alcohol extension dimer mesylate 70 to provide a tetramer compound 72.
  • a generic chain extension reaction scheme involves reacting the primary alcohol extension mono- or poly-mer 73 with the primary alcohol extension mono- or poly-mer mesylate 74 to provide the uniform polypropylene polymer 75.
  • the values of m and n may each range from 0 to 1000 or more. Preferably, m and n are each from 0 to 50.
  • An end of a primary alcohol extension mono- or poly-mer or an end of a primary alcohol extension mono- or poly-mer mesylate may be reacted with a primary alcohol capping mono- or poly-mer mesylate or a primary alcohol capping mono- or poly-mer, respectively, to provide a capped uniform polypropylene chain.
  • the primary alcohol extension dimer mesylate 70 is reacted with the primary alcohol capping monomer 60 to provide the capped/blocked primary alcohol extension trimer 71.
  • the Bi blocking moiety may be removed and the resulting capped primary alcohol extension trimer may be reacted with a primary alcohol extension mono- or poly-mer mesylate to extend the chain of the capped trimer 71.
  • An end of a secondary alcohol extension mono-or poly-mer or an end of a secondary alcohol extension mono-or poly-mer mesylate may be reacted with a secondary alcohol capping mono-or poly-mer mesylate or a secondary alcohol capping mono- or poly-mer, respectively, to provide a capped uniform polypropylene chain.
  • the secondary alcohol extension dimer mesylate 67 is reacted with the secondary alcohol capping monomer 63 to provide the capped/blocked primary alcohol extension trimer 68.
  • the B 2 blocking moiety may be removed as described above and the resulting capped secondary alcohol extension trimer may be reacted with a secondary alcohol extension mer mesylate to extend the chain of the capped trimer 68. While the syntheses illustrated in
  • Figure 12 show the reaction of a dimer with a capping monomer to provide a trimer, it is to be understood that the capping process may be performed at any point in the synthesis of a uniform polypropylene glycol moiety, or, alternatively, uniform polypropylene glycol moieties may be provided that are not capped. While the embodiments illustrated in Figure 12 show the capping of a polybutylene oligomer by synthesis with a capping monomer, it is to be understood that polybutylene oligomers of the present invention may be capped directly (i.e., without the addition of a capping monomer) using a capping reagent as described above in Figure 11.
  • Uniform polypropylene glycol moieties according to embodiments of the present invention may be coupled to an insulin drug, a lipophilic moiety such as a carboxyhc acid, and/or various other moieties by various methods as will be understood by those skilled in the art including, but not limited to, those described herein with respect to polyethylene glycol moieties.
  • the lipophilic moiety is a lipophilic moiety as will be understood by those skilled in the art.
  • the lipophilic moiety is preferably a saturated or unsaturated, linear or branched alkyl moiety or a saturated or unsaturated, linear or branched fatty acid moiety.
  • the lipophilic moiety is an alkyl moiety, it is preferably a linear, saturated or unsaturated alkyl moiety having 1 to 28 carbon atoms. More preferably, the alkyl moiety has 2 to 12 carbon atoms.
  • the lipophilic moiety is a fatty acid moiety, it is preferably a natural fatty acid moiety that is linear, saturated or unsaturated, having 2 to 18 carbon atoms. More preferably, the fatty acid moiety has 3 to 14 carbon atoms. Most preferably, the fatty acid moiety has at least 4, 5 or 6 carbon atoms.
  • the spacer moieties, G, G and G are spacer moieties as will be understood by those skilled in the art.
  • Spacer moieties are preferably selected from the group consisting of sugar, cholesterol and glycerine moieties.
  • oligomers of these embodiments do not include spacer moieties (i.e., k, m and n are preferably 0).
  • linker moiety may be used to couple the oligomer with the drag as will be understood by those skilled in the art.
  • Linker moieties are preferably selected from the group consisting of alkyl and fatty acid moieties.
  • the terminating moiety is preferably an alkyl or alkoxy moiety, and is more preferably a lower alkyl or lower alkoxy moiety. Most preferably, the terminating moiety is methyl or methoxy. While the terminating moiety is preferably an alkyl or alkoxy moiety, it is to be understood that the terminating moiety may be various moieties as will be understood by those skilled in the art including, but not limited to, sugars, cholesterol, alcohols, and fatty acids.
  • the oligomer which is represented by the bracketed portion of the structure of Formula A, is covalently coupled to the insulin drag.
  • the insulin drug is coupled to the oligomer utilizing a hydrolyzable bond (e.g., an ester or carbonate bond).
  • a hydrolyzable coupling may provide an insulin drug-oligomer conjugate that acts as a prodrug.
  • a hydrolyzable coupling may provide for a time-release or controlled-release effect, administering the insulin drag over a given time period as one or more oligomers are cleaved from their respective insulin drug-oligomer conjugates to provide the active drag.
  • the insulin drag is coupled to the oligomer utilizing a non-hydrolyzable bond (e.g., a carbamate, amide, or ether bond).
  • the bonding moiety, B may be various bonding moieties as will be understood by those skilled in the art. Bonding moieties are preferably selected from the group consisting of covalent bond(s), ester moieties, carbonate moieties, carbamate moieties, amide moieties and secondary amine moieties.
  • the variable p is an integer from 1 to the number of nucleophilic residues on the insulin drag.
  • p is greater than 1, more than one oligomer (i.e., a plurality of oligomers) is coupled to the drag.
  • the oligomers in the plurality are the same.
  • all of the bonds coupling the plurality of oligomers to the insulin drug may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more of the oligomers is rapidly removed from the insulin drag by hydrolysis in the body and one or more of the oligomers is slowly removed from the insulin drug by hydrolysis in the body.
  • the oligomer may be coupled to the insulin drug at various nucleophilic residues of the insulin drug including, but not limited to, nucleophilic hydroxyl functions and/or amino functions.
  • nucleophilic hydroxyl function may be found, for example, at serine and/or tyrosine residues
  • a nucleophilic amino function may be found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini of the polypeptide.
  • the coupling preferably forms a secondary amine.
  • the oligomer may be coupled to an amino functionality of the insulin, including the amino functionality of Gly ⁇ , the amino functionality of Phe 31 , and the amino functionality of Lys 329 .
  • the oligomer is preferably coupled to the amino functionality of Lys 329 .
  • the oligomers are preferably coupled to the amino functionality of Phe 31 and the amino functionality of Lys 329 . While more than one oligomer may be coupled to the human insulin, a higher activity (improved glucose lowering ability) is observed for the mono-conjugated human insulin.
  • Mixtures of insulin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A may be synthesized by various methods. For example, a mixture of oligomers consisting of carboxyhc acid and polyethylene glycol is synthesized by contacting a mixture of carboxyhc acid with a mixture of polyethylene glycol under conditions sufficient to provide a mixture of oligomers. The oligomers of the mixture are then activated so that they are capable of reacting with an insulin drag to provide an insulin drag-oligomer conjugate.
  • a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 3 and described in Examples 11-18 hereinbelow.
  • Another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 4 and described in Examples 19-24 hereinbelow.
  • Still another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 5 and described in Examples 25-29 hereinbelow.
  • FIG. 6 Yet another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 6 and described in Examples 30-31 hereinbelow.
  • FIG 7 Another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 7 and described in Examples 32-37 hereinbelow.
  • Still another embodiment of a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A is illustrated in Figure 8 and described in Example 38 hereinbelow.
  • the reaction conditions e.g., selected molar ratios, solvent mixtures and/or pH
  • the mixture of insulin drug-oligomer conjugates resulting from the reaction of the mixture of activated oligomers where each oligomer has the same molecular weight and has a structure of the oligomer of Formula A and the mixture of insulin drags is a mixture of conjugates where each conjugate has the same molecular weight and has the stracture Formula A.
  • conjugation at the amino functionality of lysine may be suppressed by maintaining the pH of the reaction solution below the pK a of lysine.
  • the mixture of insulin drug-oligomer conjugates may be separated and isolated utilizing, for example, HPLC as described below in Example 50 to provide a mixture of insulin drag-oligomer conjugates, for example mono-, di-, or tri-conjugates, where each conjugate in the mixture has the same number molecular weight and has the stracture of Formula A.
  • the degree of conjugation e.g., whether the isolated molecule is a mono-, di-, or tri-conjugate
  • the particular conjugate stracture e.g., whether the oligomer is at
  • Gly ⁇ , Phe 31 or Lys 329 of a human insulin monoconjugate may be determined and/or verified utilizing various techniques as will be understood by those skilled in the art including, but not limited to, sequence analysis, peptide mapping, selective enzymatic cleavage, and/or endopeptidase cleavage.
  • one or more of the reaction sites on the insulin drug may be blocked by, for example, reacting the insulin drug with a suitable blocking reagent such as N-tert-butoxycarbonyl (t-BOC), or N-(9-fluorenylmethoxycarbonyl) (N-FMOC).
  • This process may be preferred, for example, when the insulin drug is a polypeptide and it is desired to form an unsaturated conjugate (i.e., a conjugate wherein not all nucleophilic residues are conjugated) having an oligomer at the one or more N-termini of the polypeptide.
  • an unsaturated conjugate i.e., a conjugate wherein not all nucleophilic residues are conjugated
  • the mixture of blocked insulin drags may be reacted with the mixture of activated oligomers where each oligomer in the mixture has the same molecular weight and has a stracture of the oligomer of Formula A to provide a mixture of insulin drag-oligomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues.
  • the insulin drug-oligomer conjugates may be de-blocked as will be understood by those skilled in the art. If necessary, the mixture of insulin drag-oligomer conjugates may then be separated as described above to provide a mixture of insulin drag- oligomer conjugates where each conjugate in the mixture has the same number molecular weight and has the structure of Formula A. Alternatively, the mixture of insulin drag- oligomer conjugates may be separated prior to de-blocking. Mixtures of insulin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stracture of Formula A according to these embodiments of the present invention preferably have improved properties when compared with those of polydispersed mixtures.
  • a mixture of insulin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A preferably has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of insulin drag-oligomer conjugates having the same number average molecular weight as the mixture of insulin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stracture of Formula A.
  • the number average molecular weight of the mixture of insulin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography such as gel permeation cliromatography as described, for example, in H.R. Allcock & F.W. Lampe, CONTEMPORARY POLYMER CHEMISTRY 394-402 (2d. ed., 1991).
  • a mixture of insulin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stracture of Formula A preferably has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of insulin drug-oligomer conjugates having the same number average molecular weight as the mixture of insulin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stracture of Formula A.
  • the number average molecular weight of the mixture of insulin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stracture of Formula A and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • the in vitro activity of a particular mixture may be measured by various methods, as will be understood by those skilled in the art.
  • the in vitro activity is measured using a Cytosensor® Microphysiometer commercially available from Molecular Devices Corporation of Sunnyvale, California. The microphysiometer monitors small changes in the rates of extracellular acidification in response to a drag being added to cultured cells in a transwell. This response is proportional to the activity of the molecule under study.
  • the in vitro activity of the mixture of insulin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stracture of Formula A is at least about 5 percent greater than the in vitro activity of the polydispersed mixture. More preferably, the in vitro activity of the mixture of insulin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stracture of Formula A is at least about 10 percent greater than the in vitro activity of the polydispersed mixture.
  • a mixture of insulin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A preferably has an increased resistance to degradation by chymotrypsin when compared to the resistance to degradation by chymotrypsin of a polydispersed mixture of insulin drag- oligomer conjugates having the same number average molecular weight as the mixture of insulin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stracture of Formula A.
  • Resistance to chymotrypsin corresponds to the percent remaining when the molecule to be tested is digested in chymotrypsin using a procedure similar to the one outlined in Example 52 below.
  • the number average molecular weight of the mixture of insulin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • the resistance to degradation by chymotrypsin of the A mixture of insulin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A is at least about 10 percent greater than the resistance to degradation by chymotrypsin of the polydispersed mixture.
  • the resistance to degradation by chymotrypsin of the A mixture of insulin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A is at least about 20 percent greater than the resistance to degradation by chymotrypsin of the polydispersed mixture.
  • a mixture of insulin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stracture of Formula A preferably has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of insulin drug-oligomer conjugates having the same number average molecular weight as the mixture of insulin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A.
  • the number average molecular weight of the mixture of insulin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A and the number average weight of the polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • the inter-subject variability may be measured by various methods as will be understood by those skilled in the art.
  • the inter-subject variability is preferably calculated as follows.
  • the area under a dose response curve (AUC) i.e., the area between the dose-response curve and a baseline value
  • the average AUC for all subjects is determined by summing the AUCs of each subject and dividing the sum by the number of subjects.
  • the absolute value of the difference between the subject's AUC and the average AUC is then determined for each subject.
  • the absolute values of the differences obtained are then summed to give a value that represents the inter-subject variability.
  • Lower values represent lower inter-subject variabilities and higher values represent higher inter-subject variabilities.
  • the inter-subject variability of the mixture of insulin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A is at least about 10 percent less than the inter-subject variability of the polydispersed mixture.
  • the inter- subject variability of the mixture of insulin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the structure of Formula A is at least about 25 percent less than the inter-subject variability of the polydispersed mixture.
  • Mixtures of insulin drag-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stracture of Formula A according to embodiments of the present invention preferably have two or more of the above-described properties. More preferably, mixtures of insulin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stracture of Formula A according to embodiments of the present invention have three or more of the above-described properties. Most preferably, mixtures of insulin drug-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stracture of Formula A according to embodiments of the present invention have all four of the above-described properties.
  • compositions comprising a conjugate mixture according to embodiments of the present invention are also provided.
  • the mixtures of insulin drug- oligomer conjugates described above may be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (9 th Ed. 1995).
  • the mixture of insulin drag- oligomer conjugates is typically admixed with, inter alia, a pharmaceutically acceptable carrier.
  • the carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the pharmaceutical composition and should not be deleterious to the subject.
  • the carrier may be a solid or a liquid, or both, and is preferably formulated with the mixture of insulin drag-oligomer conjugates as a unit-dose formulation, for example, a tablet, which may contain from about 0.01 or 0.5% to about 95% or 99% by weight of the mixture of insulin drug-oligomer conjugates.
  • the pharmaceutical compositions may be prepared by any of the well known techniques of pharmacy including, but not limited to, admixing the components, optionally including one or more accessory ingredients.
  • compositions according to embodiments of the present invention include those suitable for oral, rectal, topical, inhalation (e.g., via an aerosol) buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, inracerebral, intraarterial, or intravenous), topical (i.e., both skin and mucosal surfaces, including airway surfaces) and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular mixture of insulin drag-oligomer conjugates which is being used.
  • buccal e.g., sub-lingual
  • vaginal e.g., parenteral (e.g., subcutaneous, intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, inracerebral, intraarterial, or intravenous)
  • parenteral e.g
  • compositions suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tables, each containing a predetermined amount of the mixture of insulin drag-oligomer conjugates; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in- water or water- in-oil emulsion.
  • Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the mixture of insulin drag-oligomer conjugates and a suitable carrier (which may contain one or more accessory ingredients as noted above).
  • the pharmaceutical composition according to embodiments of the present invention are prepared by uniformly and intimately admixing the mixture of insulin drag-oligomer conjugates with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture.
  • a tablet may be prepared by compressing or molding a powder or granules containing the mixture of insulin drug- oligomer conjugates, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing, in a suitable machine, the mixture in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s).
  • Molded tablets may be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder.
  • compositions suitable for buccal (sub-lingual) administration include lozenges comprising the mixture of insulin drag-oligomer conjugates in a flavoured base, usually sucrose and acacia or tragacanth; and pastilles comprising the mixture of insulin drug-oligomer conjugates in an inert base such as gelatin and glycerin or sucrose and acacia.
  • Pharmaceutical compositions according to embodiments of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the mixture of insulin drag-oligomer conjugates, which preparations are preferably isotonic with the blood of the intended recipient.
  • compositions may contain anti-oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended recipient.
  • Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents.
  • the compositions may be presented in unit ⁇ dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection immediately prior to use.
  • sterile liquid carrier for example, saline or water-for-injection immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • an injectable, stable, sterile composition comprising a mixture of insulin drag-oligomer conjugates in a unit dosage form in a sealed container.
  • the mixture of insulin drag-oligomer conjugates is provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject.
  • the unit dosage form typically comprises from about 10 mg to about 10 grams of the mixture of insulin drug-oligomer conjugates.
  • a sufficient amount of emulsifying agent which is physiologically acceptable may be employed in sufficient quantity to emulsify the mixture of insulin drag-oligomer conjugates in an aqueous carrier.
  • One such useful emulsifying agent is phosphatidyl choline.
  • compositions suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the mixture of insulin drug-oligomer conjugates with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.
  • Pharmaceutical compositions suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
  • compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
  • Compositions suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6): 318 (1986)) and typically take the form of an optionally buffered aqueous solution of the mixture of insulin drag-oligomer conjugates.
  • Suitable formulations comprise citrate or bis ⁇ tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2M active ingredient.
  • any mixture of insulin drag-oligomer conjugates will vary somewhat from mixture to mixture, and subject to subject, and will depend upon factors such as the age and condition of the subject and the route of delivery. Such dosages can be determined in accordance with routine pharmacological procedures known to those skilled in the art. As a general proposition, a dosage from about 0.1 to about 50 mg/kg will have therapeutic efficacy, with all weights being calculated based upon the weight of the mixture of insulin drug-oligomer conjugates. Toxicity concerns at the higher level may restrict intravenous dosages to a lower level such as up to about 10 mg/kg, with all weights being calculated based upon the weight of the active base.
  • a dosage from about 10 mg/kg to about 50 mg/kg may be employed for oral administration.
  • a dosage from about 0.5 mg/kg to 5 mg/kg may be employed for intramuscular injection.
  • the frequency of administration is usually one, two, or three times per day or as necessary to control the condition.
  • the drug-oligomer conjugates may be administered by continuous infusion. The duration of treatment depends on the type of insulin deficiency being treated and may be for as long as the life of the subject.
  • a substantially monodispersed mixture of polymers comprising polyethylene glycol moieties is provided as illustrated in reaction 1:
  • R 1 is H or a lipophilic moiety.
  • R 1 is preferably H, alkyl, aryl alkyl, an aromatic moiety, a fatty acid moiety, an ester of a fatty acid moiety, cholesteryl, or adamantyl.
  • R 1 is more preferably H, lower alkyl, or an aromatic moiety.
  • R 1 is most preferably H, methyl, or benzyl.
  • n is from 1 to 25.
  • n is from 1 to 6.
  • X* is a positive ion.
  • X + is any positive ion in a compound, such as a strong base, that is capable of ionizing a hydroxyl moiety on PEG.
  • R 2 is H or a lipophilic moiety.
  • R 2 is preferably linear or branched alkyl, aryl alkyl, an aromatic moiety, a fatty acid moiety, or an ester of a fatty acid moiety.
  • R 2 is more preferably lower alkyl, benzyl, a fatty acid moiety having 1 to 24 carbon atoms, or an ester of a fatty acid moiety having 1 to 24 carbon atoms.
  • R 2 is most preferably methyl, a fatty acid moiety having 1 to 18 carbon atoms or an ethyl ester of a fatty acid moiety having 1 to 18 carbon atoms.
  • m is from 1 to 25. Preferably m is from 1 to 6.
  • Ms is a mesylate moiety (i.e., CH 3 S(O 2 )-).
  • a mixture of compounds having the structure of Formula I is reacted with a mixture of compounds having the structure of Formula II to provide a mixture of polymers comprising polyethylene glycol moieties and having the structure of Formula III.
  • the mixture of compounds having the stracture of Formula I is a substantially monodispersed mixture.
  • at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compounds of Formula I have the same molecular weight, and, more preferably, the mixture of compounds of Formula I is a monodispersed mixture.
  • the mixture of compounds of Formula II is a substantially monodispersed mixture.
  • At least about 96, 97, 98 or 99 percent of the compounds in the mixture of compounds of Formula II have the same molecular weight, and, more preferably, the mixture of compounds of Formula II is a monodispersed mixture.
  • the mixture of compounds of Formula III is a substantially monodispersed mixture.
  • at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compound of Formula III have the same molecular weight. More preferably, the mixture of compounds of Formula III is a monodispersed mixture.
  • Reaction 1 is preferably performed between about 0°C and about 40°C, is more preferably performed between about 15°C and about 35°C, and is most preferably performed at room temperature (approximately 25°C). Reaction 1 may be performed for various periods of time as will be understood by those skilled in the art. Reaction 1 is preferably performed for a period of time between about 0.25, 0.5 or 0.75 hours and about 2, 4 or 8 hours.
  • Reaction 1 is preferably carried out in an aprotic solvent such as, but not limited to, N,N-dimethylacetamide (DMA), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexamethylphosphoric triamide, tetrahydrofuran (THF), dioxane, diethyl ether, methyl t-butyl ether (MTBE), toluene, benzene, hexane, pentane, N-methylpyrollidinone, tetrahydronaphthalene, decahydronaphthalene, 1,2-dichlorobenzene, l,3-dimethyl-2- imidazolidinone, or a mixture thereof. More preferably, the solvent is DMF, DMA or toluene.
  • DMA N,N-dimethylacetamide
  • DMF N,N-dimethylformamide
  • DMSO dimethyl sulfoxide
  • the molar ratio of the compound of Formula I to the compound of Formula II is preferably greater than about 1:1. More preferably, the molar ratio is at least about 2:1.
  • R 1 and X + are as described above and the mixture of compounds of Formula IV is substantially monodispersed; preferably, at least about 96, 91, 98 or 99 percent of the compounds in the mixture of compounds of Formula IV have the same molecular weight; and, more preferably, the mixture of compounds of Formula IV is a monodispersed mixture.
  • Various compounds capable of ionizing a hydroxyl moiety on the PEG moiety of the compound of Formula IV will be understood by those skilled in the art.
  • the compound capable of ionizing a hydroxyl moiety is preferably a strong base. More preferably, the compound capable of ionizing a hydroxyl moiety is selected from the group consisting of sodium hydride, potassium hydride, sodium t-butoxide, potassium t-butoxide, butyl lithium (BuLi), and lithium diisopropylamine.
  • the compound capable of ionizing a hydroxyl moiety is more preferably sodium hydride.
  • the molar ratio of the compound capable of ionizing a hydroxyl moiety on the PEG moiety of the compound of Formula IV to the compound of Formula IV is preferably at least about 1:1, and is more preferably at least about 2:1.
  • Reaction 2 is preferably performed between about 0°C and about 40°C, is more preferably performed between about 0°C and about 35°C, and is most preferably performed between about 0°C and room temperature (approximately 25°C).
  • Reaction 2 may be performed for various periods of time as will be understood by those skilled in the art. Reaction 2 is preferably performed for a period of time between about 0.25, 0.5 or 0.75 hours and about 2, 4 or 8 hours.
  • Reaction 2 is preferably carried out in an aprotic solvent such as, but not limited to, N,N-dimethylacetamide (DMA), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexamethylphosphoric triamide, tetrahydrofuran (THF), dioxane, diethyl ether, methyl t-butyl ether (MTBE), toluene, benzene, hexane, pentane, N-methylpyrollidinone, dichloromethane, chloroform, tetrahydronaphthalene, decahydronaphthalene, 1,2- dichlorobenzene, l,3-dimethyl-2-imidazolidinone, or a mixture thereof. More preferably, the solvent is DMF, dichloromethane or toluene.
  • DMA N,N-dimethylacetamide
  • DMF N,N-dimethylformamide
  • R and Ms are as described above and the compound of Formula V is present as a substantially monodispersed mixture of compounds of Formula V; preferably at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compounds of Formula V have the same molecular weight; and, more preferably, the mixture of compounds of Formula V is a monodispersed mixture.
  • Q is a halide, preferably chloride or fluoride.
  • CH 3 S(O 2 )Q is methanesulfonyl halide.
  • the methanesulfonyl halide is preferably methanesulfonyl chloride or methanesulfonyl fluoride. More preferably, the methanesulfonyl halide is methanesulfonyl chloride.
  • the molar ratio of the methane sulfonyl halide to the compound of Formula V is preferably greater than about 1:1, and is more preferably at least about 2 : 1.
  • Reaction 3 is preferably performed between about -10°C and about 40°C, is more preferably performed between about 0°C and about 35°C, and is most preferably performed between about 0°C and room temperature (approximately 25°C).
  • Reaction 3 may be performed for various periods of time as will be understood by those skilled in the art. Reaction 3 is preferably performed for a period of time between about 0.25, 0.5 or 0.75 hours and about 2, 4 or 8 hours.
  • Reaction 3 is preferably carried out in the presence of an aliphatic amine including, but not limited to, monomethylamhie, dimethylamine, trimethylamine, monoethylamine, diethylamine, triethylamine, monoisopropylamine, diisopropylamine, mono-n-butylamine, di- n-butylamine, tri-n-butylamine, monocyclohexylamine, dicyclohexylamine, or mixtures thereof. More preferably, the aliphatic amine is a tertiary amine such as triethylamine.
  • the compounds of Formula V are PEG or mPEG compounds, respectively, which are commercially available from Aldrich of Milwaukee, Wisconsin; Fluka of Switzerland, and/or TCI America of Portland, Oregon.
  • R is a lipophilic moiety such as, for example, higher alkyl, fatty acid, an ester of a fatty acid, cholesteryl, or adamantyl
  • the compounds of Formula V may be provided by various methods as will be understood by those skilled in the art.
  • the compounds of Formula V are preferably provided as follows:
  • R 2 is a lipophilic moiety, preferably higher alkyl, fatty acid ester, cholesteryl, or adamantyl, more preferably a lower alkyl ester of a fatty acid, and most preferably an ethyl ester of a fatty acid having from 1 to 18 carbon atoms.
  • R 3 is H, benzyl, trityl, tetrahydropyran, or other alcohol protecting groups as will be understood by those skilled in the art.
  • X + is a positive ion as described above with respect to XT 1" .
  • a mixture of compounds of Formula VI is reacted with a mixture of compounds of Formula VII under reaction conditions similar to those described above with reference to reaction 1.
  • the mixture of compounds of Formula VI is a substantially monodispersed mixture.
  • at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compounds of Formula VI have the same molecular weight.
  • the mixture of compounds of Formula VI is a monodispersed mixture.
  • the mixture of compounds of Formula VII is a substantially monodispersed mixture.
  • Formula VII have the same molecular weight. More preferably, the mixture of compounds of Formula VII is a monodispersed mixture.
  • the compound of Formula VIII may be hydrolyzed to convert the R 3 moiety into an alcohol by various methods as will be understood by those skilled in the art.
  • R is benzyl or trityl
  • the hydrolysis is preferably performed utilizing H 2 in the presence of a palladium-charcoal catalyst as is known by those skilled in the art.
  • R 3 is H
  • reaction 5 is unnecessary.
  • the compound of Formula VI may be commercially available or be provided as described above with reference to reaction 3.
  • the compound of Formula VII may be provided as described above with reference to reaction 2.
  • Substantially monodispersed mixtures of polymers comprising PEG moieties and having the structure of Formula III above can further be reacted with other substantially monodispersed polymers comprising PEG moieties in order to extend the PEG chain.
  • the following scheme may be employed: O
  • Ms, m and n are as described above with reference to reaction 1; p is similar to n and m, and X 2 is similar to X as described above with reference to reaction 1.
  • Q is as described above with reference to reaction 3.
  • R 2 is as described above with reference to reaction 1 and is preferably lower alkyl.
  • R 1 is H.
  • Reaction 6 is preferably performed in a manner similar to that described above with reference to reaction 3.
  • Reaction 7 is preferably performed in a manner similar to that described above with reference to reaction 1.
  • at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compounds of Formula III have the same molecular weight, and, more preferably, the mixture of compounds of Formula III is a monodispersed mixture.
  • the mixture of compounds of Formula X is a substantially monodispersed mixture.
  • at least about 96, 97, 98 or 99 percent of the compounds in the mixture of compounds of Formula X have the same molecular weight, and, more preferably, the mixture of compounds of Formula X is a monodispersed mixture.
  • a process according to embodiments of the present invention is illustrated by the scheme shown in Figure 1, which will now be described.
  • the synthesis of substantially monodispersed polyethylene glycol-containing oligomers begins by the preparation of the monobenzyl ether (1) of a substantially monodispersed polyethylene glycol.
  • the mesylate (5) of this alcohol may be prepared by addition of methanesulfonyl chloride and used as the electrophile in the reaction with the sodium salt of the monomethyl ether of a substantially monodispersed polyethylene glycol derivative, thereby extending the polyethylene glycol portion of the oligomer to the desired length, obtaining the elongated ester (6).
  • the ester may be hydrolyzed to the acid (7) in aqueous base and transformed into the activated ester (8) by reaction with a carbodiimide and N-hydroxysuccinimide.
  • oligomer illustrated in Figure 1 is activated using N-hydroxysuccinimide
  • various other reagents may be used to activate oligomers of the present invention including, but not limited to, active phenyl chloroformates such as r ⁇ r ⁇ -nitrophenyl chloroformate, phenyl chloroformate, 3,4-phenyldichloroformate, and 3,4-phenyldichloroformate; tresylation; and acetal formation.
  • q is from 1 to 24.
  • q is from 1 to 18, and q is more preferably from 4 to 16.
  • R 4 is a moiety capable of undergoing hydrolysis to provide the carboxyhc acid.
  • R is preferably lower alkyl and is more preferably ethyl.
  • the variables n and m are as described above with reference to reaction 1.
  • reaction mixture was diluted with another 75 mL of dichloromethane and washed successively with saturated NaHCO 3 , water and brine. The organics were dried over Na 2 SO 4 , filtered and concentrated in vacuo to give a non-polydispersed mixture of compounds 9 as a clear oil (5.3 lg, 86%).
  • the crude product mixture was purified via flash chromatography (silica gel, gradient elution: ethyl acetate to 9/1 ethyl acetate/methanol) to yield 8.099 g (70 %) of non-polydispersed 16 as a yellow oil.
  • Ethyl 6-methylsulfonyloxyhexanoate A solution of non-polydispersed ethyl 6-hydroxyhexanoate (50.76 ml, 50.41 g, 227 mmol) in dry dichloromethane (75 ml) was chilled in a ice bath and placed under a nitrogen atmosphere. Triethylamine (34.43 ml, 24.99 g, 247 mmol) was added. A solution of methanesulfonyl chloride (19.15 ml, 28.3 g, 247 mmol) in dry dichloromethane (75 ml) was added dropwise from an addition funnel.
  • the non-polydispersed mesylate 17 (19.21 g, 80.6 mmol) in 80 ml dry toluene was added to the NaH/alcohol mixture, and the combined solutions were stirred at room temperature for three days.
  • the reaction mixture was quenched with 50 ml methanol and filtered through basic alumina.
  • the filtrate was concentrated in vacuo and purified by flash chromatography (silica gel, gradient elution: 3/1 ethyl acetate/hexanes to ethyl acetate) to yield the non-polydispersed product as a pale yellow oil (16.52 g, 44 %).
  • Non-polydispersed benzyl ether 18 (1.03 g, 2.0 mmol) was dissolved in 25 ml ethanol. To this solution was added 270 mg 10 % Pd/C, and the mixture was placed under a hydrogen atmosphere and stirred for four hours, at which time TLC showed the complete disappearance of the starting material. The reaction mixture was filtered through Celite 545 to remove the catalyst, and the filtrate was concentrated in vacuo to yield the non- polydispersed title compound as a clear oil (0.67 g, 79 %). FAB MS: m/e 425 (M+H), 447 (M+Na). Example 15
  • the non-polydispersed alcohol 19 (0.835 g, 1.97 mmol) was dissolved in 3.5 ml dry dichloromethane and placed under a nitrogen atmosphere. Triethylamine (0.301 ml, 0.219 g, 2.16 mmol) was added and the mixture was chilled in an ice bath. After two minutes, the methanesulfonyl chloride (0.16 ml, 0.248 g, 2.16 mmol) was added. The mixture was stirred for 15 minutes at 0 C, then at room temperature for two hours.
  • Example 17 6-(2 ⁇ 2-[2-(2- ⁇ 2-[2-(2-methoxyethoxy)ethoxy]-ethoxy ⁇ - ethoxy)-ethoxy]-ethoxy ⁇ -ethoxy)-hexanoic acid (22)
  • Non-polydispersed ester 21 (0.25 g, 0.46 mmol) was stirred for 18 hours in 0.71 ml of I N NaOH. After 18 hours, the mixture was concentrated in vacuo to remove the alcohol and the residue dissolved in a further 10 ml of water. The aqueous solution was acidified to pH 2 with 2 N HCl and the product was extracted into dichloromethane (30 ml x 2).
  • Non-polydispersed acid 22 (0.209 g, 0.42 mmol) were dissolved in 4 ml of dry dichloromethane and added to a dry flask already containing NHS (N-hydroxysuccinimide) (57.8 mg, 0.502 mmol) and EDC (l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (98.0 mg, 0.502 mmol) under a N 2 atmosphere.
  • NHS N-hydroxysuccinimide
  • EDC l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • the crude reaction mixture was filtered through Celite (washed CH 2 C1 2 -200 mL), then washed with H 2 O (300 mL), 5% NaHCO 3 (300 mL), H O (300 mL), sat. NaCl (300 mL), dried MgSO 4 , and evaporated to dryness.
  • the oil was then placed on a vacuum line for ⁇ 2h to ensure dryness and afforded the non-polydispersed title compound as a yellow oil (29.15 g, 80% yield).
  • Example 20 Heptaethylene glycol monomethyl ether (25) To a solution of non-polydispersed tetraethylene glycol (51.5 g, 0.27 mol) in THF (IL) was added potassium t-butoxide (14.8 g, 0.13 mol, small portions over -30 min). The reaction mixture was then stirred for lh and then 24 (29.15 g, 0.12 mol) dissolved in THF (90 mL) was added dropwise and the reaction mixture was stirred overnight. The crude reaction mixture was filtered through Celite (washed CH 2 C1 2 , ⁇ 200 mL) and evaporated to dryness.
  • the oil was then dissolved in HCl (250 mL, 1 N) and washed with ethyl acetate (250 mL) to remove excess 24. Additional washings of ethyl acetate (125 mL) may be required to remove remaining 24.
  • the aqueous phase was washed repetitively with CH 2 C1 2 (125 mL volumes) until most of the 25 has been removed from the aqueous phase.
  • the first extraction will contain 24, 25, and dicoupled side product and should be back extracted with HCl (125 mL, IN).
  • the organic layers were combined and evaporated to dryness.
  • the resultant oil was then dissolved in CH 2 C1 2 (100 mL) and washed repetitively with H 2 O (50 mL volumes) until 25 was removed.
  • the aqueous fractions were combined, total volume 500 mL, and NaCl was added until the solution became cloudy and then was washed with CH 2 C1 2 (2 x 500 mL).
  • the organic layers were combined, dried MgSO 4 , and evaporated to dryness to afford a the non- polydispersed title compound as an oil (16.9 g, 41% yield). It may be desirable to repeat one or more steps of the purification procedure to ensure high purity.
  • Example 23 MPEG7-C8 acid (28) To the oil of the non-polydispersed compound 27 (0.70 g, 1.4 mmol) was added IN NaOH (2.0 mL) and the reaction mixture was stirred for 4h. The crude reaction mixture was concentrated, acidified (pH ⁇ 2), saturated with NaCl, and washed CH C1 2 (2 x 50 mL). The organic layers were combined, washed sat. NaCl, dried MgSO 4 , and evaporated to dryness to afford the non-polydispersed title compound as a clear oil (0.35 g, 53% yield).
  • Non-polydispersed mPEG7-C8-acid 28 (0.3 lg, 0.64 mmol) was dissolved in 3 ml of anhydrous methylene chloride and then solution of N-hydroxysuccinimide (0.079g, 0.69 mmol) and EDCI-HC1 (135.6 mg, 0.71 mmol) in anhydrous methylene chloride added. Reaction was stirred for several hours, then washed with IN HCl, water, dried over MgSO , filtered and concentrated. Crude material was purified by column chromatography, concentrated to afford the non-polydispersed title compound as a clear oil and dried via vacuum. Examples 25 through 29 refer to the scheme illustrated in Figure 5.
  • the crade reaction mixture was filtered through Celite (washed CH 2 C1 2 , 80 mL) and the filtrate was washed H 2 O (100 mL), 5% NaHCO 3 (2 x 100 mL), H 2 O (100 mL), sat. NaCl (100 mL), dried MgSO 4 , and evaporated to dryness to afford the non-polydispersed title compound as a yellowish oil (7.42 g, 97% yield).
  • Example 28 MPEG 7 -C ⁇ o Acid (33) To the oil of non-polydispersed mPEG -C 1 o ester 32 (0.570 g, 1.1 mmol) was added IN NaOH (1.6 mL) and the reaction mixture was stirred overnight. The crude reaction mixture was concentrated, acidified (pH ⁇ 2), saturated with NaCl, and washed CH 2 C1 (2 x 50 mL). The organic layers were combined, washed sat. NaCl (2 x 50 mL), dried MgSO 4 , and evaporated to dryness to afford the non-polydispersed title compound as a clear oil (0.340 g, 62% yield).
  • the non-polydispersed acid 33 was activated using procedures similar to those described above in Example 24.
  • Non-polydispersed stearoyl chloride 35 (0.7g, 2.31 mmol) was added slowly to a mixture of PEG6 (5 g, 17.7 mmol) and pyridine (0.97g, 12.4 mmol) in benzene. The reaction mixture was stirred for several hours (-5). The reaction was followed by TLC using ethylacetate/methanol as a developing solvent. Then the reaction mixture was washed with water, dried over MgSO 4 , concentrated and dried via vacuum. Purified non-polydispersed compound 36 was analyzed by FABMS: m/e 549/ M + H. Example 31 Activation of C18(PEG6) Oligomer
  • Non-polydispersed stearoyl-PEG636 ( 0.8 g, 1.46 mmol ) was dissolved in toluene and added to a phosgene solution (10 ml, 20 % in toluene) which was cooled with an ice bath. The reaction mixture was stirred for 1 h at 0 ° C and then for 3 h at room temperature. Then phosgene and toluene were distilled off and the remaining non-polydispersed stearoyl PEG6 chloroformate 37 was dried over P 2 O 5 overnight.
  • Example 32 Tetraethylene glycol monobenzylether (39) To the oil of non-polydispersed tetraethylene glycol (19.4 g, 0.10 mol) was added a solution of NaOH (4.0 g in 4.0 mL) and the reaction was stirred for 15 mm. Then benzyl chloride (3.54 mL, 30.8 mmol) was added and the reaction mixture was heated to 100°C and stirred overnight. The reaction mixture was cooled to room temperature, diluted with sat. NaCl (250 mL), and washed CH 2 C1 2 (2 x 200 mL). The organic layers were combined, washed sat. NaCl, dried MgSO 4 , and chromatographed (silica, ethyl acetate) to afford the non-polydispersed title compound as a yellow oil (6.21 g, 71% yield).
  • Example 33 Mesylate of tetraethylene glycol monobenzylether (40) To a solution of CH 2 CI 2 (20 mL) was added non-polydispersed tetraethylene glycol monobenzylether 39 (6.21 g, 22 mmol) and cooled to 0°C in an ice bath. Then triethylamine (3.2 mL, 24 mmol) was added and the reaction mixture was stirred for 15 min at 0°C.
  • the crude reaction mixture was filtered through Celite (washed, CH C1 2 , 250 mL) and the filtrate was washed H 2 O, dried MgSO 4 , and evaporated to dryness.
  • the resultant oil was chromatographed (silica, ethyl acetate/methanol, 10:1) and chromatographed (silica, chloroform/methanol, 25:1) to afford the non-polydispersed title compound as a clear oil (2.62 g, 34% yield).
  • non-polydispersed octaethylene glycol monobenzylether 41 (0.998 g, 2.07 mmol) and pyridine (163.9 mg, 2.07 mmol) was added non-polydispersed stearoyl chloride 42 (627.7 mg, 2.07 mmol) in benzene.
  • the reaction mixture was stirred overnight (18 hours). The next day the reaction mixture was washed with water, dried over MgSO 4 , concentrated and dried via vacuum. Then the crude product was chromatographed on flash silica gel column, using 10% methanol/90% chloroform. The fractions containing the product were combined, concentrated and dried via vacuum to afford the non-polydispersed title compound.
  • Example 36 Hydrogenolysis of Stearate-PEG8-BenzyI
  • Example 38 Synthesis of Activated Triethylene Glycol Monomethyl Oligomers The following description refers to the scheme illustrated in Figure 8.
  • a solution of toluene containing 20% phosgene (100 ml, approximately 18.7 g, 189 mmol phosgene) was chilled to 0°C under a N 2 atmosphere.
  • Non-polydispersed mTEG triethylene glycol, monomethyl ether, 7.8 g, 47.5 mmol
  • the mixture was stirred for one hour at 0°C, then allowed to warm to room temperature and stirred for another two and one half hours.
  • the remaining phosgene, ethyl acetate and toluene were removed via vacuum distillation to leave the non-polydispersed mTEG chloroformate 46 as a clear oily residue.
  • the non-polydispersed residue 46 was dissolved in 50 mL of dry dichloromethane to which was added TEA (triethyleamine, 6.62 mL, 47.5 mmol) andNHS (N-hydroxysuccinimide, 5.8 g, 50.4 mmol). The mixture was stirred at room temperature under a dry atmosphere for twenty hours during which time a large amount of white precipitate appeared. The mixture was filtered to remove this precipitate and concentrated in vacuo. The resultant oil 47 was taken up in dichloromethane and washed twice with cold deionized water, twice with IN HCl and once with brine.
  • TEA triethyleamine, 6.62 mL, 47.5 mmol
  • NHS N-hydroxysuccinimide
  • the organics were dried over MgSO 4 , filtered and concentrated to provide the non-polydispersed title compound as a clear, light yellow oil. If necessary, the NHS ester could be further purified by flash chromatography on silica gel using EtOAc as the elutant.
  • Non- polydispersed palmitic anhydride (5 g; 10 mmol) was dissolved in dry THF (20 mL) and stirred at room temperature. To the stirring solution, 3 mol excess of pyridine was added followed by non-polydispersed triethylene glycol (1.4 mL). The reaction mixture was stirred for 1 hour (progress of the reaction was monitored by TLC; ethyl acetate-chloroform; 3:7). At the end of the reaction, THF was removed and the product was mixed with 10% H 2 SO 4 acid and extracted ethyl acetate (3 x 30 mL).
  • non-polydispersed product 48 was washed sequentially with water, brine, dried over MgSO , and evaporated to give non-polydispersed product 48.
  • a solution of N,N'-disuccinimidyl carbonate (3 mmol) in DMF (-10 mL) is added to a solution of the non-polydispersed product 48 (1 mmol) in 10 mL of anydrous DMF while stirring.
  • Sodium hydride (3 mmol) is added slowly to the reaction mixture. The reaction mixture is stirred for several hours (e.g., 5 hours). Diethyl ether is added to precipitate the activated oligomer. This process is repeated 3 times and the product is finally dried.
  • Example 40 Synthesis of Activated Hexaethylene Glycol Monomethyl Oligomers The following description refers to the scheme illustrated in Figure 10.
  • Non- polydispersed activated hexaethylene glycol monomethyl ether was prepared analogously to that of non-polydispersed triethylene glycol in Example 39 above.
  • a 20% phosgene in toluene solution 35 mL, 6.66 g, 67.4 mmol phosgene was chilled under a N 2 atmosphere in an ice/salt water bath.
  • Non-polydispersed hexaethylene glycol 50 (1.85 mL, 2.0 g, 6.74 mmol) was dissolved in 5 mL anhydrous EtOAc and added to the phosgene solution via syringe. The reaction mixture was kept stirring in the ice bath for one hour, removed and stirred a further 2.5 hours at room temperature. The phosgene, EtOAc, and toluene were removed by vacuum distillation, leaving non-polydispersed compound 51 as a clear, oily residue. The non-polydispersed residue 51 was dissolved in 20 mL dry dichloromethane and placed under a dry, inert atmosphere.
  • This reaction condition produces PEG7-hexyl-insulin, monoconjugated at the B29 position (PEG7-hexyl-insulin, B29 monoconjugated) at yield 40-60%.
  • the crade reaction mixture (PEG7-hexyl-insulin, B29 monoconjugated, 40-60%), unreacted insulin 8-25%, related substances 15-35%) was dialyzed or difiltered (3000-3500 molecular weight cut off, MWCO) to remove organic solvents and small molecular weight impurities, exchanged against ammonium acetate buffer and lyophilized.
  • This analytical HPLC method used a Waters Delta-Pak C18 column, 150 x 3.9 mm I.D., 5 ⁇ m, 300 A.
  • the solvent system consisted of Solvent B: 0.1% TFA in 50/50 methanol/water, and Solvent D: 0.1% TFA in methanol.
  • the gradient system was as follows:
  • Example 41 The procedure of Example 41 is used to conjugate human insulin with the activated oligomer of Example 24.
  • Example 41 The procedure of Example 41 is used to conjugate human insulin with the activated oligomer of Example 31.
  • Example 44 The procedure of Example 41 is used to conjugate human insulin with the activated oligomer of Example 37.
  • Example 41 The procedure of Example 41 is used to conjugate human insulin with the activated oligomer of Example 38.
  • Example 47 The procedure of Example 41 is used to conjugate human insulin with the activated oligomer of Example 39.
  • Example 49 The procedure of Example 41 is used to conjugate human insulin with the activated oligomer of Example 40.
  • Example 49
  • the dispersity coefficient of a mixture of human insulin-oligomer conjugates is determined as follows.
  • a mixture of human insulin-oligomer conjugates is provided, for example as described above in Example 41.
  • a first sample of the mixture is purified via
  • HPLC HPLC to separate and isolate the various human insulin-oligomer conjugates in the sample.
  • the mixture may include one or more of the following conjugates, which are described by stating the conjugation position followed by the degree of conjugation: Gb ⁇ 1 monoconjugate; Phe B1 monoconjugate; Lys B29 monoconjugate; Gl) ⁇ 1 , Phe B1 diconjugate; Gly ⁇ , Lys B29 diconjugate; Phe B1 , Lys B29 diconjugate; and/or Gi ⁇ 1 , Phe B1 , Lys B29 triconjugate.
  • Each isolated fraction of the mixture is analyzed via mass spectroscopy to determine the mass of the fraction, which allows each isolated fraction to be categorized as a mono-, di-, or tri-conjugate and provides a value for the variable "Mj" for each conjugate in the sample.
  • a second sample of the mixture is analyzed via HPLC to provide an HPLC trace. Assuming that the molar absorptivity does not change as a result of the conjugation, the weight percent of a particular conjugate in the mixture is provided by the area under the peak of the HPLC trace corresponding to the particular conjugate as a percentage of the total area under all peaks of the HPLC trace.
  • the sample is collected and lyophilized to dryness to determine the anhydrous gram weight of the sample.
  • the gram weight of the sample is multiplied by the weight percent of each component in the sample to determine the gram weight of each conjugate in the sample.
  • Nj is determined for a particular conjugate (the i th conjugate) by dividing the gram weight of the particular conjugate in the sample by the mass of the particular conjugate and multiplying the quotient by Avagadro's number (6.02205 x 10 mole " ), Mj, determined above, to give the number of molecules of the particular conjugate, N;, in the sample.
  • the dispersity coefficient is then calculated using n, Mj as determined for each conjugate, and Nj as determined for each conjugate.
  • PEG7-hexyl-insulin, B29 monoconjugated was purified from the crude mixture of
  • Example 41 using a preparative HPLC system Lyophilized crade mixture (0.5 g, composition: PEG7-hexyl-insulin, B29 monoconjugated, 40-60%, unreacted insulin 8-25%), related substances 15-35%) was dissolved in 5-10 mL 0.01 M ammonium acetate buffer, pH
  • Example 51 Cytosensor Studies Colo 205 (colorectal adenocarcinoma cells from ATCC, catalog #CCL-222) cells that had been serum-deprived for approximately 18 hours were suspended in 3 : 1 Cytosensor low- buffer RPMI-1640 media: Cytosensor agarose entrapment media and seeded into Cytosensor capsule cups at 100,000 cells/10 ⁇ L droplet. Cells were allowed to equilibrate on the Cytosensor to the low-buffer RPMI-1640 media at a flow rate of 100 ⁇ L per minute for approximately 3 hours until baseline acidification rates were stable. Insulin drags (insulin or insulin conjugates) were diluted to 50nM in low-buffer RPMI-1640 media and applied to the cells for 20 minutes at 100 ⁇ L/minute.
  • insulin is human insulin
  • PEG4 is a non-polydispersed mixture of mPEG4-hexyl-insulin, monoconjugates
  • PEG10 is a non-polydispersed mixture of mPEGlO-hexyl-insulin, monoconjugates
  • PEG7 is a non-polydispersed mixture of mPEG7- hexyl-insulin, monoconjugates
  • PEG7 AVG is a polydispersed mixture of mPEG7 A v G -hexyl- insulin, monoconjugates.
  • Chymotrypsin digests were conducted in a phosphate buffer, pH 7.4, at 37°C in a shaking water bath.
  • the insulin/insulin conjugate concentration was 0.3 mg/niL.
  • the chymotrypsin concentration was 2 Units/mL.
  • 100 ⁇ L samples were removed at the indicated time points and quenched with 25 ⁇ L of a 1:1 mixture of 0.1% trifluoroacetic acid:isopropyl alcohol. Samples were analyzed by reverse phase HPLC and the relative concentrations of insulin/insulin conjugate were determined by calculating the areas under the curves.
  • insulin is human insulin
  • PEG4 is a non-polydispersed mixture of mPEG4-hexyl-insulin, monoconjugates
  • PEG10 is a non-polydispersed mixture of mPEGlO-hexyl-insulin, monoconjugates
  • PEG7 is a non-polydispersed mixture of mPEG7- hexyl-insulin, monoconjugates
  • PEG7 AVG is a polydispersed mixture of mPEG7 AVG -hexyl- insulin, monoconjugates.
  • An effective animal model for evaluating formulations uses normal fasted beagle dogs. These dogs are given from 0.25 mg/kg to 1.0 mg/kg of insulin conjugates to evaluate the efficacy of various formulations. This model was used to demonstrate that insulin conjugates according to the present invention provide lower glucose levels in a dose dependent manner better than polydispersed insulin conjugates, which are not part of the present invention and are provided for comparison purposes.
  • the protocol for dog experiments calls for a blood glucose measurement at time zero just before a drug is administered.
  • the formulation in solid oral dosage form is then inserted into the dog's mouth.
  • Blood is drawn at 15, 30, 60 and 120 minutes and glucose levels are measured and graphed. The lower the glucose levels, the better the activity of the insulin conjugate.
  • the glucose lowering, and thus the activity, of the conjugates of the present invention is shown to be dose dependent.
  • Figure 17 shows that the glucose lowering of polydispersed insulin conjugates in a capsule formulation, which are not a part of the present invention, is less dose dependent than conjugates of the present invention.
  • An effective animal model for evaluating formulations uses normal fasted beagle dogs. These dogs are given 0.25 mg/kg of insulin conjugates to evaluate the efficacy of various formulations. This model was used to demonstrate that insulin conjugates according to the present invention provide lower inter-subject variability and better activity than polydispersed insulin conjugates, which are not part of the present invention but are provided for comparison purposes.
  • the protocol for dog experiments calls for a blood glucose measurement at time zero just before a drug is administered.
  • the oral liquid dosage formulation is then squirted into the back of the dog's mouth.
  • the dogs received 0.25 mg/kg of this solution.
  • Blood is drawn at 15, 30, 60 and 120 minutes and glucose levels are measured and graphed. The lower the glucose levels, the better the activity of the insulin conjugate.

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US20030027748A1 (en) 2003-02-06
EP1404178A4 (en) 2010-05-26
US7084114B2 (en) 2006-08-01
JP2003113113A (ja) 2003-04-18
US20060199759A1 (en) 2006-09-07
US20040198949A1 (en) 2004-10-07
US6828297B2 (en) 2004-12-07
CN1538809A (zh) 2004-10-20
AU2002310291B2 (en) 2007-09-20
BR0106851A (pt) 2003-04-08
MXPA03011284A (es) 2004-03-26
CA2449426C (en) 2013-12-03
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US7470663B2 (en) 2008-12-30
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