WO2002098452A1 - Mixtures of growth hormone drug-oligomer conjugates compromising polyalkylene glycol, uses thereof, and methods of making same - Google Patents

Mixtures of growth hormone drug-oligomer conjugates compromising polyalkylene glycol, uses thereof, and methods of making same Download PDF

Info

Publication number
WO2002098452A1
WO2002098452A1 PCT/US2002/017504 US0217504W WO02098452A1 WO 2002098452 A1 WO2002098452 A1 WO 2002098452A1 US 0217504 W US0217504 W US 0217504W WO 02098452 A1 WO02098452 A1 WO 02098452A1
Authority
WO
WIPO (PCT)
Prior art keywords
mixture
growth hormone
moiety
oligomer
ofthe
Prior art date
Application number
PCT/US2002/017504
Other languages
English (en)
French (fr)
Inventor
Nnochiri N. Ekwuribe
Christopher H. Price
Aslam M. Ansari
Amy L. Odenbaugh
Original Assignee
Nobex Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nobex Corporation filed Critical Nobex Corporation
Priority to EP02737344A priority Critical patent/EP1404361A4/en
Priority to CA002449320A priority patent/CA2449320A1/en
Priority to KR10-2003-7015911A priority patent/KR20040004693A/ko
Priority to MXPA03011282A priority patent/MXPA03011282A/es
Priority to JP2003501490A priority patent/JP2004534783A/ja
Publication of WO2002098452A1 publication Critical patent/WO2002098452A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormones [GH] (Somatotropin)
    • 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
    • A61K38/27—Growth hormone [GH] (Somatotropin)
    • 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
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/10—Drugs for disorders of the endocrine system of the posterior pituitary hormones, e.g. oxytocin, ADH

Definitions

  • the present invention relates to drug-oligomer conjugates, and, more particularly, to growth hormone drug-oligomer conjugates.
  • Growth hormones have been used for replacement therapy in growth hormone- deficient children. Growth hormone is also under investigation as an adjunct in the treatment of several catabolic conditions such as burn injuries, surgery and malabsorption. The positive effects of growth hormone on calcium ion retention and on osteogenesis also may be of use in the treatment of osteoporosis and non-healing fractures. Growth hormone may be administered by intramuscular or subcutaneous injection.
  • subcutaneous injection may be preferred because it facilitates self- administration.
  • both of these methods may be less than optimal because ofthe psychological and/or physical trauma that may be associated with administration by injection, especially in children.
  • Pharmaceutically active molecules such as proteins and polypeptides 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.
  • 5,359,030 to Ekwuribe proposes conjugating polypeptides such as somatostatin, somatotropin and/or somatomedin with polydispersed polyethylene glycol modified glycolipid polymers and polydispersed polyethylene glycol modified fatty acid polymers.
  • the number average molecular weight of polydispersed 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 a 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 (Mford 380-420); PEG 1,000 (Mschreib 950-1,050); PEG 1,500 (M Fabric 1,400-1,600); and PEG 2,000 (Mfect 1,900-2,200). It is desirable to provide non-polydispersed mixtures of growth hormone-oligomer conjugates where the oligomer comprises polyalkylene glycol.
  • a mixture of growth hormone-oligomer conjugates comprising polyalkylene glycol according to embodiments ofthe present invention may exhibit higher in vivo activity than a polydispersed mixture of similar conjugates, where the polydispersed mixture has the same number average molecular weight as the mixture according to the present invention. This heightened activity may result in lower dosage requirements. Moreover, a mixture of growth hormone-oligomer conjugates comprising polyalkylene glycol according to embodiments of the present invention may be more effective at surviving an in vitro model of intestinal digestion than polydispersed mixtures of similar conjugates.
  • mixtures of growth hormone-oligomer conjugates comprising polyalkylene glycol may also result in less inter-subject variability than polydispersed mixtures of similar conjugates.
  • a substantially monodispersed mixture of conjugates where each conjugate includes a growth hormone drug coupled to an oligomer that comprises a polyalkylene glycol moiety is provided.
  • the polyalkylene glycol moiety preferably has at least 2, 3, or 4 polyalkylene glycol subunits and, most preferably, has at least 7 polyalkylene glycol subunits.
  • the polyalkylene glycol moiety is preferably polypropylene glycol.
  • the oligomer preferably further comprises a lipophilic moiety.
  • the growth hormone drug is preferably human growth hormone.
  • the oligomer is preferably covalently coupled to an amino function ofthe human growth hormone.
  • the conjugate is preferably amphiphilically balanced such that the conjugate is aqueously soluble and able to penetrate biological membranes.
  • the oligomer may comprise a first polyalkylene glycol moiety covalently coupled to the drug by a non-hydrolyzable bond and a second polyalkylene glycol moiety covalently coupled to the first polyalkylene glycol moiety by a hydrolyzable bond.
  • the mixture is preferably a monodispersed mixture and is most preferably a purely monodispersed mixture.
  • a substantially monodispersed mixture of conjugates where each conjugate comprises a growth hormone drug coupled to an oligomer including a polyalkylene glycol moiety, and the mixture has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of growth hormone 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 a growth hormone drug coupled to an oligomer including a polyalkylene glycol moiety, and the mixture has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of growth hormone 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 a growth hormone drug coupled to an oligomer including a polyalkylene 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 growth hormone 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 a growth hormone drug coupled to an oligomer including a polyalkylene glycol moiety, and the mixture has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of growth hormone drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture.
  • a mixture of conjugates is provided where each conjugate includes a growth hormone drug coupled to an oligomer that comprises a polyalkylene glycol moiety, and the mixture has a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • each conjugate includes a growth hormone drug coupled to an oligomer that comprises a polyalkylene glycol moiety, and the mixture has a dispersity coefficient (DC) greater than 10,000 where
  • each conjugate includes a growth hormone drug coupled to an oligomer and has the same number of polyalkylene glycol subunits.
  • each conjugate has the same molecular weight and has the formula:
  • 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; h, i, j, k, m and n are individually 0 or 1 , with the proviso that when R is the polyalkylene glycol moiety; m is 1 , and when R' is the polyalkylene glycol moiety, n is i; and p is an integer from 1 to the number of nucleophilic residues on the growth hormone drug.
  • compositions comprising conjugate mixtures ofthe present invention are also provided. Methods of treating a growth hormone deficiency in a subject in need of such treatment by administering an effective amount of such pharmaceutical compositions are also provided. Methods of accelerating the growth rate of an animal by administering to the animal an effective amount of a mixture of conjugates according to the various embodiments described above are also provided.
  • Growth hormone drug-oligomer conjugate mixtures may provide increased in vivo activity and/or lowered inter-subject variability and/or decreased degradation by chymotrypsin when compared to conventional polydispersed growth hormone drug-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 ofthe present invention
  • Figure 2 illustrates a scheme for synthesizing a mixture of mPEG according to embodiments ofthe present invention
  • Figure 3 illustrates a scheme for synthesizing a mixture of activated mPEG7-hexyl oligomers according to embodiments ofthe present invention
  • Figure 4 illustrates a scheme for synthesizing a mixture of activated mPEG7-octyl oligomers according to embodiments ofthe present invention
  • Figure 5 illustrates a scheme for synthesizing a mixture of activated mPEG-decyl oligomers according to embodiments ofthe present invention
  • Figure 6 illustrates a scheme for synthesizing a mixture of activated stearate-PEG6 oligomers according to embodiments ofthe present invention
  • Figure 7 illustrates a scheme for synthesizing a mixture of activated stearate-PEG8 oligomers according to embodiments ofthe present invention
  • Figure 8 illustrates a scheme for synthesizing a mixture of activated PEG3 oligomers according to embodiments ofthe present invention
  • Figure 9 illustrates a scheme for synthesizing a mixture of activated palmitate-PEG3 oligomers according to embodiments ofthe present invention.
  • Figure 10 illustrates a scheme for synthesizing a mixture of activated PEG6 oligomers and conjugating human growth hormone with the activated PEG6 oligomers according to embodiments ofthe 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 ofthe present invention
  • Figure 13 illustrates a scheme for synthesizing various propylene glycol polymers according to embodiments ofthe present invention
  • Figure 14 is an HPLC trace (HPLC gradient: 50% to 90% acetonitrile in 30 minutes) ofthe conjugation reaction illustrated in Figure 10 using two equivalents of activated MPEG6 oligomers and five equivalents of activated MPEG6 oligomers;
  • Figure 15 is an HPLC trace (HPLC gradient: 0% to 95% acetonitrile in 20 minutes) ofthe conjugation reaction illustrated in Figure 10 using 30 equivalents of activated MPEG6 oligomers;
  • Figure 16 is a MALDI spectra ofthe conjugation reaction illustrated in Figure 10 using two equivalents of activated MPEG6 oligomers;
  • Figure 17 is an HPLC trace (HPLC gradient: 50% to 70% acetonitrile in 30 minutes) illustrating a partial purification ofthe product ofthe conjugation reaction of Figure 10 using five equivalents of activated MPEG6 oligomers;
  • Figure 18 is a MALDI spectra of fraction B from the partial purification illustrated in Figure 17
  • Figure 19 is a MALDI spectra of fraction C from the partial purification illustrated in Figure 17;
  • Figure 20 is a MALDI spectra of fractions D and E from the partial purification illustrated in Figure 17;
  • Figure 21 is an electrospray spectra of fraction E from the partial purification illustrated in Figure 17;
  • Figure 22 is an electrospray spectra ofthe reaction mixture from the conjugation reaction illustrated in Figure 10 using 30 equivalents of activated MPEG6 oligomers;
  • Figure 23 is an HPLC trace of a conjugation reaction of human growth hormone with the activated oligomer of Figure 9;
  • Figure 24 is an HPLC trace of a conjugation reaction using one equivalent of human growth hormone and two equivalents ofthe activated oligomer of Figure 9;
  • Figure 25 is an HPLC trace of a conjugation reaction using one equivalent of human growth hormone and five equivalents ofthe activated oligomer of Figure 8;
  • Figure 26 is a MALDI spectra of the fraction corresponding to the left half of the peak in the conjugation HPLC trace of Figure 25;
  • Figure 27 is a MALDI spectra ofthe fraction corresponding to the right half of the peak in the conjugation HPLC trace of Figure 25;
  • Figure 28 is an HPLC trace of a conjugation reaction using one equivalent of human growth hormone and nine equivalents ofthe activated oligomer of Figure 8;
  • Figure 29 illustrates a bar graph denoting the activity as determined by luciferase assay of mixtures of growth hormone conjugates according to embodiments ofthe present invention compared with the activity of human growth hormone standards, which are provided for comparison purposes only and do not form part ofthe present invention
  • Figure 30 illustrates a bar graph denoting the activity as determined by luciferase assay of mixtures of growth hormone conjugates according to embodiments ofthe present invention compared with the activity of human growth hormone standards, which are provided for comparison purposes only and do not form part ofthe present invention.
  • 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. 5,359,030 to Ekwuribe.
  • substantially monodispersed is used to describe a mixture of compounds wherein at least about 95 percent ofthe compounds in the mixture have the same molecular weight.
  • the term "monodispersed" is used to describe a mixture of compounds wherein about 100 percent ofthe compounds in the mixture have the same molecular weight.
  • substantially purely monodispersed is used to describe a mixture of compounds wherein at least about 95 percent ofthe 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.
  • 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.
  • a purely monodispersed mixture is a monodispersed mixture, but a monodispersed mixture is not necessarily a purely monodispersed mixture.
  • weight average molecular weight is defined as the sum of the products ofthe weight fraction for a given molecule in the mixture times the mass ofthe 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
  • Mj is the mass ofthe i- molecule.
  • 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.
  • growth hormone drug means a drug possessing all or some ofthe biological activity of growth hormone peptides.
  • growth hormone peptides means human growth hormone, human growth hormone-releasing hormone, animal growth hormone or animal growth hormone-releasing hormone any of which may be provided by natural, synthetic, or genetically engineered sources.
  • growth hormone peptide analog means growth hormone peptide wherein one or more ofthe amino acids have been replaced while retaining some or all ofthe activity ofthe growth hormone peptide.
  • the analog is described by noting the replacement amino acids with the position ofthe replacement as a superscript followed by a description ofthe growth hormone.
  • Pro 41 growth hormone, human means that the amino acid typically found at the 41 position of a human growth hormone molecule has been replaced with proline.
  • Growth hormone analogs may be obtained by various means, as will be understood by those skilled in the art.
  • certain amino acids may be substituted for other amino acids in the growth hormone 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 interactive capacity and nature of growth hormone defines its biological functional activity
  • certain amino acid sequence substitutions can be made in the amino acid sequence and nevertheless remain a polypeptide with like properties.
  • the hydropathic index of amino acids may be considered. The importance ofthe hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art.
  • the relative hydropathic character ofthe amino acid contributes to the secondary structure ofthe resultant polypeptide, which in turn defines the interaction ofthe polypeptide 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 ofthe 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.
  • growth hormone peptide fragment means a segment ofthe amino acid sequence found in the growth hormone that retains some or all ofthe activity of the growth hormone polypeptide.
  • growth hormone peptide fragment analog means a segment ofthe amino acid sequence found in the growth hormone peptide wherein one or more ofthe amino acids in the segment has been replaced while retaining some or all ofthe activity ofthe growth hormone polypeptide.
  • polyalkylene glycol refers to straight or branched polyalkylene glycol polymers.
  • polyalkylene glycol subunit refers to a single polyalkylene glycol unit.
  • a polyethylene glycol subunit is — (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 growth hormone drug-oligomer conjugates is provided.
  • Each growth hormone drug-oligomer conjugate in the monodispersed mixture includes a growth hormone drug coupled to an oligomer that comprises a polyalkylene glycol moiety.
  • at least about 96, 97, 98 or 99 percent ofthe 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 ofthe conjugates in the mixture have the same molecular weight and have the same molecular structure.
  • the mixture is a purely monodispersed mixture.
  • the growth hormone drug is preferably human growth hormone.
  • the growth hormone drug may be selected from various growth hormone drugs known to those skilled in the art including, for example, growth hormone peptides, growth hormone peptide analogues, growth hormone peptide fragments, and growth hormone peptide fragment analogues.
  • Growth hormone peptides include, but are not limited to, growth hormone, human (hGH); growth hormone, porcine; growth hormone, bovine; growth hormone, chicken; growth hormone, rat; growth hormone, mouse; growth hormone, ovine; growth hormone releasing factor, human; growth hormone pro-releasing factor, human; growth hormone releasing factor, mouse; growth hormone releasing factor, ovine; growth hormone releasing factor, rat; growth hormone releasing factor, bovine; growth hormone releasing factor, porcine; and growth hormone releasing factor, chicken.
  • Growth hormone peptide analogs may be provided as described above by substituting one or more amino acids in a growth hormone peptide.
  • Growth hormone peptide fragments include, but are not limited to, growth hormone 1-43, human; growth hormone 6-13; growth hormone releasing factor 1-37, human; growth hormone releasing factor 1-40, human; growth hormone releasing factor 1-40, amide, human; growth hormone releasing factor 30-44, amide, human; growth hormone releasing factor 1-29, amide, rat; hexarelin (growth hormone releasing hexapeptide); and growth hormone releasing factor 1 -29, amide, human.
  • Growth hormone peptide fragment analogues include, but are not limited to, [D-Ala ]-growth hormone releasing factor 1-29, amide, human; [N-Ac-Tyr , D-Arg ]-growth hormone releasing factor 1-29, amide; [His 1 , Nle 27 ] -growth hormone releasing factor 1-32, amide; growth hormone releasing peptide-6 ([His 1 , Lys 6 ]-GHRP); and [D-Lys 3 ]-GHRP-6.
  • the oligomer may be various oligomers comprising a polyalkylene glycol moiety as will be understood by those skilled in the art.
  • the polyalkylene glycol moiety 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. Most preferably, the polyalkylene glycol moiety ofthe oligomer has at least 7 polyalkylene glycol subunits.
  • the polyalkylene glycol moiety ofthe oligomer is preferably a lower alkyl polyalkylene glycol moiety such as a polyethylene glycol moiety, a polypropylene glycol moiety, or a polybutylene glycol moiety.
  • the polyalkylene glycol moiety is more preferably a polypropylene glycol moiety having a uniform 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 growth hormone drug-oligomer conjugates without the use of lipophilic polymer moieties.
  • coupling the secondary alcohol moiety ofthe polypropylene glycol moiety with a growth hormone drug may provide the growth hormone drug (e.g., human growth hormone) with improved resistance to degradation caused by enzymes such as trypsin and chymotrypsin found, for example, in the gut.
  • Uniform polypropylene glycol according to embodiments ofthe present invention is preferably synthesized as illustrated in Figures 11 through 13, which will now be described.
  • a 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 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 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 Bi 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 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 B ⁇ 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 and provide a secondary alcohol capping monomer 63.
  • the B 2 de-blocking reagent may be various de- blocking 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 B ⁇ blocking moiety on the dimer compound 65 may be removed using a Bj 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 2 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.
  • 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 Bj 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 ofthe 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 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 ofthe capped trimer 68.
  • Uniform polypropylene glycol moieties may be coupled to a growth hormone drug, a lipophilic moiety such as a carboxylic 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 oligomer may comprise one or more other moieties as will be understood by those skilled in the art including, but not limited to, 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 hydrophilic moieties including, but not limited to, sugars, polyalkylene glycols, and polyamine/PEG copolymers. Adjacent polyalkylene glycol moieties will be considered to be the same moiety if they are coupled by an ether bond and have the same alkyl structure. For example, the 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 — is a single polyethylene glycol moiety having six polyethylene glycol subunits. Adjacent polyalkylene glycol moieties will be considered to be different moieties if they are coupled by a bond other than an ether bond or if they have different alkyl structures. For example, the moiety
  • oligomers according to embodiments ofthe present invention comprise a polyalkylene glycol moiety and do not further comprise a hydrophilic moiety.
  • 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 growth hormone 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 growth hormone 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 ofthe oligomer which are not coupled to the growth hormone 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 growth hormone drug.
  • the growth hormone drug is coupled to the oligomer utilizing a hydrolyzable bond (e.g., an ester or carbonate bond).
  • a hydrolyzable coupling may provide a growth hormone drug-oligomer conjugate that acts as a prodrug.
  • a hydrolyzable coupling may provide for a time-release or controlled-release effect, administering the growth hormone drug over a given time period as one or more oligomers are cleaved from their respective growth hormone drug-oligomer conjugates to provide the active drug.
  • the growth hormone 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 growth hormone 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 growth hormone 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 growth hormone drug. While the oligomer is preferably covalently coupled to the growth hormone drug, it is to be understood that the oligomer may be non-covalently coupled to the growth hormone drug to form a non-covalently conjugated growth hormone 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 growth hormone 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 ofthe oligomers in the plurality may be the same and some may be different.
  • all ofthe bonds coupling the plurality of oligomers to the growth hormone drug may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more ofthe oligomers is rapidly removed from the growth hormone drug by hydrolysis in the body and one or more ofthe oligomers is slowly removed from the growth hormone drug by hydrolysis in the body.
  • the oligomer may be coupled to the growth hormone drug at various nucleophilic residues ofthe drug including, but not limited to, nucleophilic hydroxyl functions and/or amino functions.
  • Nucleophilic hydroxyl functions may be found, for example, at serine and/or tyrosine residues, and nucleophilic amino functions may be found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini ofthe polypeptide.
  • the coupling preferably forms a secondary amine.
  • the oligomer may be coupled to an amino functionality of Phe 1 , Lys 38 , Lys 41 , Lys 70 , Lys 115 , Lys 140 , Lys 145 , Lys 158 , Lys 168 , and/or Lys 172 .
  • Substantially monodispersed mixtures of growth hormone drug-oligomer conjugates ofthe present invention may be synthesized by various methods. For example, a substantially monodispersed mixture of oligomers consisting of carboxylic 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 ofthe substantially monodispersed mixture are then activated so that they are capable of reacting with a growth hormone drug to provide a growth hormone 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 growth hormone drugs under conditions sufficient to provide a mixture of growth hormone drug-oligomer conjugates.
  • Exemplary syntheses are described hereinbelow in Examples 40 through 42.
  • the reaction conditions e.g., selected molar ratios, solvent mixtures and/or pH
  • the reaction conditions may be controlled such that the mixture of growth hormone drug-oligomer conjugates resulting from the reaction ofthe substantially monodispersed mixture of activated oligomers and the substantially monodispersed mixture of growth hormone drugs is a substantially monodispersed mixture.
  • conjugation at the amino functionality of lysine may be suppressed by maintaining the pH ofthe reaction solution below the pK a of lysine.
  • the mixture of growth hormone drug-oligomer conjugates may be separated and isolated utilizing, for example, HPLC to provide a substantially monodispersed mixture of growth hormone 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
  • 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 Phe , Lys , Lys 41 , Lys 70 , Lys 115 , Lys 140 , Lys 145 , Lys 158 , Lys 168 or Lys 172 of a human growth hormone 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 ofthe reaction sites on the growth hormone drug may be blocked by, for example, reacting the growth hormone 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 substantially monodispersed mixture of blocked growth hormone drugs may be reacted with the substantially monodispersed mixture of activated oligomers to provide a mixture of growth hormone drug-oligomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues.
  • the growth hormone drug-oligomer conjugates may be de-blocked as will be understood by those skilled in the art. If necessary, the mixture of growth hormone drug-oligomer conjugates may then be separated as described above to provide a substantially monodispersed mixture of growth hormone drug-oligomer conjugates. Alternatively, the mixture of growth hormone drug-oligomer conjugates may be separated prior to de-blocking.
  • Substantially monodispersed mixtures of growth hormone drug-oligomer conjugates according to embodiments ofthe present invention preferably have improved properties when compared with those of conventional mixtures.
  • a substantially monodispersed mixture of growth hormone drug-oligomer conjugates preferably has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of growth hormone drug- oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture.
  • the number average molecular weight ofthe substantially monodispersed mixture and the number average weight ofthe 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 growth hormone drug- oligomer conjugates preferably has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of growth hormone drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture.
  • the number average molecular weight ofthe substantially monodispersed mixture and the number average weight ofthe 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 drug being added to cultured cells in a Transwell (Corning, Inc., Acton, Massachusetts). This response is proportional to the activity ofthe molecule under study.
  • a substantially monodispersed mixture of growth hormone drug-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 growth hormone drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture.
  • the number average molecular weight ofthe substantially monodispersed mixture and the number average weight ofthe polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • a substantially monodispersed mixture of growth hormone drug-oligomer conjugates has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of growth hormone drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture.
  • the number average molecular weight ofthe substantially monodispersed mixture and the number average weight ofthe 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 ofthe difference between the subject's AUC and the average AUC is then determined for each subject.
  • the absolute values ofthe 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.
  • Substantially monodispersed mixtures of growth hormone drug-oligomer conjugates according to embodiments ofthe present invention preferably have two or more ofthe above- described improved properties. More preferably, substantially monodispersed mixtures of growth hormone drug-oligomer conjugates according to embodiments ofthe present invention have three or more ofthe above-described improved properties. Most preferably, substantially monodispersed mixtures of growth hormone drug-oligomer conjugates according to embodiments ofthe present invention have all four ofthe above-described improved properties.
  • a mixture of conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons is provided.
  • Each conjugate in the mixture includes a growth hormone drug coupled to an oligomer that comprises a polyalkylene 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 ofthe molecular weight distribution may then be determined by statistical methods as will be understood by those skilled in the art.
  • the growth hormone drug is preferably human growth hormone.
  • the growth hormone drug may be selected from various growth hormone drugs known to those skilled in the art including, for example, growth hormone peptides, growth hormone peptide analogues, growth hormone peptide fragments, and growth hormone peptide fragment analogues.
  • Growth hormone peptides include, but are not limited to, growth hormone, human (hGH); growth hormone, porcine; growth hormone, bovine; growth hormone, chicken; growth hormone, rat; growth hormone, mouse; growth hormone, ovine; growth hormone releasing factor, human; growth hormone pro-releasing factor, human; growth hormone releasing factor, mouse; growth hormone releasing factor, ovine; growth hormone releasing factor, rat; growth hormone releasing factor, bovine; growth hormone releasing factor, porcine; and growth hormone releasing factor, chicken.
  • Growth hormone peptide analogs may be provided as described above by substituting one or more amino acids in a growth hormone peptide.
  • Growth hormone peptide fragments include, but are not limited to, growth hormone 1-43, human; growth hormone 6-13; growth hormone releasing factor 1-37, human; growth hormone releasing factor 1-40, human; growth hormone releasing factor 1-40, amide, human; growth hormone releasing factor 30-44, amide, human; growth hormone releasing factor 1-29, amide, rat; hexarelin (growth hormone releasing hexapeptide); and growth hormone releasing factor 1 -29, amide, human.
  • Growth hormone peptide fragment analogues include, but are not limited to, [D-Ala ] -growth hormone releasing factor 1-29, amide, human; [N-Ac-Tyr 1 , D- Arg 2 ] -growth hormone releasing factor 1-29, amide; [His 1 , Nle 27 ]-growth hormone releasing factor 1-32, amide; growth hormone releasing peptide-6 ([His 1 , Lys 6 ]-GHRP); and [D-Lys 3 ]-GHRP-6.
  • the oligomer may be various oligomers comprising a polyalkylene glycol moiety as will be understood by those skilled in the art.
  • the polyalkylene glycol moiety 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. Most preferably, the polyalkylene glycol moiety ofthe oligomer has at least 7 polyalkylene glycol subunits.
  • the polyalkylene glycol moiety ofthe oligomer is preferably a lower alkyl polyalkylene glycol moiety such as a polyethylene glycol moiety, a polypropylene glycol moiety, or a polybutylene glycol moiety.
  • the polyalkylene glycol moiety is more preferably a polypropylene glycol moiety having a uniform 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 growth hormone drug-oligomer conjugates without the use of lipophilic polymer moieties.
  • coupling the secondary alcohol moiety ofthe polypropylene glycol moiety with a growth hormone drug may provide the growth hormone drug (e.g., human growth hormone) with improved resistance to degradation caused by enzymes such as trypsin and chymotrypsin found, for example, in the gut.
  • a growth hormone drug e.g., human growth hormone
  • enzymes such as trypsin and chymotrypsin found, for example, in the gut.
  • Uniform polypropylene glycol according to embodiments ofthe 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 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 B] de-blocking reagent to remove the blocking moiety Bi 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 de-blocking reagent to remove the blocking moiety B 2 and provide a secondary alcohol capping monomer 63.
  • the B 2 de-blocking reagent may be various de- blocking agents as will be understood by those skilled in the art including, but not limited to, H 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 Bj blocking moiety on the dimer compound 65 may be removed using a Bi 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 2 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 ofthe 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 ofthe 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 ofthe 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 ofthe present invention may be coupled to a growth hormone drug, a lipophilic moiety such as a carboxylic 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 oligomer may comprise one or more other moieties as will be understood by those skilled in the art including, but not limited to, 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 hydrophilic moieties including, but not limited to, sugars, polyalkylene glycols, and polyamine/PEG copolymers. Adjacent polyalkylene glycol moieties will be considered to be the same moiety if they are coupled by an ether bond and have the same alkyl structure. For example, the moiety
  • O II — O— C 2 H 4 — 0— C 2 H 4 — O— C 2 H 4 — O— C 2 H 4 — C-O— C 2 H 4 — O— C 2 H 4 — is a polyethylene glycol moiety having four polyethylene glycol subunits and a hydrophilic moiety having two polyethylene glycol subunits.
  • oligomers according to embodiments ofthe present invention comprise a polyalkylene glycol moiety and do not further comprise a hydrophilic moiety.
  • 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 growth hormone 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 growth hormone 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 ofthe oligomer which are not coupled to the growth hormone 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 growth hormone drug.
  • the growth hormone drug is coupled to the oligomer utilizing a hydrolyzable bond (e.g., an ester or carbonate bond).
  • a hydrolyzable coupling may provide a growth hormone drug-oligomer conjugate that acts as a prodrug.
  • a hydrolyzable coupling may provide for a time-release or controlled-release effect, administering the growth hormone drug over a given time period as one or more oligomers are cleaved from their respective growth hormone drug-oligomer conjugates to provide the active drug.
  • the growth hormone 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 growth hormone drug, the oligomer further comprises one or more bonding moieties that are used to covalently couple the oligomer with the growth hormone 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 growth hormone drug.
  • oligomer is preferably covalently coupled to the growth hormone drug
  • the oligomer may be non-covalently coupled to the growth hormone drug to form a non-covalently conjugated growth hormone 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 growth hormone 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 ofthe oligomers in the plurality may be the same and some may be different.
  • all ofthe bonds coupling the plurality of oligomers to the growth hormone drug may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more ofthe oligomers is rapidly removed from the growth hormone drug by hydrolysis in the body and one or more ofthe oligomers is slowly removed from the growth hormone drug by hydrolysis in the body.
  • the oligomer may be coupled to the growth hormone drug at various nucleophilic residues ofthe drug including, but not limited to, nucleophilic hydroxyl functions and/or amino functions. Nucleophilic hydroxyl functions may be found, for example, at serine and/or tyrosine residues, and nucleophilic amino functions may be found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini ofthe polypeptide. When an oligomer is coupled to the one or more N-termini ofthe growth hormone polypeptide, the coupling preferably forms a secondary amine.
  • the oligomer may be coupled to an amino functionality of Phe 1 , Lys 38 , Lys 41 , Lys 70 , Lys 115 , Lys 140 , Lys 145 , Lys 158 , Lys 168 , and/or Lys 172 .
  • Mixtures of growth hormone 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 carboxylic acid and polyethylene glycol is synthesized by contacting a mixture of carboxylic 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 ofthe 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 a growth hormone drug to provide a growth hormone drug-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 growth hormone drugs having a molecular weight distribution with a standard deviation of less than about 22 Daltons under conditions sufficient to provide a mixture of growth hormone drug-oligomer conjugates.
  • Exemplary syntheses are described hereinbelow in Examples 40 through 42.
  • the reaction conditions e.g., selected molar ratios, solvent mixtures and/or pH
  • the mixture of growth hormone drug-oligomer conjugates resulting from the reaction ofthe mixture of activated oligomers having a molecular weight distribution with a standard deviation of less than about 22 Daltons and the mixture of growth hormone 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 growth hormone drug-oligomer conjugates may be separated and isolated utilizing, for example, HPLC to provide a mixture of growth hormone drug-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 Phe 1 , Lys 38 , Lys 41 , Lys 70 , Lys 115 , Lys 140 , Lys 145 , Lys 158 , Lys 168 or Lys 172 of a human growth hormone 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 ofthe reaction sites on the growth hormone drug may be blocked by, for example, reacting the growth hormone 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 growth hormone 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 growth hormone drug-oligomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues.
  • the growth hormone drug-oligomer conjugates may be de-blocked as will be understood by those skilled in the art.
  • the mixture of growth hormone drug-oligomer conjugates may then be separated as described above to provide a mixture of growth hormone drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • the mixture of growth hormone drug-oligomer conjugates may be separated prior to de-blocking.
  • Mixtures of growth hormone drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons according to embodiments ofthe present invention preferably have improved properties when compared with those of conventional mixtures.
  • a mixture of growth hormone 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 growth hormone drug-oligomer conjugates having the same number average molecular weight as the mixture of growth hormone drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • the number average molecular weight ofthe mixture of growth hormone drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons and the number average weight ofthe 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 growth hormone 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 growth hormone drug-oligomer conjugates having the same number average molecular weight as the mixture of growth hormone drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • the number average molecular weight ofthe mixture of growth hormone drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons and the number average weight ofthe 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 drug being added to cultured cells in a transwell. This response is proportional to the activity ofthe molecule under study.
  • a mixture of growth hormone drug-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 growth hormone drug-oligomer conjugates having the same number average molecular weight as the mixture of growth hormone drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • the number average molecular weight ofthe mixture of growth hormone drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons and the number average weight ofthe polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • a mixture of growth hormone drug-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 growth hormone drug-oligomer conjugates having the same number average molecular weight as the mixture of growth hormone drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons.
  • the number average molecular weight ofthe mixture of growth hormone drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons and the number average weight ofthe 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 ofthe 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.
  • Mixtures of growth hormone drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons according to embodiments ofthe present invention preferably have two or more ofthe above-described improved properties. More preferably, mixtures of growth hormone drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons according to embodiments ofthe present invention have three or more of the above-described improved properties. Most preferably, mixtures of growth hormone drug-oligomer conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons according to embodiments ofthe present invention have all four of the above-described improved properties.
  • each conjugate includes a growth hormone drug coupled to an oligomer that comprises a polyalkylene 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
  • ⁇ j is the number of i- molecules in the sample
  • Mj is the mass ofthe i ⁇ molecule.
  • the mixture of conjugates preferably has a dispersity coefficient greater than 100,000. More preferably, the dispersity coefficient ofthe conjugate mixture is greater than 500,000 and, most preferably, the dispersity coefficient is greater than 10,000,000.
  • the variables n, ⁇ i, 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 44.
  • the growth hormone drug is preferably human growth hormone.
  • the growth hormone drug may be selected from various growth hormone drugs known to those skilled in the art including, for example, growth hormone peptides, growth hormone peptide analogues, growth hormone peptide fragments, and growth hormone peptide fragment analogues.
  • Growth hormone peptides include, but are not limited to, growth hormone, human (hGH); growth hormone, porcine; growth hormone, bovine; growth hormone, chicken; growth hormone, rat; growth hormone, mouse; growth hormone, ovine; growth hormone releasing factor, human; growth hormone pro-releasing factor, human; growth hormone releasing factor, mouse; growth hormone releasing factor, ovine; growth hormone releasing factor, rat; growth hormone releasing factor, bovine; growth hormone releasing factor, porcine; and growth hormone releasing factor, chicken.
  • Growth hormone peptide analogs may be provided as described above by substituting one or more amino acids in a growth hormone peptide.
  • Growth hormone peptide fragments include, but are not limited to, growth hormone 1-43, human; growth hormone 6-13; growth hormone releasing factor 1-37, human; growth hormone releasing factor 1-40, human; growth hormone releasing factor 1-40, amide, human; growth hormone releasing factor 30-44, amide, human; growth hormone releasing factor 1-29, amide, rat; hexarelin (growth hormone releasing hexapeptide); and growth hormone releasing factor 1 -29, amide, human.
  • Growth hormone peptide fragment analogues include, but are not limited to, [D-Ala 2 ]-growth hormone releasing factor 1-29, amide, human; [N-Ac-Tyr , D-Arg ]-growth hormone releasing factor 1-29, amide; [His 1 , Nle 27 ]-growth hormone releasing factor 1-32, amide; growth hormone releasing peptide-6 ([His 1 , Lys 6 ]-GHRP); and [D-Lys 3 ]-GHRP-6.
  • the oligomer may be various oligomers comprising a polyalkylene glycol moiety as will be understood by those skilled in the art.
  • the polyalkylene glycol moiety 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. Most preferably, the polyalkylene glycol moiety ofthe oligomer has at least 7 polyalkylene glycol subunits.
  • the polyalkylene glycol moiety ofthe oligomer is preferably a lower alkyl polyalkylene glycol moiety such as a polyethylene glycol moiety, a polypropylene glycol moiety, or a polybutylene glycol moiety.
  • the polyalkylene glycol moiety is more preferably a polypropylene glycol moiety having a uniform 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 growth hormone drug-oligomer conjugates without the use of lipophilic polymer moieties.
  • a growth hormone drug e.g., human growth hormone
  • Uniform polypropylene glycol according to embodiments ofthe present invention is preferably synthesized as illustrated in Figures 11 through 13, which will now be described. As illustrated in Figure 11, 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 ⁇ 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).
  • a base e.g., potassium carbonate
  • the Bi de-blocking reagent is preferably tetrabutylammonium fluoride (TBAF).
  • TBAF tetrabutylammonium fluoride
  • 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 de- blocking 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 B ⁇ blocking moiety on the dimer compound 65 may be removed using a Bi 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 2 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.
  • 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 B ⁇ 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 ofthe 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 ofthe capped trimer 68.
  • Uniform polypropylene glycol moieties according to embodiments ofthe present invention may be coupled to a growth hormone drug, a lipophilic moiety such as a carboxylic 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 oligomer may comprise one or more other moieties as will be understood by those skilled in the art including, but not limited to, 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 hydrophilic moieties including, but not limited to, sugars, polyalkylene glycols, and polyamine/PEG copolymers. Adjacent polyalkylene glycol moieties will be considered to be the same moiety if they are coupled by an ether bond and have the same alkyl structure. For example, the moiety
  • oligomers according to embodiments ofthe present invention comprise a polyalkylene glycol moiety and do not further comprise a hydrophilic moiety.
  • 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 growth hormone 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 growth hormone 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 ofthe oligomer which are not coupled to the growth hormone 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 growth hormone drug.
  • the growth hormone drug is coupled to the oligomer utilizing a hydrolyzable bond (e.g., an ester or carbonate bond).
  • a hydrolyzable coupling may provide a growth hormone drug-oligomer conjugate that acts as a prodrug.
  • a hydrolyzable coupling may provide for a time-release or controlled-release effect, administering the growth hormone drug over a given time period as one or more oligomers are cleaved from their respective growth hormone drug-oligomer conjugates to provide the active drug.
  • the growth hormone 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 growth hormone drug, the oligomer further comprises one or more bonding moieties that are used to covalently couple the oligomer with the growth hormone 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 growth hormone drug.
  • oligomer is preferably covalently coupled to the growth hormone drug
  • the oligomer may be non-covalently coupled to the growth hormone drug to form a non-covalently conjugated growth hormone 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 growth hormone 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 ofthe oligomers in the plurality may be the same and some may be different.
  • all ofthe bonds coupling the plurality of oligomers to the growth hormone drug may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more ofthe oligomers is rapidly removed from the growth hormone drug by hydrolysis in the body and one or more ofthe oligomers is slowly removed from the growth hormone drug by hydrolysis in the body.
  • the oligomer may be coupled to the growth hormone drug at various nucleophilic residues ofthe drug including, but not limited to, nucleophilic hydroxyl functions and/or amino functions. Nucleophilic hydroxyl functions may be found, for example, at serine and/or tyrosine residues, and nucleophilic amino functions may be found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini ofthe polypeptide. When an oligomer is coupled to the one or more N-termini ofthe growth hormone polypeptide, the coupling preferably forms a secondary amine.
  • the oligomer may be coupled to an amino functionality of Phe 1 , Lys 38 , Lys 41 , Lys 70 , Lys 115 , Lys 140 , Lys 145 , Lys 158 , Lys 168 , and/or Lys 172 .
  • Mixtures of growth hormone drug-oligomer conjugates having a dispersity coefficient greater than 10,000 may be synthesized by various methods.
  • a mixture of oligomers having a dispersity coefficient greater than 10,000 consisting of carboxylic acid and polyethylene glycol is synthesized by contacting a mixture of carboxylic 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 ofthe mixture having a dispersity coefficient greater than 10,000 are then activated so that they are capable of reacting with a growth hormone drug to provide a growth hormone drug-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.
  • the mixture of activated oligomers having a dispersity coefficient greater than 10,000 is reacted with a mixture of growth hormone drugs having a dispersity coefficient greater than 10,000 under conditions sufficient to provide a mixture of growth hormone drug- oligomer conjugates.
  • Exemplary syntheses are described hereinbelow in Examples 40 through 42.
  • the reaction conditions e.g., selected molar ratios, solvent mixtures and/or pH
  • the reaction conditions may be controlled such that the mixture of growth hormone drug-oligomer conjugates resulting from the reaction ofthe mixture of activated oligomers having a dispersity coefficient greater than 10,000 and the mixture of growth hormone 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 ofthe reaction solution below the pK a of lysine.
  • the mixture of growth hormone drug-oligomer conjugates may be separated and isolated utilizing, for example, HPLC to provide a mixture of growth hormone 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 Phe 1 , Lys 38 , Lys 41 , Lys 70 , Lys 115 , Lys 140 , Lys 145 , Lys 158 , Lys 168 or Lys 17 of a human growth hormone 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 ofthe reaction sites on the growth hormone drug may be blocked by, for example, reacting the growth hormone 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 growth hormone 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 growth hormone drug-oligomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues.
  • the growth hormone drug-oligomer conjugates may be de-blocked as will be understood by those skilled in the art. If necessary, the mixture of growth hormone drug-oligomer conjugates may then be separated as described above to provide a mixture of growth hormone drug-oligomer conjugates having a dispersity coefficient greater than 10,000.
  • the mixture of growth hormone drug-oligomer conjugates may be separated prior to de-blocking.
  • Mixtures of growth hormone drug-oligomer conjugates having a dispersity coefficient greater than 10,000 according to embodiments ofthe present invention preferably have improved properties when compared with those of conventional mixtures.
  • a mixture of growth hormone drug-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 growth hormone drug-oligomer conjugates having the same number average molecular weight as the mixture of growth hormone drug-oligomer conjugates having a dispersity coefficient greater than 10,000.
  • the number average molecular weight ofthe mixture of growth hormone drug- oligomer conjugates having a dispersity coefficient greater than 10,000 and the number average weight ofthe 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 growth hormone drug-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 growth hormone drug-oligomer conjugates having the same number average molecular weight as the mixture of growth hormone drug-oligomer conjugates having a dispersity coefficient greater than 10,000.
  • the number average molecular weight ofthe mixture of growth hormone drug-oligomer conjugates having a dispersity coefficient greater than 10,000 and the number average weight ofthe 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 drug being added to cultured cells in a transwell. This response is proportional to the activity ofthe molecule under study.
  • a mixture of growth hormone drug-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 growth hormone drug-oligomer conjugates having the same number average molecular weight as the mixture of growth hormone drug- oligomer conjugates having a dispersity coefficient greater than 10,000.
  • the number average molecular weight ofthe mixture of growth hormone drug-oligomer conjugates having a dispersity coefficient greater than 10,000 and the number average weight ofthe polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • a mixture of growth hormone 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 growth hormone drug-oligomer conjugates having the same number average molecular weight as the mixture of growth hormone drug-oligomer conjugates having a dispersity coefficient greater than 10,000.
  • the number average molecular weight ofthe mixture of growth hormone drug-oligomer conjugates having a dispersity coefficient greater than 10,000 and the number average weight ofthe 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 ofthe difference between the subject's AUC and the average AUC is then determined for each subject.
  • the absolute values ofthe 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.
  • Mixtures of growth hormone drug-oligomer conjugates having a dispersity coefficient greater than 10,000 according to embodiments ofthe present invention preferably have two or more ofthe above-described improved properties. More preferably, mixtures of growth hormone drug-oligomer conjugates having a dispersity coefficient greater than 10,000 according to embodiments ofthe present invention have three or more ofthe above-described improved properties. Most preferably, mixtures of growth hormone drug-oligomer conjugates having a dispersity coefficient greater than 10,000 according to embodiments of the present invention have all four ofthe above-described improved properties.
  • a mixture of conjugates in which each conjugate includes a growth hormone drug coupled to an oligomer and has the same number of polyalkylene glycol subunits is provided.
  • the growth hormone drug is preferably human growth hormone.
  • the growth hormone drug may be selected from various growth hormone drugs known to those skilled in the art including, for example, growth hormone peptides, growth hormone peptide analogues, growth hormone peptide fragments, and growth hormone peptide fragment analogues.
  • Growth hormone peptides include, but are not limited to, growth hormone, human (hGH); growth hormone, porcine; growth hormone, bovine; growth hormone, chicken; growth hormone, rat; growth hormone, mouse; growth hormone, ovine; growth hormone releasing factor, human; growth hormone pro-releasing factor, human; growth hormone releasing factor, mouse; growth hormone releasing factor, ovine; growth hormone releasing factor, rat; growth hormone releasing factor, bovine; growth hormone releasing factor, porcine; and growth hormone releasing factor, chicken.
  • Growth hormone peptide analogs may be provided as described above by substituting one or more amino acids in a growth hormone peptide.
  • Growth hormone peptide fragments include, but are not limited to, growth hormone 1-43, human; growth hormone 6-13; growth hormone releasing factor 1-37, human; growth hormone releasing factor 1-40, human; growth hormone releasing factor 1-40, amide, human; growth hormone releasing factor 30-44, amide, human; growth hormone releasing factor 1-29, amide, rat; hexarelin (growth hormone releasing hexapeptide); and growth hormone releasing factor 1-29, amide, human.
  • Growth hormone peptide fragment analogues include, but are not limited to, [D-Ala ] -growth hormone releasing factor 1-29, amide, human; [N-Ac-Tyr , D-Arg ] -growth hormone releasing factor 1-29, amide; [His 1 , Nle 27 ] -growth hormone releasing factor 1-32, amide; growth hormone releasing peptide-6 ([His 1 , Lys 6 ]-GHRP); and [D-Lys 3 ]-GHRP-6.
  • the oligomer may be various oligomers comprising a polyalkylene glycol moiety as will be understood by those skilled in the art.
  • the polyalkylene glycol moiety 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. Most preferably, the polyalkylene glycol moiety ofthe oligomer has at least 7 polyalkylene glycol subunits.
  • the polyalkylene glycol moiety ofthe oligomer is preferably a lower alkyl polyalkylene glycol moiety such as a polyethylene glycol moiety, a polypropylene glycol moiety, or a polybutylene glycol moiety.
  • the polyalkylene glycol moiety is more preferably a polypropylene glycol moiety having a uniform 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 growth hormone drug-oligomer conjugates without the use of lipophilic polymer moieties.
  • coupling the secondary alcohol moiety ofthe polypropylene glycol moiety with a growth hormone drug may provide the growth hormone drug (e.g., human growth hormone) with improved resistance to degradation caused by enzymes such as trypsin and chymotrypsin found, for example, in the gut.
  • Uniform polypropylene glycol according to embodiments ofthe present invention is preferably synthesized as illustrated in Figures 11 through 13, which will now be described.
  • a 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 Bi and provide a primary alcohol extension monomer 57.
  • the B ⁇ de-blocking reagent may be selected from various de-blocking reagents as will be understood by one skilled in the art.
  • the B ⁇ de-blocking reagent is a de- esterification reagent, such as a base (e.g., potassium carbonate).
  • a base e.g., potassium carbonate
  • the Bi de-blocking reagent is preferably tetrabutylammonium fluoride (TBAF).
  • TBAF tetrabutylammonium fluoride
  • 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 B ⁇ 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 and provide a secondary alcohol capping monomer 63.
  • the B 2 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 Bj 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 2 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 B] 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 ofthe 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 ofthe 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 ofthe 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 ofthe present invention may be coupled to a growth hormone drug, a lipophilic moiety such as a carboxylic 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 oligomer may comprise one or more other moieties as will be understood by those skilled in the art including, but not limited to, 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 hydrophilic moieties including, but not limited to, sugars, polyalkylene glycols, and polyamine/PEG copolymers. Adjacent polyalkylene glycol moieties will be considered to be the same moiety if they are coupled by an ether bond and have the same alkyl structure. For example, the moiety
  • O II — O— C 2 H 4 — O— C 2 H 4 — O— C 2 H1— O— C 2 H 4 — C-O— C 2 H 4 — O-C 2 H 4 is a polyethylene glycol moiety having four polyethylene glycol subunits and a hydrophilic moiety having two polyethylene glycol subunits.
  • oligomers according to embodiments ofthe present invention comprise a polyalkylene glycol moiety and do not further comprise a hydrophilic moiety.
  • 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 growth hormone 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 growth hormone 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 ofthe oligomer which are not coupled to the growth hormone 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 growth hormone drug.
  • the growth hormone drug is coupled to the oligomer utilizing a hydrolyzable bond (e.g., an ester or carbonate bond).
  • a hydrolyzable coupling may provide a growth hormone drug-oligomer conjugate that acts as a prodrug.
  • a hydrolyzable coupling may provide for a time-release or controlled-release effect, administering the growth hormone drug over a given time period as one or more oligomers are cleaved from their respective growth hormone drug-oligomer conjugates to provide the active drug.
  • the growth hormone 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 growth hormone drug, the oligomer further comprises one or more bonding moieties that are used to covalently couple the oligomer with the growth hormone 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 growth hormone drug.
  • oligomer is preferably covalently coupled to the growth hormone drug
  • the oligomer may be non-covalently coupled to the growth hormone drug to form a non-covalently conjugated growth hormone 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 growth hormone 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 ofthe oligomers in the plurality may be the same and some may be different.
  • all ofthe bonds coupling the plurality of oligomers to the growth hormone drug may be hydrolyzable, but have varying degrees of hydrolyzability such that, for example, one or more ofthe oligomers is rapidly removed from the growth hormone drug by hydrolysis in the body and one or more ofthe oligomers is slowly removed from the growth hormone drug by hydrolysis in the body.
  • the oligomer may be coupled to the growth hormone drug at various nucleophilic residues ofthe drug including, but not limited to, nucleophilic hydroxyl functions and/or amino functions. Nucleophilic hydroxyl functions may be found, for example, at serine and/or tyrosine residues, and nucleophilic amino functions may be found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini ofthe polypeptide. When an oligomer is coupled to the one or more N-termini ofthe growth hormone polypeptide, the coupling preferably forms a secondary amine.
  • the oligomer may be coupled to an amino functionality of Phe 1 , Lys 38 , Lys 41 , Lys 70 , Lys 115 , Lys 140 , Lys 145 , Lys 158 , Lys 168 , and/or Lys 172 .
  • Mixtures of growth hormone drug oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits may be synthesized by various methods.
  • a mixture of oligomers consisting of carboxylic 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 carboxylic 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 ofthe 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 a growth hormone drug to provide a growth hormone 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.
  • FIG. 6 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.
  • the mixture of activated oligomers where each oligomer in the mixture has the same number of polyethylene glycol subunits is reacted with a mixture of growth hormone drugs under conditions sufficient to provide a mixture of growth hormone drug-oligomer conjugates.
  • Exemplary syntheses are described hereinbelow in Examples 40 through 42.
  • reaction conditions e.g., selected molar ratios, solvent mixtures and/or pH
  • 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 growth hormone drug-oligomer conjugates may be separated and isolated utilizing, for example, HPLC to provide a mixture of growth hormone 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 Phe 1 , Lys 38 , Lys 41 , Lys 70 , Lys 115 , Lys 140 , Lys 145 , Lys 158 , Lys 168 or Lys 172 of a human growth hormone 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 ofthe reaction sites on the growth hormone drug may be blocked by, for example, reacting the growth hormone 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 growth hormone drugs 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 growth hormone drug-oligomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues.
  • the growth hormone drug-oligomer conjugates may be de-blocked as will be understood by those skilled in the art. If necessary, the mixture of growth hormone drug-oligomer conjugates may then be separated as described above to provide a mixture of growth hormone drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits. Alternatively, the mixture of growth hormone drug-oligomer conjugates may be separated prior to de-blocking.
  • Mixtures of growth hormone drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits preferably have improved properties when compared with those of conventional mixtures.
  • a mixture of growth hormone 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 growth hormone drug-oligomer conjugates having the same number average molecular weight as the mixture of growth hormone drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits.
  • the number average molecular weight ofthe mixture of growth hormone drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits and the number average weight ofthe 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 growth hormone 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 growth hormone drug-oligomer conjugates having the same number average molecular weight as the mixture of growth hormone drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits.
  • the number average molecular weight ofthe mixture of growth hormone drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits and the number average weight ofthe 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 drug being added to cultured cells in a transwell. This response is proportional to the activity ofthe molecule under study.
  • a mixture of growth hormone drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits has an increased resistance to degradation by chymotrypsin when compared to the resistance to degradation by chymotrypsin of a polydispersed mixture of growth hormone drug-oligomer conjugates having the same number average molecular weight as the mixture of growth hormone drug- oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits.
  • the number average molecular weight ofthe mixture of growth hormone drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits and the number average weight ofthe polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • a mixture of growth hormone drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of growth hormone drug-oligomer conjugates having the same number average molecular weight as the mixture of growth hormone drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits.
  • the number average molecular weight ofthe mixture of growth hormone drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits and the number average weight ofthe 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 ofthe difference between the subject's AUC and the average AUC is then determined for each subject.
  • the absolute values ofthe 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.
  • Mixtures of growth hormone drug-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 ofthe above-described improved properties. More preferably, mixtures of growth hormone drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits according to embodiments ofthe present invention have three or more ofthe above-described improved properties. Most preferably, mixtures of growth hormone drug-oligomer conjugates where each conjugate in the mixture has the same number of polyethylene glycol subunits according to embodiments ofthe present invention have all four ofthe above-described improved properties.
  • each conjugate has the same molecular weight and has the formula:
  • 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; h, i, j, k, m and n are individually 0 or 1 , with the proviso that when R is the polyalkylene glycol moiety; m is 1 , and when R' is the polyalkylene glycol moiety, n is i; and p is an integer from 1 to the number of nucleophilic residues on the growth hormone drug.
  • the growth hormone drug is preferably human growth hormone.
  • the growth hormone drug may be selected from various growth hormone drugs known to those skilled in the art including, for example, growth hormone peptides, growth hormone peptide analogues, growth hormone peptide fragments, and growth hormone peptide fragment analogues.
  • Growth hormone peptides include, but are not limited to, growth hormone, human (hGH); growth hormone, porcine; growth hormone, bovine; growth hormone, chicken; growth hormone, rat; growth hormone, mouse; growth hormone, ovine; growth hormone releasing factor, human; growth hormone pro-releasing factor, human; growth hormone releasing factor, mouse; growth hormone releasing factor, ovine; growth hormone releasing factor, rat; growth hormone releasing factor, bovine; growth hormone releasing factor, porcine; and growth hormone releasing factor, chicken.
  • Growth hormone peptide analogs may be provided as described above by substituting one or more amino acids in a growth hormone peptide.
  • Growth hormone peptide fragments include, but are not limited to, growth hormone 1-43, human; growth hormone 6-13; growth hormone releasing factor 1-37, human; growth hormone releasing factor 1-40, human; growth hormone releasing factor 1-40, amide, human; growth hormone releasing factor 30-44, amide, human; growth hormone releasing factor 1-29, amide, rat; hexarelin (growth hormone releasing hexapeptide); and growth hormone releasing factor 1 -29, amide, human.
  • Growth hormone peptide fragment analogues include, but are not limited to, [D-Ala ]-growth hormone releasing factor
  • R or R' is a polyalkylene moiety.
  • the polyalkylene glycol moiety 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. Most preferably, the polyalkylene glycol moiety ofthe oligomer has at least 7 polyalkylene glycol subunits.
  • the polyalkylene glycol moiety ofthe oligomer is preferably a lower alkyl polyalkylene glycol moiety such as a polyethylene glycol moiety, a polypropylene glycol moiety, or a polybutylene glycol moiety.
  • the polyalkylene glycol moiety is more preferably a polypropylene glycol moiety having a uniform 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 growth hormone drug-oligomer conjugates without the use of lipophilic polymer moieties (i.e., the sum of m + n is 1).
  • coupling the secondary alcohol moiety ofthe polypropylene glycol moiety with a growth hormone drug may provide the growth hormone drug (e.g., human growth hormone) with improved resistance to degradation caused by enzymes such as trypsin and chymotrypsin found, for example, in the gut.
  • Uniform polypropylene glycol according to embodiments ofthe present invention is preferably synthesized as illustrated in Figures 11 through 13, which will now be described.
  • a 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 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 B ⁇ de-blocking reagent to remove the blocking moiety B[ 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).
  • a base e.g., potassium carbonate
  • the Bi de-blocking reagent is preferably tetrabutylammonium fluoride (TBAF).
  • TBAF tetrabutylammonium fluoride
  • 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 2 de-blocking reagent may be various deblocking agents as will be understood by those skilled in the art including, but not limited to, H 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.
  • 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 ofthe 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 ofthe capped trimer 68.
  • Uniform polypropylene glycol moieties according to embodiments ofthe present invention may be coupled to a growth hormone drug, a lipophilic moiety such as a carboxylic 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.
  • R or R' 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., i, j and k are preferably 0).
  • the linker moiety, L may be used to couple the oligomer with the growth hormone 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.
  • 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 ofthe structure of Formula A, is covalently coupled to the growth hormone drug.
  • the growth hormone drug is coupled to the oligomer utilizing a hydrolyzable bond (e.g., an ester or carbonate bond).
  • a hydrolyzable coupling may provide a growth hormone drug-oligomer conjugate that acts as a prodrug.
  • a hydrolyzable coupling may provide for a time-release or controlled-release effect, administering the growth hormone drug over a given time period as one or more oligomers are cleaved from their respective growth hormone drug-oligomer conjugates to provide the active drug.
  • the growth hormone drug is coupled to the oligomer utilizing a non-hydrolyzable bond (e.g., a carbamate, amide, or ether bond).
  • the bonding moiety may be various bonding moieties that may be used to covalently couple the oligomer with the growth hormone 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.
  • variable p is an integer from 1 to the number of nucleophilic residues on the growth hormone drug.
  • p is greater than 1, more than one oligomer (i.e., a plurality of oligomers) is coupled to the drug.
  • the oligomers in the plurality are the same.
  • the oligomer may be coupled to the growth hormone drug at various nucleophilic residues ofthe drug including, but not limited to, nucleophilic hydroxyl functions and/or amino functions. Nucleophilic hydroxyl functions may be found, for example, at serine and/or tyrosine residues, and nucleophilic amino functions may be found, for example, at histidine and/or lysine residues, and/or at the one or more N-termini ofthe polypeptide. When an oligomer is coupled to the one or more N-termini ofthe growth hormone polypeptide, the coupling preferably forms a secondary amine.
  • the oligomer may be coupled to an amino functionality of Phe 1 , Lys 38 , Lys 41 , Lys 70 , Lys 115 , Lys 140 , Lys 145 , Lys 158 , Lys 168 , and/or Lys 172 .
  • Mixtures of growth hormone 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 carboxylic acid and polyethylene glycol is synthesized by contacting a mixture of carboxylic acid with a mixture of polyethylene glycol under conditions sufficient to provide a mixture of oligomers. The oligomers ofthe mixture are then activated so that they are capable of reacting with a growth hormone drug to provide a growth hormone drug-oligomer conjugate.
  • a synthesis route for providing a mixture of activated oligomers where each oligomer has the same molecular weight and has a structure ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe oligomer of Formula A is illustrated in Figure 8 and described in Example 38 hereinbelow.
  • the mixture of activated oligomers where each oligomer has the same molecular weight and has a structure ofthe oligomer of Formula A is reacted with a mixture of growth hormone drugs where each dmg in the mixture has the same molecular weight under conditions sufficient to provide a mixture of growth hormone drug-oligomer conjugates.
  • Exemplary syntheses are described hereinbelow in Examples 40 through 42.
  • the reaction conditions e.g., selected molar ratios, solvent mixtures and/or pH
  • the mixture of growth hormone drug-oligomer conjugates resulting from the reaction ofthe mixture of activated oligomers where each oligomer has the same molecular weight and has a structure ofthe oligomer of Formula A and the mixture of growth hormone drugs is a mixture of conjugates where each conjugate has the same molecular weight and has the structure 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 growth hormone drug-oligomer conjugates may be separated and isolated utilizing, for example, HPLC to provide a mixture of growth hormone drug-oligomer conjugates, for example mono-, di-, or tri-conjugates, where each conjugate in the mixture has the same number molecular weight and has the structure of Formula A.
  • the degree of conjugation e.g., whether the isolated molecule is a mono-, di-, or tri-conjugate
  • the particular conjugate structure e.g., whether the oligomer is at Phe 1 , Lys 38 , Lys 41 , Lys 70 , Lys 115 , Lys 140 , Lys 145 , Lys 158 ,
  • Lys or Lys of a human growth hormone 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 ofthe reaction sites on the growth hormone dmg may be blocked by, for example, reacting the growth hormone dmg 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 growth hormone d gs may be reacted with the mixture of activated oligomers where each oligomer in the mixture has the same molecular weight and has a stmcture ofthe oligomer of Formula A to provide a mixture of growth hormone dmg-oligomer conjugates having oligomer(s) coupled to one or more nucleophilic residues and having blocking moieties coupled to other nucleophilic residues.
  • the growth hormone dmg-oligomer conjugates may be de-blocked as will be understood by those skilled in the art.
  • the mixture of growth hormone dmg-oligomer conjugates may then be separated as described above to provide a mixture of growth hormone dmg-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stmcture of Formula A.
  • the mixture of growth hormone dmg-oligomer conjugates may be separated prior to de-blocking.
  • Mixtures of growth hormone dmg-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stmcture of Formula A according to embodiments ofthe present invention preferably have improved properties when compared with those of conventional mixtures.
  • a mixture of growth hormone dmg- oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stmcture of Formula A preferably has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of growth hormone dmg-oligomer conjugates having the same number average molecular weight as the mixture of growth hormone dmg-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stmcture of Formula A.
  • the number average molecular weight ofthe mixture of growth hormone dmg-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stmcture of Formula A and the number average weight ofthe 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 growth hormone dmg-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stmcture of Formula A preferably has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of growth hormone dmg-oligomer conjugates having the same number average molecular weight as the mixture of growth hormone dmg-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stmcture of Formula A.
  • the number average molecular weight ofthe mixture of growth hormone dmg-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stmcture of Formula A and the number average weight ofthe 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 dmg being added to cultured cells in a transwell. This response is proportional to the activity ofthe molecule under study.
  • a mixture of growth hormone dmg-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stmcture 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 growth hormone dmg-oligomer conjugates having the same number average molecular weight as the mixture of growth hormone dmg-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stmcture of Formula A.
  • the number average molecular weight ofthe mixture of growth hormone dmg-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stmcture of Formula A and the number average weight ofthe polydispersed mixture may be measured by various methods including, but not limited to, size exclusion chromatography.
  • a mixture of growth hormone dmg-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stmcture of Formula A preferably has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of growth hormone dmg-oligomer conjugates having the same number average molecular weight as the mixture of growth hormone dmg-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stmcture of Formula A.
  • the number average molecular weight ofthe mixture of growth hormone dmg-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stmcture of Formula A and the number average weight ofthe 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.
  • 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 ofthe 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.
  • Mixtures of growth hormone dmg-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stmcture of Formula A according to embodiments ofthe present invention preferably have two or more ofthe above-described improved properties.
  • mixtures of growth hormone dmg-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stmcture of Formula A according to embodiments ofthe present invention have three or more ofthe above-described improved properties.
  • mixtures of growth hormone dmg-oligomer conjugates where each conjugate in the mixture has the same molecular weight and has the stmcture of Formula A according to embodiments ofthe present invention have all four ofthe above-described improved properties.
  • Pharmaceutical compositions comprising a conjugate mixture according to embodiments ofthe present invention are also provided.
  • the mixtures of growth hormone dmg-oligomer conjugates described above may be formulated for administration in a pharmaceutical carrier in accordance with known techniques.
  • the mixture of growth hormone dmg-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 patient.
  • the carrier may be a solid or a liquid, or both, and is preferably formulated with the mixture of growth hormone dmg-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 ofthe mixture of growth hormone dmg-oligomer conjugates.
  • the pharmaceutical compositions may be prepared by any ofthe well known techniques of pharmacy including, but not limited to, admixing the components, optionally including one or more accessory ingredients.
  • compositions according to embodiments ofthe 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 ofthe condition being treated and on the nature ofthe particular mixture of growth hormone dmg-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
  • compositions suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tables, each containing a predetermined amount ofthe mixture of growth hormone dmg-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 growth hormone dmg-oligomer conjugates and a suitable carrier (which may contain one or more accessory ingredients as noted above).
  • the pharmaceutical composition according to embodiments ofthe present invention are prepared by uniformly and intimately admixing the mixture of growth hormone dmg-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 growth hormone dmg-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 growth hormone drug-oligomer conjugates in a flavoured base, usually sucrose and acacia or tragacanth; and pastilles comprising the mixture of growth hormone dmg-oligomer conjugates in an inert base such as gelatin and glycerin or sucrose and acacia.
  • compositions according to embodiments ofthe present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the mixture of growth hormone dmg-oligomer conjugates, which preparations are preferably isotonic with the blood ofthe intended recipient. These preparations may contain anti-oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood ofthe intended recipient.
  • Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents.
  • 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 ofthe 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 ofthe kind previously described.
  • an injectable, stable, sterile composition comprising a mixture of growth hormone dmg-oligomer conjugates in a unit dosage form in a sealed container may be provided.
  • the mixture of growth hormone dmg- 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 ofthe mixture of growth hormone dmg-oligomer conjugates.
  • a sufficient amount of emulsifying agent which is physiologically acceptable may be employed in sufficient quantity to emulsify the mixture of growth hormone dmg-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 growth hormone dmg-oligomer conjugates with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.
  • 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 ofthe 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 ofthe mixture of growth hormone dmg-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 growth hormone dmg-oligomer conjugates will vary somewhat from mixture to mixture, and patient to patient, and will depend upon factors such as the age and condition ofthe patient 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 ofthe mixture of growth hormone dmg-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 ofthe active base.
  • a dosage from about 10 mg/kg to about 50 mg/kg may be employed for oral administration. Typically, 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 dmg-oligomer conjugates may be administered by continuous infusion.
  • the duration of treatment depends on the type of growth hormone deficiency being treated.
  • Methods of accelerating the growth rate of an animal comprise administering to the animal an effective amount of mixture of conjugates according to the various embodiments described above.
  • the effective amount of growth hormone will, of course, depend upon the animal undergoing administration and can be determined by one of skill in the art.
  • 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. Preferably 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.
  • positive ions include, but are not limited to, sodium ions, potassium ions, lithium ions, cesium ions, and thallium ions. 9
  • R is H or a lipophilic moiety.
  • R 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 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.
  • n is from 1 to 25. Preferably m is from 1 to 6.
  • Ms is a mesylate moiety (i.e., CH 3 S(O )-).
  • reaction 1 a mixture of compounds having the structure of Formula I is reacted with a mixture of compounds having the stmcture of Formula II to provide a mixture of polymers comprising polyethylene glycol moieties and having the stmcture of Formula III.
  • the mixture of compounds having the stmcture of Formula I is a substantially monodispersed mixture.
  • at least about 96, 97, 98 or 99 percent ofthe 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 ofthe 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 ofthe 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 ofthe 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 1" are as described above and the mixture of compounds of Formula IV is substantially monodispersed; preferably, at least about 96, 97, 98 or 99 percent ofthe 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 ofthe 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.
  • 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 ofthe compound capable of ionizing a hydroxyl moiety on the PEG moiety ofthe 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
  • (V) ° (II) 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 ofthe 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 ofthe 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, monomethylamine, 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.
  • various substantially monodispersed mixtures of compounds of Formula V are commercially available. For example, when R 2 is H or methyl, 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 2 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 2 + is a positive ion as described above with respect to X + .
  • 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.
  • at least about 96, 97, 98 or 99 percent ofthe compounds in the mixture of compounds of 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 3 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.
  • 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 stmcture 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:
  • 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 ofthe 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 ofthe 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.
  • substantially monodispersed polyethylene glycol-containing oligomers begins by the preparation ofthe monobenzyl ether (1) of a substantially monodispersed polyethylene glycol.
  • An excess of a commercially available substantially monodispersed polyethylene glycol is reacted with benzyl chloride in the presence of aqueous sodium hydroxide as described by Coudert et al (Synthetic Communications, 16(1): 19-26 (1986)).
  • the sodium salt of 1 is then prepared by the addition of NaH, and this sodium salt is allowed to react with the mesylate synthesized from the ester of a hydroxyalkanoic acid (2).
  • the product (3) ofthe displacement ofthe mesylate is debenzylated via catalytic hydrogenation to obtain the alcohol (4).
  • 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 ofthe monomethyl ether of a substantially monodispersed polyethylene glycol derivative, thereby extending the polyethylene glycol portion ofthe 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 ofthe present invention including, but not limited to, active phenyl chloroformates such as ? ⁇ ra-niuOphenyl 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 is a moiety capable of undergoing hydrolysis to provide the carboxylic acid.
  • R 4 is preferably lower alkyl and is more preferably ethyl.
  • the variables n and m are as described above with reference to reaction 1.
  • Example 1 8-Methoxy-l-(methylsulfonyl)oxy-3,6-dioxaoctane (9)
  • a solution of non-polydispersed triethylene glycol monomethyl ether molecules (4.00 mL, 4.19 g, 25.5 mmol) and triethylamine (4.26 mL, 3.09 g, 30.6 mmol) in dry dichloromethane (50 mL) was chilled in an ice bath and place under a nitrogen atmosphere.
  • a solution of methanesulfonyl chloride (2.37 mL, 3.51 g, 30.6 mmol) in dry dichloromethane (20 mL) was added dropwise from an addition funnel.
  • reaction mixture was removed from the ice bath and allowed to come to room temperature. The mixture was stirred for an additional hour, at which time TLC (CHC1 3 with 15% MeOH as the elutant) showed no remaining triethylene glycol monomethyl ether.
  • non-polydispersed compound 11 (35.7 mmol) in dry DMF (25.7 mL), under N 2 was added in portion a 60% dispersion of NaH in mineral oil, and the mixture was stirred at room temperature for 1 hour.
  • this salt 12 was added a solution of non-polydispersed mesylate 9 (23.36) in dry DMF (4 ml) in a single portion, and the mixture was stirred at room temperature for 3.5 hours. Progress ofthe reaction was monitored by TLC (12%) CH 3 OH-CHCl 3 ). The reaction mixture was diluted with an equal amount of IN HCl, and extracted with ethyl acetate (2 x 20 ml) and discarded. Extraction of aqueous solution and work-up gave non-polydispersed polymer 10 (82 -84% yield).
  • the non-polydispersed compounds 15 were prepared from a diol by using the procedure described above for compound 10.
  • 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.
  • 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 ofthe 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).
  • 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 o for 15 minutes at 0 C, then at room temperature for two hours.
  • Non-polydispersed ester 21 (0.25 g, 0.46 mmol) was stirred for 18 hours in 0.71 ml of 1 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). The combined organics were then washed with brine (25 ml x 2), dried over Na SO 4 , filtered and concentrated in vacuo to yield the non-polydispersed title compound as a yellow oil (0.147 g, 62 %). FAB MS: m/e 499 (M+H), 521 (M+Na).
  • 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. The solution was stirred at room temperature overnight and filtered through silica gel to remove excess reagents and the urea formed from the EDC. The filtrate was concentrated in vacuo to provide the non- polydispersed product as a dark yellow oil (0.235 g, 94 %).
  • FAB MS m/e 596 (M+H), 618 (M+Na).
  • 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 2 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).
  • ethyl acetate 125 mL
  • the aqueous phase was washed repetitively with CH 2 C1 2 (125 mL volumes) until most ofthe 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.
  • 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 EDCTHC1 (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 4 , filtered and concentrated. Cmde 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 cmde 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 ⁇ o ester 32 (0.570 g, 1.1 mmol) was added
  • Example 29 Activation of MPEG 7 -C ⁇ 0 Acid (34)
  • 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.
  • Non-polydispersed C18(PEG6) oligomer was accomplished in two steps: 1) Non-polydispersed stearoyl-PEG6 36 ( 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.
  • 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 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 (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.
  • 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 cmde 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 37 Activation of C18(PEG8) Oligomer Two step activation of non-polydispersed stearate-PEG8 oligomer was performed as described for stearate-PEG6 in Example 31 above to provide the non-polydispersed activated C18(PEG8) oligomer 45.
  • 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) and NHS (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. 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.
  • Example 39 Synthesis of Activated Palmitate-TEG Oligomers The following description refers to the scheme illustrated in Figure 9.
  • Non- polydispersed palmitic anhydride (5 g; 10 mmol) was dissolved in dry THF (20 mL) and stirred at room temperature.
  • 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 ofthe reaction was monitored by TLC; ethyl acetate-chloroform; 3:7).
  • 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 activated hexaethylene glycol monomethyl ether was prepared analogously to that of non-polydispersed triethylene glycol in Example 38 above.
  • a 20% phosgene in toluene solution 35 mL, 6.66 g, 67.4 mmol phosgene
  • 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.
  • 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. Triethylamine (0.94 mL, 0.68 g, 6.7 mmol) and then NHS (N-hydroxy succinimide, 0.82 g, 7.1 mmol) were added, and the reaction mixture was stirred at room temperature for 18 hours. The mixture was filtered through silica gel to remove the white precipitate and concentrated in vacuo. The residue was taken up in dichloromethane and washed twice with cold water, twice with 1 N HCl and once with brine. The organics were dried over Na 2 SO 4 , filtered and concentrated. Final purification was done via flash chromatography (silica gel, EtOAc) to obtain the UV active non-polydispersed NHS ester 52.
  • Human growth hormone (somatropin (rDNA origin) for injection), available under the trade name SaizenTM from Serono of Randolph, Massachusetts, was dissolved in DMSO such that the hGH was at a 0.58 mmol concentration.
  • TEA (278 equivalents) was added and the solution was stirred for approximately ten minutes.
  • Two equivalents, five equivalents or thirty equivalents of non-polydispersed activated hexaethylene glycol 52 was added from a 0.2 M solution ofthe activated oligomer in dry THF. Reactions were stirred at room temperature for 45 minutes to one hour. Aliquots of each reaction mixture were quenched in 600 ⁇ L of 0.1% TFA in water.
  • HPLC comparison ofthe 2 polymer equivalent and the 5 polymer equivalent reaction mixtures vs. unconjugated hGH is shown in Figure 14.
  • HPLC analysis ofthe thirty polymer equivalent reaction is shown in Figure 15.
  • Samples ofthe conjugates for mass spectroscopy were purified via analytical HPLC using a re versed-phase C 18 column and a water/acetonitrile gradient. The entire peak from the 2 equivalent reaction mixture was collected, concentrated and analyzed using MALDI mass spectroscopy. The mass spectra of this material showed evidence ofthe presence of mono-conjugated, di-conjugated, tri-conjugated and tetra-conjugated hGH as well as some remaining unreacted hGH ( Figure 16).
  • the pooled fraction was lyophilized into a white powder.
  • the mass spectra for the compound are illustrated in Figures 26 and 27.
  • a similar procedure utilizing nine equivalents TEA and nine equivalents ofthe activated oligomer of Example 39 was performed.
  • the conjugated product was purified by prep. HPLC using C18 column as illustrated in Figure 28.
  • the dispersity coefficient of a mixture of human growth hormone-oligomer conjugates is determined as follows.
  • a mixture of human growth hormone-oligomer conjugates is provided, for example, as described above in Example 40.
  • a first sample ofthe mixture is purified via HPLC to separate and isolate the various human growth hormone- oligomer conjugates in the sample. Assuming that each isolated fraction contains a purely monodispersed mixture of conjugates, "n" is equal to the number of fractions collected.
  • the mixture may include one or more conjugates, including mono-, di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, and/or nona-conjugates.
  • Each isolated fraction ofthe mixture is analyzed via mass spectroscopy to determine the mass ofthe fraction, which allows each isolated fraction to be categorized by its degree of conjugation and provides a value for the variable "Mi" for each conjugate in the sample.
  • a second sample ofthe mixture is analyzed via HPLC to provide an HPLC trace. Assuming that the molar absorptivity does not change as a result ofthe conjugation, the weight percent of a particular conjugate in the mixture is provided by the area under the peak ofthe HPLC trace corresponding to the particular conjugate as a percentage ofthe total area under all peaks ofthe HPLC trace.
  • the sample is collected and lyophilized to dryness to determine the anhydrous gram weight ofthe sample.
  • the gram weight ofthe sample is multiplied by the weight percent of each component in the sample to determine the gram weight of each conjugate in the sample.
  • the variable "Ni" is determined for a particular conjugate (the i th conjugate) by dividing the gram weight ofthe particular conjugate in the sample by the mass ofthe particular conjugate and multiplying the quotient by Avagadro's number (6.02205 x 10 mole " ), Mi, determined above, to give the number of molecules of the particular conjugate, Nj, in the sample.
  • the dispersity coefficient is then calculated using n, Mj as determined for each conjugate, and Nj as determined for each conjugate.
  • Transcription assays were performed in 293 GHR cells transiently transfected with a reported construct containing a Stat5 -binding element (LHRE) fused to a minimal TK promoter and luciferase.
  • a ⁇ -galactosidase expression vector was co-transfected as a transfection control and luciferase values corrected for ⁇ -galactosidase activity.
  • the maximal activity stimulated by GH is the fold induction stimulated by GH, i.e.
  • Genotropin is human growth hormone (standard, not part ofthe present invention)
  • GH-002 is a 2 equivalent mTEG conjugate
  • GH-003 is a 5 equivalent mTEG conjugate
  • GH-004 is a 5 equivalent mTEG conjugate
  • Prot hGH is human growth hormone (standard, not part ofthe present invention)
  • hGH-TEG is a 9 equivalent mTEG conjugate.
PCT/US2002/017504 2001-06-04 2002-06-04 Mixtures of growth hormone drug-oligomer conjugates compromising polyalkylene glycol, uses thereof, and methods of making same WO2002098452A1 (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP02737344A EP1404361A4 (en) 2001-06-04 2002-06-04 MIXTURES OF OLIGOMER-ACTIVE GROWTH HORMONE CONJUGATED CONJUGATES COMPRISING POLYALKYLENE GLYCOL, USES THEREOF AND METHODS OF MAKING THEM
CA002449320A CA2449320A1 (en) 2001-06-04 2002-06-04 Mixtures of growth hormone drug-oligomer conjugates compromising polyalkylene glycol, uses thereof, and methods of making same
KR10-2003-7015911A KR20040004693A (ko) 2001-06-04 2002-06-04 폴리알킬렌 글리콜을 포함하는 성장 호르몬 약물-올리고머접합체들의 혼합물, 그 용도 및 그 제조 방법
MXPA03011282A MXPA03011282A (es) 2001-06-04 2002-06-04 Mezclas de conjugados de farmaco de hormona de crecimiento-oligomero que comprenden polialquilenglicol, uso de los mismos y metodos para su elaboracion.
JP2003501490A JP2004534783A (ja) 2001-06-04 2002-06-04 ポリアルキレングリコールを含む成長ホルモン剤−オリゴマー抱合体の混合物、それらの使用、およびそれらの製造方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US09/873,757 2001-06-04
US09/873,757 US6828305B2 (en) 2001-06-04 2001-06-04 Mixtures of growth hormone drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same

Publications (1)

Publication Number Publication Date
WO2002098452A1 true WO2002098452A1 (en) 2002-12-12

Family

ID=25362249

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/017504 WO2002098452A1 (en) 2001-06-04 2002-06-04 Mixtures of growth hormone drug-oligomer conjugates compromising polyalkylene glycol, uses thereof, and methods of making same

Country Status (8)

Country Link
US (1) US6828305B2 (US20030027995A1-20030206-C00005.png)
EP (1) EP1404361A4 (US20030027995A1-20030206-C00005.png)
JP (1) JP2004534783A (US20030027995A1-20030206-C00005.png)
KR (1) KR20040004693A (US20030027995A1-20030206-C00005.png)
CN (1) CN1538852A (US20030027995A1-20030206-C00005.png)
CA (1) CA2449320A1 (US20030027995A1-20030206-C00005.png)
MX (1) MXPA03011282A (US20030027995A1-20030206-C00005.png)
WO (1) WO2002098452A1 (US20030027995A1-20030206-C00005.png)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005000359A2 (en) * 2002-11-20 2005-01-06 Pharmacia Corporation Chemically-modified human growth hormone conjugates
EP1594440A2 (en) * 2003-02-14 2005-11-16 Quanta Biodesign, Ltd. The selective and specific preparation of discrete peg compounds
US9012469B2 (en) 2010-09-30 2015-04-21 Astrazeneca Ab Crystalline naloxol-peg conjugate
EP2905033A1 (en) * 2003-12-16 2015-08-12 Nektar Therapeutics Monodisperse PEGylated naloxol compositions
US9308263B2 (en) 2011-10-21 2016-04-12 Seachaid Pharmaceuticals, Inc. Pharmaceutical compositions and uses thereof
US11129794B2 (en) 2003-12-16 2021-09-28 Nektar Therapeutics Chemically modified small molecules

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6703381B1 (en) * 1998-08-14 2004-03-09 Nobex Corporation Methods for delivery therapeutic compounds across the blood-brain barrier
US6858580B2 (en) * 2001-06-04 2005-02-22 Nobex Corporation Mixtures of drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6713452B2 (en) 2001-06-04 2004-03-30 Nobex Corporation Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6835802B2 (en) 2001-06-04 2004-12-28 Nobex Corporation Methods of synthesizing substantially monodispersed mixtures of polymers having polyethylene glycol moieties
US6828297B2 (en) * 2001-06-04 2004-12-07 Nobex Corporation Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US7713932B2 (en) 2001-06-04 2010-05-11 Biocon Limited Calcitonin drug-oligomer conjugates, and uses thereof
US7196059B2 (en) * 2001-09-07 2007-03-27 Biocon Limited Pharmaceutical compositions of insulin drug-oligomer conjugates and methods of treating diseases therewith
US7611700B2 (en) * 2002-09-09 2009-11-03 Hanall Pharmaceuticals, Co., Ltd. Protease resistant modified interferon alpha polypeptides
AU2003297583B2 (en) * 2002-11-26 2010-01-14 Biocon, Ltd Modified naturetic compounds, conjugates, and uses thereof
US8329958B2 (en) 2004-07-02 2012-12-11 Biocon Limited Combinatorial synthesis of PEG oligomer libraries
RU2393168C2 (ru) 2004-07-19 2010-06-27 Биокон Лимитед Инсулин-олигомерные конъюгаты, их препараты и применения
US7998930B2 (en) 2004-11-04 2011-08-16 Hanall Biopharma Co., Ltd. Modified growth hormones
WO2006076471A2 (en) * 2005-01-12 2006-07-20 Nobex Corporation Bnp conjugates and methods of use
WO2006102659A2 (en) * 2005-03-23 2006-09-28 Nektar Therapeutics Al, Corporation CONJUGATES OF AN hGH MOIETY AND A POLYMER
US20070015689A1 (en) * 2005-06-23 2007-01-18 Alza Corporation Complexation of metal ions with polypeptides
MX2010003979A (es) * 2007-10-16 2010-06-02 Biocon Ltd Composicion farmaceutica oralmente administrable y proceso para su preparacion.
US9849188B2 (en) 2009-06-08 2017-12-26 Amunix Operating Inc. Growth hormone polypeptides and methods of making and using same
CN104592382A (zh) * 2015-01-19 2015-05-06 中国科学院过程工程研究所 一种peg-长链脂肪烷定点修饰的人生长激素及其制备方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) * 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US5349052A (en) * 1988-10-20 1994-09-20 Royal Free Hospital School Of Medicine Process for fractionating polyethylene glycol (PEG)-protein adducts and an adduct for PEG and granulocyte-macrophage colony stimulating factor
US5359030A (en) * 1993-05-10 1994-10-25 Protein Delivery, Inc. Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic formulations comprising same, and method of making and using the same
US5597797A (en) * 1990-06-07 1997-01-28 Genentech, Inc. Method for treatment or prevention of obesity
WO1997014740A1 (en) * 1995-10-19 1997-04-24 Receptagen Corporation Discrete-length polyethylene glycols
US6057292A (en) * 1995-09-21 2000-05-02 Genentech, Inc. Method for inhibiting growth hormone action

Family Cites Families (160)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3256153A (en) * 1963-02-08 1966-06-14 Smith Kline French Lab Method of stabilizing wax-fat coating materials and product thereof
US4003792A (en) * 1967-07-01 1977-01-18 Miles Laboratories, Inc. Conjugates of acid polysaccharides and complex organic substances
US3950517A (en) * 1970-05-08 1976-04-13 National Research Development Corporation Insulin derivatives
GB1381274A (en) * 1971-01-28 1975-01-22 Nat Res Dev Insulin derivatives
US3919411A (en) 1972-01-31 1975-11-11 Bayvet Corp Injectable adjuvant and compositions including such adjuvant
US4044196A (en) * 1972-03-30 1977-08-23 Bayer Aktiengesellschaft Crosslinked copolymers of α,β-olefinically unsaturated dicarboxylic anhydrides
FR2408387A2 (fr) 1975-06-30 1979-06-08 Oreal Compositions a base de dispersions aqueuses de spherules lipidiques
US4087390A (en) * 1977-02-02 1978-05-02 Eli Lilly And Company Somatostatin analogs and intermediates thereto
US4093574A (en) * 1977-02-02 1978-06-06 Eli Lilly And Company Somatostatin analogs and intermediates thereto
GB1492997A (en) 1976-07-21 1977-11-23 Nat Res Dev Insulin derivatives
US4223163A (en) 1976-12-10 1980-09-16 The Procter & Gamble Company Process for making ethoxylated fatty alcohols with narrow polyethoxy chain distribution
JPS53116315A (en) 1977-03-17 1978-10-11 Ueno Seiyaku Oyo Kenkyujo Kk Powder or granular containing improved sorbinic acid
US4100117A (en) * 1977-04-21 1978-07-11 Eli Lilly And Company Somatostatin analogs and intermediates thereto
US4253998A (en) * 1979-03-09 1981-03-03 American Home Products Corporation Peptides related to somatostatin
JPS54148722A (en) 1978-05-12 1979-11-21 Takeda Chem Ind Ltd Nonapeptide and its preparation
US4277394A (en) * 1979-04-23 1981-07-07 Takeda Chemical Industries, Ltd Tetrapeptidehydrazide derivatives
GB2051574B (en) * 1979-05-10 1984-01-18 Kyoto Pharma Ind Adjuvant for promoting absorption of pharmacologically active substances through the rectum
US4348387A (en) 1979-07-31 1982-09-07 The Rockefeller University Method and system for the controlled release of biologically active substances to a body fluid
US4469681A (en) 1979-07-31 1984-09-04 The Rockefeller University Method and system for the controlled release of biologically active substances to a body fluid
FR2465486A1 (fr) 1979-09-21 1981-03-27 Roussel Uclaf Nouvelle application utilisant la lh-rh ou des agonistes
JPS5692846A (en) 1979-12-27 1981-07-27 Takeda Chem Ind Ltd Tetrapeptide derivative and its preparation
US4554101A (en) 1981-01-09 1985-11-19 New York Blood Center, Inc. Identification and preparation of epitopes on antigens and allergens on the basis of hydrophilicity
US4698264A (en) 1982-08-02 1987-10-06 Durkee Industrial Foods, Corp. Particulate composition and process for making same
IL68769A (en) * 1983-05-23 1986-02-28 Hadassah Med Org Pharmaceutical compositions containing insulin for oral administration
US4585754A (en) * 1984-01-09 1986-04-29 Valcor Scientific, Ltd. Stabilization of proteins and peptides by chemical binding with chondroitin
US4684524A (en) * 1984-03-19 1987-08-04 Alza Corporation Rate controlled dispenser for administering beneficial agent
US4717566A (en) * 1984-03-19 1988-01-05 Alza Corporation Dosage system and method of using same
US4849405A (en) * 1984-05-09 1989-07-18 Synthetic Blood Corporation Oral insulin and a method of making the same
US4963367A (en) 1984-04-27 1990-10-16 Medaphore, Inc. Drug delivery compositions and methods
US4963526A (en) 1984-05-09 1990-10-16 Synthetic Blood Corporation Oral insulin and a method of making the same
US4839341A (en) * 1984-05-29 1989-06-13 Eli Lilly And Company Stabilized insulin formulations
US4622392A (en) 1984-06-21 1986-11-11 Health Research Inc. (Roswell Park Division) Thiophospholipid conjugates of antitumor agents
US4629621A (en) * 1984-07-23 1986-12-16 Zetachron, Inc. Erodible matrix for sustained release bioactive composition
US4797288A (en) * 1984-10-05 1989-01-10 Warner-Lambert Company Novel drug delivery system
US4946828A (en) * 1985-03-12 1990-08-07 Novo Nordisk A/S Novel insulin peptides
US5157021A (en) 1985-03-15 1992-10-20 Novo Nordisk A/S Insulin derivatives and pharmaceutical preparations containing these derivatives
US4917888A (en) * 1985-06-26 1990-04-17 Cetus Corporation Solubilization of immunotoxins for pharmaceutical compositions using polymer conjugation
SE457326B (sv) * 1986-02-14 1988-12-19 Lejus Medical Ab Foerfarande foer framstaellning av en snabbt soenderfallande kaerna innehaallande bl a mikrokristallin cellulosa
US4801575A (en) * 1986-07-30 1989-01-31 The Regents Of The University Of California Chimeric peptides for neuropeptide delivery through the blood-brain barrier
ZA877505B (en) 1986-10-14 1989-05-30 Lilly Co Eli Process for transforming a human insulin precursor to human insulin
GB8706313D0 (en) 1987-03-17 1987-04-23 Health Lab Service Board Treatment & prevention of viral infections
US5093198A (en) * 1987-06-19 1992-03-03 Temple University Adjuvant-enhanced sustained release composition and method for making
DE3721721C1 (de) * 1987-07-01 1988-06-09 Hoechst Ag Verfahren zur Umhuellung von Granulaten
US5080891A (en) 1987-08-03 1992-01-14 Ddi Pharmaceuticals, Inc. Conjugates of superoxide dismutase coupled to high molecular weight polyalkylene glycols
JPH01308231A (ja) 1988-06-03 1989-12-12 Takeda Chem Ind Ltd 安定化された医薬組成物および製造法
US5055300A (en) 1988-06-17 1991-10-08 Basic Bio Systems, Inc. Time release protein
DK336188D0 (da) * 1988-06-20 1988-06-20 Nordisk Gentofte Propeptider
US5162430A (en) * 1988-11-21 1992-11-10 Collagen Corporation Collagen-polymer conjugates
US5306500A (en) * 1988-11-21 1994-04-26 Collagen Corporation Method of augmenting tissue with collagen-polymer conjugates
AU641631B2 (en) 1988-12-23 1993-09-30 Novo Nordisk A/S Human insulin analogues
US4994439A (en) * 1989-01-19 1991-02-19 California Biotechnology Inc. Transmembrane formulations for drug administration
US5089261A (en) * 1989-01-23 1992-02-18 Cetus Corporation Preparation of a polymer/interleukin-2 conjugate
US5182258A (en) * 1989-03-20 1993-01-26 Orbon Corporation Systemic delivery of polypeptides through the eye
US5122614A (en) 1989-04-19 1992-06-16 Enzon, Inc. Active carbonates of polyalkylene oxides for modification of polypeptides
US5324844A (en) * 1989-04-19 1994-06-28 Enzon, Inc. Active carbonates of polyalkylene oxides for modification of polypeptides
US5286637A (en) * 1989-08-07 1994-02-15 Debiopharm, S.A. Biologically active drug polymer derivatives and method for preparing same
US5112614A (en) * 1989-09-14 1992-05-12 Alza Corporation Implantable delivery dispenser
US5013556A (en) * 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
DE3937797A1 (de) * 1989-11-14 1991-05-16 Basf Ag Verfahren zur herstellung von polyetherglykolen
US5312808A (en) * 1989-11-22 1994-05-17 Enzon, Inc. Fractionation of polyalkylene oxide-conjugated hemoglobin solutions
US5650388A (en) * 1989-11-22 1997-07-22 Enzon, Inc. Fractionated polyalkylene oxide-conjugated hemoglobin solutions
CA2030174C (en) * 1990-01-10 1996-12-24 Anthony H. Cincotta Process for the long term reduction of body fat stores, insulin resistance, hyperinsulinemia and hypoglycemia in vertebrates
US5545618A (en) * 1990-01-24 1996-08-13 Buckley; Douglas I. GLP-1 analogs useful for diabetes treatment
IE912365A1 (en) * 1990-07-23 1992-01-29 Zeneca Ltd Continuous release pharmaceutical compositions
DD297249A5 (de) 1990-08-07 1992-01-02 Veb Mineralwollewerk Flechtingen Bereich F/E Mineralwolle,De Verfahren zur automatischen ueberwachung des aushaertegrades an materialbahnen
IL99699A (en) * 1990-10-10 2002-04-21 Autoimmune Inc Drug with the option of oral, intra-intestinal, or inhaled dosing for suppression of autoimmune response associated with type I diabetes
US5595732A (en) 1991-03-25 1997-01-21 Hoffmann-La Roche Inc. Polyethylene-protein conjugates
DK0580778T3 (da) * 1991-04-19 2000-01-31 Lds Technologies Inc Konvertible mikroemulsionsformuleringer
FR2675807B1 (fr) 1991-04-23 1994-07-01 Medgenix Group Sa Conjugue de calcitonine et de polyethylene glycol.
US5304473A (en) * 1991-06-11 1994-04-19 Eli Lilly And Company A-C-B proinsulin, method of manufacturing and using same, and intermediates in insulin production
ES2093269T3 (es) 1991-07-26 1996-12-16 Smithkline Beecham Corp Microemulsiones del tipo agua en aceite.
US5206219A (en) * 1991-11-25 1993-04-27 Applied Analytical Industries, Inc. Oral compositions of proteinaceous medicaments
US5693769A (en) 1991-12-13 1997-12-02 Transcell Technologies, Inc. Glycosylated steroid derivatives for transport across biological membranes and process for making and using same
EP0701815B1 (de) 1992-01-17 1999-06-30 ALFATEC-PHARMA GmbH Verfahren zur Herstellung von Wirkstoff enthaltenden Pulvern, Granulaten oder Pellets mit einem Gerüst aus hydrophilen Makromolekülen und ihre Verwendung
GB9212511D0 (en) 1992-06-12 1992-07-22 Cortecs Ltd Pharmaceutical compositions
US5262172A (en) * 1992-06-19 1993-11-16 Digestive Care Inc. Compositions of gastric acid-resistant microspheres containing buffered bile acids
US5415872A (en) * 1992-06-22 1995-05-16 Digestive Care Inc. Compositions of gastric acid-resistant microspheres containing salts of bile acids
TW278088B (US20030027995A1-20030206-C00005.png) * 1992-06-24 1996-06-11 Himont Inc
US6093391A (en) * 1992-10-08 2000-07-25 Supratek Pharma, Inc. Peptide copolymer compositions
GB9316895D0 (en) 1993-08-13 1993-09-29 Guy S And St Thomas Hospitals Hepatoselective insulin analogues
US5298643A (en) * 1992-12-22 1994-03-29 Enzon, Inc. Aryl imidate activated polyalkylene oxides
US5349001A (en) 1993-01-19 1994-09-20 Enzon, Inc. Cyclic imide thione activated polyalkylene oxides
US5321095A (en) 1993-02-02 1994-06-14 Enzon, Inc. Azlactone activated polyalkylene oxides
US5298410A (en) * 1993-02-25 1994-03-29 Sterling Winthrop Inc. Lyophilized formulation of polyethylene oxide modified proteins with increased shelf-life
US5681811A (en) 1993-05-10 1997-10-28 Protein Delivery, Inc. Conjugation-stabilized therapeutic agent compositions, delivery and diagnostic formulations comprising same, and method of making and using the same
US6191105B1 (en) * 1993-05-10 2001-02-20 Protein Delivery, Inc. Hydrophilic and lipophilic balanced microemulsion formulations of free-form and/or conjugation-stabilized therapeutic agents such as insulin
US5621039A (en) 1993-06-08 1997-04-15 Hallahan; Terrence W. Factor IX- polymeric conjugates
AU7113594A (en) * 1993-06-21 1995-01-17 Enzon, Inc. Site specific synthesis of conjugated peptides
US5506203C1 (en) * 1993-06-24 2001-02-06 Astra Ab Systemic administration of a therapeutic preparation
US5830853A (en) 1994-06-23 1998-11-03 Astra Aktiebolag Systemic administration of a therapeutic preparation
IS1796B (is) 1993-06-24 2001-12-31 Ab Astra Fjölpeptíð lyfjablanda til innöndunar sem einnig inniheldur eykjaefnasamband
US5747445A (en) * 1993-06-24 1998-05-05 Astra Aktiebolag Therapeutic preparation for inhalation
TW402506B (en) 1993-06-24 2000-08-21 Astra Ab Therapeutic preparation for inhalation
US6342225B1 (en) * 1993-08-13 2002-01-29 Deutshces Wollforschungsinstitut Pharmaceutical active conjugates
WO1995007931A1 (en) * 1993-09-17 1995-03-23 Novo Nordisk A/S Acylated insulin
US5919455A (en) 1993-10-27 1999-07-06 Enzon, Inc. Non-antigenic branched polymer conjugates
US5605976A (en) 1995-05-15 1997-02-25 Enzon, Inc. Method of preparing polyalkylene oxide carboxylic acids
US5643575A (en) * 1993-10-27 1997-07-01 Enzon, Inc. Non-antigenic branched polymer conjugates
US5951974A (en) * 1993-11-10 1999-09-14 Enzon, Inc. Interferon polymer conjugates
ATE317397T1 (de) * 1993-11-17 2006-02-15 Athena Neurosciences Inc Transparente flüssigkeit zur verabreichung von verkapselten medikamenten
GB9406094D0 (en) 1994-03-28 1994-05-18 Univ Nottingham And University Polymer microspheres and a method of production thereof
EP0761683B1 (en) * 1994-05-20 2005-02-02 Hisamitsu Pharmaceutical Co., Inc. Protein or polypeptide, process for producing the same, and intermediate compound tehrefor
US5461031A (en) * 1994-06-16 1995-10-24 Eli Lilly And Company Monomeric insulin analog formulations
US5504188A (en) * 1994-06-16 1996-04-02 Eli Lilly And Company Preparation of stable zinc insulin analog crystals
US6165976A (en) 1994-06-23 2000-12-26 Astra Aktiebolag Therapeutic preparation for inhalation
US5730990A (en) * 1994-06-24 1998-03-24 Enzon, Inc. Non-antigenic amine derived polymers and polymer conjugates
GB9417524D0 (en) 1994-08-31 1994-10-19 Cortecs Ltd Pharmaceutical compositions
US5738846A (en) * 1994-11-10 1998-04-14 Enzon, Inc. Interferon polymer conjugates and process for preparing the same
US5646242A (en) * 1994-11-17 1997-07-08 Eli Lilly And Company Selective acylation of epsilon-amino groups
US5693609A (en) 1994-11-17 1997-12-02 Eli Lilly And Company Acylated insulin analogs
CA2206852A1 (en) * 1994-12-07 1996-06-13 Novo Nordisk A/S Polypeptide with reduced allergenicity
GB9424902D0 (en) 1994-12-09 1995-02-08 Cortecs Ltd Solubilisation Aids
SE9404468D0 (sv) 1994-12-22 1994-12-22 Astra Ab Powder formulations
US5932462A (en) 1995-01-10 1999-08-03 Shearwater Polymers, Inc. Multiarmed, monofunctional, polymer for coupling to molecules and surfaces
US5907030A (en) * 1995-01-25 1999-05-25 University Of Southern California Method and compositions for lipidization of hydrophilic molecules
KR0150565B1 (ko) 1995-02-15 1998-08-17 김정재 유전자 조환에 의한 사람 인슐린 전구체의 제조 및 이를 이용한 인슐린의 제조방법
US6251856B1 (en) * 1995-03-17 2001-06-26 Novo Nordisk A/S Insulin derivatives
YU18596A (sh) 1995-03-31 1998-07-10 Eli Lilly And Company Analogne formulacije monomernog insulina
US5606038A (en) * 1995-04-10 1997-02-25 Competitive Technologies, Inc. Amphiphilic polyene macrolide antibiotic compounds
ES2093593T1 (es) * 1995-05-05 1997-01-01 Hoffmann La Roche Proteinas obesas (ob) recombinantes.
US5824638A (en) 1995-05-22 1998-10-20 Shire Laboratories, Inc. Oral insulin delivery
US5700904A (en) 1995-06-07 1997-12-23 Eli Lilly And Company Preparation of an acylated protein powder
US5631347A (en) * 1995-06-07 1997-05-20 Eli Lilly And Company Reducing gelation of a fatty acid-acylated protein
GB9516268D0 (en) * 1995-08-08 1995-10-11 Danbiosyst Uk Compositiion for enhanced uptake of polar drugs from the colon
US5766620A (en) * 1995-10-23 1998-06-16 Theratech, Inc. Buccal delivery of glucagon-like insulinotropic peptides
US5639705A (en) * 1996-01-19 1997-06-17 Arco Chemical Technology, L.P. Double metal cyanide catalysts and methods for making them
US5948751A (en) 1996-06-20 1999-09-07 Novo Nordisk A/S X14-mannitol
US5866538A (en) * 1996-06-20 1999-02-02 Novo Nordisk A/S Insulin preparations containing NaCl
GB9613858D0 (en) * 1996-07-02 1996-09-04 Cortecs Ltd Hydrophobic preparations
US5905140A (en) 1996-07-11 1999-05-18 Novo Nordisk A/S, Novo Alle Selective acylation method
US5856369A (en) 1996-07-30 1999-01-05 Osi Specialties, Inc. Polyethers and polysiloxane copolymers manufactured with double metal cyanide catalysts
US5874111A (en) * 1997-01-07 1999-02-23 Maitra; Amarnath Process for the preparation of highly monodispersed polymeric hydrophilic nanoparticles
US6011008A (en) * 1997-01-08 2000-01-04 Yissum Research Developement Company Of The Hebrew University Of Jerusalem Conjugates of biologically active substances
US5830918A (en) 1997-01-15 1998-11-03 Terrapin Technologies, Inc. Nonpeptide insulin receptor agonists
US5898028A (en) * 1997-03-20 1999-04-27 Novo Nordisk A/S Method for producing powder formulation comprising an insulin
US6043214A (en) * 1997-03-20 2000-03-28 Novo Nordisk A/S Method for producing powder formulation comprising an insulin
US6310038B1 (en) 1997-03-20 2001-10-30 Novo Nordisk A/S Pulmonary insulin crystals
CO4750643A1 (es) * 1997-06-13 1999-03-31 Lilly Co Eli Formulacion estable de la insulina que contiene l-arginina y protamina
IL134901A0 (en) * 1997-10-24 2001-05-20 Lilly Co Eli Insoluble insulin compositions
ZA989744B (en) * 1997-10-31 2000-04-26 Lilly Co Eli Method for administering acylated insulin.
US5981709A (en) 1997-12-19 1999-11-09 Enzon, Inc. α-interferon-polymer-conjugates having enhanced biological activity and methods of preparing the same
US5985263A (en) 1997-12-19 1999-11-16 Enzon, Inc. Substantially pure histidine-linked protein polymer conjugates
JP2002518408A (ja) 1998-06-12 2002-06-25 キングス・カレツジ・ロンドン インスリン類似体
US6211144B1 (en) * 1998-10-16 2001-04-03 Novo Nordisk A/S Stable concentrated insulin preparations for pulmonary delivery
DE19908041A1 (de) 1999-02-24 2000-08-31 Hoecker Hartwig Kovalent verbrückte Insulindimere
US6248363B1 (en) * 1999-11-23 2001-06-19 Lipocine, Inc. Solid carriers for improved delivery of active ingredients in pharmaceutical compositions
US6309633B1 (en) 1999-06-19 2001-10-30 Nobex Corporation Amphiphilic drug-oligomer conjugates with hydroyzable lipophile components and methods for making and using the same
DE19932440C2 (de) 1999-07-12 2003-04-10 Bauer Maschinen Gmbh Bohrzahn zur Erdbearbeitung
KR100345214B1 (ko) * 1999-08-17 2002-07-25 이강춘 생체적합성 고분자가 수식된 펩타이드의 비점막 전달
US6323311B1 (en) 1999-09-22 2001-11-27 University Of Utah Research Foundation Synthesis of insulin derivatives
US6638906B1 (en) * 1999-12-13 2003-10-28 Nobex Corporation Amphiphilic polymers and polypeptide conjugates comprising same
CN1430623A (zh) 2000-05-26 2003-07-16 日本淀粉工业株式会社 新糖、其制备方法和用途
US6867183B2 (en) 2001-02-15 2005-03-15 Nobex Corporation Pharmaceutical compositions of insulin drug-oligomer conjugates and methods of treating diseases therewith
US7060675B2 (en) 2001-02-15 2006-06-13 Nobex Corporation Methods of treating diabetes mellitus
US6828297B2 (en) 2001-06-04 2004-12-07 Nobex Corporation Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6858580B2 (en) * 2001-06-04 2005-02-22 Nobex Corporation Mixtures of drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6713452B2 (en) 2001-06-04 2004-03-30 Nobex Corporation Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6835802B2 (en) * 2001-06-04 2004-12-28 Nobex Corporation Methods of synthesizing substantially monodispersed mixtures of polymers having polyethylene glycol moieties
US6913903B2 (en) 2001-09-07 2005-07-05 Nobex Corporation Methods of synthesizing insulin polypeptide-oligomer conjugates, and proinsulin polypeptide-oligomer conjugates and methods of synthesizing same
US6770625B2 (en) 2001-09-07 2004-08-03 Nobex Corporation Pharmaceutical compositions of calcitonin drug-oligomer conjugates and methods of treating diseases therewith

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) * 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US5349052A (en) * 1988-10-20 1994-09-20 Royal Free Hospital School Of Medicine Process for fractionating polyethylene glycol (PEG)-protein adducts and an adduct for PEG and granulocyte-macrophage colony stimulating factor
US5597797A (en) * 1990-06-07 1997-01-28 Genentech, Inc. Method for treatment or prevention of obesity
US5359030A (en) * 1993-05-10 1994-10-25 Protein Delivery, Inc. Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic formulations comprising same, and method of making and using the same
US6057292A (en) * 1995-09-21 2000-05-02 Genentech, Inc. Method for inhibiting growth hormone action
WO1997014740A1 (en) * 1995-10-19 1997-04-24 Receptagen Corporation Discrete-length polyethylene glycols

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1404361A4 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005000359A2 (en) * 2002-11-20 2005-01-06 Pharmacia Corporation Chemically-modified human growth hormone conjugates
WO2005000359A3 (en) * 2002-11-20 2005-06-16 Pharmacia Corp Chemically-modified human growth hormone conjugates
EP1594440A2 (en) * 2003-02-14 2005-11-16 Quanta Biodesign, Ltd. The selective and specific preparation of discrete peg compounds
EP1594440A4 (en) * 2003-02-14 2006-12-27 Quanta Biodesign Ltd SELECTIVE AND SPECIFIC PREPARATION OF DISCRETE PEG COMPOUNDS
EP2905033A1 (en) * 2003-12-16 2015-08-12 Nektar Therapeutics Monodisperse PEGylated naloxol compositions
US11129794B2 (en) 2003-12-16 2021-09-28 Nektar Therapeutics Chemically modified small molecules
US9012469B2 (en) 2010-09-30 2015-04-21 Astrazeneca Ab Crystalline naloxol-peg conjugate
US9149539B1 (en) 2010-09-30 2015-10-06 Astrazeneca Ab Crystalline naloxol-PEG conjugate
US9308263B2 (en) 2011-10-21 2016-04-12 Seachaid Pharmaceuticals, Inc. Pharmaceutical compositions and uses thereof

Also Published As

Publication number Publication date
KR20040004693A (ko) 2004-01-13
US20030027995A1 (en) 2003-02-06
CN1538852A (zh) 2004-10-20
MXPA03011282A (es) 2004-03-26
EP1404361A4 (en) 2005-03-16
EP1404361A1 (en) 2004-04-07
JP2004534783A (ja) 2004-11-18
US6828305B2 (en) 2004-12-07
CA2449320A1 (en) 2002-12-12

Similar Documents

Publication Publication Date Title
US6828305B2 (en) Mixtures of growth hormone drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6713452B2 (en) Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6828297B2 (en) Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
AU2002310291A1 (en) Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US8030269B2 (en) Calcitonin drug-oligomer conjugates, and uses thereof
AU2002310278A1 (en) Mixtures of growth hormone drug-oligomer conjugates compromising polyalkylene glycol, uses thereof, and methods of making same
JP4829783B2 (ja) カルシトニン薬−オリゴマーコンジュゲートの混合物および疼痛治療における使用方法
AU2002303961A1 (en) Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US20090281023A9 (en) Mixtures Of Calcitonin Drug-Oligomer Conjugates And Methods Of Use In Pain Treatment

Legal Events

Date Code Title Description
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2449320

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: PA/a/2003/011282

Country of ref document: MX

Ref document number: 2003501490

Country of ref document: JP

Ref document number: 1020037015911

Country of ref document: KR

WWE Wipo information: entry into national phase

Ref document number: 2002310278

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2002737344

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 20028153014

Country of ref document: CN

WWP Wipo information: published in national office

Ref document number: 2002737344

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642