WO2017210077A1 - Promédicaments à base d'insuline monocaténaire - Google Patents

Promédicaments à base d'insuline monocaténaire Download PDF

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WO2017210077A1
WO2017210077A1 PCT/US2017/034444 US2017034444W WO2017210077A1 WO 2017210077 A1 WO2017210077 A1 WO 2017210077A1 US 2017034444 W US2017034444 W US 2017034444W WO 2017210077 A1 WO2017210077 A1 WO 2017210077A1
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chain
seq
insulin
group
alkyl
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Richard D. Dimarchi
Fa Liu
Fa Zhang
Alexander Zaykov
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Indiana University Research and Technology Corp
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Indiana University Research and Technology Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 19 kilobytes acii (text) file named "265651seqlist_ST25.txt,” created on May 19, 2017.
  • Insulin is a peptide hormone comprised of a two chain heterodimer that is biosynthetically derived from a low potency single chain proinsulin precursor through enzymatic processing.
  • Human insulin is comprised of two peptide chains (an "A chain” (SEQ ID NO: 1) and "B chain” (SEQ ID NO: 2)) bound together by disulfide bonds and having a total of 51 amino acids.
  • the C-terminal region of the B-chain and the two terminal regions of the A-chain associate in a three-dimensional structure to assemble a site for high affinity binding to the insulin receptor.
  • Insulin demonstrates unparalleled ability to lower glucose in virtually all forms of diabetes. Unfortunately, its pharmacology is not glucose sensitive and as such it is capable of excessive action that can lead to life-threatening hypoglycemia. Inconsistent pharmacology is a hallmark of insulin therapy such that it is extremely difficult to normalize blood glucose without occurrence of hypoglycemia.
  • native insulin is of short duration of action and requires modification to render it suitable for use in control of basal glucose.
  • Established approaches to delay the onset of insulin action include reduction in solubility, and albumin binding.
  • insulin derivatives For example, two commercially available insulin derivatives have been prepared to provide a longer action profile. More particularly, the insulin derivative [GlyA21, ArgB31, ArgB32]insulin was prepared to shift insulin's pi from 5.4 to 6.7 resulting in the peptide being precipitated at physiological pH and thus delaying adsoption and time of action (see Bolli et al., Diabetologia 1999, 42, 1151-1167). However, this insulin derivative has enhanced IGF-1 affinity, leading to increased proliferative actions and the possibility of tumorigenesis.
  • Another commercially available insulin derivative is [LysB29-tetradecanoyl, des(B30)]insulin, wherein LysB29 has been acylated with a C 14 fatty acid (Mayer et al., Peptide Science, 88, 5, 687-713).
  • the presence of the fatty acid chain enhances binding of the peptide to serum albumin, resulting in increased plasma half life.
  • this derivative suffers the disadvantage of having reduced potency in vivo.
  • both insulin derivatives exhibit variability in biological action from one patient to the next.
  • Prodrug chemistry offers the opportunity to precisely control the onset and duration of insulin action after clearance from the site of administration and equilibration in the plasma at a highly defined concentration.
  • the central virtue of such an approach, relative to current long-acting insulin analogs and formulations, is that the insulin reservoir is not the subcutaneous fatty tissue where injection occurs, but rather the blood compartment. This removes the variability in absorption encountered with prior art delayed onset insulin derivatives. It also enables administration of the peptide hormone by routes other than a subcutaneous injection.
  • Binding of insulin to its receptor will result in biological stimulation, but will also initiate the subsequent deactivation of insulin induced pharmacology through the enzymatic degradation of the insulin peptide.
  • An added advantage of using a prodrug derivative of insulin is that such an approach also extends insulin's biological half life based on a strategy of inhibiting recognition of the prodrug by the corresponding receptor.
  • the complex nature of preparing such prodrugs has, until now, prevented the preparation of an efficacious prodrug derivative of insulin.
  • the insulin prodrugs disclosed herein are chemically converted to structures that can be recognized by the receptor, wherein the speed of this chemical conversion will determine the time of onset and duration of in vivo biological action.
  • the prodrug chemistry disclosed in this application relies upon an intramolecular chemical reaction that is not dependent upon additional chemical additives, or enzymes or enzyme inhibitors.
  • the ideal prodrug should be soluble in water at physiological conditions (for example, a pH of 7.2 and 37 °C), and it should be stable in the powder form for long term storage. It should also be immunologically silent and exhibit a low activity relative to the parent drug.
  • prodrugs of insulin that exhibits less than 5%, about 1%, or less than 1% activity relative to the parent drug.
  • Peptide-based drugs are highly effective medicines with relatively short duration of action and variable therapeutic index.
  • the present disclosure is directed to insulin prodrugs wherein the prodrug derivative is designed to delay onset of action and extend the half life of the drug.
  • the delayed onset of action is advantageous in that it allows systemic distribution of the prodrug prior to its activation. Accordingly, the administration of prodrugs eliminates complications caused by peak activities upon administration and increases the therapeutic index of the parent drug.
  • Single chain insulins having the carboxy terminus of the B chain directly linked to the amino terminus of the A chain are inactive. Cleavage of this inactive single chain analog to produce a two chain insulin having intact A and B chains restores activity.
  • single chain insulin analogs are provided wherein the carboxy terminus of an insulin B chain is linked to the amino terminus of an insulin A chain via a dipeptide that is inherently metastable under physiological conditions. Such single-chain insulins are inactive until the point the prodrug decomposes to a two chain insulin through controlled diketopiperazine or diketomorpholine formation and release of the A chain.
  • the degree of stability of the single chain structure can be engineered by the selection of the chemical structure of the dipeptide that eventually forms the diketopiperazine.
  • an insulin prodrug having the general structure of B-U-O-A, wherein B represents an insulin B chain, U is an amino acid or hydroxyl acid, O is an N-alkylated amino acid and A represents an insulin A chain, wherein U is covalently linked to the carboxy terminus of the B chain via the side chain of U, and O is linked to U and the N-terminus of the A chain via amide bonds.
  • the structure of U-0 is selected, in one embodiment, to chemically cleave from the insulin A chain at a rate that is at least about 90% complete within about 12 to about 720 hours in PBS under physiological conditions.
  • the chemical cleavage half-life (ti /2 ) of U-0 from the insulin A chain is at least about 1 hour to about 1 week in PBS under physiological conditions.
  • the dipeptide prodrug element U-0 comprises a compound having the general structure of Formula I:
  • Ri is (C1-C4 alkyl)OH, (C1-C4 alkyl)SH or (C1-C4 alkyl)NH 2;
  • R 2 and R 8 are independently H or Ci-C 8 alkyl
  • R 4 is selected from the group consisting of H, Ci-Ci 8 alkyl, C 2 -Ci 8 alkenyl, (C 1 -C18 alkyl)OH, (Ci-Cis alkyl)SH, (C 2 -C 3 alkyl)SCH 3 , (C 1 -C4 alkyl)CONH 2 , (C 1 -C4 alkyl)COOH, (C1-C4 alkyl)NH 2 , (C1-C4 alkyl)NHC(NH 2 + )NH 2 , (C 0 -C 4 alkyl)(C 3 -C 6 cycloalkyl), (C 0 -C 4 alkyl)(C 2 -C 5 heterocyclic), (C 0 -C 4 alkyl)(C 6 -Cio aryl)R 7 , (C1-C4 alkyl)(C 3 -C9 heteroaryl), and C 1 -C 12 alkyl(W)Ci-C
  • R 3 is selected from the group consisting of Ci-Ci 8 alkyl, (Ci-Ci 8 alkyl)OH, (Ci-Cis alkyl)NH 2, and (Ci-Ci 8 alkyl)SH, or R 4 and R 3 together with the atoms to which they are attached form a pyrrolidine ring;
  • R 5 is NH 2 or OH
  • R 7 is selected from the group consisting of H and OH.
  • Ri is (C1-C4 alkyl)NH 2;
  • R 2 and R 8 are both H
  • R 3 is selected from the group consisting of Ci-C 6 alkyl
  • R 5 is NH 2 or OH.
  • a single chain insulin prodrug comprising an A chain and a B chain
  • the A chain comprises the sequence GIVEQCCX 8 SICSLYQLENYCX 2 i (SEQ ID NO: 43)
  • the B chain comprises the sequence of R 22 -X 25 LCGX 29 X 30 LVEALYLVCG ERGFX 45 -R 23 (SEQ ID NO: 14), wherein the carboxy terminus of the B chain is linked to the amino terminus of the A chain via a metastable dipeptide linker U-O, and wherein
  • X 8 is selected from the group consisting of threonine and histidine;
  • X 2 i is selected from the group consisting of asparagine, ornathine, glycine, alanine, threonine, and serine;
  • X 2 5 is selected from the group consisting of histidine and threonine;
  • X 2 9 is selected from the group consisting of alanine, glycine and serine;
  • X 3 o is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid;
  • X 4 5 is tyrosine or phenylalanine
  • R 22 is selected from the group consisting of FVNQ (SEQ ID NO: 12), FVKQ (SEQ ID NO: 11), a tripeptide glycine-proline-glutamic acid, a tripeptide valine- asparagine-glutamine, a dipeptide proline-glutamic acid, a dipeptide asparagine- glutamine, glutamine, glutamic acid and a bond; and
  • R 23 is selected from the group consisting of R 23 is selected from the group consisting of YTX 28 KT (SEQ ID NO: 20), YTKPT (SEQ ID NO: 23), YTX 28 K (SEQ ID NO: 24), YTKP (SEQ ID NO: 29), YTPK (SEQ ID NO: 32),YTX 28 , YT, Y and a bond.
  • GIVEQCCTSICSLYQLENYCN SEQ ID NO: 1
  • B chain comprising a sequence selected from the group consisting of
  • R 23 is selected from the group consisting of YTEKT (SEQ ID NO: 27), YTDKT (SEQ ID NO: 28), YTKPT (SEQ ID NO: 23), YTEK (SEQ ID NO: 30), YTDK (SEQ ID NO: 31), YTKP (SEQ ID NO: 29), YTPK (SEQ ID NO: 32), YTE, YTD, YTK, YT, Y or a bond wherein the C-terminus of the B chain is linked to the N-terminus of the A chain via a dipeptide linker comprising the structure of Formula
  • X is NH, S, O or CH 2 ;
  • R 2 is H or Ci-C 4 alkyl
  • R 3 is Ci-C 6 alkyl
  • R 4 is H or Ci-C 4 alkyl, or R 3 and R4 together with the atoms to which they are attached form a pyrrolidine ring;
  • R 5 is NH 2 or OH
  • n is an integer selected from 0 to 4.
  • X is NH
  • R 4 is H
  • R5 is NH 2 .
  • a first end of the linker of Formula II is linked to the carboxy terminus of the B chain and a second end of the linker of Formula II is linked to the N-terminus of the A chain wherein the linkages are amide bonds.
  • the solubility of the single chain insulin prodrugs is enhanced by the covalent linkage of a hydrophilic moiety to the peptide.
  • the hydrophilic moiety is linked to either the N-terminal amino acid of the B chain or to the side chain of a lysine at a position C-terminal to position 27 relative to native insulin B chain.
  • the hydrophilic moiety is a polyethylene glycol (PEG) chain, having a molecular weight selected from the range of about 500 to about 40,000 Daltons.
  • the polyethylene glycol chain has a molecular weight selected from the range of about 500 to about 5,000 Daltons.
  • the polyethylene glycol chain has a molecular weight of about 10,000 to about 20,000 Daltons.
  • Acylation or alkylation can increase the half-life of the insulin peptides in circulation. Acylation or alkylation can advantageously delay the onset of action and/or extend the duration of action at the insulin receptors upon activation of the prodrug.
  • the insulin analogs may be acylated or alkylated at the same amino acid position where a hydrophilic moiety is linked, or at a different amino acid position.
  • the N-terminal alpha amine of the B chain is acylated with a C 14 to C 18 alkyl group optionally with a 1 to six amino acid spacer linking the acyl group to the N-terminal alpha amine of the B chain.
  • a pharmaceutical composition comprising any of the novel single chain insulin prodrugs disclosed herein, preferably at a purity level of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and a pharmaceutically acceptable diluent, carrier or excipient.
  • compositions may contain an insulin analog as disclosed herein at a concentration of at least 0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml, 20 mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 24 mg/ml, 25 mg/ml or higher.
  • the pharmaceutical compositions comprise aqueous solutions that are sterilized and optionally stored at a pH of 6.0 or less and contained within various package containers. In other embodiments the pharmaceutical compositions comprise a lyophilized powder.
  • the pharmaceutical compositions can be further packaged as part of a kit that includes a disposable device for administering the composition to a patient.
  • the containers or kits may be labeled for storage at ambient room temperature or at refrigerated temperature.
  • an improved method of regulating blood glucose levels in insulin dependent patients comprises the steps of administering a single chain insulin prodrug of the present disclosure in an amount therapeutically effective for the control of diabetes.
  • the single chain insulin prodrug is pegylated with a PEG chain having a molecular weight selected from the range of about 5,000 to about 40,000 Daltons
  • Fig. 1 is a schematic overview of the two step synthetic strategy for preparing human insulin. Details of the procedure are provided in Example 1.
  • Fig. 2 is a graph comparing insulin receptor specific binding of synthetic human insulin relative to purified native insulin. As indicated by the data presented in the graph, the two molecules have similar binding activities.
  • Fig. 3 is a schematic drawing demonstrating one synthetic scheme for preparing the single chain insulin prodrugs of the present invention, wherein the A chain comprises SEQ ID NO: 1 and the B chain comprises SEQ ID NO: 40.
  • Fig. 4 provides the structure of two acylated single chain insulin (SCI) prodrugs.
  • the prodrugs are acylated at the N-terminal alpha amine of the B chain via a gamma Glu-Gly-Gly-Gly linker.
  • the DTI (Des-Tetrapeptide (B27-B30) Insulin) Analog represents a native A chain (SEQ ID NO: 1) linked to a B chain analog having the C-terminal 4 amino acids deleted (SEQ ID NO: 40).
  • the DPI (Des-Pentapeptide (B26-B30) Insulin) Analog represents a native A chain (SEQ ID NO: 1) linked to a B chain analog having the C-terminal 5 amino acids deleted (SEQ ID NO: 26).
  • Fig. 5 provides data for the in vitro insulin activity for native insulin and three single chain analogs.
  • CIU-158 is a single chain insulin analog having an insulin B chain with the last two amino acids deleted covalently linked to the N-terminus of the native A chain).
  • CIU-235 and CIU-199 are single chain insulin analog comprising a metastable dipeptide linker joining the C-terminus of a B l acetylated, C-terminal truncated B chain to the N-terminus of the A chain.
  • Fig. 6 provides data for the in vitro insulin activation kinetics (chemical cleavage half-life) using physiological conditions (PBS, 37°C) for various single chain insulin prodrugs.
  • Fig. 8 PD Profiling of Single Chain Insulin Prodrug in Mice.
  • Fig. 9 provides data comparing the activity of CIU-202 (Tl/2: 2.5 d) vs CIU- 209 (Tl/2: 1 d) in Pigs.
  • Fig. 10 provides data demonstrating the activity of CIU-207 (Tl/2: 3 d) in Pigs at 6 nmol/kg.
  • Fig. 11 provides data demonstrating the activity of the prodrug vs active drug by IV in Pigs. The data demonstrate the extended duration of action achieved with the prodrug analogs.
  • Fig. 12 provides data demonstrating the activity of the prodrug vs active drug by IV in Pigs presented in linear and logarithmic scales.
  • Fig. 13 provides data demonstrating the activity of the prodrugs CIU-202 (Tl/2: 2.5 d) vs CIU-211 (Tl/2: 2 d) by IV in Pigs.
  • Fig. 14 provides data demonstrating the activity of the prodrugs CIU-207 (Tl/2: 3 d) vs CIU-214 (Tl/2: 5 d) by IV in Pigs.
  • Fig. 15 provides data comparing the clearance of prodrug CIU-183 vs active drug CIU-201.
  • Fig. 16 provides data comparing the absorption phase of prodrug & active drug.
  • Fig. 17 provides data comparing the absorption phase of prodrug & active drug.
  • Fig. 18 provides data regarding the chemical stability of the indicated single chain insulin prodrugs at pH 6.0.
  • prodrug is defined as any compound that undergoes chemical modification before exhibiting its pharmacological effects.
  • bioactive polypeptide refers to polypeptides which are capable of exerting a biological effect in vitro and/or in vivo.
  • amino acid encompasses any molecule containing both amino and carboxyl functional groups, wherein the amino and carboxylate groups are attached to the same carbon (the alpha carbon).
  • the alpha carbon optionally may have one or two further organic substituents. Designation of an amino acid without specifying its stereochemistry is intended to encompass either the L or D form of the amino acid or a racemic mixture.
  • the D form of the amino acid is specified by inclusion of a lower case d before the three letter code and superscript number (e.g., dLys "1 ), wherein the designation lacking the lower case d (e.g., Lys "1 ) is intended to specify the native L form of the amino acid.
  • the inclusion of the superscript number designates the position of the amino acid relative to the native insulin sequence, wherein amino acids that are located within the corresponding native insulin sequence are designated by positive superscript numbers numbered consecutively from the N-terminus.
  • Additional amino acids linked to the insulin peptide either at the N-terminus or through a side chain are numbered starting with 0 and increasing in negative integer value as they are further removed from the corresponding native insulin sequence.
  • the position of an amino acid linked to the N-terminus of an insulin B chain is designated Baa "1 .
  • hydroxyl acid refers to amino acids that have been modified to replace the alpha carbon amino group with a hydroxyl group.
  • non-coded amino acid encompasses any amino acid that is not an L-isomer of any of the following 20 amino acids: Ala, Cys, Asp, Glu,
  • a “dipeptide” is a compound formed by linkage of an alpha amino acid or an alpha hydroxyl acid to another amino acid, through a peptide bond.
  • chemical cleavage absent any further designation encompasses a non-enzymatic reaction that results in the breakage of a covalent chemical bond.
  • bioactive polypeptide refers to polypeptides which are capable of exerting a biological effect in vitro and/or in vivo.
  • a general reference to a peptide is intended to encompass peptides that have modified amino and carboxy termini.
  • an amino acid sequence designating the standard amino acids is intended to encompass standard amino acids at the N- and C- terminus as well as a corresponding hydroxyl acid at the N-terminus and/or a corresponding C-terminal amino acid modified to comprise an amide group in place of the terminal carboxylic acid.
  • an "acylated" amino acid is an amino acid comprising an acyl group which is non-native to a naturally-occurring amino acid, regardless by the means by which it is produced.
  • exemplary methods of producing acylated amino acids and acylated peptides are known in the art and include acylating an amino acid before inclusion in the peptide or peptide synthesis followed by chemical acylation of the peptide.
  • the acyl group causes the peptide to have one or more of (i) a prolonged half-life in circulation, (ii) a delayed onset of action, (iii) an extended duration of action, (iv) an improved resistance to proteases, such as DPP-IV, and (v) increased potency at the insulin peptide receptor.
  • an "alkylated” amino acid is an amino acid comprising an alkyl group which is non-native to a naturally-occurring amino acid, regardless of the means by which it is produced.
  • exemplary methods of producing alkylated amino acids and alkylated peptides are known in the art and including alkylating an amino acid before inclusion in the peptide or peptide synthesis followed by chemical alkylation of the peptide.
  • alkylation of peptides will achieve similar, if not the same, effects as acylation of the peptides, e.g., a prolonged half-life in circulation, a delayed onset of action, an extended duration of action, an improved resistance to proteases, such as DPP-IV, and increased potency at the insulin peptide receptor.
  • the term "pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.
  • pharmaceutically acceptable salt refers to salts of compounds that retain the biological activity of the parent compound, and which are not biologically or otherwise undesirable. Many of the compounds disclosed herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.
  • Pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases, include by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines.
  • Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid, and the like.
  • treating includes prophylaxis of the specific disorder or condition, or alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms.
  • treating diabetes will refer in general to maintaining glucose blood levels near normal levels and may include increasing or decreasing blood glucose levels depending on a given situation.
  • an "effective" amount or a “therapeutically effective amount” of a prodrug refers to a nontoxic but sufficient amount of the prodrug to provide the desired effect.
  • one desired effect would be the prevention or treatment of hyperglycemia.
  • the amount that is "effective” will vary from subject to subject, depending on the age and general condition of the individual, mode of administration, and the like. Thus, it is not always possible to specify an exact “effective amount.” However, an appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • parenteral means not through the alimentary canal but by some other route such as intranasal, inhalation, subcutaneous, intramuscular, intraspinal, or intravenous.
  • the term “native insulin peptide” is intended to designate the 51 amino acid heterodimer comprising the A chain of SEQ ID NO: 1 and the B chain of SEQ ID NO: 2, as well as single-chain insulin analogs that comprise SEQ ID NOS: 1 and 2.
  • insulin peptide as used herein, absent further descriptive language is intended to encompass the 51 amino acid heterodimer comprising the A chain of SEQ ID NO: 1 and the B chain of SEQ ID NO: 2, as well as single-chain insulin analogs thereof (including for example those disclosed in published international application W096/34882 and US Patent No.
  • single-chain insulin analog encompasses a group of structurally-related proteins wherein the insulin A and B chains are covalently linked.
  • an amino acid "modification” refers to a substitution, addition or deletion of an amino acid, or the derivation of an amino acid by the addition and/or removal of chemical groups to/from the amino acid, and includes substitution with or addition of any of the 20 amino acids commonly found in human proteins, as well as atypical or non-naturally occurring amino acids.
  • Commercial sources of atypical amino acids include Sigma-Aldrich (Milwaukee, WI), ChemPep Inc. (Miami, FL), and Genzyme Pharmaceuticals (Cambridge, MA).
  • Atypical amino acids may be purchased from commercial suppliers, synthesized de novo, or chemically modified or derivatized from naturally occurring amino acids.
  • amino acid substitution refers to the replacement of one amino acid residue by a different amino acid residue.
  • all references to a particular amino acid position by letter and number refer to the amino acid at that position of either the A chain (e.g. position A5) or the B chain (e.g. position B5) in the respective native human insulin A chain (SEQ ID NO: 1) or B chain (SEQ ID NO: 2), or the corresponding amino acid position in any analogs thereof.
  • a reference herein to "position B28" absent any further elaboration would mean the corresponding position B27 of the B chain of an insulin analog in which the first amino acid of SEQ ID NO: 2 has been deleted.
  • polyethylene glycol chain refers to mixtures of condensation polymers of ethylene oxide and water, in a branched or straight chain, represented by the general formula ⁇ ( ⁇ 3 ⁇ 4 3 ⁇ 4) ⁇ , wherein n is at least 9. Absent any further characterization, the term is intended to include polymers of ethylene glycol with an average total molecular weight selected from the range of 500 to 80,000 Daltons. "Polyethylene glycol chain” or “PEG chain” is used in combination with a numeric suffix to indicate the approximate average molecular weight thereof. For example, PEG-5,000 refers to polyethylene glycol chain having a total molecular weight average of about 5,000 Daltons.
  • pegylated refers to a compound that has been modified from its native state by linking a polyethylene glycol chain to the compound.
  • a “pegylated polypeptide” is a polypeptide that has a PEG chain covalently bound to the polypeptide.
  • Linker is a bond, molecule or group of molecules that binds two separate entities to one another. Linkers may provide for optimal spacing of the two entities or may further supply a labile linkage that allows the two entities to be separated from each other. Labile linkages include photocleavable groups, acid-labile moieties, base-labile moieties and enzyme-cleavable groups.
  • insulin dimer is a complex comprising two insulin peptides covalently bound to one another via a linker.
  • the term insulin dimer when used absent any qualifying language, encompasses both insulin homodimers and insulin heterodimers.
  • An insulin homodimer comprises two identical subunits (each comprising an A and B chain), whereas an insulin heterodimer comprises two subunits that differ, although the two subunits are substantially similar to one another.
  • Ci-C n alkyl wherein n can be from 1 through 6, as used herein, represents a branched or linear alkyl group having from one to the specified number of carbon atoms.
  • Typical Ci-C 6 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.
  • C2-C n alkenyl wherein n can be from 2 through 6, as used herein, represents an olefinically unsaturated branched or linear group having from 2 to the specified number of carbon atoms and at least one double bond.
  • C 2 -C n alkynyl wherein n can be from 2 to 6, refers to an unsaturated branched or linear group having from 2 to n carbon atoms and at least one triple bond. Examples of such groups include, but are not limited to, 1-propynyl, 2- propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, and the like.
  • aryl refers to a mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like.
  • the size of the aryl ring and the presence of substituents or linking groups are indicated by designating the number of carbons present.
  • (Ci-C 3 alkyl)(C 6 -Cio aryl) refers to a 5 to 10 membered aryl that is attached to a parent moiety via a one to three membered alkyl chain.
  • heteroaryl refers to a mono- or bi- cyclic ring system containing one or two aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring.
  • the size of the heteroaryl ring and the presence of substituents or linking groups are indicated by designating the number of carbons present.
  • (Ci-C n alkyl)(C5-C 6 heteroaryl) refers to a 5 or 6 membered heteroaryl that is attached to a parent moiety via a one to "n" membered alkyl chain.
  • C 3 -C n cycloalkyl refers to a non-aromatic, monocyclic or polycyclic ring comprising carbon and hydrogen atoms with the subscript number indicating the number of carbon atoms present.
  • C 3 -C 8 cycloalkyl represents the compounds cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • C 3 -C n heterocyclic refers to a cycloalkyl ring system containing from one to "n - 1 " heteroatoms wherein the heteroatoms are selected from the group consisting of oxygen, sulfur, and nitrogen.
  • the phrase "5-membered heterocycle” or “C5 heterocycle” includes, but is not limited to, 5-membered heterocycles having one hetero atom (e.g. thiophenes, pyrroles, furans); 5-membered heterocycles having two heteroatoms in 1,2 or 1,3 positions (e.g. oxazoles, pyrazoles, imidazoles, thiazoles, purines); 5-membered heterocycles having three heteroatoms (e.g. triazoles, thiadiazoles).
  • C3-C n membered ring refers to a saturated or unsaturated hydrocarbon ring structure comprising a total of three to "n" number of elements linked to one another to form a ring, wherein the ring elements are selected from the group consisting of C, O, S and N.
  • the term is intended to encompass cycloalkyls, heterocyles, aryls and heteroaryls.
  • halo refers to one or more members of the group consisting of fluorine, chlorine, bromine, and iodine.
  • patient without further designation is intended to encompass any warm blooded vertebrate domesticated animal (including for example, but not limited to livestock, horses, cats, dogs and other pets) and humans.
  • the present disclosure provides insulin prodrug derivatives that are formulated to delay onset of action and enhance the half life of the insulin peptide, thus improving the therapeutic index of the underlying insulin peptide.
  • the insulin prodrug chemistry disclosed herein allows for activation of the prodrug via a non- enzymatic degradation mechanism.
  • the disclosed prodrug chemistry can be chemically conjugated to link the insulin A and B chains to form an inactive single chain insulin analog. Chemical cleavage of the dipeptide linker to form a
  • diketopiperazine or diketomorpholine releases the A chain to form the active two chain insulin.
  • This novel biologically friendly prodrug chemistry spontaneously degrades under physiological conditions (e.g. pH of about 7, at 37 °C in an aqueous environment) and is not reliant on enzymatic degradation.
  • the duration of the prodrug derivative is determined by the selection of the substituents of the amino acids comprising the dipeptide linker, and thus allows for flexibility in prodrug formulation.
  • a prodrug having a non-enzymatic activation half time (tl/2) of between 1-100 hrs under physiological conditions.
  • Physiological conditions as disclosed herein are intended to include a temperature of about 35 to 40 °C and a pH of about 7.0 to about 7.4 and more typically include a pH of 7.2 to 7.4 and a temperature of 36 to 38 °C in an aqueous environment.
  • a dipeptide capable of undergoing diketopiperazine or diketomorpholine formation under physiological conditions, is used as a linking peptide joining the insulin A chain to the insulin B chain to form a single chain analog.
  • the rate of cleavage, and thus activation of the prodrug depends on the structure and stereochemistry of the dipeptide pro-moiety and also on the strength of the nucleophile.
  • the prodrugs disclosed herein will ultimately be chemically converted to structures that can be recognized by the native receptor of the drug, wherein the speed of this chemical conversion will determine the time of onset and duration of in vivo biological action.
  • the prodrug chemistry disclosed in this application relies upon an intramolecular chemical reaction that is not dependent upon additional chemical additives, or enzymes.
  • the speed of conversion is controlled by the chemical nature of the dipeptide substituent and its cleavage under physiological conditions. Since physiological pH and temperature are tightly regulated within a highly defined range, the speed of conversion from prodrug to drug will exhibit high intra and interpatient reproducibility.
  • prodrugs wherein the bioactive polypeptides have extended half lives of at least 1 hour, and more typically greater than 20 hours and less than 100 hours, or less than 250 hours, and are converted to the active form at physiological conditions through a non-enzymatic reaction driven by inherent chemical instability.
  • the a non-enzymatic activation tl/2 time of the prodrug is between 1-250 hrs, and more typically between 1 and 100 hrs, 24 and 120 hours, 12 and 72 hours, and in one embodiment the tl/2 is between 24-48 hours as measured by incubating the prodrug in a phosphate buffer solution (e.g., PBS) at 37 °C and pH of 7.2.
  • a phosphate buffer solution e.g., PBS
  • activation of the prodrug occurs after cleavage of an amide bond releasing the insulin A chain and resulting in the formation of a diketopiperazine or diketomorpholine, and the active two chain insulin peptide.
  • a single chain insulin prodrug having the general structure of B-U-O-A, wherein B represents an insulin B chain, A represents an insulin A chain and U-0 is a dipeptide that links the B chain to the A chain. More particularly, U is an amino acid or hydroxyl acid, and O is an N- alkylated amino acid, wherein U is covalently linked to the carboxy terminus of the B chain via the side chain of U, and O is linked to both U and the N-terminus of the A chain via amide bonds.
  • the structure of U-0 is selected, in one embodiment, to chemically cleave from the insulin A chain at a rate that is at least about 90% complete within about 12 to about 720 hours in PBS under physiological conditions.
  • the chemical cleavage half-life (tl/2) of U-0 from the insulin A chain is at least about 1 hour to about 168 hours (1 week) in PBS under physiological conditions
  • U is an amino acid in the D stereoisomer configuration and O is an amino acid in the L stereoisomer configuration. In some exemplary embodiments, U is an amino acid in the L stereoisomer configuration and O is an amino acid in the D stereoisomer configuration. In some exemplary embodiments, U is an amino acid in the D stereoisomer configuration and O is an amino acid in the D stereoisomer configuration.
  • the N-alkylated group of amino acid O is a Ci-Cis alkyl, and in one embodiment N-alkylated group is Ci-C 6 alkyl. In one embodiment U-0 is a dipeptide comprising the structure of Formula I as defined herein.
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide is linked to the carboxy terminus of the insulin B chain and the carboxy terminus of the second amino acid of the metastable linking dipeptide is linked to the N-terminal alpha amine of the insulin A chain to form an amide bond.
  • the dipeptide linker is designed to spontaneously cleave its amide bond linkage to the insulin A chain and generating an active two chain insulin.
  • the metastable dipeptide linker comprises a C-alkylated amino acid as the first amino acid (U) and an N-alkylated amino acid as the second amino acid (O).
  • Ri and R 8 are independently selected from the group consisting of H or Ci-C 4 alkyl
  • R 2, and R4 are independently selected from the group consisting of H, Ci-Ci 8 alkyl, C 2 -Ci 8 alkenyl, (Ci-Cis alkyl)OH, (Ci-Cis alkyl)SH, (C 2 -C 3 alkyl)SCH 3 , (C1-C4 alkyl)CONH 2 , (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH 2 , (d-C 4 alkyl)NHC(NH 2 + )NH 2 , (Co-C 4 alkyl)(C 3 -C 6 cycloalkyl), (C 0 -C 4 alkyl)(C 2 -C 5 heterocyclic), (C 0 -C 4 alkyl)(C 6 - C10 aryl)R 7 , (C1-C4 alkyl)(C 3 -C 9 heteroaryl), and C1-C12 alkyl(W)Ci-Ci 2 alkyl
  • R 3 is selected from the group consisting of Ci-Ci 8 alkyl, (Ci-Ci 8 alkyl)OH, (Ci-Cis alkyl)NH 2, (Ci-Cis alkyl)SH, (C 0 -C 4 alkyl)(C 3 -C 6 )cycloalkyl, (C 0 -C 4 alkyl)(C 2 -C 5 heterocyclic), (C 0 -C 4 alkyl)(C 6 -Cio aryl)R 7 , and (C1-C4 alkyl)(C 3 -C 9 heteroaryl) or R 4 and R 3 together with the atoms to which they are attached form a 4, 5 or 6 member heterocyclic ring;
  • R 5 is NHR 6 or OH
  • R 6 is H, Ci-C 8 alkyl or R 6 and R 2 together with the atoms to which they are attached form a 4, 5 or 6 member heterocyclic ring;
  • R 7 is selected from the group consisting of H and OH, optionally with the proviso that Ri and R 2 are not both H, and when R 4 and R 3 together with the atoms to which they are attached form a 4, 5 or 6 member heterocyclic ring, both Ri and R 2 are other than H.
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide i.e., the R 2 substituent
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide is linked to the carboxy terminus of the insulin B chain and the carboxy terminus of the second amino acid of the metastable linking dipeptide is linked to the N-terminal alpha amine of the insulin A chain to form an amide bond.
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide i.e., the R2 substituent
  • Ri and R 8 are independently H or Ci-C 8 alkyl
  • R 2 and R 4 are independently selected from the group consisting of H, Ci-C 8 alkyl, C 2 -C 8 alkenyl, (C1-C4 alkyl)OH, (C1-C4 alkyl)SH, (C 2 -C 3 alkyl)SCH 3 , (C1-C4 alkyl)CONH 2 , (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH 2 , (C1-C4 alkyl)NHC(NH 2 +) NH 2 , (Co-C 4 alkyl)(C 3 -C 6 cycloalkyl), (C 0 -C 4 alkyl)(C 2 -C 5 heterocyclic), (C 0 -C 4 alkyl)(C 6 -Cio aryl)R 7 , and CH 2 (C 3 -C 9 heteroaryl);
  • R 3 is selected from the group consisting of Ci-C 8 alkyl, or R 4 and R 3 together with the atoms to which they are attached form a pyrrolidine ring;
  • R 5 is NH 2 or OH
  • R 7 is hydrogen or OH, optionally with the proviso that when R 4 and R 3 together with the atoms to which they are attached form a pyrrolidine ring, both Ri and R 2 are other than H.
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide i.e., the R 2 substituent
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide is linked to the carboxy terminus of the insulin B chain and the carboxy terminus of the second amino acid of the metastable linking dipeptide is linked to the N-terminal alpha amine of the insulin A chain to form an amide bond.
  • Ri is Ci-Cg alkyl
  • R 2 is selected from the group consisting of Ci-C 8 alkyl, C 2 -C 8 alkenyl, (Ci-C 4 alkyl)OH, (d-C 4 alkyl)SH, (C 2 -C 3 alkyl)SCH 3 , (d-C 4 alkyl)CONH 2 , (d-C 4 alkyl)COOH, (C1-C4 alkyl)NH 2 , (C1-C4 alkyl)NHC(NH 2 +) NH 2 , (C 0 -C 4 alkyl)(C 3 -C 6 cycloalkyl), (C 0 -C 4 alkyl)(C 2 -C 5 heterocyclic), (C 0 -C 4 alkyl)(C 6 -Ci 0 aryl)R 7 , and CH 2 (C -C 9 heteroaryl);
  • R 4 is selected from the group consisting of H, Ci-C 8 alkyl, C 2 -C 8 alkenyl, (Q- C 4 alkyl)OH, (C1-C4 alkyl)SH, (C 2 -C 3 alkyl)SCH 3 , (C1-C4 alkyl)CONH 2 , (C 1-C4 alkyl)COOH, (C1-C4 alkyl)NH 2 , (C1-C4 alkyl)NHC(NH 2 +) NH 2 , (C 0 -C 4 alkyl)(C 3 -C 6 cycloalkyl), (Co-C 4 alkyl)(C 2 -C5 heterocyclic), (Co-C 4 alkyl)(C6-Cio aryl)R 7 , and CH 2 (C 3 -C 9 heteroaryl);
  • R 3 is selected from the group consisting of Ci-C 8 alkyl, or R 4 and R 3 together with the atoms to which they are attached form a pyrrolidine ring;
  • R 5 is NH 2 or OH
  • R 7 is hydrogen or OH
  • R 8 is H.
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide i.e., the R 2 substituent
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide is linked to the carboxy terminus of the insulin B chain and the carboxy terminus of the second amino acid of the metastable linking dipeptide is linked to the N-terminal alpha amine of the insulin A chain to form an amide bond.
  • Ri and R 8 are each H;
  • R 2 is selected from the group consisting of Ci-C 8 alkyl, (Ci-C 4 alkyl)OH, (Ci- C 4 alkyl)SH, (d-C 4 alkyl)CONH 2 , (d-C 4 alkyl)COOH, (d-C 4 alkyl)NH 2 , (C1-C4 alkyl)NHC(NH 2 +) NH 2 , (C 0 -C 4 alkyl)(C 2 -C 5 heterocyclic), and (CH 3 )(C 6 aryl)R 7 ;
  • R 4 is selected from the group consisting of H, Ci-C 8 alkyl, (C1-C4 alkyl)OH,
  • R 3 is Ci-Cg alkyl
  • R 5 is NH 2 or OH
  • R 7 is hydrogen or OH.
  • R5 is NH 2 .
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide i.e., the R 2 substituent
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide is linked to the carboxy terminus of the insulin B chain and the carboxy terminus of the second amino acid of the metastable linking dipeptide is linked to the N-terminal alpha amine of the insulin A chain to form an amide bond.
  • the dipeptide prodrug element comprises the general structure:
  • Ri , R 4 and R 8 are each H;
  • R 2 is selected from the group consisting of Ci-C 8 alkyl, (Ci-C 4 alkyl)OH, (Q- C 4 alkyl)SH, and (C C 4 alkyl)NH 2 ;
  • R 3 is Ci-Cg alkyl
  • R5 is NH 2 or OH. In a further embodiment, R5 is NH 2 .
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide i.e., the R 2 substituent
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide is linked to the carboxy terminus of the insulin B chain and the carboxy terminus of the second amino acid of the metastable linking dipeptide is linked to the N-terminal alpha amine of the insulin A chain to form an amide bond.
  • prodrug element of Formula I wherein Ri is selected from the group consisting of H and Ci-C 8 alkyl; and
  • R 2 and R 4 are independently selected from the group consisting of H, Ci-C 8 alkyl, C 2 -C 8 alkenyl, (d-C 4 alkyl)OH, (d-C 4 alkyl)SH, (C 2 -C 3 alkyl)SCH 3 , (d-C 4 alkyl)CONH 2 , (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH 2 , (C1-C4 alkyl)NHC(NH 2 + ) NH 2 , (Co-C 4 alkyl)(C 3 -C 6 cycloalkyl), (C 0 -C 4 alkyl)(C 6 -Ci 0 aryl)R 7 , and CH 2 (C 5 -C 9 heteroaryl), or Ri and R 2 together with the atoms to which they are attached form a C 3 -C 8 cycloalkyl ring;
  • R 3 is selected from the group consisting of Ci-C 8 alkyl or R 4 and R 3 together with the atoms to which they are attached form a pyrrolidine ring;
  • R 5 is NH 2 or OH
  • R 7 is selected from the group consisting of hydrogen and OH; and R 8 is H, provided that when R 4 and R 3 together with the atoms to which they are attached form a pyrrolidine ring, both Ri and R 2 are not H.
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide i.e., the R 2 substituent
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide is linked to the carboxy terminus of the insulin B chain and the carboxy terminus of the second amino acid of the metastable linking dipeptide is linked to the N-terminal alpha amine of the insulin A chain to form an amide bond.
  • dipeptide prodrug element has the structure of Formula I, wherein
  • Ri and R 8 are independently H or Ci-C 8 alkyl
  • R 2 and R 4 are independently selected from the group consisting of H, Ci-C 8 alkyl, C 2 -C 8 alkenyl, (d-C 4 alkyl)OH, (d-C 4 alkyl)SH, (C 2 -C 3 alkyl)SCH 3 , (d-C 4 alkyl)CONH 2 , (C C 4 alkyl)COOH, (C C 4 alkyl)NH 2 , (C C 4 alkyl)NHC(NH 2 +) NH 2 , (Co-C 4 alkyl)(C 3 -C 6 cycloalkyl), (C 0 -C 4 alkyl)(C 2 -C 5 heterocyclic), (C 0 -C 4 alkyl)(C 6 -Cio aryl)R 7 , and CH 2 (C 3 -C 9 heteroaryl);
  • R 3 is Ci-Cis alkyl
  • R 5 is NHR 6 ;
  • R 6 is H or Ci-C 8 alkyl
  • R 7 is selected from the group consisting of hydrogen, Ci-Ci 8 alkyl, C 2 -Ci 8 alkenyl, (C 0 -C 4 alkyl)CONH 2, (C 0 -C 4 alkyl)COOH, (C 0 -C 4 alkyl)NH 2 , (C 0 -C 4 alkyl)OH, and halo and R 8 is H.
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide (i.e., the R 2 substituent) is linked to the carboxy terminus of the insulin B chain and the carboxy terminus of the second amino acid of the metastable linking dipeptide is linked to the N-terminal alpha amine of the insulin A chain to form an amide bond.
  • dipeptide prodrug element has the structure of Formula I, wherein
  • Ri and R 2 are independently Ci-Cis alkyl, (Ci-C 4 alkyl)CONH 2 , or (Ci-C 4 alkyl)(C 6 aryl)R 7 ;
  • R 3 is Ci-Cis alkyl
  • R 4 and R 8 are each hydrogen
  • R 5 is NH 2 ;
  • R 7 is selected from the group consisting of hydrogen and OH.
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide i.e., the R 2 substituent
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide is linked to the carboxy terminus of the insulin B chain and the carboxy terminus of the second amino acid of the metastable linking dipeptide is linked to the N-terminal alpha amine of the insulin A chain to form an amide bond.
  • the dipeptide prodrug element has the structure of Formula I, wherein
  • Ri is H or Ci-Cg alkyl
  • R 2 is selected from the group consisting of (Ci-C 4 alkyl)NH 2 and (Ci-C 4 alkyl)SH;
  • R 3 is Ci-Cg alkyl or R 3 and R 4 together with the atoms to which they are attached form a pyrrolidine ring;
  • R 4 is selected from the group consisting of hydrogen and Ci-C 8 alkyl
  • R 8 is hydrogen
  • R5 is NH 2 optionally with the proviso that Ri is Ci-C 8 alkyl when R 3 and R 4 together with the atoms to which they are attached form a pyrrolidine ring.
  • Ri and R4 are each H and R 3 is Ci-C 8 alkyl.
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide i.e., the R 2 substituent
  • the side chain of the first amino acid/hydroxyl acid of the metastable linking dipeptide is linked to the carboxy terminus of the insulin B chain and the carboxy terminus of the second amino acid of the metastable linking dipeptide is linked to the N-terminal alpha amine of the insulin A chain to form an amide bond.
  • dipeptide linker U-0 comprises or consists of the general structure of Formula II:
  • X is NH, S, O or CH 2 ;
  • R 2 is H or Ci-C 4 alkyl
  • R 3 is Ci-Ce alkyl
  • R 4 is H, Ci-Cg alkyl, C 2 -C 8 alkenyl, (C1-C4 alkyl)OH, (C1-C4 alkyl)SH, (C 2 -C 3 alkyl)SCH 3 , (C 1 -C4 alkyl)CONH 2 , (C 1 -C4 alkyl)COOH, (C 1 -C4 alkyl)NH 2 ,
  • R 5 is NH 2 or OH
  • R 7 is H or OH; and n is an integer selected from a range of 0 to 4 or 3 to 4. In one embodiment n is 3.
  • the dipeptide linker U-0 comprises or consists of the general structure of Formula II:
  • X is NH, S or O
  • R 2 is H or Ci-C 4 alkyl
  • R 3 is Ci-Ce alkyl
  • R 4 is H, Q-C 6 alkyl, CH 2 (C6 aryl)R 7 , or R 3 and R 4 together with the atoms to which they are attached form a pyrrolidine ring.
  • R 5 is NH 2 or OH
  • R 7 is H or OH
  • n is an integer selected from 1 to 4. In one embodiment n is 3.
  • dipeptide linker U-0 comprises or consists of the general structure of Formula II:
  • X is NH, S or O
  • R 2 is H or Ci-C 4 alkyl
  • R 3 is Ci-C 6 alkyl
  • R 4 is H, CrC 6 alkyl, CH 2 (C 6 aryl)R 7 , or R 3 and R 4 together with the atoms to which they are attached form a pyrrolidine ring;
  • R 5 is NH 2 ;
  • R 7 is H or OH
  • n is an integer selected from 1 to 4.
  • dipeptide linker U-0 comprises or consists of the general structure of Formula II:
  • X is NH or S
  • R 2 is H or Ci-C 4 alkyl
  • R 3 is Ci-C 6 alkyl
  • R 4 is H, Ci-C 6 alkyl, or CH 2 (C 6 aryl)R 7 ;
  • R 7 is H or OH
  • n is an integer selected from 1 to 4.
  • dipeptide linker U-0 comprises or consists of the general structure of Formula II:
  • X is NH or S
  • R 2 is Ci-C 4 alkyl
  • R 3 is Ci-C 6 alkyl
  • R 4 is H, or R 3 and R 4 together with the atoms to which they are attached form a pyrrolidine ring;
  • R 5 is NH 2 ;
  • n is an integer selected from 1 to 4.
  • dipeptide linker U-0 comprises or consists of the general structure of Formula II:
  • R 2 is H
  • R 3 is Ci-C 6 alkyl
  • R 4 is H, C1-C4 alkyl, or CH 2 (C 6 aryl)R 7 ;
  • R 5 is NH 2 ;
  • R 7 is H or OH
  • n is an integer selected from 1 to 4.
  • a single chain insulin prodrug analog comprising an insulin A chain and an insulin B chain linked as a linear chain via a metastable dipeptide linker. More particularly, the single chain insulin prodrug analog comprises any of the dipeptide linker likers disclosed herein, including those of formula I and II, in combination with any know insulin agonist peptide.
  • the single chain insulin prodrugs in one embodiment comprise the native sequence of the respective A and B chain peptides (i.e., SEQ ID NO: 1 and SEQ ID NO: 2) or may comprise a derivative of SEQ ID NO: 1 and/or SEQ ID NO: 2 wherein the derivative includes modification of the amino acid at position A 19, B 16 or B25 to a 4-amino phenylalanine and/or one or more amino acid substitutions at positions selected from A5, A8, A9, A10, A14, A15, A17, A18, A19 and A21, B l, B2, B3, B4, B5, B9, B 10, B 13, B 14, B 17, B20, B22, B23, B26, B27, B28, B29 and B30 or deletions of any or all of positions B l-4 and B26-30.
  • a single chain insulin prodrug comprising the structure B-U-O-A, wherein
  • A is an insulin A chain
  • B is an insulin B chain
  • U-0 is a dipeptide that links the carboxy terminus of said B chain to the amino terminus of the A chain, wherein
  • said A chain comprises the sequence
  • said B chain comprises the sequence
  • X 4 is glutamic acid or aspartic acid
  • X5 is glutamine or glutamic acid
  • X 8 is histidine, threonine or phenylalanine
  • X9 is serine, arginine, lysine, ornithine or alanine
  • X10 is isoleucine or serine
  • X12 is serine or aspartic acid
  • Xi 4 is tyrosine, arginine, lysine, ornithine or alanine;
  • Xi 5 is glutamine, glutamic acid, arginine, alanine, lysine, ornithine or leucine;
  • Xi 7 is glutamine, glutamic acid, arginine, aspartic acid, ornithine or lysine;
  • Xis is methionine, asparagine, glutamine, aspartic acid, glutamic acid or threonine;
  • X19 is tyrosine, 4-methoxy-phenylalanine or 4-amino phenylalanine
  • X21 is selected from the group consisting of alanine, glycine, serine, valine, threonine, isoleucine, leucine, glutamine, glutamic acid, asparagine, aspartic acid, histidine, tryptophan, tyrosine, and methionine;
  • X25 is histidine or threonine
  • X29 is selected from the group consisting of alanine, glycine and serine;
  • X30 is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid;
  • X33 is selected from the group consisting of aspartic acid, glutamine and glutamic acid;
  • X 34 is selected from the group consisting of alanine and threonine;
  • X 4 i is selected from the group consisting of glutamic acid, aspartic acid or asparagine;
  • X 4 2 is selected from the group consisting of alanine, lysine, ornithine and arginine;
  • X 4 5 is tyrosine or phenylalanine
  • R 22 is selected from the group consisting of FVNQ (SEQ ID NO: 12), FVKQ (SEQ ID NO: 11), a tripeptide glycine-proline-glutamic acid, a tripeptide valine-asparagine-glutamine, a dipeptide proline-glutamic acid, a dipeptide asparagine-glutamine, glutamine, glutamic acid and a bond;
  • R2 3 is selected from the group consisting of R2 3 is selected from the group consisting of YTX 28 KT (SEQ ID NO: 20), YTKPT (SEQ ID NO: 23), YTX 28 K (SEQ ID NO: 24), YTKP (SEQ ID NO: 29), YTPK (SEQ ID NO: 32), YTX 28 , YT, Y and a bond;
  • X 28 is proline, aspartic acid or glutamic acid; and Ri3 is COOH or CONH 2 wherein U is an amino acid or a hydroxyl acid and O is an N-alkylated amino acid, and O is linked to the N-terminus of the A chain through formation of an amide bond, and U is linked to the carboxy terminus of the B chain via the amino acid side chain of U, further wherein the chemical cleavage half- life (ti /2 ) of O from the A chain is at least about 1 hour to about 1 week in PBS under physiological conditions.
  • a single chain insulin prodrug comprising the structure B-U-O-A, wherein
  • A is an insulin A chain
  • B is an insulin B chain
  • U-0 is a dipeptide that links the carboxy terminus of said B chain to the amino terminus of the A chain, wherein
  • said A chain comprises the sequence
  • said B chain comprises the sequence
  • X 4 is glutamic acid or aspartic acid
  • X5 is glutamine or glutamic acid
  • X 8 is histidine, threonine or phenylalanine
  • X9 is serine
  • X10 is isoleucine
  • Xi 2 is serine or aspartic acid
  • Xi 4 is tyrosine
  • Xi 5 is glutamine
  • Xi 7 is glutamine, glutamic acid, arginine, aspartic acid, ornithine or lysine;
  • Xis is methionine, asparagine, glutamine, aspartic acid, glutamic acid or threonine;
  • X19 is tyrosine
  • X21 is selected from the group consisting of alanine, glycine, serine, valine, threonine, isoleucine, leucine, glutamine, glutamic acid, asparagine, aspartic acid, histidine, tryptophan, tyrosine, and methionine;
  • X25 is histidine or threonine
  • X29 is selected from the group consisting of alanine, glycine and serine;
  • X30 is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid;
  • X33 is selected from the group consisting of aspartic acid, glutamine and glutamic acid;
  • X 34 is selected from the group consisting of alanine and threonine
  • X 4 i is selected from the group consisting of glutamic acid, aspartic acid or asparagine;
  • X 4 2 is selected from the group consisting of alanine, lysine, ornithine and arginine;
  • X 4 5 is tyrosine or phenylalanine
  • R 22 is FVNQ (SEQ ID NO: 12), or FVKQ (SEQ ID NO: 11);
  • P 2 3 is selected from the group consisting of P 2 3 is selected from the group consisting of YTX 28 KT (SEQ ID NO: 20), YTKPT (SEQ ID NO: 23), YTX 28 K (SEQ ID NO: 24), YTKP (SEQ ID NO: 29), YTPK (SEQ ID NO: 32), YTX 28 , YT, Y and a bond;
  • X2 8 is proline, aspartic acid or glutamic acid
  • GIVEQCCX 8 SICSLYQLENYCX 2 i (SEQ ID NO: 43) or a sequence that differs from SEQ ID NO: 3 by 1, 2, 3 or 4 amino acid modifications, selected from positions A5, A9, A10, A14, A15, A17, A18, and said B chain sequence comprises a sequence of FVNQHLCGX 29 X 3 oLVEALYLVCGERGFF-R 23 (SEQ ID NO: 41) or a sequence that differs from SEQ ID NO: 41 by 1, 2, 3, 4 or 5 amino acid modifications, selected from positions B l, B2, B3, B4, B5, B 13, B 14, B 17, B20, B21, B22, or B23, wherein X 8 is selected from the group consisting of threonine and histidine;
  • X21 is selected from the group consisting of asparagine, glycine and alanine
  • X29 is selected from the group consisting of alanine, glycine and serine
  • X30 is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid
  • R 23 is selected from the group consisting of YTX 28 KT (SEQ ID NO: 20), YTKPT (SEQ ID NO: 23), YTX 28 K (SEQ ID NO: 21), YTKP (SEQ ID NO: 29), YTPK (SEQ ID NO: 32, YTX 28 , YT, Y and a bond.
  • a single chain insulin prodrug comprising the structure B-U-O-A, wherein
  • A is an insulin A chain
  • B is an insulin B chain
  • U-0 is a dipeptide that links the carboxy terminus of said B chain to the amino terminus of the A chain, wherein the A chain of the insulin peptide comprises the sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1) and the B chain comprises a sequence selected from the group consisting of
  • R23 is selected from the group consisting of YTEKT (SEQ ID NO: 27), YTDKT (SEQ ID NO: 28), YTKPT (SEQ ID NO: 23), YTEK (SEQ ID NO: 30), YTDK (SEQ ID NO: 31), YTKP (SEQ ID NO: 29), YTPK (SEQ ID NO: 32), YTE, YTD, YTK, YT, Y or a bond wherein the C-terminus of the B chain is linked to the N-terminus of the A chain via a dipeptide linker comprising the structure of Formula
  • X is NH, S or O
  • R 2 is H or Ci-C 4 alkyl
  • R 3 is Ci-Ce alkyl
  • R 4 is H, or R3 and R 4 together with the atoms to which they are attached form a pyrrolidine ring;
  • R 5 is NH 2 or OH; and n is an integer selected from 1 to 4.
  • X is NH; R 2 is H; R 3 is Q-C 6 alkyl; R 4 is H; and R5 is NH 2 and n is 3.
  • R 3 is CH 3 , isopropyl, n-propyl t-butyl or iso-butyl.
  • a single chain insulin prodrug comprising the structure B-U-O-A, wherein
  • A is an insulin A chain
  • B is an insulin B chain
  • U-0 is a dipeptide that links the carboxy terminus of said B chain to the amino terminus of the A chain, wherein the A chain of the insulin peptide comprises the sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1) and the B chain comprising a sequence selected from the group consisting of
  • R 23 is selected from the group consisting of YTEKT (SEQ ID NO: 27), YTDKT (SEQ ID NO: 28), YTKPT (SEQ ID NO: 23), YTEK (SEQ ID NO: 30), YTDK (SEQ ID NO: 31), YTKP (SEQ ID NO: 29), YTPK (SEQ ID NO: 32), YTE, YTD, YTK, YT, Y or a bond wherein the C-terminus of the B chain is linked to the N-terminus of the A chain via a dipeptide linker comprising the structure of Formula
  • X is NH, S or O
  • R 2 is Ci-C 4 alkyl
  • R 3 and R 4 together with the atoms to which they are attached form a pyrrolidine ring
  • R 5 is NH 2 ;
  • n is an integer selected from 1 to 4.
  • X is NH; R 2 Ci-C 4 alkyl; R 3 and R 4 together with the atoms to which they are attached form a pyrrolidine ring; and R5 is NH 2 and n is 3.
  • the single chain insulin prodrug comprises the structure of DTI (Des-Tetrapeptide (B27-B30) Insulin) or DPI (Des-Pentapeptide (B26-B30) Insulin) Analog as depicted in Fig. 4, wherein R is Ci-C 4 alkyl.
  • GIVEQCCX1SICSLYQLENX2CX3-R13 (SEQ ID NO: 3), or an analog thereof comprising a sequence that differs from SEQ ID NO: 3 by 1 to 8, 1 to 5 or 1 to 3 amino acid modifications, selected from positions A5, A8, A9, A10, A14, A15, A17, A18 (relative to the native insulin A chain), and the B chain sequence comprises the sequence of X 25 LCGX 29 X 3 oLVEX 34 LYLVCGERGFX 45 (SEQ ID NO: 42), or an analog thereof comprising a sequence that differs from SEQ ID NO: 42 sequence by 1 to 10, 1 to 5 or 1 to 3 amino acid modifications, selected from positions B l, B2, B3, B4, B5, B 13, B 14, B 17, B20, B22, B23, B26, B27, B28, B29 and B30 (relative to the native insulin B chain; i.e., amino acid X 4 of SEQ ID NO: 14 corresponds to position B5 in native
  • GIVEQCCX8SICSLYQLENYCX 21 (SEQ ID NO: 43); and the B chain of the single chain insulin prodrug comprises the sequence
  • X 21 is selected from the group consisting of asparagine, glycine and alanine; and Ri 4 is COOH or CONH 2 , and in one embodiment R 14 is CONH 2 .
  • the single chain insulin prodrugs disclosed herein may be part of a dimer, trimer or higher order multimer comprising at least two, three, or more peptides bound via a linker.
  • the dimer may be a homodimer (comprising two identical single chain insulin analogs) or heterodimer (wherein the two single chain insulin analogs differ in structure for each other).
  • the dimerizing linker is selected from the group consisting of a bifunctional thiol crosslinker and a bi- functional amine crosslinker.
  • the dimerization linker is PEG, e.g., a 5 kDa PEG, 20 kDa PEG.
  • the dimerizing linker is a disulfide bond.
  • each monomer of the dimer may comprise a Cys residue (e.g., a terminal or internally positioned Cys) and the sulfur atom of each Cys residue participates in the formation of the disulfide bond.
  • Each monomer of the dimer represents a heterodimer of an A and B chain.
  • the monomers are connected via terminal amino acids (e.g., N-terminal or C-terminal), via internal amino acids, or via a terminal amino acid of at least one monomer and an internal amino acid of at least one other monomer. In specific aspects, the monomers are not connected via an N-terminal amino acid.
  • the monomers of the multimer are attached together in a "tail-to-tail" orientation in which the C- terminal amino acids of each monomer are attached together.
  • a conjugate moiety may be covalently linked to any of the insulin peptides described herein, including a dimer, trimer or higher order multimer.
  • the prodrugs disclosed herein can be further modified to improve the peptide's solubility in aqueous solutions at physiological pH, while enhancing the effective duration of the peptide by preventing renal clearance of the peptide. Peptides are easily cleared because of their relatively small molecular size when compared to plasma proteins. Increasing the molecular weight of a peptide above 40 kDa exceeds the renal threshold and significantly extends duration in the plasma. Accordingly, in one embodiment the peptide prodrugs are further modified to comprise a covalently linked hydrophilic moiety. In one embodiment the hydrophilic moiety is a plasma protein polyethylene oxide chain or the Fc portion of an immunoglobin. Therefore, in one embodiment the presently disclosed prodrugs are further modified to comprise one or more hydrophilic groups covalently linked to the side chains of amino acids.
  • the insulin prodrugs disclosed herein are further modified by linking a hydrophilic moiety to either the N-terminal amino acid of the B chain or to the side chain of a lysine amino acid located at the carboxy terminus of the B chain.
  • the hydrophilic group is a polyethylene oxide chain, and in one embodiment two or more polyethylene oxide chains are covalently attached to two or more amino acid side chains of the single chain insulin prodrug.
  • the hydrophilic moiety is covalently attached to an amino acid side chain of a single chain insulin prodrug disclosed herein at a position selected from the group consisting of A9, A14, A15, or B22, B28, B29 and the N-terminus of the B chain.
  • the polyethylene oxide chains can be attached at the N-terminal amino acid of the B chain or to the side chain of a lysine amino acid located at the carboxy terminus of the B chain.
  • any of the single chain insulin prodrugs disclosed herein further comprises a polyethylene oxide, alkyl or acyl group.
  • one or more polyethylene oxide chains are linked to N-terminal alpha amine of the A or B chain or linked to the side chain of an amino acid of the A or B chain wherein the combined molecular weight of the polyethylene oxide chains ranges from about 20,000 to about 80,000 Daltons, or 40,000 to 80,000 Daltons or 40,000 to 60,000 Daltons.
  • the polyethylene oxide is polyethylene glycol.
  • at least one polyethylene glycol chain having a molecular weight of about 40,000 Daltons is linked to the single chain insulin prodrug.
  • one or more alkyl or acyl groups are linked to N-terminal alpha amine of the A or B chain or linked to the side chain of an amino acid of the A or B chain.
  • the single chain insulin analog is acylated with an acyl group of sufficient size to bind serum albumin.
  • the N- terminal alpha amine of the B chain is acylated with a C18 to C28 hydrocarbon chain.
  • the acyl group can be linear or branched, and in one embodiment is a C 16 to C30 fatty acid or a C 16 to C30 diacid.
  • the acyl group can be any of a C 16 fatty acid, C18 fatty acid, C20 fatty acid, C22 fatty acid, C24 fatty acid, C26 fatty acid, C28 fatty acid, or a C30 fatty acid.
  • the acyl group is a C 16 to C20 fatty acid, e.g., a C18 fatty acid or a C20 fatty acid.
  • the acyl group is a C16 to C20 diacid, e.g., a C18 diacid or a C20 diacid.
  • Hydrophilic moieties can be attached to the insulin analogs under any suitable conditions used to react a protein with an activated polymer molecule. Any means known in the art can be used, including via acylation, reductive alkylation, Michael addition, thiol alkylation or other chemoselective conjugation/ligation methods through a reactive group on the PEG moiety (e.g., an aldehyde, amino, ester, thiol, a- haloacetyl, maleimido or hydrazino group) to a reactive group on the target compound (e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group).
  • a reactive group on the PEG moiety e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group
  • Activating groups which can be used to link the water soluble polymer to one or more proteins include without limitation sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine, oxirane and 5-pyridyl. If attached to the peptide by reductive alkylation, the polymer selected should have a single reactive aldehyde so that the degree of polymerization is controlled. See, for example, Kinstler et al., Adv. Drug. Delivery Rev. 54: 477-485 (2002); Roberts et al., Adv. Drug Delivery Rev. 54: 459- 476 (2002); and Zalipsky et al., Adv. Drug Delivery Rev. 16: 157-182 (1995).
  • Suitable hydrophilic moieties include polyethylene glycol (PEG),
  • polypropylene glycol polyoxyethylated polyols (e.g., POG), polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG),
  • POG polyoxyethylated polyols
  • POG polyoxyethylated glycerol
  • polyoxyalkylenes polyethylene glycol propionaldehyde, copolymers of ethylene glycol/propylene glycol, monomethoxy-polyethylene glycol, mono-(Cl-ClO) alkoxy- or aryloxy-polyethylene glycol, carboxymethylcellulose, polyacetals, polyvinyl alcohol (PVA), polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, poly (.beta.-amino acids) (either
  • polysaccharide polymers Ficoll or dextran and mixtures thereof.
  • the hydrophilic moiety e.g., polyethylene glycol chain in accordance with one embodiment has a molecular weight selected from the range of about 500 to about 40,000 Daltons.
  • the hydrophilic moiety, e.g. PEG has a molecular weight selected from the range of about 500 to about 5,000 Daltons, or about 1,000 to about 5,000 Daltons.
  • the hydrophilic moiety, e.g., PEG has a molecular weight of about 10,000 to about 20,000 Daltons.
  • the hydrophilic moiety, e.g., PEG has a molecular weight of about 20,000 to about 40,000 Daltons.
  • dextrans are used as the hydrophilic moiety.
  • Dextrans are polysaccharide polymers of glucose subunits, predominantly linked by al-6 linkages.
  • Dextran is available in many molecular weight ranges, e.g., about 1 kD to about 100 kD, or from about 5, 10, 15 or 20 kD to about 20, 30, 40, 50, 60, 70, 80 or 90 kD. Linear or branched polymers are contemplated.
  • Resulting preparations of conjugates may be essentially monodisperse or polydisperse, and may have about 0.5, 0.7, 1, 1.2, 1.5 or 2 polymer moieties per peptide.
  • the single chain insulin prodrugs disclosed herein are further modified by amino acid substitutions or the addition of 1 to 3, or 1 to 6 amino acids at the N-terminus of the B chain, wherein the substituting amino acid comprises a side chain suitable for crosslinking with hydrophilic moieties, including for example, polyethylene glycol and/or acyl or alkyl groups.
  • the amino acid at the position of the single chain insulin prodrug where the hydrophilic moiety is to be linked is substituted (or added at the C-terminus) with a natural or synthetic amino acid to introduce, or allow for ease in attaching, the hydrophilic moiety.
  • a native amino acid at a position selected from A5, A8, A9, A10, A12, A14, A15, A17, A18, B l, B2, B3, B4, B5, B 13, B 14, B 17, B21, B22, B26, B27, B28, B29 and B30 is substituted with a lysine, cysteine or acetyl phenylalanine residue (or a lysine, cysteine or acetyl phenylalanine residue is added to the C-terminus) to allow for the covalent attachment of a polyethylene glycol chain.
  • the single chain insulin prodrug has a single cysteine residue added to the carboxy terminus of the B chain, or the single chain insulin prodrug is substituted with at least one cysteine residue, wherein the side chain of the cysteine residue is further modified with a thiol reactive reagent, including for example, maleimido, vinyl sulfone, 2-pyridylthio, haloalkyl, and haloacyl.
  • thiol reactive reagents may contain carboxy, keto, hydroxyl, and ether groups as well as other hydrophilic moieties such as polyethylene glycol units.
  • the single chain insulin prodrug has a single lysine residue added to the carboxy terminus of the B chain, or the single chain insulin prodrug is substituted with lysine, and the side chain of the substituting lysine residue is further modified using amine reactive reagents such as active esters (succinimido, anhydride, etc) of carboxylic acids or aldehydes of hydrophilic moieties such as polyethylene glycol.
  • active esters succinimido, anhydride, etc
  • the polyethylene glycol chain may be in the form of a straight chain or it may be branched.
  • the polyethylene glycol chain has an average molecular weight selected from the range of about 20,000 to about 60,000 Daltons.
  • Multiple polyethylene glycol chains can be linked to the single chain insulin prodrug to provide a single chain insulin prodrug with optimal solubility and blood clearance properties.
  • the single chain insulin prodrug is linked to a single polyethylene glycol chain that has an average molecular weight selected from the range of about 20,000 to about 60,000 Daltons.
  • the single chain insulin prodrug is linked to two polyethylene glycol chains wherein the combined average molecular weight of the two chains is selected from the range of about 40,000 to about 80,000 Daltons.
  • a single polyethylene glycol chain having an average molecular weight of 20,000 or 60,000 Daltons is linked to the single chain insulin prodrug.
  • a single polyethylene glycol chain is linked to the single chain insulin prodrug and has an average molecular weight selected from the range of about 40,000 to about 50,000 Daltons.
  • two polyethylene glycol chains are linked to the single chain insulin prodrug wherein the first and second polyethylene glycol chains each have an average molecular weight of 20,000 Daltons.
  • two polyethylene glycol chains are linked to the single chain insulin prodrug wherein the first and second polyethylene glycol chains each have an average molecular weight of 40,000 Daltons.
  • a single chain insulin prodrug comprising two or more polyethylene glycol chains covalently bound to the peptide is provided, wherein the total molecular weight of the polyethylene glycol chains is about 40,000 to about 60,000 Daltons.
  • the pegylated single chain insulin prodrug comprises a polyethylene glycol chain linked to one or more amino acids selected from the N-terminus of the B chain and/or position 28 of SEQ ID NO: 9 or at position 29 of SEQ ID NO: 8, wherein the combined molecular weight of the PEG chain(s) is about 40,000 to about 80,000 Daltons.
  • an insulin peptide, or prodrug/depot derivative thereof is fused to an accessory peptide which is capable of forming an extended conformation similar to chemical PEG (e.g., a recombinant PEG (rPEG) molecule), such as those described in International Patent Application Publication No. WO2009/023270 and U.S. Patent Application Publication No. US2008/0286808.
  • the rPEG molecule is not polyethylene glycol.
  • a single chain insulin prodrug is provided wherein a plasma protein has been covalently linked to an amino acid side chain of the peptide to improve the solubility, stability and/or pharmacokinetics of the single chain insulin prodrug.
  • serum albumin can be covalently bound to the single chain insulin prodrugs presented herein.
  • the plasma protein is covalently bound to the N-terminus of the B chain and/or to an amino acid corresponding to position 28 of SEQ ID NO: 9 or at position 29 of SEQ ID NO: 8.
  • a single chain insulin prodrug wherein a linear amino acid sequence representing the Fc portion of an immunoglobin molecule has been covalently linked to an amino acid side chain of a single chain insulin prodrug disclosed herein to improve the solubility, stability and/or pharmacokinetics of the single chain insulin prodrug.
  • the amino acid sequence representing the Fc portion of an immunoglobin molecule can be covalently bound to the N-terminus of the B chain or the C-terminus of the A chain.
  • the single chain insulin prodrug is modified to comprise an alkyl or acyl group by direct alkylation or acylation of an amine, hydroxyl, or thiol of a side chain of an amino acid of the single chain insulin prodrug or the N-terminal alpha amine of the B chain.
  • the single chain insulin prodrug is directly acylated through the side chain amine, hydroxyl, or thiol of an amino acid.
  • acylation is at one or more positions selected from A9, A14, A15, B22, B28 or B29.
  • acylation is at the N-terminal alpha amine of B 1.
  • the acylated single chain insulin prodrug can comprise an A chain amino acid sequence of SEQ ID NO: 3 and a B chain of SEQ ID NO: 5, or a modified amino acid sequence of SEQ ID NO: 3 and/or SEQ ID NO: 5 with at least one of the amino acids at positions A9, A14, A15, B22, B28 or B29 modified to any amino acid comprising a side chain amine, hydroxyl, or thiol.
  • the direct acylation of the single chain insulin prodrug occurs through the side chain amine, hydroxyl, or thiol of the amino acid at position B28 or B29.
  • the single chain insulin prodrug comprises an acyl group of a carboxylic acid with 1-24 carbon atoms bound to the epsilon-amino group of a Lys present at position B28 or B29.
  • a single-chain insulin prodrug analog is provided wherein one of the amino acids of the peptide linker is modified to comprise an acyl group by direct acylation of an amine, hydroxyl, or thiol of a side chain of an amino acid of the peptide linker.
  • the acylating group comprises a 1-5, 10-12 or 12- 24 carbon chain.
  • the present disclosure also encompasses other conjugates in which single chain insulin prodrugs of the invention are linked, optionally via covalent bonding, and optionally via a linker, to a conjugate.
  • Linkage can be accomplished by covalent chemical bonds, physical forces such electrostatic, hydrogen, ionic, van der Waals, or hydrophobic or hydrophilic interactions.
  • a variety of non-covalent coupling systems may be used, including biotin-avidin, ligand/receptor, enzyme/substrate, nucleic acid/nucleic acid binding protein, lipid/lipid binding protein, cellular adhesion molecule partners; or any binding partners or fragments thereof which have affinity for each other.
  • conjugates include but are not limited to a heterologous peptide or polypeptide (including for example, a plasma protein), a targeting agent, an immunoglobulin or portion thereof (e.g. variable region, CDR, or Fc region), a diagnostic label such as a radioisotope, fluorophore or enzymatic label, a polymer including water soluble polymers, or other therapeutic or diagnostic agents.
  • a conjugate is provided comprising a single chain insulin prodrug of the present disclosure and a plasma protein, wherein the plasma protein is selected from the group consisting of albumin, transferin and fibrinogen.
  • the plasma protein moiety of the conjugate is albumin or transferin.
  • the linker comprises a chain of atoms from 1 to about 60, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms long.
  • the chain atoms are all carbon atoms.
  • the chain atoms in the backbone of the linker are selected from the group consisting of C, O, N, and S. Chain atoms and linkers may be selected according to their expected solubility (hydrophilicity) so as to provide a more soluble conjugate.
  • the linker provides a functional group that is subject to cleavage by an enzyme or other catalyst or hydrolytic conditions found in the target tissue or organ or cell.
  • the length of the linker is long enough to reduce the potential for steric hindrance.
  • the linker is a covalent bond or a peptidyl bond and the conjugate is a polypeptide
  • the entire conjugate can be a fusion protein.
  • peptidyl linkers may be any length. Exemplary linkers are from about 1 to 50 amino acids in length, 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids in length.
  • Such fusion proteins may alternatively be produced by recombinant genetic engineering methods known to one of ordinary skill in the art. Conjugates and fusions
  • the present disclosure also encompasses other conjugates in which the insulin analogs disclosed herein are linked, optionally via covalent bonding and optionally via a linker, to a conjugate moiety.
  • Linkage can be accomplished by covalent chemical bonds, physical forces such electrostatic, hydrogen, ionic, van der Waals, or hydrophobic or hydrophilic interactions.
  • a variety of non-covalent coupling systems may be used, including biotin-avidin, ligand/receptor, enzyme/substrate, nucleic acid/nucleic acid binding protein, lipid/lipid binding protein, cellular adhesion molecule partners; or any binding partners or fragments thereof which have affinity for each other.
  • the peptide can be linked to conjugate moieties via direct covalent linkage by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of these targeted amino acids.
  • Reactive groups on the peptide or conjugate include, e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group.
  • Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride or other agents known in the art.
  • the conjugate moieties can be linked to the peptide indirectly through intermediate carriers, such as polysaccharide or polypeptide carriers.
  • polysaccharide carriers include aminodextran.
  • suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixed polymers of these amino acids and others, e.g., serines, to confer desirable solubility properties on the resultant loaded carrier. Cysteinyl residues most commonly are reacted with a-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives.
  • Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, alpha-bromo- -(5- imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2- pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2- chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa- 1 ,3-diazole.
  • Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Para- bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • Lysinyl and amino-terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O- methylisourea, 2,4-pentanedione, and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon- amino group.
  • tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane.
  • aromatic diazonium compounds or tetranitromethane Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • R and R' are different alkyl groups, such as l-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or l-ethyl-3-(4- azonia-4,4-dimethylpentyl) carbodiimide.
  • aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Other modifications include hydroxylation of proline and
  • Sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or
  • conjugate moieties that can be linked to any of the insulin analogs described herein include but are not limited to a heterologous peptide or polypeptide (including for example, a plasma protein), a targeting agent, an immunoglobulin or portion thereof (e.g. variable region, CDR, or Fc region), a diagnostic label such as a radioisotope, fluorophore or enzymatic label, a polymer including water soluble polymers, or other therapeutic or diagnostic agents.
  • a conjugate comprising an insulin analog disclosed herein and a plasma protein, wherein the plasma protein is selected from the group consisting of albumin, transferin, fibrinogen and globulins.
  • the linker comprises a chain of atoms from 1 to about 60, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms long.
  • the chain atoms are all carbon atoms.
  • the chain atoms in the backbone of the linker are selected from the group consisting of C, O, N, and S. Chain atoms and linkers may be selected according to their expected solubility (hydrophilicity) so as to provide a more soluble conjugate.
  • the linker provides a functional group that is subject to cleavage by an enzyme or other catalyst or hydro lytic conditions found in the target tissue or organ or cell.
  • the length of the linker is long enough to reduce the potential for steric hindrance.
  • the linker is a covalent bond or a peptidyl bond and the conjugate is a polypeptide
  • the entire conjugate can be a fusion protein.
  • peptidyl linkers may be any length. Exemplary linkers are from about 1 to 50 amino acids in length, 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids in length.
  • Such fusion proteins may alternatively be produced by recombinant genetic engineering methods known to one of ordinary skill in the art.
  • the single chain insulin prodrugs are conjugated, e.g., fused to an immunoglobulin or portion thereof (e.g. variable region, CDR, or Fc region).
  • immunoglobulins e.g. variable region, CDR, or Fc region.
  • immunoglobulins include IgG, IgA, IgE, IgD or IgM.
  • the Fc region is a C-terminal region of an Ig heavy chain, which is responsible for binding to Fc receptors that carry out activities such as recycling (which results in prolonged half-life), antibody dependent cell-mediated cytotoxicity (ADCC), and complement dependent cytotoxicity (CDC).
  • the human IgG heavy chain Fc region stretches from Cys226 to the C-terminus of the heavy chain.
  • the "hinge region” generally extends from Glu216 to Pro230 of human IgGl (hinge regions of other IgG isotypes may be aligned with the IgGl sequence by aligning the cysteines involved in cysteine bonding).
  • the Fc region of an IgG includes two constant domains, CH2 and CH3.
  • the CH2 domain of a human IgG Fc region usually extends from amino acids 231 to amino acid 341.
  • the CH3 domain of a human IgG Fc region usually extends from amino acids 342 to 447.
  • the Fc region may comprise one or more native or modified constant regions from an
  • immunoglobulin heavy chain other than CHI, for example, the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4 regions of IgE.
  • Suitable conjugate moieties include portions of immunoglobulin sequence that include the FcRn binding site.
  • FcRn a salvage receptor, is responsible for recycling immunoglobulins and returning them to circulation in blood.
  • the region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379).
  • the major contact area of the Fc with the FcRn is near the junction of the CH2 and CH3 domains.
  • Fc-FcRn contacts are all within a single Ig heavy chain.
  • the major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain.
  • FcyR are responsible for ADCC and CDC.
  • positions within the Fc region that make a direct contact with FcyR are amino acids 234-239 (lower hinge region), amino acids 265-269 (B/C loop), amino acids 297-299 (C'/E loop), and amino acids 327-332 (F/G) loop (Sondermann et al., Nature 406: 267-273, 2000).
  • the lower hinge region of IgE has also been implicated in the FcRI binding (Henry, et al., Biochemistry 36, 15568-15578, 1997).
  • Such variant Fc regions comprise at least one amino acid modification in the CH3 domain of the Fc region (residues 342-447) and/or at least one amino acid modification in the CH2 domain of the Fc region (residues 231-341).
  • Mutations believed to impart an increased affinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).
  • Other mutations may reduce binding of the Fc region to FcyRI, FcyRIIA, FcyRIIB, and/or FcyRIIIA without significantly reducing affinity for FcRn.
  • substitution of the Asn at position 297 of the Fc region with Ala or another amino acid removes a highly conserved N-glycosylation site and may result in reduced immunogenicity with concomitant prolonged half- life of the Fc region, as well as reduced binding to FcyRs (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68: 1632; Shields et al. 1995, J. Biol. Chem. 276:6591).
  • Amino acid modifications at positions 233-236 of IgGl have been made that reduce binding to FcyRs (Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613).
  • Some exemplary amino acid substitutions are described in US Patents 7,355,008 and 7,381,408, each incorporated by reference herein in its entirety.
  • the single chain insulin prodrugs disclosed herein are modified to comprise an acyl group or alkyl group.
  • Acylation or alkylation can increase the half-life of the insulin analogs in circulation.
  • Acylation or alkylation can advantageously delay the onset of action and/or extend the duration of action at the insulin and/or IGF-1 receptors and/or improve resistance to proteases such as DPP-IV and/or improve solubility.
  • Insulin analogs may be acylated or alkylated at the same amino acid position where a hydrophilic moiety is linked, or at a different amino acid position.
  • the invention provides an insulin analog modified to comprise an acyl group or alkyl group covalently linked to the amino acid at a position corresponding to A10, B28, B29 of native insulin, or at the C-terminus or N- terminus of the A or B chain.
  • the acylation or alkylation is at the N-terminal alpha amine of the insulin B chain.
  • the insulin analog may further comprise a spacer between the insulin analog amino acid and the acyl group or alkyl group.
  • the acyl group is a fatty acid or bile acid, or salt thereof, e.g.
  • the spacer is any moiety with suitable reactive groups for attaching acyl or alkyl groups.
  • the spacer comprises an amino acid, a dipeptide, or a tripeptide, or a hydrophilic bifunctional spacer.
  • the spacer is selected from the group consisting of: Gly, Trp, Glu, Asp, Cys and a spacer comprising
  • the acylation or alkylation is at the N- terminal alpha amine of the insulin B chain and a spacer of 1, 2, 3, 4, 5 or 6 amino acids (e.g. Gly, where acylation occurs at the N-terminal alpha amine of the spacer amino acids) that links the acyl or alkyl group to the N-terminus.
  • the spacer of 1, 2, 3, 4, 5 or 6 amino acids can be a homopolymer of a single amino acid (e.g., Gly) or a mixture of any of the 20 amino acids.
  • Such acylated or alkylated insulin peptides may also further comprise a hydrophilic moiety, optionally a polyethylene glycol.
  • a hydrophilic moiety optionally a polyethylene glycol.
  • Any of the foregoing insulin analogs may comprise two acyl groups or two alkyl groups, or a combination thereof.
  • Acylation can be carried out at any positions within the single chain insulin prodrug, provided that insulin agonist activity is retained.
  • the acyl group can be covalently linked directly to an amino acid of the insulin analog, or indirectly to an amino acid of the insulin analog via a spacer, wherein the spacer is positioned between the amino acid of the insulin peptide and the acyl group.
  • the insulin analog is modified to comprise an acyl group by direct acylation of an amine, hydroxyl, or thiol of a side chain of an amino acid of the insulin peptide.
  • the insulin analog is directly acylated through the side chain amine, hydroxyl, or thiol of an amino acid.
  • acylation is at a position corresponding to A10, B28, B29 of native insulin, or at the C-terminus or N-terminus of the A or B chain.
  • the amino acid to be acylated is an amino acid of Formula
  • the amino acid of Formula IV is the amino acid wherein n is 4 (Lys) or n is 3 (Orn).
  • the amino acid comprising a side chain hydroxyl is an amino acid of Formula V:
  • the amino acid of Formula V is the amino acid wherein n is 1 (Ser).
  • the amino acid comprising a side chain thiol is an amino acid of Formula VI:
  • the amino acid of Formula VI is the amino acid wherein n is 1 (Cys).
  • the insulin analog is modified to comprise an acyl group by acylation of an amine, hydroxyl, or thiol of a spacer, which spacer is attached to a side chain of an amino acid at position A10, B28 or B29 (according to the amino acid numbering of wild type insulin).
  • the acylation can occur through the alpha amine of the amino acid or a side chain amine.
  • the spacer amino acid can be any amino acid.
  • the spacer amino acid can be a hydrophobic amino acid, e.g., Gly, Ala, Val, Leu, He, Trp, Met, Phe, Tyr.
  • the spacer amino acid can be an acidic residue, e.g., Asp and Glu.
  • the spacer amino acid is an amino acid comprising a side chain amine, e.g., an amino acid of Formula I (e.g., Lys or Orn).
  • an amino acid of Formula I e.g., Lys or Orn
  • both the alpha amine and the side chain amine of the spacer amino acid to be acylated, such that the insulin peptide is diacylated.
  • the present disclosure further contemplates diacylated insulin analogs.
  • the amino acid or one of the amino acids of the dipeptide or tripeptide can be an amino acid of Formula II.
  • the amino acid is Ser.
  • the amino acid or one of the amino acids of the dipeptide or tripeptide can be an amino acid of Formula III.
  • the amino acid is Cys.
  • the spacer comprises a hydrophilic bifunctional spacer.
  • the spacer comprises an amino poly(alkyloxy)carboxylate.
  • the spacer can comprise, for example, NH 2 (CH 2 CH 2 0) n (CH 2 ) m COOH, wherein m is any integer from 1 to 6 and n is any integer from 2 to 12, such as, e.g., 8- amino-3,6-dioxaoctanoic acid, which is commercially available from Peptides International, Inc. (Louisville, KY).
  • the acyl group of the acylated insulin peptide can be of any size, e.g., any length carbon chain, and can be linear or branched.
  • the acyl group is a C4 to C30 fatty acid or a C4 to C30 diacid.
  • the acyl group can be any of a C4 fatty acid, C6 fatty acid, C8 fatty acid, CIO fatty acid, C12 fatty acid, C14 fatty acid, C16 fatty acid, C18 fatty acid, C20 fatty acid, C22 fatty acid, C24 fatty acid, C26 fatty acid, C28 fatty acid, or a C30 fatty acid.
  • the acyl group is a C8 to C20 fatty acid, e.g., a C14 fatty acid or a C16 fatty acid. In one embodiment, the acyl group is a C8 to C20 diacid, e.g., a C14 diacid or a C 16 diacid.
  • the acyl group is a bile acid.
  • the bile acid can be any suitable bile acid, including, but not limited to, cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, and cholesterol acid.
  • the insulin analog comprises a cholesterol acid, which is linked to a Lys residue of the insulin analog through an alkylated des-amino Cys spacer, i.e., an alkylated 3-mercaptopropionic acid spacer.
  • the alkylated des- amino Cys spacer can be, for example, a des-amino-Cys spacer comprising a dodecaethylene glycol moiety.
  • the insulin analog comprises the structure:
  • the acylated insulin analogs described herein can be further modified to comprise a hydrophilic moiety.
  • the hydrophilic moiety can comprise a polyethylene glycol (PEG) chain.
  • PEG polyethylene glycol
  • the incorporation of a hydrophilic moiety can be accomplished through any suitable means, such as any of the methods described herein.
  • the acylated insulin peptide can comprise a spacer, wherein the spacer is both acylated and modified to comprise the hydrophilic moiety.
  • suitable spacers include a spacer comprising one or more amino acids selected from the group consisting of Cys, Lys, Orn, homo-Cys, and Ac-Phe.
  • the insulin analog is modified to comprise an alkyl group which is attached to the insulin analog via an ester, ether, thioether, amide, or alkyl amine linkage for purposes of prolonging half-life in circulation and/or delaying the onset of and/or extending the duration of action and/or improving resistance to proteases such as DPP-IV.
  • the alkyl group of the alkylated insulin peptide can be of any size, e.g., any length carbon chain, and can be linear or branched.
  • the alkyl group is a CI to C30 alkyl.
  • the alkyl group can be any of a CI alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C6 alkyl, C8 alkyl, CIO alkyl, C12 alkyl, C14 alkyl, C16 alkyl, C18 alkyl, C20 alkyl, C22 alkyl, C24 alkyl, C26 alkyl, C28 alkyl, or a C30 alkyl.
  • the alkyl group is a C8 to C20 alkyl, e.g., a C14 alkyl or a C 16 alkyl.
  • the alkyl group comprises a steroid moiety of a bile acid, e.g., cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, and cholesterol acid.
  • a bile acid e.g., cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, and cholesterol acid.
  • a pharmaceutical composition comprising any of the novel single chain insulin prodrugs disclosed herein, preferably at a purity level of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and a pharmaceutically acceptable diluent, carrier or excipient.
  • compositions may contain an A19 insulin analog as disclosed herein at a concentration of at least 0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml, 20 mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 24 mg/ml, 25 mg/ml or higher.
  • the pharmaceutical compositions comprise aqueous solutions that are sterilized and optionally stored contained within various package containers. In other embodiments the pharmaceutical compositions comprise a lyophilized powder.
  • the pharmaceutical compositions can be further packaged as part of a kit that includes a disposable device for administering the composition to a patient.
  • the single chain insulin prodrug is stored in an aqueous solution at a pH of 6 or less.
  • the containers or kits may be labeled for storage at ambient room temperature or at refrigerated temperature.
  • the disclosed single chain insulin prodrugs are believed to be suitable for any use that has previously been described for insulin peptides. Accordingly, the single chain insulin prodrugs described herein can be used to treat hyperglycemia, or treat other metabolic diseases that result from high blood glucose levels. Accordingly, the present invention encompasses pharmaceutical compositions comprising a single chain insulin prodrug of the present disclosure, and a pharmaceutically acceptable carrier for use in treating a patient suffering from high blood glucose levels. In accordance with one embodiment the patient to be treated using the single chain insulin prodrugs disclosed herein is a domesticated animal, and in another
  • the patient to be treated is a human.
  • One method of treating hyperglycemia in accordance with the present disclosure comprises the steps of administering the presently disclosed single chain insulin prodrug to a patient using any standard route of administration, including parenterally, such as intravenously, intraperitoneally, subcutaneously or
  • composition is administered subcutaneously or
  • composition is administered parenterally and the single chain insulin prodrug composition is prepackaged in a syringe.
  • the single chain insulin prodrugs of the invention may be administered alone or in combination with other anti-diabetic agents.
  • Anti-diabetic agents known in the art or under investigation include native insulin, native glucagon and functional derivatives thereof, sulfonylureas, such as tolbutamide (Orinase), acetohexamide (Dymelor), tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide (Glucotrol), glyburide (Diabeta, Micronase, Glynase), glimepiride (Amaryl), or gliclazide
  • native insulin native glucagon and functional derivatives thereof
  • sulfonylureas such as tolbutamide (Orinase), acetohexamide (Dymelor), tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide (Glucotrol), glyburide (Diabet
  • Diamicron meglitinides, such as repaglinide (Prandin) or nateglinide (Starlix); biguanides such as metformin (Glucophage) or phenformin; thiazolidinediones such as rosiglitazone (Avandia), pioglitazone (Actos), or troglitazone (Rezulin), or other PPARy inhibitors; alpha glucosidase inhibitors that inhibit carbohydrate digestion, such as miglitol (Glyset), acarbose (Precose/Glucobay); exenatide (Byetta) or pramlintide; Dipeptidyl peptidase-4 (DPP-4) inhibitors such as vildagliptin or sitagliptin; SGLT (sodium-dependent glucose transporter 1) inhibitors; or FBPase (fructose 1,6-bisphosphatase) inhibitors.
  • DPP-4 Dipeptid
  • compositions comprising the single chain insulin prodrugs disclosed herein can be formulated and administered to patients using standard pharmaceutically acceptable carriers and routes of administration known to those skilled in the art. Accordingly, the present disclosure also encompasses
  • compositions comprising one or more of the single chain insulin prodrugs disclosed herein, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises a lmg/ml concentration of the single chain insulin prodrug at pH of about 4.0 to about 6.0 or about 4.0 to about 7.0 in a phosphate buffer system.
  • the pharmaceutical compositions may comprise the single chain insulin prodrug as the sole pharmaceutically active component, or the single chain insulin prodrug can be combined with one or more additional active agents.
  • a pharmaceutical composition comprising one of the single chain insulin prodrugs disclosed herein, preferably sterile and preferably at a purity level of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and a pharmaceutically acceptable diluent, carrier or excipient.
  • compositions may contain an single chain insulin prodrug wherein the resulting active peptide is present at a concentration of at least 0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml, 20 mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 24 mg/ml, 25 mg/ml or higher.
  • the pharmaceutical compositions comprise aqueous solutions that are sterilized and optionally stored within various containers.
  • the compounds of the present invention can be used in accordance with one embodiment to prepare pre- formulated solutions ready for injection.
  • the pharmaceutical compositions comprise a lyophilized powder.
  • the pharmaceutical compositions can be further packaged as part of a kit that includes a disposable device for administering the composition to a patient.
  • the containers or kits may be labeled for storage at ambient room temperature or at refrigerated temperature.
  • the kit is provided with a device for administering the single chain insulin prodrug composition to a patient.
  • the kit may further include a variety of containers, e.g., vials, tubes, bottles, and the like.
  • the kits will also include instructions for use.
  • the device of the kit is an aerosol dispensing device, wherein the composition is prepackaged within the aerosol device.
  • the kit comprises a syringe and a needle, and in one embodiment the insulin analog composition is prepackaged within the syringe.
  • the compounds of this invention may be prepared by standard synthetic methods, recombinant DNA techniques, or any other methods of preparing peptides and fusion proteins. Although certain non-natural amino acids cannot be expressed by standard recombinant DNA techniques, techniques for their preparation are known in the art. Compounds of this invention that encompass non-peptide portions may be synthesized by standard organic chemistry reactions, in addition to standard peptide chemistry reactions when applicable.
  • any of the insulin A or B chains disclosed herein or any known insulin agonist analogs can be linked to form a single chain insulin prodrug as disclosed herein using any of the dipeptide linkers as described herein.
  • a single chain insulin analog comprising the structure B-U-O-A, wherein
  • A is an insulin A chain
  • B is an insulin B chain
  • U-0 is a dipeptide that links the carboxy terminus of said B chain to the amino terminus of the A chain, wherein
  • said A chain comprises the sequence
  • X 4 is glutamic acid or aspartic acid
  • X5 is glutamine or glutamic acid
  • X 8 is histidine, threonine or phenylalanine
  • X9 is serine, arginine, lysine, ornithine or alanine
  • X10 is isoleucine or serine
  • Xi 2 is serine or aspartic acid
  • Xi 4 is tyrosine, arginine, lysine, ornithine or alanine;
  • Xi 5 is glutamine, glutamic acid, arginine, alanine, lysine, ornithine or leucine;
  • Xi 7 is glutamine, glutamic acid, arginine, aspartic acid, ornithine or lysine;
  • X 18 is methionine, asparagine, glutamine, aspartic acid, glutamic acid or threonine;
  • Xi 9 is tyrosine, 4-methoxy-phenylalanine or 4-amino phenylalanine;
  • X21 is selected from the group consisting of alanine, glycine, serine, valine, threonine, isoleucine, leucine, glutamine, glutamic acid, asparagine, aspartic acid, histidine, tryptophan, tyrosine, and methionine;
  • X25 is histidine or threonine
  • X29 is selected from the group consisting of alanine, glycine and serine;
  • X30 is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid;
  • X33 is selected from the group consisting of aspartic acid, glutamine and glutamic acid;
  • X 34 is selected from the group consisting of alanine and threonine
  • X 4 i is selected from the group consisting of glutamic acid, aspartic acid or asparagine;
  • X 4 2 is selected from the group consisting of alanine, lysine, ornithine and arginine;
  • X 4 5 is tyrosine or phenylalanine
  • R 22 is selected from the group consisting of FVNQ (SEQ ID NO: 12), FVKQ (SEQ ID NO: 11), a tripeptide glycine-proline-glutamic acid, a tripeptide valine- asparagine-glutamine, a dipeptide proline-glutamic acid, a dipeptide asparagine- glutamine, glutamine, glutamic acid and a bond; and
  • R 23 is selected from the group consisting of YTX 28 KT (SEQ ID NO: 20), YTKPT (SEQ ID NO: 23), YTX 28 K (SEQ ID NO: 24), YTKP (SEQ ID NO: 29), YTPK (SEQ ID NO: 32), YTX 28 , YT, Y and a bond;
  • X2 8 is proline, aspartic acid or glutamic acid
  • Ri 3 is COOH or CONH 2 .
  • U is an amino acid or a hydroxyl acid and O is an N-alkylated amino acid
  • O is linked to the N-terminus of the A chain through formation of an amide bond
  • U is linked to the carboxy terminus of the B chain via the amino acid side chain of U
  • the chemical cleavage half- life (ti/2) of O from the A chain is at least about 1 hour to about 1 week in PBS under physiological conditions.
  • U- O comprises the general structure of Formula II:
  • X is NH, S or O
  • R 2 is H or Ci-C 4 alkyl
  • R 3 is Ci-C 6 alkyl
  • R 4 is H, Ci-C 6 alkyl, (C1-C4 alkyl)NH 2 , CH 2 (C 6 aryl)R 7 , or R 3 and R 4 together with the atoms to which they are attached form a pyrrolidine ring;
  • R 5 is NH 2 or OH
  • n is an integer selected from 1 to 4.
  • the insulin analog of embodiment 2 is provided wherein R 2 is Ci-C 4 alkyl;
  • R 3 and R 4 together with the atoms to which they are attached form a pyrrolidine ring
  • R 5 is NH 2 .
  • R 3 is C1-C4 alkyl
  • R 4 is H
  • R 5 is NH 2 .
  • an insulin analog of any one of claims 2-5 is provided wherein X is NH.
  • an insulin analog of any one of claims 1-6 is provided further comprising an acyl or alkyl group covalently linked to the insulin analog, wherein said acyl or alkyl group comprises 16 to 30 carbon atoms.
  • an insulin analog of embodiment 7 is provided wherein the acyl group is a C18 to C28 fatty acid.
  • an insulin analog of claim 7 or 8 is provided wherein said insulin analog is acylated at one or more positions selected from Al, A9, A14, A15, B 1, B22, B28 or B29, optionally through a spacer.
  • anvinsulin analog of embodiment 9 wherein the spacer is one to six amino acids in length and the acyl group is linked via a gamma glutamic acid of the linker.
  • an insulin analog of any one of embodiments 1-10 is provided wherein said A chain comprises a sequence of
  • GIVEQCCXgSICSLYQLENYCXii (SEQ ID NO: 43) and the B chain sequence comprises the sequence FVX 23 QX 25 LCGX 2 9X 30 LVEALYLVCGERGFF-R 23 (SEQ ID NO: 33), wherein
  • X 8 is selected from the group consisting of threonine and histidine;
  • X 21 is selected from the group consisting of asparagine, glycine and alanine;
  • X 23 is asparagine or lysine
  • X 25 is selected from the group consisting of histidine and threonine
  • X 2 9 is selected from the group consisting of alanine, glycine and serine;
  • X30 is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid;
  • R 23 is selected from the group consisting of YTX 28 KT (SEQ ID NO: 20), YTKPT (SEQ ID NO: 23), YTX 28 K (SEQ ID NO: 21), YTKP (SEQ ID NO: 29), YTPK (SEQ ID NO: 32), YTX 28 , YT, Y and a bond.
  • an insulin analog of any of embodiments 1 to 11 is provided wherein X 8 is threonine and X 21 is asparagine or glycine.
  • an insulin analog of any one of embodiments 1-12 is provided wherein the B chain sequence comprises the sequence
  • X 25 is selected from the group consisting of histidine and threonine.
  • R 23 is selected from the group consisting of YTX 28 KT (SEQ ID NO: 20), YTKPT (SEQ ID NO: 23), YTX 28 K (SEQ ID NO: 21), YTKP (SEQ ID NO: 29), YTPK (SEQ ID NO: 32), YTX 28 , YT, Y and a bond
  • YTX 28 KT SEQ ID NO: 20
  • YTKPT SEQ ID NO: 23
  • YTX 28 K SEQ ID NO: 21
  • YTKP SEQ ID NO: 29
  • YTPK SEQ ID NO: 32
  • an insulin analog of any one of embodiments 1-13 is provided
  • B chain sequence comprises the sequence
  • FVNQX 2 5LCGS HLVE ALYLVC GERGFF YTPKT (SEQ ID NO: 19) wherein X 25 is selected from the group consisting of histidine and threonine.
  • hydrophilic moiety is linked to the insulin analog.
  • an insulin analog of claim 15 wherein the hydrophilic moiety is polyethylene glycol.
  • an insulin analog of any one of embodiments 1-16 wherein a hydrophilic moiety is linked to the insulin analog at the N- terminal alpha amine of the B chain, or at the side chain of a lysine located after position 27 relative to native insulin.
  • an insulin analog of any one of embodiments 1-17 is provided wherein the single chain insulin analog comprises the structure B-U-O-A, wherein
  • A is an insulin A chain
  • B is an insulin B chain
  • U-0 is a dipeptide that links the carboxy terminus of said B chain to the amino terminus of the A chain, wherein
  • said A chain comprises the sequence GIVEQCCX 8 SICSLYQLENYCX 2 i (SEQ ID NO: 43);
  • said B chain comprises the sequence the sequence
  • X 8 is selected from the group consisting of threonine and histidine;
  • X 21 is selected from the group consisting of asparagine, glycine and alanine; X25 is selected from the group consisting of histidine and threonine; and
  • X is NH
  • R 2 is H or Ci-C 4 alkyl
  • R 3 is Ci-C 6 alkyl
  • R 4 is H, or Ci-C 6 alkyl
  • R 5 is NH 2 ;
  • an insulin analog of any one of embodiments 1-18 wherein the insulin analog is a dimer or multimer comprising an insulin analog of any of embodiments 1 to 18.
  • a pharmaceutical composition comprising an insulin analog of any one of embodiments 1-19 and a pharmaceutically acceptable carrier is provided
  • an insulin analog of any one of embodiments 1-20 is provided for treating diabetes, optionally wherein said method comprises
  • an insulin analog of any one of embodiments 1-18 or a dimer or multimer of embodiment 19, is used in the manufacture of a medicament for the treatment of hyperglycemia.
  • a compound any of claims 1 to 18 or a dimer or multimer of embodiment 19, is used to treat diabetes.
  • Insulin A & B chains were synthesized on 4-methylbenzhyryl amine (MB HA) resin or 4-Hydroxymethyl-phenylacetamidomethyl (PAM) resin using Boc chemistry.
  • the peptides were cleaved from the resin using HF/p-cresol 95:5 for 1 hour at 0°C. Following HF removal and ether precipitation, peptides were dissolved into 50% aqueous acetic acid and lyophilized. Alternatively, peptides were synthesized using Fmoc chemistry.
  • the peptides were cleaved from the resin using Trifluoroacetic acid (TFA)/ Triisopropylsilane (TIS)/ H 2 0 (95:2.5:2.5), for 2 hour at room temperature.
  • TSA Trifluoroacetic acid
  • TIS Triisopropylsilane
  • H 2 0 95:2.5:2.5
  • the peptide was precipitated through the addition of an excessive amount of diethyl ether and the pellet solubilized in aqueous acidic buffer.
  • the quality of peptides were monitored by RP-HPLC and confirmed by Mass Spectrometry (ESI or MALDI).
  • Insulin A chains were synthesized with a single free cysteine at amino acid 7 and all other cysteines protected as acetamidomethyl A-(SH) 7 (Acm) 6 ' U ' 20 .
  • Insulin B chains were synthesized with a single free cysteine at position 7 and the other cysteine protected as acetamidomethyl B-(SH) 7 (Acm) 19.
  • the crude peptides were purified by conventional RP-HPLC.
  • the synthesized A and B chains were linked to one another through their native disulfide bond linkage in accordance with the general procedure outlined in Fig. 1.
  • the respective B chain was activated to the Cys -Npys derivative through dissolution in DMF or DMSO and reacted with 2,2'-Dithiobis (5-nitropyridine) (Npys) at a 1: 1 molar ratio, at room temperature.
  • the activation was monitored by RP-HPLC and the product was confirmed by ESI-MS.
  • the first B7-A7 disulfide bond was formed by dissolution of the respective A-
  • Insulin peptides comprising a modified amino acid can also be synthesized in vivo using a system that allows for incorporation of non-coded amino acids into proteins, including for example, the system taught in US Patent Nos. 7,045,337 and 7,083,970.
  • Insulin or an insulin analog
  • mPEG20k-Aldyhyde or an insulin analog
  • NaBH 3 CN in a molar ratio of 1:2:30, were dissolved in acetic acid buffer at a pH of 4.1-4.4.
  • the reaction solution was composed of 0.1 N NaCl, 0.2 N acetic acid and 0.1 N Na 2 C0 .
  • the insulin peptide concentration was approximately 0.5 mg/ml.
  • the reaction occurs over six hours at room temperature. The degree of reaction was monitored by RP-HPLC and the yield of the reaction was approximately 50%.
  • the reaction mixture was diluted 2-5 fold with 0.1% TFA and applied to a preparative RP-HPLC column.
  • HPLC condition C4 column; flow rate 10 ml/min; A buffer 10% ACN and 0.1% TFA in water; B buffer 0.1% TFA in ACN; A linear gradient B% from 0-40% (0-80 min); PEG-insulin or analogues was eluted at approximately 35% buffer B.
  • the desired compounds were verified by MALDI-TOF, following chemical modification through sulftolysis or trypsin degradation.
  • reaction mixture was diluted 2-5 fold and loaded to RP-HPLC.
  • HPLC condition C4 column; flow rate 10 ml/min; A buffer 10% ACN and 0.1% TFA in water; B buffer 0.1% TFA in ACN; A linear gradient B% from 0-40% (0-80 min); PEG-insulin or analogues was collected at approximately 35% B. .
  • the desired compounds were verified by MAIDI-TOF, following chemical modification through sulftolysis or trypsin degradation.
  • Insulin (or an insulin analogue), mPEG20k-Hydrazide, and NaBH 3 CN in a molar ratio of 1:2:20 were dissolved in acetic acid buffer (pH of 4.1 to 4.4).
  • the reaction solution was composed of 0.1 N NaCl, 0.2 N acetic acid and 0.1 N Na 2 C0 3 .
  • Insulin or insulin analogue concentration was approximately 0.5 mg/ml. at room temperature for 24h.
  • the reaction process was monitored by HPLC. The conversion of the reaction was approximately 50%. (calculated by HPLC)
  • reaction mixture was diluted 2-5 fold and loaded to RP-HPLC.
  • HPLC condition C4 column; flow rate 10 ml/min; A buffer 10% ACN and 0.1% TFA in water; B buffer 0.1% TFA in ACN; A linear gradient B% from 0-40% (0-80 min); PEG-insulin, or the PEG-insulin analogue was collected at approximately 35%B. .
  • the desired compounds were verified by MAIDI-TOF, following chemical modification through sulftolysis or trypsin degradation.
  • each peptide for the insulin or IGF-1 receptor was measured in a competition binding assay utilizing scintillation proximity technology.
  • Serial 3-fold dilutions of the peptides were made in Tris-Cl buffer (0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.1% w/v bovine serum albumin) and mixed in 96 well plates (Corning Inc., Acton, MA) with 0.05 nM (3-[125I]-iodotyrosyl) A TyrA14 insulin or (3-[125I]- iodotyrosyl) IGF-1 (Amersham Biosciences, Piscataway, NJ).
  • % Specific Binding (Bound-NSB / Total bound-NSB) x 100. IC50 values were determined by using Origin software (OriginLab, Northampton, MA).
  • receptor transfected HEK293 cells were plated in 96 well tissue culture plates (Costar #3596, Cambridge, MA) and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 100 IU/ml penicillin, 100 g/ml streptomycin, 10 mM HEPES and 0.25% bovine growth serum (HyClone SH30541, Logan, UT) for 16-20 hrs at 37 °C, 5% C0 2 and 90% humidity. Serial dilutions of insulin or insulin analogs were prepared in DMEM supplemented with 0.5% bovine serum albumin (Roche Applied Science #100350, Indianapolis, IN) and added to the wells with adhered cells.
  • DMEM Dulbecco's modified Eagle medium
  • TMB single solution substrate (Invitrogen, #00- 2023, Carlbad, CA) was added to each well. Color development was stopped 5 min later by adding 0.05 ml 1 N HC1. Absorbance at 450 nm was measured on Titertek Multiscan MCC340 (ThermoFisher, Pittsburgh, PA). Absorbance vs. peptide concentration dose response curves were plotted and EC 50 values were determined by using Origin software (OriginLab, Northampton, MA).
  • HSRGTF-NH 2 A specific hexapeptide (HSRGTF-NH 2 ; SEQ ID NO: 36) was used as a model peptide upon which the rate of cleavage of dipeptide N-terminal extensions could be studied.
  • the dipeptide-extended model peptides were prepared Boc-protected sarcosine and lysine were successively added to the model peptide-bound resin to produce peptide A (Lys-Sar-HSRGTF-NH 2 ; SEQ ID NO: 37).
  • Peptide A was cleaved by HF and purified by preparative HPLC.
  • the rate of cleavage was determined for the respective propeptides.
  • the concentrations of the propeptides and the model parent peptide were determined by their respective peak areas.
  • the first order dissociation rate constants of the prodrugs were determined by plotting the logarithm of the concentration of the prodrug at various time intervals. The slope of this plot provides the rate constant 'k' .
  • the half life of the Lys-Sar extension to this model peptide HSRGTF-NH 2 (SEQ ID NO: 36) was determined to be 14.0h.
  • the rate of cleavage was determined for the respective propeptides.
  • the concentrations of the propeptides and the model parent peptide were determined by their respective peak areas.
  • the first order dissociation rate constants of the prodrugs were determined by plotting the logarithm of the concentration of the prodrug at various time intervals. The slope of this plot provides the rate constant 'k' .
  • the half life of the Lys-Sar extension to this model peptide dHdTdRGdTdF-NH 2 (SEQ ID NO: 38) was determined to be 18.6h.
  • Table 2 Cleavage of the Dipeptides O-U that are linked to the side chain of an N- terminal para-amino-Phe from the Model Hexapeptide (HSRGTF-NH 2 ; SEQ ID NO: 36) in PBS
  • Insulin A-chain was assembled on Rink amide-chemmatrix (0.15 mmol, 0.47 mmol/g) with Symphony peptide synthesizer through Fmoc-SPPS. Fmoc-Aspi /Bu)- OH was used as the first amino acid residue.
  • the resulting resin was ready for B- chain assembly after removal of the Fmoc group with 20 % piperidine/DMF. Small amount of resin was cleaved with 90 % TFA, 2.5 % TIS, 2.5 % H 2 0, 5 % DODT. After cold ether precipitation, the residue was taken up into 50 mM aqueous NaHC0 3 /ACN (1: 1) and analyzed with LC-MS.
  • the resin was treated with 90 % TFA, 2.5 % TIS, 2.5 % H 2 0, 5 % DODT. After cold ether precipitation, the precipitate was collected by centrifugation and dried in vacuum. An aliquot was taken up into 50 mM aqueous NaHC0 3 and treated with NaOH solution and a little bit ACN to get clear solution. The crude product was characterized by RP-HPLC and mass spectrometry (negative Mass).
  • the crude peptide was taken up into 20 mM glycine solution (200 mL) followed by Cysteine HQ (0.75 mmol). The pH of reaction mixture was adjusted to 10.5 with 1 N NaOH. Sonication was used to accelerate the dissolving process. Then the resulting solution was stirred vigorously in cold room for 2 d. To the reaction mixture was added 20 mL of ACN. The pH was lowered to 7.0. The resulting solution was purified with prep HPLC (Kinetex® 5 ⁇ C8 100 A LC Column 250 X 21.2 mm, 10-25 % B over 60 min) and lyophilized.
  • the peptide was treated with TFA/thioanisole (6/1) for 3 h at room
  • the single chain insulin was synthesized follow the procedure described before. Only difference was that Boc-Lys(Fmoc)-OH was used during construction of dipeptide. After cleavage, the crude peptide was taken up into 50 mM NaHC0 3 solution and pH was adjusted to 8.0. To the mixture was added 3,4,5,6- tetrahydrophthalic anhydride (3 mmol). The resulting mixture was sonicated and monitored with LC-MS.
  • the THP-protected single insulin was diluted with 20 mM glycine solution, and then Cysteine HQ (0.75 mmol) was added. The pH of reaction mixture was adjusted to 10.5 with 1 N NaOH. The resulting solution was stirred vigorously in cold room for 2 d.
  • mice Single administration of six- to eight-week-old male C57BL/6 mice were maintained at 23'C, constant humidity, and a 12-h light— dark cycle.
  • the acute in vivo effects of select peptides were evaluated by subcutaneously injecting peptides solubilized in physiologically buffered saline or a vehicle control to normal or diabetic mice. Blood glucose was measured at various time points through the course of a 24hr period following administration of the peptides. Each group of mice contained 8 animals per group. The average body weight was 25 g. Mice were made diabetic by administration of streptozotocin.

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Abstract

L'invention concerne des formulations de promédicaments d'insuline et d'analogues d'insuline, dans lesquelles le peptide d'insuline a été modifié par liaison de l'extrémité carboxy de la chaîne B à l'extrémité N de la chaîne A par l'intermédiaire d'un dipeptide métastable pour former un analogue monocaténaire inactif. Les promédicaments décrits dans la description de l'invention présentent des demi-vies prolongées d'au moins 10 heures, et plus généralement supérieures à 24 heures et sont convertis en une forme bicaténaire active dans des conditions physiologiques par une réaction non enzymatique stimulée par une instabilité chimique.
PCT/US2017/034444 2016-06-02 2017-05-25 Promédicaments à base d'insuline monocaténaire Ceased WO2017210077A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112789290A (zh) * 2018-08-08 2021-05-11 株式会社大熊制药 使用梭菌蛋白酶制备长效胰岛素类似物缀合物的活性形式的方法
CN113166222A (zh) * 2018-08-08 2021-07-23 株式会社大熊制药 长效胰岛素类似物及其复合物

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030104981A1 (en) * 1995-11-03 2003-06-05 Jelena Mandic Human insulin analogues
US20090304814A1 (en) * 2006-01-06 2009-12-10 Case Western Reserve University Fibrillation resistant proteins
US20110195896A1 (en) * 2008-04-22 2011-08-11 Case Western Reserve University Isoform-specific insulin analogues
US20130130967A1 (en) * 2010-07-02 2013-05-23 University Of Southern California Method for Uses of Protein Precursors as Prodrugs
US20150274802A1 (en) * 2012-09-26 2015-10-01 Indiana University Research And Technology Corporation Insulin analog dimers

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030104981A1 (en) * 1995-11-03 2003-06-05 Jelena Mandic Human insulin analogues
US20090304814A1 (en) * 2006-01-06 2009-12-10 Case Western Reserve University Fibrillation resistant proteins
US20110195896A1 (en) * 2008-04-22 2011-08-11 Case Western Reserve University Isoform-specific insulin analogues
US20130130967A1 (en) * 2010-07-02 2013-05-23 University Of Southern California Method for Uses of Protein Precursors as Prodrugs
US20150274802A1 (en) * 2012-09-26 2015-10-01 Indiana University Research And Technology Corporation Insulin analog dimers

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112789290A (zh) * 2018-08-08 2021-05-11 株式会社大熊制药 使用梭菌蛋白酶制备长效胰岛素类似物缀合物的活性形式的方法
CN113166222A (zh) * 2018-08-08 2021-07-23 株式会社大熊制药 长效胰岛素类似物及其复合物
CN113166222B (zh) * 2018-08-08 2024-04-02 株式会社大熊制药 长效胰岛素类似物及其复合物
CN112789290B (zh) * 2018-08-08 2024-05-17 株式会社大熊制药 使用梭菌蛋白酶制备长效胰岛素类似物缀合物的活性形式的方法

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