Encapsulation of ultra-stable insulin analogues with polymer melts

Abstract

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° 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.

Claims

1. An insulin composition comprising an insulin analogue and a polymer blend, wherein the insulin analogue contains cysteine substitutions at positions corresponding to residues B4 and A10 relative to wild type insulin, a connecting domain of 5-11 amino acids between A and B domains of the insulin analogue, wherein the insulin analogue comprises Glutamic acid at at least one of the first two positions of the connecting domain, and wherein the insulin analogue contains at least one of: (a) an alanine, histidine, glutamic acid or arginine substitution at a position corresponding to position A8 of wild type insulin; and (b) an alanine, glutamic acid or arginine substitution at a position corresponding to position A14 of wild type insulin.

2. The insulin composition of claim 1 wherein the polymer is comprised of poly(lactic-co-glycolic acid) (PL-GA).

3. The insulin composition of claim 2 wherein the polymer is comprised of poly(lactic-co-glycolic acid) (PL-GA) such that the percentage of poly lactic acid is between 25 and 75%.

4. The insulin composition of claim 3 wherein the polymer is comprised of poly(lactic-co-glycolic acid) (PL-GA) such that the percentage of poly lactic acid is 50%.

5. The insulin composition of claim 2, additionally comprising free Polyethylene glycol.

6. The insulin composition of claim 1 wherein the insulin analogue has a sequence selected from the group consisting of SEQ ID NOs: 10, 12 and 15.

7. The insulin composition of claim 1, wherein the insulin analogue comprises SEQ ID NO: 5.

8. The insulin composition of claim 7, additionally comprising SEQ ID NO: 6.

9. The insulin composition of claim 1, wherein the insulin composition is fabricated into a microneedle patch adapted for topical application.

10. The insulin composition of claim 1, wherein the insulin composition is fabricated into a suspension of microbeads adapted for subcutaneous injection.

11. A method for the treatment of diabetes mellitus in a human patient or a mammal, the method comprising administration of an insulin composition comprising an insulin analogue and a polymer blend, wherein the insulin analogue contains cysteine substitutions at positions corresponding to residues B4 and A10 relative to wild type insulin, a connecting domain of 5-11 amino acids between A and B domains of the insulin analogue, wherein the insulin analogue comprises Glutamic acid at at least one of the first two positions of the connecting domain, and wherein the insulin analogue contains at least one of: (a) an alanine, histidine, glutamic acid or arginine substitution at a position corresponding to position A8 of wild type insulin; (b) an alanine, glutamic acid or arginine substitution at a position corresponding to position A14 of wild type insulin.

12. The method of claim 11 wherein the insulin composition is administered by a device attached to the skin.

13. The method of claim 11 wherein the insulin composition is administered by the subcutaneous injection.

14. The method of claim 11, wherein the insulin analogue has a sequence selected from the group consisting of SEQ ID NOs: 10, 12 and 15.

15. The method of claim 11, wherein the insulin analogue comprises SEQ ID NO: 5.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

(1) FIG. 1A is a schematic representation of the sequence of human proinsulin (SEQ ID NO: 1) including the A- and B-chains and the connecting region shown with flanking dibasic cleavage sites (filled circles) and C-peptide (open circles).

(2) FIG. 1B is a structural model of proinsulin, consisting of an insulin-like moiety and a disordered connecting peptide (dashed line).

(3) FIG. 1C is a schematic representation of the sequence of wild type human insulin A-Chain (SEQ ID NO:2) and B-Chain (SEQ ID NO:3) indicating the location of the disulfide bridges and the position of residue B24 in the B-chain.

(4) FIG. 2. 3D model of an SCI stabilized by a fourth disulfide bridge. The asterisk indicates B4-A10 disulfide bridge. The corresponding engineered cystine in a two-chain insulin analogue (i.e., between positions B4 and A10 in the two individual polypeptide chains) may be combined with at least one additional stabilizing modification at position A8, A14, B24 or B29.

(5) FIG. 3A is a photograph of a vial containing protein-polymer blends of modified insulin including a SCI with fourth disulfide bridge blend strips (thumb/finger at bottom for scale)

(6) FIG. 3B is a graph showing blood glucose levels of rats in response to recovered SCI not containing a fourth disulfide bridge from the protein-polymer blends shown in FIG. 3A.

(7) FIG. 3C is a graph showing blood glucose levels of rats in response to recovered modified SCI containing the fourth disulfide bridge from the protein-polymer blends shown in FIG. 3A.

(8) FIG. 4 is a graph showing elution of ultra-stable single-chain analogue (SCI) from PLGA polymer (50-50%) in phosphate-buffered saline (pH 7.4 and 37° C.). Horizontal axis denotes soaking time. Vertical axis denotes nanograms of insulin analogue (dark gray triangles) or relative Bradford-active material in control polymers containing hen egg white lysozyme (light gray squares) or no protein (dark gray diamonds). The latter represents background signal in the Bradford assay due to non-protein material. Horizontal plateau at days 10-15 represents no further elution of protein from the polymers. Samples were made with 5% PEG (mean molecular mass 8 kDa) to optimize the near-linear rate of SCI elution between 1-10 days. Immediate burst release of a portion of the material is not shown.

(9) FIG. 5 is a graph showing blood glucose levels over time for 57mer SCIs with (squares; SEQ ID NO: 15) and without (gray diamonds; SEQ ID NO: 16) a fourth disulfide bridge (between residues B4 and A10) over time following subcutaneous injection in diabetic male Lewis rats.

DETAILED DESCRIPTION OF THE INVENTION

(10) The present invention is directed toward compositions containing polymer melts of a two-chain or single-chain insulin analogue that exhibits so marked an increase in thermal and thermodynamic stability that it may be subjected to a polymer melt process within the temperature range 90-120° C. for at least ten minutes (a) without loss of biological activity on dissolution of the polymer within the dermis or within the subcutaneous space of a mammal or (b) without loss of biological activity on dissolution of the polymer in vitro in a physiological buffer or on a dilute acidic solution on incubation with gentle agitation at 37° C.

(11) It is a feature of the present invention that the isoelectric point of the insulin analogues may either (i) be in the range 3.0-6.0 so as to permit a soluble manufacturing intermediate as a solution at neutral pH or (ii) be in the range 6.5 and 8.0 such that a soluble formulation may be obtained under acidic conditions (pH 3.0-5.5). The latter analogues, when released in the body from a polymer melt, would be expected to undergo isoelectric precipitation in the subcutaneous depot due to a shift of pH to near neutrality. Such precipitation could enhance the safety of a polymeric device in the advent that one or more microneedles or micropellets dissolve suddenly or more rapidly than expected based on the bulk properties of the parent polymer melts.

(12) In one embodiment, the polymer may be selected from the group consisting of Poly(lactic-co-glycolic acid) (PLGA), Poly(caprolactone), Polylactic acid, Polyglycolic acid, Poly(hydroxybutyric acid), chitosan, poly(sebacic acid), polyanhydrides, polyphosphazenes, poly(orthoesters, Poly (lactic acid-co-caprolactone), Poly(hydroxybutyrate-valerate) and mixtures and copolymers thereof. In addition or in the alternative, porogens such as polyethylene glycol, NaCl and/or sugars may optionally also be present to regulate the rate of release of insulin from the polymer composition.

(13) The polymer molecular weight may be chosen according to the requirements of a particular application and the desired rate of release of insulin. In one embodiment, the polymer molecular weight, such as the molecular weight of polyethylene glycol, may have an average molecular weight less than 200 daltons, between 200 and 1000 daltons, between 1000 and 4500 daltons, between 4500 and 9000 daltons, between 9000 and 15000 daltons, between 15000 daltons and 25000 daltons, or greater than 25000 daltons. In one particular embodiment, PEG of about 8000 daltons may be used.

(14) It is envisioned that single-chain or two-chain insulin analogues may be made with A- and B-chain sequences derived from animal insulins, such as porcine, bovine, equine, and canine insulins, by way of non-limiting examples. In addition or in the alternative, the insulin analogue of the present invention may contain a deletion of residue B1, residues B1-B2, or residues B1-B3 or may be combined with a variant B chain lacking Lysine to avoid Lys-directed proteolysis of a precursor polypeptide in yeast biosynthesis in Pichia pastoris, Saccharomyces cerevisciae, or other yeast expression species or strains. While not wishing to be constrained by theory, we envision that non-beta-branched substitutions at position A8 would protect the two-chain insulin analogues and SCIs from both physical and chemical degradation due to their more optimal properties within an alpha-helix and/or at the C-terminal position of an alpha-helix. Examples of stabilizing A8 substitutions are provided by, but not limited to, Arginine, Glutamic Acid and Histidine. While not wishing to be constrained by theory, we envision that charged or polar non-beta-branched substitutions at position A14 would protect the two-chain insulin analogues and SCIs from both physical and chemical degradation due to mitigation of the reverse-hydrophobic effect associated with solvent exposure of Tyr.sup.A14 in wild-type human insulin. Among the proscribed set of stabilizing elements, we also envision that a halogen modification at the 2 ring position of Phe.sup.B24 (i.e., ortho-F-Phe.sup.B24, ortho-Cl-Phe.sup.B24, or ortho-Br-Phe.sup.B24; intended to enhance thermodynamic stability and resistance to fibrillation) provides a molecular mechanism that protects from both chemical degradation and physical degradation. We likewise envision that removal of the naturally occurring positive charge at position B29 (as provided by Lys.sup.B29) would incrementally enhance the resistance of a two-chain insulin analogue containing cystine B4-A10 or of an SCI containing cystine B4-A10 to fibrillation at elevated temperatures. The B29 substitution may be Glutamic Acid or a neutral aliphatic standard or non-standard amino acid. A standard neutral aliphatic residue would be chosen from the group consisting of Ala, Val, Ile, or Leu; a nonstandard such residue would be chosen from the group amino-propionic acid, amino-butryic acid, or norleucine).

(15) Furthermore, in view of the similarity between human and animal insulins, and use in the past of animal insulins in human patients with diabetes mellitus, it is also envisioned that other minor modifications in the sequence of insulin may be introduced, especially those substitutions considered “conservative.” For example, additional substitutions of amino acids may be made within groups of amino acids with similar side chains, without departing from the present invention. These include the neutral hydrophobic amino acids: Alanine (Ala or A), Valine (Val or V), Leucine (Leu or L), Isoleucine (Ile or I), Proline (Pro or P), Tryptophan (Trp or W), Phenylalanine (Phe or F) and Methionine (Met or M). Likewise, the neutral polar amino acids may be substituted for each other within their group of Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T), Tyrosine (Tyr or Y), Cysteine (Cys or C), Glutamine (Glu or Q), and Asparagine (Asn or N). Basic amino acids are considered to include Lysine (Lys or K), Arginine (Arg or R) and Histidine (His or H). Acidic amino acids are Aspartic acid (Asp or D) and Glutamic acid (Glu or E). Unless noted otherwise or wherever obvious from the context, the amino acids noted herein should be considered to be L-amino acids. Standard amino acids may also be substituted by non-standard amino acids belong to the same chemical class. By way of non-limiting example, the basic side chain Lys may be replaced by basic amino acids of shorter side-chain length (Ornithine, Diaminobutyric acid, or Diaminopropionic acid). Lys may also be replaced by the neutral aliphatic isostere Norleucine (Nle), which may in turn be substituted by analogues containing shorter aliphatic side chains (Aminobutyric acid or Aminopropionic acid).

(16) By way of example, protein-PG-LA polymer blends were prepared containing insulin lispro (SEQ ID NOs: 2 and 13), an analogue of lispro insulin (Lys.sup.B28, Pro.sup.B29-human insulin) additionally containing Cys.sup.A10, Cys.sup.B4 substitutions (SEQ ID NOs: 2 and 14), an SCI of 59 amino-acid residues (SEQ ID NO: 9) and a corresponding 59-mer SCI modified with Cys.sup.A10, Cys.sup.B4 substitutions to contain the fourth disulfide bridge between residues B4 and A10 (SEQ ID NO: 10). These SCI contained a C domain of sequence EEGSRRSR. The A domain was modified at A8 to contain Arginine instead of Threonine. The B domain was modified to contain Arginine instead of Lysine to avoid protease digestion in the yeast Pichia pastoris. The isoelectric point of this SCI thus lies in the range 6.5-7.5 but is readily soluble in the pH range 2-4. Its affinity for the A- and B isoforms of the insulin receptor lies within the range 10-150% relative to wild-type human insulin, whereas its affinity for the Type 1 IGF receptor is tenfold lower than that of wild-type insulin. A three-dimensional model of this SCI and the predicted position of cysteine B4-A10 is shown in FIG. 2.

(17) The above three polymer blends were cast in strips (FIG. 3A). The polymers were dissolved over 2 days in 0.01% trifluoroacetic acid at 20° C. to assess potential recovery of functional hormone. Whereas no active lispro was recovered, rat testing demonstrated ˜40% recovery of the SCI (FIG. 3B) and essentially complete recovery of functional modified SCI containing the fourth disulfide bridge (FIG. 3C). The robustness of this “hyper-stable” insulin analogue to high-temperature protein-polymer blend extrusion is of exceptional promise as a technology to provide a long-term dissolvable therapeutic polymer blend for the treatment of diabetes mellitus. It should be noted that in this example utilizes a single-chain insulin analogue that exhibits an isoelectric point of near pH 7.4. This indicates that it is possible to take advantage of its basal PK/PD properties as a future safety mechanism: should a micro-needle crumble and dissolve rapidly subcutaneously, the released analogue would undergo precipitation, avoiding acute hypoglycemia.

(18) Elution of insulin analogue protein from a polymer melt in phosphate buffered saline (PBS) at pH 7.4 was also examined. Test cylindrical polymers were prepared with 50%-50% PLGA containing 25% weight/weight of a single-chain insulin (SCI) analogue stabilized by a fourth disulfide bridge between residues B4 and A10. The analogue (designated 4SS-81-06; SEQ ID NO: 12) contains a six-residue linker of sequence EEGPRR, two substitutions in the A domain (substitution of Thr.sup.A8 by His and substitution of Tyr.sup.A14 by Glu) and one substitution in the B domain (substitution of Lys.sup.B29 by Glu). The mixed powder was heated to 95 degrees centigrade for 10 minutes and then extruded rapidly by force through using a special syringe extruder. The extruded polymeric cylinders (1 mm diameter and 8 mm in length; 10 mg) were prepared using mixtures of PL, GA and SCI powders containing 0, 5 or 10% polyethylene glycol (PEG; mean molecular mass 8 kDa). To test the effect of the free PEG molecule on the rate of release of the SCI from the polymer, the cylinders were placed in phosphate-buffered saline at pH 7.4 and 37° C. with gentle rocking with daily replacement of the buffer. 500 microliters of solution was collected daily and replaced with 500 uL of fresh PBS with (0.1%) sodium azide. Polymer was placed in solution during the afternoon of day 0 and samples were collected roughly every 24 hours after. The day 0 sample was collected immediately (<5 minutes) after polymer was immersed in solution.

(19) Whereas little protein was released in the absence of PEG (0%) over the course of 10 days, addition of 10% PEG led to substantial release over 1-2 days. Addition of 5% PEG resulted in a near-linear release of ca. half of the loaded protein over a 10-day period (triangles in FIG. 4; insulin analogue concentrations measured by the Bradford assay as calibrated by ELISA). A similar elution profile was observed in control studies of an analogous co-polymer containing hen egg-white lysozyme instead of an insulin analogue (squares in FIG. 4; arbitrary units). In both cases a horizontal plateau was observed during days 10-15, indicating no further release of the protein from the polymer. The cumulative amounts of protein released are also provided below in Table 1. In this table the micrograms of insulin corresponding to the Bradford assay was verified by ELISA whereas the readings for lysozyme are uncalibrated by an independent assay.

(20) TABLE-US-00001 TABLE 1 Cumulative Amount of Protein Released Daily A (Neat − B (Lysozyme + C (8106-4SS + Day PLGA) 5% PEG) 5% PEG) 0 0.0104 0.0432 0.024 1 −0.0071 0.135 0.1766 2 0.0036 0.2289 0.2504 3 0.0127 0.2759 0.2896 4 0.0213 0.3104 0.3184 5 0.0365 0.3383 0.3574 6 0.0546 0.3978 0.4316 7 0.0482 0.3987 0.455 8 0.0574 0.44 0.5197 9 0.0679 0.5212 0.6054 10 0.0809 0.6783 0.7418 11 0.0828 0.7366 0.7452 12 0.0795 0.7297 0.7185 13 0.0802 0.7556 0.7246 14 0.0802 0.7556 0.7246 15 0.0915 0.7718 0.7387

(21) The biological activity of the released SCI hormone analogue (in the polymer melts prepared with 5% PEG) was tested in diabetic male Lewis rats (mean weight ca. 300 grams; rendered diabetic by streptozotocin with mean glycemia ca. 400 mg/dl); the blood glucose-lowering activity of the virgin SCI was compared to the activities of the protein eluted after day 1 and day 5. The biological activities of these three samples were indistinguishable, demonstrating that the process of thermal-melt extrusion and graduate release in a physiological buffer at body temperature is not associated with loss of potency.

(22) TABLE-US-00002 TABLE 2 Delta per Delta per hour for hour for first half first Number Insulin hour SE hour SE of Animals C-1 (PLGA + −320.10 48.90 −315.11 6.96 2 25% 4SS- 81-06 + 5% PEG; 20 μg) C-5 (PLGA + −312.60 22.20 −224.36 2.36 2 25% 4SS- 81-06 + 5% PEG; 5 μg) Percentage Percentage of baseline of baseline Number Insulin at 30 min SE at 60 min SE of Animals C-1 (PLGA + 0.6296 0.0504 0.2964 0.0014 2 25% 4SS- 81-06 + 5% PEG; 20 μg) C-5 (PLGA + 0.950 0.005 0.814 0.043 2 25% 4SS- 81-06 + 5% PEG; 5 μg)

(23) The biological activities of a 57mer SCI (noted as 81-04 herein; SEQ ID NO: 16) and its derivative containing a fourth disulfide bridge (4SS 81-04; SEQ ID NO: 15) were compared. Providing a dose of 20 micrograms per 300 gram rat, the biological activities are essentially identical (see FIG. 5) The sequence of 81-04 is similar to that of SEQ ID NO: 12 except for the absence of Cys.sup.A10, Cys.sup.B4; further, residue B28 is Aspartic Acid and residue B29 is Proline. This demonstrates that the introduction of a fourth disulfide bridge into a single-chain insulin analogue molecule does not alter the underlying biological activity of the SCI. This is surprising in view of the prior art, which indicated that in two-chain analogs a marked prolongation of the pharmacodynamic response occurs when introducing the 4th disulfide bridge.

(24) The receptor-binding affinity of analogue 81-04 and analogue 4SS 81-04 was also determined. The affinity of 4SS 81-04 for the A isoform of the insulin receptor was determined to be 120±20 percent relative to human insulin (and may in fact be the same as wild-type human insulin given the error present; data not shown). Its affinity for the B isoform of the insulin receptor is reduced by between fivefold and tenfold relative to wild-type human insulin. This preference for the A isoform is similar to that of the 81-04 parent analogue. Furthermore, the affinity of 4SS 81-04 for the mitogenic IGF Type I receptor (IGF-1R) is reduced by between fivefold and tenfold relative to wild-type human insulin (data not shown). Such impaired binding to IGF-1R is desirable from the perspective of potential carcinogenesis on long-term use.

(25) The sequences of the polypeptides disclosed herein are provided as follows. The amino-acid sequence of human proinsulin is provided, for comparative purposes, as SEQ ID NO: 1.

(26) TABLE-US-00003 (human proinsulin) SEQ ID NO: 1 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Pro-Lys-Thr-Arg-Arg-Glu-Ala-Glu-Asp- Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro- Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly- Ser-Leu-Gln-Lys-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys- Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr- Cys-Asn

(27) The amino-acid sequence of the A chain of human insulin is provided as SEQ ID NO: 2.

(28) TABLE-US-00004 (human A chain) SEQ ID NO: 2 Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser- Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn

(29) The amino-acid sequence of the B chain of human insulin is provided as SEQ ID NO: 3.

(30) TABLE-US-00005 (human B chain) SEQ ID NO: 3 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Pro-Lys-Thr

(31) The amino-acid sequence of single-chain insulin analogues of the present invention are given in SEQ ID NO 4, containing a fourth cysteine at positions B4 and A10 and corresponding to polypeptides of length 56, 57, 57, 58, 59, 60, 61, and 62, such that the SCI contains at least one other stabilizing modification at one or more of the indicated positions.

(32) TABLE-US-00006 SEQ ID NO: 4 Phe-Val-Asn-Cys-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Xaa.sub.1- Phe-Tyr-Thr-Pro-Xaa.sub.2-Thr-[foreshortened C domain]-Gly-Ile-Val-Glu-Gln-Cys-Cys-Xaa.sub.3- Ser-Cys-Cys-Ser-Leu-Xaa.sub.4-Gln-Leu-Glu-Asn- Tyr-Cys-Xaa.sub.5

(33) Where Xaa.sub.1 indicates Phe or a modification of Phe by a halogen atom (F, Cl or Br) at the ortho or 2-ring position; Xaa.sub.2 indicates Glu, Ala, Ile, Leu, Val, Norleucine, amino-propionic acid or amino-butryic acid; where Xaa.sub.3 is His, Glu, Lys, Arg, or another non-beta-branched polar or charged amino acid; where Xaa.sub.4 is Tyr (as in wild-type insulin), Glu or another non-beta-branched polar or charged amino acid; and optionally where Xaa.sub.5 is Gly, Ala, Asp or Ser. The bracketed term “[foreshortened C domain]” designates a connecting peptide domain of length 5-11 residues that contains an acidic residue at either the first (N-terminal) or second peptide position (i.e., residues 31 or 32 of the single-chain insulin analogue). Optionally, Phe.sup.B1 may be deleted to yield a des-B1 analogue or both Phe.sup.B1 and Val.sup.B2 may be omitted to yield a des-[B1, B2] analogue.

(34) The amino-acid sequence of two-chain insulin analogues of the present invention are given in SEQ ID NO 5-8, corresponding to a B chain containing Cysteine at position B4 (SEQ ID NOS: 5, 7 and 8) and an A chain containing Cysteine at position A10 (SEQ ID NO: 6) such that the intact insulin analogue contains a fourth disulfide bridge between positions B4 and A10 and at least one other stabilizing modification at the designated positions.

(35) TABLE-US-00007 (Variant B chain): SEQ ID NO: 5 Phe-Val-Asn-Cys-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Xaa.sub.1- Phe-Tyr-Thr-Pro-Xaa.sub.2-Thr

(36) Where Xaa.sub.1 indicates Phe or a modification of Phe by a halogen atom (F, Cl or Br) at the ortho or 2-ring position; Xaa.sub.2 indicates Glu, Ala, Ile, Leu, Val, Norleucine, amino-propionic acid or amino-butryic acid;

(37) TABLE-US-00008 (Variant A chain): SEQ ID NO: 6 Gly-Ile-Val-Glu-Gln-Cys-Cys-Xaa.sub.3-Ser-Cys-Cys- Ser-Leu-Xaa.sub.4-Gln-Leu-Glu-Asn-Tyr-Cys-Xaa.sub.5

(38) where Xaa.sub.3 is His, Glu, Lys, Arg, or another non-beta-branched polar or charged amino acid; where Xaa.sub.4 is Tyr (as in wild-type insulin), Glu or another non-beta-branched polar or charged amino acid; and optionally where Xaa.sub.5 is Gly, Ala, Asp or Ser.

(39) TABLE-US-00009 (Variant des-[B1]-B chain): SEQ ID NO: 7 Val-Asn-Cys-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu- Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Xaa.sub.1- Phe-Tyr-Thr-Pro-Xaa.sub.2-Thr

(40) Where Xaa.sub.1 indicates Phe or a modification of Phe by a halogen atom (F, Cl or Br) at the ortho or 2-ring position; Xaa.sub.2 indicates Glu, Ala, Ile, Leu, Val, Norleucine, amino-propionic acid or amino-butryic acid.

(41) TABLE-US-00010 (Variant des-[B1, B2]-B chain): SEQ ID NO: 8 Asn-Cys-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala- Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Xaa.sub.1- Phe-Tyr-Thr-Pro-Xaa.sub.2-Thr

(42) Where Xaa.sub.1 indicates Phe or a modification of Phe by a halogen atom (F, Cl or Br) at the ortho or 2-ring position; Xaa.sub.2 indicates Glu, Ala, Ile, Leu, Val, Norleucine, amino-propionic acid or amino-butryic acid.

(43) Single-Chain Insulin (SCI) analogues are provided as SEQ ID NOs: 9-12, 15 and 16.

(44) TABLE-US-00011 SEQ ID NO: 9 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Pro-Arg-Thr-Glu-Glu-Gly-Ser-Arg-Arg- Ser-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys-Arg-Ser-Ile- Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn SEQ ID NO: 10 Phe-Val-Asn-Cys-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Pro-Arg-Thr-Glu-Glu-Gly-Ser-Arg-Arg- Ser-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys-Arg-Ser-Cys- Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn SEQ ID NO: 11 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Pro-Glu-Thr-Glu-Glu-Gly-Pro-Arg-Arg- Gly-Ile-Val-Glu-Gln-Cys-Cys-Glu-Ser-Ile-Cys-Ser- Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn (81-066-4SS) SEQ ID NO: 12 Phe-Val-Asn-Cys-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Pro-Glu-Thr-Glu-Glu-Gly-Pro-Arg-Arg- Gly-Ile-Val-Glu-Gln-Cys-Cys-His-Ser-Cys-Cys-Ser- Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn (human B chain, KP) SEQ ID NO: 13 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Lys-Pro-Thr (human B chain, Cys B4, KP) SEQ ID NO: 14 Phe-Val-Asn-Cys-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Lys-Pro-Thr (4SS 81-04; 57mer) SEQ ID NO: 15 Phe-Val-Asn-Cys-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Asp-Pro-Thr-Glu-Glu-Gly-Pro-Arg-Arg- Gly-Ile-Val-Glu-Gln-Cys-Cys-His-Ser-Cys-Cys-Ser- Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn (81-04; 57mer) SEQ ID NO: 16 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Asp-Pro-Thr-Glu-Glu-Gly-Pro-Arg-Arg- Gly-Ile-Val-Glu-Gln-Cys-Cys-His-Ser-Ile-Cys-Ser- Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn

(45) Based upon the foregoing disclosure, it should now be apparent that the single-chain insulin analogues provided will carry out the objects set forth hereinabove. Namely, these insulin analogues exhibit enhanced resistance to fibrillation while retaining desirable pharmacokinetic and pharmacodynamic features (conferring prolonged action) and maintaining at least a fraction of the biological activity of wild-type insulin. It is, therefore, to be understood that any variations evident fall within the scope of the claimed invention and thus, the selection of specific component elements can be determined without departing from the spirit of the invention herein disclosed and described.

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