Case Western Reserve University

Thermalin, Inc.

Thermalin Diabetes, Inc.

Michael A Weiss

Bruce H Frank

Thomas Hattier

Gregory Dubé

Richard William Berenson

Zhiqiang Chen

Jeffrey I Joseph

Jonathan Pokorski

A two-chain insulin analogue contains a modified A-chain polypeptide and a modified B-chain polypeptide. The A-chain polypeptide comprises one or more of: a His or Glu substitution at position A8, a Glu substitution at position A14; and a Gln or Arg substitution at position A17. The B-chain polypeptide comprises one or more of: a deletion of the amino acids at position B1, B1-B2, B1-B3, B30 or a combination thereof; an Ala or Glu substitution at position B2; a Glu substitution at position B3. The analogue exhibits thermodynamic stability in a zinc-free solution, decreased self-association, maintains biological potency, and no increased mitogenicity. The analogue exhibits resistance to chemical degradation and physical degradation. A method of treating a patient with diabetes mellitus or obesity comprises administering a physiologically effective amount of the insulin analogue or a physiologically acceptable salt thereof to a patient.

Rapid-acting insulin analogues of enhanced stability

An insulin composition comprises an insulin analogue and polymer blend. The insulin analogue contains cysteine substitutions at positions B4 and A10 (to form cystine B4-A10), and one or more additional substitutions selected from the group consisting of: a connecting domain of 5-11 amino acids between insulin A- and B domains; a non-beta-branched amino-acid substitution at position A8; a non-beta-branched acidic or polar side chain at position A14; a halogenic modification of PheB24 at the ortho position; and substitution of lysine at position B29 by Glu, Ala, Val, Ile, Leu, amino-propionic acid, amino-butryic acid, or Norleucine. The insulin analogue is compatible with a process of manufacture that includes one or more steps within the temperature range 90-120&#xb0; C. The encapsulated insulin analogue may optionally contain free PEG or be PEGylated. The insulin analogue-encapsulated polymer blend may be cast as a microneedle patch for topical administration or as micropellets for subcutaneous injection.

Encapsulation of ultra-stable insulin analogues with polymer melts

A two-chain insulin analogue contains a modified A-chain polypeptide and a modified B-chain polypeptide. The A-chain polypeptide comprises one or more of: a His or Glu substitution at position A8, a Glu substitution at position A14; and a Gln or Arg substitution at positon A17. The B-chain polypeptide comprises one or more of: a deletion of the amino acids at position B1, B1-B2, B1-B3, B30 or a combination thereof; an Ala or Glu substitution at position B2; a Glu substitution at position B3. The analogue exhibits thermodynamic stability in a zinc-free solution, decreased self-association, maintains biological potency, and no increased mitogenicity. The analogue exhibits resistance to chemical degradation and physical degradation. A method of treating a patient with diabetes mellitus or obesity comprises administering a physiologically effective amount of the insulin analogue or a physiologically acceptable salt thereof to a patient.

A single-chain insulin analogue comprises a B-chain insulin polypeptide connected to an A-chain insulin polypeptide by a C-domain polypeptide. The B-chain insulin polypeptide contains a Cysteine substitution at position B4. The A-chain insulin polypeptide contains a Cysteine substitution at position A10. The C-domain polypeptide is 4 to 11 amino acids long. The analogue mitigates the unfavorable activity of this 4th disulfide bridge in conventional two-chain insulin analogues resulting in a duration of insulin signaling similar to that of wild-type insulin. A method of treating a patient with diabetes mellitus comprises the administration of a physiologically effective amount of the protein or a physiologically acceptable salt thereof to a patient. Use of a single-chain insulin analogue of the present invention in an insulin delivery device (such as a pump or pen) or as part of a high-temperature polymer-melt manufacturing process.

Single-chain insulin analogues stabilized by a fourth disulfide bridge

A single-chain insulin comprises a C-domain of 6 to 11 amino acid residues comprising at least two acidic residues at the N-terminal side of the C-domain and at least two basic residues at the C-terminal side of the C-domain peptide, a basic amino acid residue at the position corresponding to A8 of human insulin, and an acidic amino acid residue at the position corresponding to A14 of human insulin. The C-domain may contain a 2 to 4 amino acid joint region between the acidic and basic residues. Residues C1 and C2 may have a net negative charge of &#x2212;1 or &#x2212;2; and the remaining C-domain segment may culminates with two basic residues. A pharmaceutical composition comprises the single-chain insulin, formulated at a pH within the range 7.0 to 8.0, and may be formulated at a concentration of 0.6 mM to 5.0 mM and/or at a strength of U-100 to U-1000.

Biphasic single-chain insulin analogues

An insulin analogue comprises an insulin A-chain polypeptide and an insulin B-chain polypeptide. The A-chain polypeptide contains a Glu substitution at a position corresponding to position A8, and an Ala, Glu, Gln, His, Tyr, Phe or Trp substitution at a position A13, relative to wild type insulin. The B-chain polypeptide contains a cyclohexanylalanine substitution at position B24, relative to wild type insulin. The analogue may be an analogue of a mammalian insulin, such as human insulin. A nucleic acid encoding such an insulin analogue is also provided. A method of lowering the blood sugar of a patient comprises administering a physiologically effective amount of the insulin analogue or a physiologically acceptable salt thereof to a patient.

Non-standard insulin analogues

A polypeptide comprises an insulin B-chain sequence having the substitutions: Asp at position B10, Ala at position B12, and Glu at position B29, relative to wild type insulin. The polypeptide may additionally comprise a substitution of a halogenated phenylalanine at position B24, such as ortho-fluoro-phenylalanine. Optionally, the polypeptide may additionally comprise a C-terminal dipeptide extension wherein at least one amino acid in the dipeptide contains an acidic side chain, such as Glu-Glu, and/or an N-terminal deletion of one, two or three residues from the B chain. An insulin analogue may comprise any of these polypeptides with an insulin A-chain polypeptide that optionally contains a Glu A8 substitution. The A-chain sequence may be a separate polypeptide or it may be joined to the B-chain polypeptide by a two amino acid linker. The linker may be Trp-Lys or Ala-Lys. The insulin analogue may be used to treat a patient with diabetes mellitus.

Insulin analogues with enhanced stability and reduced mitogenicity

An insulin analogue comprises a B-chain polypeptide containing a cyclohexanylalanine substitution at position B24 and optionally containing additional amino-acid substitutions at positions A8, B28, and/or B29. A proinsulin analogue or single-chain insulin analogue contains a B domain containing a cyclohexanylalanine substitution at position B24 and optionally contains additional amino-acid substitutions at positions A8, B28, and/or B29. The analogue may be an analogue of a mammalian insulin, such as human insulin. A nucleic acid encoding such an insulin analogue is also provided. A method of lowering the blood sugar of a patient comprises administering a physiologically effective amount of the insulin analogue or a physiologically acceptable salt thereof to a patient. A method of semi-synthesis using an unprotected octapeptide by means of modification of an endogenous tryptic site by non-standard amino-acid substitutions.

A two-chain insulin analogue contains Aspartic Acid at position B10 and penta-fluoro-Phenylalanine at position B24, optionally Histidine or Glutamic Acid at position A8, optionally additional substitutions or modifications at positions A13 and/or A14 and/or B28 and/or B29. The analogue may be an analogue of a mammalian insulin, such as human insulin, may optionally include (i) N-terminal deletion of one, two or three residues from the B chain, (ii) a mono-peptide or dipeptide C-terminal extension of the B-chain containing at least one acidic residue, and (iii) other modifications known in the art to enhance the stability of insulin. Formulations of the above analogues at successive strengths U-100 to U-1000 in soluble solutions at at least pH value in the range 7.0-8.0 in the absence or presence of zinc ions at a molar ratio of 0.00-0.10 zinc ions per insulin analogue monomer.

Insulin analogues with selective signaling properties and reduced mitogenicity

A two-chain insulin analogue contains an A chain modified by (i) a monomeric glucose-binding element at or near its N terminus and (ii) a B chain modified by at or near its C terminus by an element that reversibly binds to the monomeric glucose-binding element such that this linkage is displaceable by glucose. The monomeric glucose-binding element may be phenylboronic acid derivative (optionally halogenated). The B chain may be modified by a diol-containing element derived from a monosaccharide, disaccharide or oligosaccharide, a non-saccharide diol-containing moiety or a &#x3b1;-hydroxycarboxylate-containing moiety. The analogue can be manufactured by trypsin-mediated semi-synthesis. Formulations can be at strengths U-10 to U-1000 in soluble solutions at pH 7.0-8.0 with or without zinc ions at a molar ratio of 0.0-3.0 ions per insulin analogue monomer. A patient with diabetes mellitus may be treated with subcutaneous, intraperitoneal, or oral administration of a physiologically effective amount of the insulin analogue.

Insulin analogues with a glucose-regulated conformational switch

The present invention provides rapid-acting insulin and insulin analogue formulations. The invention further provides delivery devices, particularly infusion sets, which allow for the rapid absorption of insulin and insulin analogues, as well as other active agents. Methods of using the insulin and insulin analogue formulations as well as the insulin delivery devices for treating subjects with diabetes mellitus are also provided.

Rapid action insulin formulations and pharmaceutical delivery systems

A two-chain insulin analog contains Aspartic Acid at position B10 and penta-fluoro-Phenylalanine at position B24, optionally Histidine or Glutamic Acid at position A8, optionally additional substitutions or modifications at positions A13 and/or A14 and/or B28 and/or B29. The analog may be an analog of a mammalian insulin, such as human insulin, may optionally include (i) N-terminal deletion of one, two or three residues from the B chain, (ii) a mono-peptide or dipeptide C-terminal extension of the B-chain containing at least one acidic residue, and (iii) other modifications known in the art to enhance the stability of insulin. Formulations of the above analogs at successive strengths U-100 to U-1000 in soluble solutions at least pH value in the range 7.0-8.0 in the absence or presence of zinc ions at a molar ratio of 0.00-0.10 zinc ions per insulin analog monomer.

An insulin analog comprises a B-chain polypeptide containing a cyclohexanylalanine substitution at position B24 and optionally containing additional amino-acid substitutions at positions A8, B28, and/or B29. A proinsulin analog or single-chain insulin analog containing a B domain containing a cyclohexanylalanine substitution at position B24 and optionally containing additional amino-acid substitutions at positions A8, B28, and/or B29. The analog may be an analog of a mammalian insulin, such as human insulin. A nucleic acid encoding such an insulin analog is also provided. A method of lowering the blood sugar of a patient comprises administering a physiologically effective amount of the insulin analog or a physiologically acceptable salt thereof to a patient. A method of semi-synthesis using an unprotected octapeptide by means of modification of an endogenous tryptic site by non-standard amino-acid substitutions.

A single-chain insulin analogue containing a basic side chain at position A8 (Arginine, Histidine, Lysine, or Ornithine), a basic side chain at position B29 (Arginine, Histidine, Lysine, or Ornithine), and a foreshortened C-domain of length 6-11 residues is provided. Residues C1 and C2 of the C-domain have a net negative charge of &#x2212;1 or &#x2212;2; C3 is chosen from a group consisting of Gly, Ala, Pro, or Ser; and the remaining C-domain segment is successively derived from the C-domain of IGF-II (RRSR (SEQ ID NO: 18), SRRSR (SEQ ID NO: 17), VSRRSR (SEQ ID NO: 16), RVSRRSR (SEQ ID NO: 15), or SRVSRRSR (SEQ ID NO: 13)). A method of treating a patient with diabetes mellitus or obesity comprises administering a physiologically effective amount of the insulin analogue or a physiologically acceptable salt thereof to a patient.

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Moreland Hills, OH, United States of America

Michael A Weiss