AU711428B2 - Monomeric insulin analog formulations - Google Patents

Description

X-9418 FOR 1 Monomeric Insulin Analog Formulations The present invention relates to monomeric analogs of human insulin. More specifically, the present invention relates to various parenteral formulations, which comprise a monomeric insulin analog, zinc, protamine, and a phenolic derivative. The formulations provide a prolonged duration of action. A process for preparing insulin analog-protamine formulations is also described.

Since the introduction of insulin in the 1920's, continuous strides have been made to improve the treatment of diabetes mellitus. Major advances have been made in insulin purity and availability with the development of recombinant DNA technology. Various formulations with different timeactions have also been developed. Currently, there are generally seven commercially available insulin formulations: Regular insulin, semilente insulin, globin insulin, isophane insulin, insulin zinc suspension, protamine zinc insulin, and Ultralente insulin.

Despite the array of formulations available, subcutaneous injection therapy still falls short of providing a patient with convenient regulation and normalized glycemic control. Frequent excursions from normal glycemia levels 25 over a patient's lifetime lead to hyper-or hypoglycemia, and long term complications including retinopathy, neuropathy, nephropathy, and micro- and macroangiopathy.

To help avoid extreme glycemic levels, diabetics often practice multiple injection therapy whereby insulin is administered with each meal. However, this therapy has not yet been optimized. The most rapid-acting insulin commercially available peaks too late after injection and lasts too long to optimally control glucose levels.

Therefore, considerable effort has been devoted to create 35 insulin formulations and insulin analog formulations that alter the kinetics of the subcutaneous absorption process.

Because all commercial pharmaceutical formulations of insulin contain insulin in the self-associated state and predominately in the hexamer form, it is believed that the ratelimiting step for the absorption of insulin from the subcutaneous injection depot to the bloodstream is the dissociation of the self-aggregated insulin hexamer. Recently, monomeric insulin analogs have been developed that are less prone to association to higher molecular weight forms than human insulin. This lack of self-association is due to modifications in the amino acid sequence of human insulin that decrease association by primarily disrupting the formation of dimers. See, Brems et al., Protein Engineering, 5:6, 527-533 (1992) and Brange et al., Nature, 333:679-682 (1988).

Accordingly, monomeric insulin analogs possess a comparatively more rapid onset of activity while retaining the biological activity of native human insulin. These insulin analogs provide a rapid absorption to place injection time and peak action of insulin into closer proximity with postprandial glucose excursion associated in the response to a meal.

The physical properties and characteristics of monomeric analogs are not analogous to insulin. For example, Brems et al., disclose that various monomeric analogs have little, or no, Zn-induced association. Any association that is observed is to a multitude of higher molecular weight forms. This differs dramatically from insulin, which is almost exclusively in an ordered, hexamer conformation in the presence of zinc. Brange et al., Diabetes Care 13: 923-954 (1990). The lack of association attributes to the fast acting 20 characteristics of the analogs. Because the analogs have lower tendency to associate, it is o S. quite surprising that a monomeric insulin analog can be formulated to provide an intermediate duration of action.

The present application describes a monomeric insulin analog formulation that yields upon use an intermediate duration of action. The application further describes a novel protamine crystal called insulin analog-NPD. The present application also describes a mixture of insulin analog-NPD and soluble monomeric insulin analog. This mixture provides a rapid onset of action and an intermediate duration of action. Accordingly, the mixture possesses advantages over both insulin and the monomeric analog. The present application further describes for a process for preparing uniform crystals of insulin 30 analog-NPD.

This application describes an insulin analog-protamine formulation, which comprises: a monomeric insulin analog, protamine, zinc, and a phenolic derivative.

The application further describes a crystalline insulin analog-protamine complex.

This complex has been defined as insulin analog-NPD. LysB 28 ProB 29 -human insulin-NPD comprises: a LysB 28 ProB 29 -human insulin, about 0.27 to about 0.32mg protamine/100 U of insulin analog, about 0.35 to about 0.9% zinc by weight, and a phenolic derivative.

This application additionally describes a process for preparing LysB 28 ProB 29 -human Sinsulin-NPD, which comprises: [N:\LIBaa]01271:ssd combining an aqueous solution of LysB 2 8 proB 29 -human insulin in a hexamer association state, and a protamine solution at a temperature from about 8 to about 220C; said aqueous solution comprising from about 0.35 to about 0.9% zinc by weight, LysB 28 proB 29 -human insulin, and a phenolic derivative at a pH of about 7.1 to about 7.6; said protamine solution comprising protamine at a pH of about 7.1 to about 7.6 such that the final concentration of protamine is about 0.27 to about 0.32 mg protamine/100 U of insulin analog.

The application also describes formulations that are both rapid and intermediate acting. The formulations are mixtures of monomeric insulin analog and crystalline insulin analog-NPD, wherein the ratio by weight of the two components is about 1-99:99-1.

Finally, the application describes a method of treating a patient suffering from diabetes mellitus, which comprises administering to said patient a pharmaceutical composition containing insulin analog-protamine crystals.

According to a first embodiment of the present invention there is provided an insulin analogue-protamine complex, which comprises: human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val, or Ala, and Lys at position B29 is Lys or Pro, des(B28-B30)-human insulin, or des(B27)-human insulin; protamine; zinc; and a phenolic derivative selected from m-cresol, phenol, and mixtures thereof; provided that 2 the insulin analogue is not AspB 28 -human insulin.

20 According to a second embodiment of the present invention there is provided a process for preparing the complex of the first embodiment, which process comprises: S combining an insulin analogue, protamine, zinc and a phenolic derivative in an aqueous solvent and allowing the complex to form.

According to a third embodiment of the present invention there is provided a complex prepared by the process of the second embodiment.

According to a fourth embodiment of the present invention there is provided a parenteral formulation which comprises the complex of the first or third embodiment and which further comprises about 0.2 to about 1.5 mg protamine/100 IU insulin analogue; about 0.35 to about 0.9% zinc by weight; and a phenolic derivative.

30 According to a fifth embodiment of the present invention there is provided a parenteral pharmaceutical insulin analogue-protamine formulation, which comprises the complex of the first or third or fourth embodiment together with a pharmaceutically acceptable diluent or carrier.

According to a sixth embodiment of the present invention there is provided a parenteral pharmaceutical formulation, which comprises: LysB 28 proB 29 -human insulin, about 0.3 mg protamine/100 IU of insulin analogue, about 0.7% zinc by weight, about 1.7 mg/mL m-cresol, about 0.7 mg/mL phenol, about 16 mg/mL glycerin and about 3.78 mg/mL dibasic sodium phosphate.

According to a seventh embodiment of the present invention there is provided a Sarenteral pharmaceutical formulation, which comprises: zinc; a phenolic derivative [N:\LIBaa]01271:ssd selected from m-cresol, phenol and mixtures thereof; and a mixture of soluble insulin analogue and insulin analogue-protamine crystals; wherein the ratio by weight of the soluble insulin analogue to the insulin analogue-protamine crystals is about 1:99 to 99:1; said insulin analogue is human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val, or Ala; and Lys at position B29 is Lys or Pro; des(B28-B30)-human insulin; or des(B27)-human insulin; provided that the insulin analogue is not AspB 28 -human insulin.

According to an eighth embodiment of the present invention there is provided a parenteral pharmaceutical formulation which comprises the formulation of the seventh embodiment combined with a pharmaceutically acceptable diluent or carrier.

According to a ninth embodiment of the present invention there is provided a process of preparing a parenteral pharmaceutical formulation of the fifth or eighth embodiment, which process comprises: suspending insulin analogue-protamine crystals in a pharmaceutically acceptable diluent or carrier.

According to a tenth embodiment of the present invenion there is provided a parenteral pharmaceutical formulation prepared by the process of the ninth embodiment.

According to an eleventh embodiment of the present invention there is provided a process for preparing a LysB 28 ProB 29 -human insulin-protamine crystal, which comprises: combining an aqueous solution of LysB 28 ProB 29 -human insulin in a hexamer 20 association state, and a protamine solution at a temperature from about 8 0 C to about 22°C; 9said aqueous solution comprising from about 0.35 to about 0.9% zinc by weight, LysB 28 ProB 2 9 -human insulin, and a phenolic derivative at a pH of about 7.1 to about 7.6; said protamine solution comprising protamine at a pH of about 7.1 to about 7.6 such that the final concentration of protamine is about 0.27 to about 0.32 mg protamine/100 IU of insulin analogue.

According to a twelfth embodiment of the present invention there is provided a LysB 28 proB 29 -human insulin-protamine crystal prepared by the process of the eleventh embodiment.

According to a thirteenth embodiment of the present invention there is provided a parenteral pharmaceutical formulation comprising the LysB 2 8 ProB 2 9 -human insulinprotamine crystal of the twelfth embodiment together with a pharmaceutically acceptable diluent or carrier.

According to a fourteenth embodiment of the present invention there is provided a process for preparing an AspB 2 8-human insulin protamine crystal, which process comprises: combining an aqueous solution of AspB 28 -human insulin in a hexamer association state, and a protamine solution at a temperature from about 50 to 22 0

C;

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9 said aqueous solution comprising about 0,35 to about 0.9% zinc by weight, AspB 2 8-human insulin, and a phenolic derivative at a pH of about 7.1 to about 7.6; said protamine solution comprising protamine at a pH of about 7.1 to about 7.6 such that the final concentration of protamine is about 0.27 to about 0.32 mg protamine/100 IU of insulin analogue.

According to a fifteenth embodiment of the present invention there is provided an AspB 2 8-human insulin protamine crystal prepared by the process of the fourteenth embodiment.

According to a sixteenth embodiment of the present invention there is provided a parenteral pharmaceutical formulation comprising the AspB2 8 -human insulin protamine crystal of the fifteenth embodiment together with a pharmaceutically acceptable diluent or carrier.

According to a seventeenth embodiment of the present invention there is provided a method of treating a patient suffering from diabetes mellitus, which method comprises administering to said patient an insulin analogue-protamine complex of the first or third embodiment or a formulation of the fourth, fifth, sixth, seventh, eighth or tenth embodiment, 15 According to an eighteenth embodiment of the present invention there is provided a method of treating a patient suffering from diabetes mellitus, which method comprises administering to said patient an insulin analogue-protamine complex, which comprises: human insulin wherein Pro at position B28 is substituted with .Asp, Lys, Leu, Val, or Ala, and Lys at position B29 is Lys or Pro, des(B28-B30)-human insulin, or des(B27)-human insulin; protamine; zinc; and a phenolic derivative selected from m-cresol, phenol, and mixtures thereof, provided that when the insulin is Asp; 8 -human insulin the concentration of protamine is less than 10% by weight.

According to a nineteenth embodiment of the present invention there is provided a method of treating a patient suffering from diabetes mellitus, which method comprises administering to said patient a LysB28PrOB29hurman insulin of the twelfth embodiment or a formulation of the thirteenth embodiment.

According to a twentieth embodiment of the present invention there is provided a method of treating a patient suffering from diabetes mellitus, which method comprises administering to said patient a parenteral pharmaceutical formulation, which comprises: zinc; a phenolic derivative selected from m-cresol, phenol and mixtures thereof; and a mixture of soluble insulin analogue and insulin analogue-protamine crystals; wherein the ratio by weight of the soluble insulin analogue to the insulin analogue-protamine crystals is about 1:99 to 99:1; said insulin analogue is human insulin wherein Pro at position 828 is substituted with Asp, Lys, Leu, Val, or Ala; and Lys at position B29 is Lys or Pro; des(B28-B30)-human insulin; or des(B27)-human insulin, provided that when the insulin is AspB2 6 human insulin the concentration of protaminc-is less than 10% by weight.

According to a twenty first embodiment of the present invention there is provided a method of treating a patient suffering from diabetes mellitus, which method comprises administering to said patient an AspB 28 -human insulin-protamine crystal of the fifteenth embodiment or a formulation of the sixteenth embodiment.

24/08 '99 TUE 18:28 [TX/RX NO 9682] U016 24, AUG. 1999 18:28 SPRUSON FERGUSON 61 2 92615486 NO, 5794 P. 17/39 According to a twenty second embodiment of the present invention there is provided use of an insulin analogue-protamine complex of the first or third embodiment for the manufacture of a medicament to treat a patient suffering from diabetes mellitus.

According to a twenty third embodiment of the present invention there is provided an insulin analogue-protamine complex of the first or third embodiment when used for the treatment of a patient suffering from diabetes mellitus.

According to a twenty fourth embodiment of the present invention there is provided use of an insulin analogue-protamine compiex, which comprises: human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val, or Ala, and Lys at position B29 is Lys or Pro, des(B28- B30)-human insulin, or des(B27)-human insulin; protamine; zinc; and a phenolic derivative selected from m-cresol, phenol, and mixtures thereof, provided that when the insulin is AspB 2 8-human insulin the concentration of protamine is less than 10% by weight for the manufacture of a medicament to treat a patient suffering from diabetes mellitus.

According to a twenty fifth embodiment of the present invention there is provided an 15 insulin analogue-protamine complex, which comprises: human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val, or Ala, and Lys at position B29 is Lys or Pro, des(B28-B30)human insulin, or des(B27)-human insulin; protamine; zinc; and a phenolic derivative selected from mcresol, phenol, and mixtures thereof, provided that when the insulin is AspB 28 -human insulin the concentration of protamine is less than 10% by weight when used for the treatment of a patient suffering from diabetes mellitus.

According to a twenty sixth embodiment of the present invention there is provided use of a LysB 2 Pro 8 29-human insulin of the twelfth embodiment for the manufacture of a medicament to treat a oatient suffering from diabetes mellitus.

According to a twenty seventh embodiment of the present invention there is provided a 25 LysB 2 8ProB29-human insulin of the twelfth embodiment when used to treat a patient suffering from diabetes mellitus, S* According to a twenty eighth embodiment of the present invention there is provided use S. of zinc; a phenolic derivative selected from m-cresol, phenol and mixtures thereof; and a mixture of soluble insulin analogue and insulin analogue-protamine crystals; wherein the ratio by weight of the 30 soluble insulin analogue to the insulin analogue-protamine crystals is about 1:99 to 99:1; said insulin analogue is human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val, or Ala; and Lys at position 829 is Lys or Pro; des(B28-B30)-human insulin; or des(B27)-human insulin, provided that when the insulin is AspB 2 8 -human insulin the concentration of protamine is less than by weight for the manufacture of a medicament to treat a patient suffering from diabetes mellitus.

According to a twenty ninth embodiment of the present invention there is provided zinc; a phenolic derivative selected from m-cresol, phenol and mixtures thereof; and a mixture of soluble insulin analogue and insulin analogue-protamine crystals; wherein the ratio by weight of the soluble insulin analogue to the insulin analogue-protamine crystals is about 1:99 to 99:1; said insulin analogue is human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val, or Ala; and Lys at position B29 is Lys or Pro;des(B28-B30)-human insulin; or des(B27)-human insulin, 24/08 '99 TUE 18:28 [TX/RX NO 9682] 1017 24. AUG. 1999 18:29 SPRUSON FERGUSON 61 2 92615486 NO, 5794 P. 18/39 4provided that when the insulin is AspB2.-human insulin the, concentration of protamine is less than by weight when used for treating a patient suffering from diabetes mellitus.

According to a thirtieth embodiment of the present invention there is provided use of an AspB 28 -human insulin-protamine crystal of the fifteenth embodiment for the manufacture of a medicament to treat a patient suffering from diabetes mellitus.

According to a thirty first embodiment of the present invention there is provided an Asp828-human insulin-protamine crystal of the fifteenth embodiment when used to treat a patient suffering from diabetes mellitus, According to a thirty second embodiment of the present invention there is provided an insulin analog-protamine complex, with comprises: human insulin wherein Pro at position B28 is substituted with: Asp, Lys, Leu, Val, or Ala, and Lys at position B29 is Lys or Pro, Des(B28-B30)human insulin, or des(B27)-human insulin; protamine; zinc; and a phenolic derivative; provided that when the insulin is AspBa2-human insulin the concentration of protamine is less than 10% by weight.

According to a thirty third embodiment of the present invention there is provided a S. 15 parenteral pharmaceutical insulin analog-protamine formulation, which comprises the complex of the thirty second embodiment.

According to a thirty fourth embodiment of the present invention there is provided a parenteral pharmaceutical formulation which comprises: LysB 2 8 ProB29-human insulin, 0.27 to 0.32mg protamine per 100 IU insulin analog, and 0.35 to 0.9% zinc by weight; and a phenolic derivative.

According to a thirty fifth embodiment of the present invention there is provided a parenteral pharmaceutical formulation which comprises: AspE 2 8 -human insulin, 0.27 to 0.35mg prolamine per 100 IU insulin analog, and 0.35 to 0.9% zinc by weight and a phenolic derivative.

According to a thirty sixth embodiment of the present invention there is provided a parenteral pharmaceutical formulation, which comprises: a mixture of soluble insulin analog solution 25 comprising a human insulin analog, zinc and a phenolic derivative; and insulin analog-protamine *crystals; wherein the ratio by weight of the two components is 1:99 to 99:1 insulin analog to insulin analog-protamine crystals; said insulin analog is: human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val or Ala and Lys at position B29 is Lys or Pro, des(B28-B30)-human insulin, or des(B27)-human insulin; provided that when the insulin is AspB 2 e-human insulin the 30 concentration of protamine is less than 10% by weight.

According to a thirty seventh embodiment of the present invention there is provided a parenteral pharmaceutical formulation, which comprises: a mixture of soluble insulin analog solution comprising a human insulin analog, zinc and a phenolic derivative; and insulin analog-protamine crystals; wherein the ratio by weight of the two components is 1:99 to 99:1 insulin analog to insulin analog-protamine crystals; and wherein said insulin analog is LyseZoProB 2 -human insulin.

According to a thirty eighth embodiment of the present invention there is provided a parenteral pharmaceutical insulin analog-protamine formulation, which comprises: a mixture of a soluble human insulin analog solution comprising a human insulin analog, zinc and a phenolic derivative characterized in that substantially all of the soluble analog is in a hexamer complex and an insulin analog-protamine complex; wherein the ratio by weight of the two components is 1:99 to 99:1 24/08 '99 TUE 18:28 [TX/RX NO 9682] IM018 S 24 AUG. 1999 18:29 SPRUSON FERGUSON 61 2 92615486 NO, 5794 P. 19/39 4c.

soluble insulin analog to insulin analog-protamine complex; said insulin analog is: human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val or Ala, and Lys at position B29 is Lys or Pro, des(B28-B30)-human insulin, or des(B27)-human insulin.

According to an thirty ninth embodiment of the present invention there is provided a process for preparing the complex of the thirty second embodiment which comprises: combining a monomeric insulin analog, protamine, zinc and a phenolic derivative in an aqueous solvent and allowing the complex to form, According ;o a fortieth embddiment of "the present invention there is provided an insulin analogue-protamine complex of the first or third embodiment for use in the treatment of a patient suffering from diabetes mellitus.

According to a forty first embodiment of the present invention there is provided an insulin analogue-protamine complex, which comprises: human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val, or Ala, and Lys at position B29 is Lys or Pro, des(B28-B30)human insulin, or des(B27)-human insulin; protamine; zinc; and a phenolic derivative selected from m- 15 cresol, phenol, and mixtures thereof, provided that when the insulin is AspB28-human insulin the Sconcentration of protamine is less than 10% by weight for use in the treatment of a patient suffering from diabetes mellitus.

SAccording to a forty second embodiment of the present invention there is provided a S LysB28ProB29-human insulin of the twelfth embodiment for use in treating a patient suffering from diabetes mellitus, According to a forty third embodiment of the present invention there is provided zinc; a phenolic derivative selected from m-cresol, phenol and mixtures thereof; and a mixture of soluble insulin analogue and insulin analogue-protamine crystals; wherein the ratio by weight of the soluble insulin analogue to the insulin analogue-protamine crystals is about 1:99 to 99:1; said insulin analogue is S 25 human insulin wherein Pro at position 828 is substituted with Asp, Lys, Leu, Val, or Ala; and Lys at position B29 is Lys or Pro; des(B28-B30)-human insulin; or des(B27)-human insulin, provided that Swhen the insulin is AspB 2 8 -human insulin the concentration of protamine is less than 10% by weight for S* use in treating a patient suffering from diabetes mellitus, According to a forty fourth embodiment of the present invention there is provided an AspB2 8 S 30 human insulin-protamine crystal of the fifteenth embodiment for use in treating a patient suffering from diabetes mellitus According to a forty fifth embodiment of the present invention there is provided a medicament to treat a patient suffering from diabetes mellitus when manufactured by the use according to any one of the twenty second, twenty fourth, twenty sixth, twenty eighth or thirtieth embodiment, FIGURE 1 is a graphical representation of the profile of action of LysB 2 8roB29-hI.NPD and human insulin-NPH. The graph is ,gU/ml versus the Time of Infusion, The figure demonstrates the advantages of the present invention, FIGURE 2 presents a picture of AspB 28 -human insulin-protamine crystals of the present invention. The picture was taken at 1000x magnification with differential phase contrast.

24/08 '99 TUE 18:28 [TX/RX NO 9682] 019 *24. AUG. 1999 18:30 SPRUSON FERGUSON 61 2 92515486 NO 5794 P. 20/39 4b FIGURE 3 presents a Picture of LysB 2 8proB29hlprotamine crystals of the present invention, The picture was taken at J0Q0X magnification with differential phase contrast, As noted above, the invention provides various formulations of a monomeric insulin analog. The term "monomeric insulin analog" or "insulin analog" as used herein is a fast-acting insulin analog that is less prone to dimerization or self-association, Monomeric insulin analog is human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val, or Ala, and Lys at position B29 is Lysine or Proline; des(B28-B30): or des(827). Monomeric insulin analogs are described in Chance et al., EPO publication number 383 472, and Brange, et at., EPO publication 214 826, and are herein incorporated by reference.

One skilled in the art would recognize that other modifications to the monomeric insulin analog are possible. These modifications are widely accepted in the art and include replacement of the histidine residue at position 1310 with aspartic acid; replacement of the phenylalanine residue at position 81 with aspartic acid; replacement of the threonine residue at position 830 with alanine;- :replacement 00 4 so 0 000 00 24/08 '99 TUE 18:28 [TX/RX NO 9682] 191020 X-9418 FOR 5 of the serine residue at position B9 with aspartic acid; deletion of amino acids at position B1 alone or in combination with a deletion at position B2; and deletion of threonine from position All amino acid abbreviations used in this disclosure are those accepted by the United States Patent *Trademark Office as set forth in 37 C.F.R. 1.822(b)(2).

Particularly preferred monomeric insulin analogs are LysB 28 ProB 29 -human insulin (B28 is Lys; B29 is Pro) and AspB28-human insulin (B28 is Asp).

The term "monomeric insulin analog-NPD" or "insulin analog-NPD" is a suspension of crystalline insulin analog and protamine in a formulation. NPD is Neutral Protamine formulation according to DeFelippis. The composition is prepared in accordance to the claimed process described herein. A related term "insulin analog NPD crystals," "crystalline insulin analog-NPD," or "LysB 2 8ProB 2 9 -human insulin-protamine crystals" refer to the insulin analogprotamine crystals in the NPD formulation.

The term "treating," as used herein, describes the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of a compound of present invention to prevent the onset of the symptoms or complications, alleviating the 25 symptoms or complications, or eliminating the disease, I condition, or disorder.

The term "isotonicity agent" refers to an agent that is physiologically tolerated and embarks a suitable tonicity to the formulation to prevent the net flow of water across the cell membrane. Compounds, such as glycerin, are commonly used for such purposes at known concentrations. The concentration of the isotonicity agent is in the range known in the art for insulin formulations.

The term "phenolic derivative" is m-cresol, phenol 35 or preferably a mixture of m-cresol and phenol.

The term "free base basis" indicates the amount of protamine in the formulation. Free base basis corrects for X-9418 FOR 6 the water and salt content of the protamine salts commercially available and commonly used in parenteral formulations. The preferred protamine, protamine sulfate, is approximately 80% protamine.

The term "IU" or is international unit.

The term "isophane ratio" is the equilibrium amount of protamine necessary to complex with the analog as taught by Krayenbihl and Rosenberg, STENO MEMORIAL HOSPITAL REPORT (COPENHAGEN), 1:60 (1946). The isophane ratio is determined by titration in a manner well known in the art and described in Krayenbihl, et al.

The present invention provides an insulin analogprotamine formulation, which comprises: a monomeric insulin analog, protamine, zinc, and a phenolic derivative. The concentration of protamine is preferably about 0.2 to about mg of protamine to 100 U of insulin analog on a free base basis. Most preferably, the range of protamine is about 0.27 mg/100 U to about 0.35 mg/100 U. The concentration of zinc is from about 0.35 to about 0.9 on a weight basis.

Preferably, the concentration of zinc is about 0.7 The phenolic derivative is m-cresol, phenol or a mixture of m-cresol and phenol. Preferably the phenolic derivative is m-cresol and phenol. The concentration of the phenolic derivative is known to one skilled in the art. The 25 concentrations must be sufficient to maintain preservative effectiveness, retard microbial growth. In general, the concentration of phenolic is, for example in the range of 1.0 mg/mL to 6.0 mg/mL; preferably greater than about 2.5 mg/mL. The most preferred concentration is about 3 mg/mL. The presence of a phenolic derivative is critical because it acts to complex the analog, protamine and zinc in addition to serving as a preservative. However, it is believed that only one molecule of phenol per molecule of insulin analog is bound to the crystal structure.

Preferably, an isotonicity agent is added to the formulation. The preferred isotonicity agent is glycerin.

X-9418 FOR 7 The concentration of the isotonicity agent is, for example 14 mg/mL to 18 mg/mL, preferably about 16 mg/mL.

The pH of the formulation may be buffered with a physiologically tolerated buffer, preferably a phosphate buffer, like dibasic sodium phosphate. Other physiologically tolerated buffers include TRIS, sodium acetate, or sodium citrate. The selection and concentration of buffer is known in the art. Generally, the concentration is, for example, about 1.5 mg/mL to 5.0 mg/mL; preferably 3.8 mg/mL.

The present invention further provides specific conditions under which the insulin analog-protamine exists as a stable crystal. Formulations of these crystals are defined as insulin analog-NPD. Insulin analog-NPD is a formulated suspension of insulin analog-NPD crystals and yields upon use an intermediate duration of action. The profile of activity of insulin analog-NPD is quite surprising in view of the lack of self-association of the monomeric analog.

The ability to form an intermediate acting formulation with a monomeric analog is demonstrated in FIGURE I. FIGURE I discloses a profile of action for LysB 2 8proB 2 9_ hI-NPD and human insulin-NPH. The NPD profile is similar to insulin-NPH. The duration of action for the NPD formulation and the insulin-NPH formulation are approximately equal.

However, most significantly, the present formulation rises 25 more rapidly and remains stable for a longer period than insulin-NPH. This difference is quite unexpected in view of the fast-acting profile of the monomeric analog.

A particularly preferred insulin analog-protamine formulation, LysB28proB29_human insulin-NPD, comprises: LysB28proB29-human insulin, about 0.27 to about 0.32 mg protamine/100 U of insulin analog, about 0.35 to about 0.9 zinc by weight, and a phenolic derivative. The concentration of protamine is preferably 0.3 mg/100 U on a free base basis.

The invention also provides the process for 35 preparing LysB28proB29-huma n insulin-protamine crystals, which comprises: X-9418 FOR 8 combining an aqueous solution of LysB 2 8ProB 29 -human insulin in a hexamer association state, and a protamine solution at a temperature from about 80 to about 22 0

C;

said aqueous solution comprising from about 0.35 to about 0.9% zinc by weight, LysB 2 8ProB 29 -human insulin, and a phenolic derivative at a pH of about 7.1 to about 7.6; said protamine solution comprising protamine at a pH of about 7.1 to about 7.6 such that the final concentration of protamine is about 0.27 to about 0.32 mg protamine/100 U of insulin analog.

At the time of invention it was known that monomeric insulin analogs have a lesser tendency to associate and form hexamers. The conditions necessary to cause the monomeric insulin analogs to associate with protamine to form crystals were previously unknown in the art. Previous studies relate to insulin. The teachings regarding the preparation of insulin-NPH (neutral protamine formulation according to Hagedorn) or isophane insulin formulations by KrayenbUhl and Rosenberg, STENO MEMORIAL HOSPITAL REPORT 20 (COPENHAGEN), 1:60 (1946) are not relevant in view of the distinct properties of the monomeric insulin analogs. In fact, the commercial process of producing Humulin-N

T

M

(insulin-NPH), an acid-neutral process, does not produce crystalline insulin analog-NPD.

Most significantly, it has been found that the parameters in the present process namely, the temperature of the crystallization and the formation of a hexamer complex of the insulin analog, zinc, and the phenolic derivative are critical limitations to the formation of stable, 30 LysB28Pro29-hI-NPD crystals.

The temperature of the crystallization must be from about 8 0 C to about 22 0 C, preferably from 13 0 C to 17 0 C. If the temperature is outside of this range, a largely amorphous insulin analog-protamine formulation results.

It is also critical that the insulin analog be transformed to a hexamer state prior to the crystallization.

X-9418 FOR 9 The crystallization results in an amorphous product when the process is carried out with a monomeric association state.

Crystals form without agitation in five to thirty-six hours hours. Good quality crystals are generally formed in 24 hours.

Soluble monomeric insulin analog is complexed to a hexamer association state by suspending solid monomeric analog in a diluent containing the phenolic derivative and adding zinc until the concentration is from about 0.35 to about 0.9 on a weight basis. Zinc is preferably added as a salt. Representative examples of zinc salts include zinc acetate, zinc bromide, zinc chloride, zinc fluoride, zinc iodide and zinc sulfate. The skilled artisan will recognize that there are many other zinc salts that also might be used in the process of the present invention. Preferably, zinc acetate or zinc chloride is used.

:Dissolution of the insulin analog in the diluent may be aided by what is commonly known as an acid dissolution, In an acid dissolution, the pH is lowered to •20 about 3.0 to 3.5 with a physiologically tolerated acid, preferably HCI, to increase solubility of the analog. Other physiologically tolerated acids include acetic acid, citric acid, and phosphoric acid. The pH is then adjusted with a physiologically tolerated base, preferably NaOH to about 7.1 to 7.6 for the crystallization. Other physiologically tolerated bases include KOH and ammonium hydroxide.

Most significantly, the process of producing LysB28proB29-hI-NPD complex is sensitive to the concentration of NaCl. If the concentration exceeds about 4 mg/mL, the 30 insulin analog-NPD crystals become mixed with amorphous product. Accordingly, it is preferred that the monomeric analog is dissolved at neutral pH to avoid the formation of salt ions. Alternatively, the analog may be dissolved in the diluent at an acid pH prior to the addition of the buffer.

This reduces the concentration of salts generated due to the pH adjustment. However, the order that the constituents are X-9418 FOR 10 added is not critical to the formation of the hexamer or the amorphous formulation.

As previously disclosed, an isotonicity agent may be added to the formulations of the present invention. The addition of the isotonicity agent can be to the analog solution, to the protamine solution, or to the final insulin analog-NPD formulation. Likewise, the addition of the physiologically tolerated buffer may be added to the analog solution, to the protamine solution, or to the final insulin analog-NPD formulation. However, it is preferred that both the analog solution and the protamine solution contain the isotonicity agent and the buffer prior to combining the aqueous solution and the protamine. Because of the NaCl effects on the process for producing crystalline insulinanalog-NPD, glycerin is the preferred isotonicity agent.

The invention also provides insulin analog formulations, which comprise mixtures of insulin analog-NPD as a crystalline solid and soluble insulin analog. These mixtures are prepared in a range of about 1:99 to 99:1, by 20 volume suspended insulin analog-NPD to soluble insulin analog. The soluble insulin analog is a monomeric insulin analog dissolved in an aqueous diluent comprising: zinc, a phenolic derivative, an isotonicity agent, and buffer. The concentrations described in the diluent are the same as previously disclosed herein. Preferably the ratio of insulin analog-NPD to soluble insulin analog is 25:75 to 75:25; and more preferably, 50:50. The mixtures are readily prepared by mixing the individual constituents.

The mixed formulations of the present invention are 30 especially suitable for the treatment of diabetes mellitus because of the combination of a rapid onset of action and prolonged duration. These mixtures allow "fine control" by varying the amount of each individual constituent based on the needs, diet, and physical activity of the patient. The mixture of suspended insulin-analog-NPD and soluble insulin analog are also advantageous because they are homogeneous, X-9418 FOR 11 any equilibrium exchange between the suspended crystals and soluble insulin analog is transparent.

The insulin analogs of the present invention can be prepared by any of a variety of recognized peptide synthesis techniques including classical (solution) methods, solid phase methods, semi synthetic methods, and more recent recombinant DNA methods. For example, Chance et al., EPO publication number 383 472, and Brange et al., EPO 214 826, disclose the preparation of various monomeric analogs.

The following examples are provided merely to further illustrate the preparation of the insulin analogs and the invention. The scope of the invention is not construed as merely consisting of the following examples.

Example 1 Preparation of LysB 2 8 ProB 29 hI-NPD A solution of LysB28proB29-human insulin (LysB 2 8proB 2 9 -hI) at 200 IU/mL (U200) concentration was prepared by dissolving zinc containing crystals of 20 LysB 2 8proB 29 -hI in a preservative/buffer system containing: 1.6 mg/mL m-cresol, 0.73 mg/mL phenol (equivalent to 0.65 mg/mL phenol calculated as 89 16 mg/mL glycerin, and 3.78 mg/mL of dibasic sodium phosphate buffer. The endogenous zinc level in the crystals was supplemented by adding an appropriate volume of an acidic ZnO solution (10 mg/mL) to achieve a final concentration of 0.025 mg/100 IU Dissolution of LysB 2 8proB 29 -hi was accomplished at ambient temperature by lowering the pH to about 3 with gL volumes of 5 M HC1. After the solution had clarified, the pH was 30 readjusted to 7.5 with pL volumes of 5 M NaOH.

A protamine solution was prepared by dissolving enough solid protamine sulfate in the preservative/buffer solution to achieve a final concentration of 0.6 mg/100 IU calculated on a free base basis. The pH of this solution was adjusted to 7.5 and equilibrated at 15 0

C.

Both solutions were diluted to final concentration with water for injection and filtered. 5 mL aliquots of the X-9418 FOR 12 LysB 2 8proB29-hI subsection were filled into separate clean glass vials, and the samples were incubated in a water bath at 15 OC. After appropriate time for equilibration minutes), precipitation was induced by rapidly adding 5 mL of the protamine solution to the LysB 2 8proB29-hi samples. The crystallization was allowed to proceed about 24 hours at oC.

Example 2 Preparation of LysB28proB29-hi-NPD The process is identical to Example 1, except that the dissolution of LysB 28 proB 29 -hi occurs at neutral pH. The process was carried out such that the final pH was 7.4.

Examle 3 Preparation of LysB28roB29-hI-NPD SInsulin analog-NPD was prepared in a manner analogous to Example 1, but the acid dissolution of LysB 2 8proB 2 9-hI was carried out in the presence of all 20 excipients except the dibasic sodium phosphate buffer. Solid dibasic sodium phosphate is added after the insulin analog solution was returned to pH 7.4. The addition of dibasic sodium phosphate clarified the solution.

Example 4 Preparation of insulin analog-NPD mixture formulations Mixtures of intermediate and rapid acting LysB 2 8proB29-hI formulations are prepared as follows. The intermediate acting, suspension preparation is prepared by 30 the methods described in Example 3 and serves as the intermediate acting section for the mixture. A separate solution of LysB 2 8proB 29 _hi (100 IU) is prepared by dissolving zinc-containing LysB 2 8 proB 2 9 _hi crystals at ambient temperature in the diluent described in Example 1.

The endogenous zinc level of LysB 28 proB 29 -hi in this solution is supplemented by the addition of acidic ZnO solution to match the level in the suspension section 0.025 mg/100

I

X-9418 FOR 13 IU Water for injection is used to dilute the solution to final concentration after the pH is adjusted to 7.4 using 10% solutions of HC1 and/or NaOH. This solution is the rapid acting section of the mixtures. The final mixture is prepared by combining appropriate volumes of the intermediate and rapid acting subsections to achieve the desired ratio. A 50/50 mixture is prepared by combining 1 part of the intermediate acting section with 1 part of the rapid acting section by volume.

Example Effect of ionic strength on LysB 2 8ProB 2 9-hI protamine crystallization The effect of ionic strength on the crystallization was evaluated by the addition of NaC1 to the LysB 2 8proB 29 -hI section prior to mixing with protamine. NaC1 was added so that the total concentration was 20, 30, and 40 mM 1.8, and 2.3 mg/ml). The volume particle size displayed multimodal behaviour (additional peaks at small particle sizes), 20 as the NaCl concentration was increased. The volume mean particle size decreased as NaC1 concentration was increased indicating an increase in amorphous material. Results of particle size vs. NaC1 concentration are as follows: a rNaCl1 Volume Mean Particle Size 13 mM 3.9 mM 30 mM 3.3 40 mM 3.2 The microscope analysis showed that all samples contained a mixture of amorphous and crystalline material. The sample containing 40 mM NaC1 had mostly amorphous material and very few crystals.

23. AUG. 999 17:32 SPRIJSGN FERGUSON 61 292815486 NO. 5517 P. 6/27 14 Example 6 Comparative dynamics of Lys "2BProB29-hI-NPD and human insulin-NPH This study was carried out in a conscious dog model. Prior 10 the commencement Of the srudy, three basal samples were taken, An infuision Of somnatostatin (0.3 V~tIg/Kg- F) min.) was initiated. After a 10 minute interval, a subcutaneous injection of either NPD or NPI-I wits administered. Frequent monitoring of plasma glucose was initiated and a v"Wiahle gjlucose infuision was given so as to maintain near-norma] glycemia.

Samples were taken thiroughout and were analyzed for imrnunoreactive insulin (Linco 0* antibodY) and glucose, The results are illustrated in FIGURE~ 1.

U Example 7 0 *Preparation of Asp(B328) Analog Protamine CrySTas :A subsection of Asp(B28)-hI at 200 IU/rnL (U200) concentration was prepared by dissolving lyophilized bulk (95% purity) in at preservative/buffer systmn containing: 1.6 ing/mL mn-cresol, 0,731ng/mL phenol (equivalent to 0.65mg/mL phenol calculated as i F 89 l61rng/mL glycer-in. and 3.78mg/tnL dibasic sodium phosphate- Zinc was added to t he system using atappropriate volume of an acidic ZnO solution (l0mg/mL) to obtain a finial concentration of 0.025mg/100 1U. Dissolution of Asp(B28) was achieved at ambient temperature at neutral p1. The final pH- of [he section was 7.4.

A crystallization was carried ats described in Example 2. Final protamine concentrations of O.3rng/100U, 0.35amg/100U, and 0.41ng/1OUJ were investigated. These protamine concentration correspond to 9.3% and 10,5%7 respectively on a weiglht/weigh! basis. Incubation temperatures included 5 0 C (.3mg/1OU only), 15'C and so 22'C, After 24 hr. at these temperatures, samples were analyzed for crystal formation- Results as determined by microscopy illustrate a mixture of a few crystals and amorphous product.

r-y "P1 [N;\LI~aa]127 :SSD 23/08 '99 MON 17:36 [TX/RX NO 9662] SX-9418 FOR 15 Example 8 Preparation of Asp(B28) Analog-Protamine Crystals The crystallization Asp(B28) Protamine was performed as described in Example 7, except that the protein was first dissolved in a buffer-free diluent. The addition of the acidic ZnO stock was sufficient to acidify the sample to pH 2.0-2.5. After the solution had clarified, the pH was readjusted to approximately pH 7 with gL volumes of 5 N NaOH.

Sodium phosphate, dibasic, was added using a concentrated stock solution at 47.25 mg/mL to achieve the final concentration of 3.78 mg/mL. The subsection was adjusted to pH 7.4 using gL quantities of HC1.

Crystallization was initiated by combining the Asp(B28) and protamine sections, as described in previous examples.

Final protamine concentrations of 0.3 mg/100U, 0.35 mg/100U, and 0.4 mg/100U were investigated. Incubation temperatures included 15 °C and 22 After 24 hr. at these temperatures, samples were analyzed for crystal formation.

Results as determined by microscopy illustrate a mixture of a 20 crystals and amorphous material.

Example 9 Preparation of Leu(B28)Pro(B29) Analog-Protamine Crystals A subsection of Leu(B28)Pro(B29) (93% purity) at 200 IU/mL (U200) concentration was prepared as described in Example 8 using an acid dissolution of the bulk followed by pH adjustment with 5N NaOH to pH 7.4. Crystallization was as described above. Final protamine concentrations of 0.3 :mg/100U, 0.35 mg/100U, and 0.4 mg/100U were investigated.

30 Incubation temperatures included 5 OC, 15 OC and 22 oC.

After 24 hr. at these temperatures, all samples contain some crystals, but were primarily amorphous as determined by microscopy.

16 Example Des(B27)hI-protamine crystals A subsection of DesThr(B27) (97.37 purity) at 200 IU/mL (U200) concentration was prepared as described in Example 8 using an acid dissolution of the bulk followed by pH adjustment with 5N NaOH to pH 7.4. A crystallization was carried out as described in Example 8. Final protamine concentrations of 0.3 mg/100U, 0.35 mg/100U, and 0.4 mg/100U were investigated. Incubation temperatures included 15 OC and 22 OC. After 24 hr. at these temperatures, all samples were primarily amorphous as determined by microscopy.

Qualitatively, crystals were observed.

Example 11 Des(B28-B30)hI-protamine A subsection of Des(28-30) (96.3 purity) at 200 IU/mL (U200) concentration was prepared as described in :Example 8 using an acid dissolution of the bulk followed by S. pH adjustment with 5N NaOH to pH 7.4. A crystallization was S 20 attempted using the neutral/neutral combination method of the protein and protamine sections as described above. Final protamine concentrations of 0.3 mg/100U, 0.35 mg/100U, and 0.4 mg/100U were investigated. Incubation temperatures Sincluded 15 oC and 22 oC. After 24 hr. at these 25 temperatures, all samples were primarily amorphous as determined by microscopy. Qualitatively, crystals were observed. The crystals were well defined.

Example 12 30 Asp(B28) Analog-Protamine A insulin Asp(B28)-human insulin analog solution was prepared by dissolving 16.6 mg of the protein in 1 mL of a solution containing 3.2 mg/mL m-cresol, 1.3 mg/mL phenol and 32 mg/mL glycerin. A 14.4 UIL aliquot of an acidic zinc stock solution (10 mg/mL in Zn 2 prepared by dissolving 0.311 g of zinc oxide in 5 mL of 10% HC1 and diluting to mL with water) was added. The solution pH was 2.3 which allowed for X-9418 FOR 17 complete dissolution of the protein. A 10 gL aliquot of NaOH was added to adjust the pH to 7.06. To the solution was added 100 gL of 0.28 M dibasic sodium phosphate, pH 7.0 which increased the solution pH to 7.27. A 870 gL aliquot of water for injection was added to the solution. Additional 10% HC1 (1 gL) and NaOH (0.7 gL) were added, and the final volume of the solution was brought to 2 mL with water for injection resulting in a final pH of 7.26. The solution was filtered through a 0.2 Jim Supor® Acrodisc® 13, Gelman Sciences) filter before use.

Protamine stock solutions were prepared by dissolving protamine sulfate in a solution containing 1.6 mg/mL m-cresol, 0.65 mg/mL phenol, 16 mg/mL glycerin and 14 mM dibasic sodium phosphate. The final pH of the solution was adjusted to 7.3. The final protamine concentration was 0.60 mg/100U on a free base basis. Both solutions were filtered through 0.22 pm (Millipore Sterivex-GV) filter units before use.

Crystallization was achieved by mixing the 20 Asp(B28)-human insulin solution in a 1:1 ratio at controlled temperature as outlined in Table 1. The final mixture conditions were 3.94 mg/mL Asp(B28)-human insulin, 0.0359 mg/mL zinc ions, 1.6 mg/mL m-cresol, 0.65 mg/mL phenol, 16 mg/mL glycerin, 14 mM dibasic sodium phosphate and 0.30 mg/100U of protamine at pH 7.3. Specifically, 50-200 RL portions of the AspB 2 8-human insulin solution were transferred to glass vials, and the samples were equilibrated to 4, 8, 15 or 23 (ambient temperature) OC. Portions of both protamine solutions were also equilibrated at these 30 temperatures. After 15-20 minutes, an equivalent volume of either protamine solution was pipetted into the Asp(B28)human insulin samples. The mixture was gently swirled, capped and then left quiescent at controlled temperature during the crystallization period. All of the samples were examined by microscopy after 24 hours and found to be predominantly amorphous. After 48 hours, the sample containing 0.30 mg/100 U of protamine and incubated at 15 °C 18 showed extensive amounts of needle-like crystals and some amorphous material.

Example 13 A insulin Asp(B28)-human insulin analog solution was prepared by dissolving 10.62 mg of the protein in 0.71 mL of a solution containing 3.2 mg/mL m-cresol, 1.3 mg/mL phenol and 32 mg/mL glycerin. A 10.2 RL aliquot of an acidic zinc stock solution (10 mg/mL in Zn 2 prepared by dissolving 0.311 g of zinc oxide in 5 mL of 10% HC1 and diluting to mL with water) was added. The solution pH was 2.3 which allowed for complete dissolution of the protein. A 6.5 pL aliquot of NaOH was added to adjust the pH to 7.00. To the solution was added 71 AL of 0.28 M dibasic sodium phosphate, pH 7.0 which increased the solution pH to 7.26. A 620 gL aliquot of water for injection was added to the solution. Additional 10% HC1 (0.2 gL) and NaOH (0.6 gL) were added, and the final volume of the solution was brought to 1.42 mL with water for injection resulting in a final pH of 7.42. The solution was 20 filtered through a 0.2 m Supor® Acrodisc® 13, Gelman Sciences) filter before use.

A protamine stock solution was prepared by dissolving protamine sulfate in a solution containing 1.6 mg/mL m-cresol, 0.65 mg/mL phenol, 16 mg/mL glycerin and 14 mM dibasic sodium phosphate. The final pH of the solution was adjusted to 7.4, and the final protamine concentration was 0.60 mg/100U on a free base basis. The solution was filtered through a 0.22 pRm (Millipore Sterivex™-GV) filter unit before use.

30 Crystallization was achieved by mixing the Asp(B28)-human insulin solution in a 1:1 ratio with the protamine solution as described in Example 12 at controlled temperatures 130C, 150C, 17°C and 230C. The results are presented in Table 1. The final mixture conditions were 3.74 mg/mL Asp(B28)-human insulin, 0.0359 mg/mL zinc ions, 1.6 mg/mL m-cresol, 0.65 mg/mL phenol, 16 mg/mL glycerin, 14 mM dibasic sodium phosphate and 0.30 mg/100U of protamine at 19pH 7.4. Four different crystallization temperatures were evaluated. A 1 mL aliquot of the AspB 28 -human insulin equilibrated at 15 °C was mixed with 1 mL of the protamine solution adjusted to the same temperature. After gentle swirling the preparation was left quiescent at 15 oC.

Another sample was prepared by equilibrating 100 JL of the Asp(B28)-human insulin solution to 13 and then combining with 100 gL of the protamine solution adjusted to the same temperature. The final mixture was incubated at 13 OC. The third sample was prepared in a similar manner except that the two 100 yL aliquots were equilibrated, combined and then incubated at 17 OC. The final solution was prepared by mixing ambient temperature equilibrated, 80 gL aliquots of the Asp(B28)-human insulin and protamine solutions and incubating at ambient temperature (23 All samples were evaluated by microscopy after 24 hours and other time intervals thereafter as listed in Table 1.

Table 1 20 Crystallization Conditionsa and Results from Microscopy [AspB28] [Protamine] Temperature Time Microscopy Resultb (m/mL) (mg/100 U) (hours) 3.94 0.30 15 24 amorphous 48 crystalline 72 crystalline 96 crystalline S..120 crystalline 3.94 0.30 23 24 amorphous 48 amorphous 72 amorphous 3.74 0.30 13 24 amorphous S* 40 crystalline/amorphous 48 crystalline/amorphous 69 crystalline/amorphous 3.74 0.30 15 24 amorphous/crystalline crystalline 48 crystalline 69 crystalline 3.74 0.30 17 24 amorphous/few crystals crystalline/amorphous X-9418 FOR 20 48 crystalline/amorphous 69 crystalline/amorphous 3.74 0.30 23 24 amorphous amorphous/few crystals 48 amorphous/few crystals 69 amorphous/few crystals a All solutions also contained 0.9% zinc ions, 1.6 mg/mL mcresol, 0.65 mg/mL phenol, 16 mg/mL glycerin and 14 mM dibasic sodium phosphate, pH 7.4.

b Crystallization outcome was evaluated by microscopy at 600X (Nikon Optiphot 66 microscope) or 1000X (Zeiss Axioplan microscope with differential interface contrast) magnification. Both microscopes were equipped with accessories for photography.

Crystals prepared in accordance with the above examples are illustrated in Figure 2 and Figure 3.

*e o oO*o *o

Claims (63)

1. An insulin analogue-protamine complex, which comprises; human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val, or Ala, and Lys at position B29 is Lys or Pro, des(B28-B30)-human insulin, or des(B27)-human insulin; protamine; zinc; and a phenolic derivative s selected from m-cresol, phenol, and mixtures thereof; provided that the insulin analogue is not AspB 2

8-human insulin. 2. The complex of claim 1, which is LysB 2 sProB29-human insulin, about 0.27 to about 0.32mg protamine/1001U of insulin analogue, about 0.35 to about 0.9% zinc by weight, and a phenolic derivative. 3. The complex of claim 1, comprising about 0.2 to about 1.5mg of protamine/1001U of insulin analogue; about 0.35 to about 0.9% zinc by weight; and a phenolic derivative. 4. The complex of any one of claims 1 to 3, in which the complex is crystalline. 5. An insulin analogue-protamine complex, substantially as hereinbefore described with reference to any one of examples 1-6 or 9-11. 15 6. A process for preparing the complex of any one of claims 1 to 3, which process comprises: combining an insulin analogue, protamine, zinc and a phenolic derivative in an aqueous solvent and allowing the complex to form. 7. A process for preparing an insulin-analogue protamine complex, substantially as hereinbefore described with reference to any one of the examples. 8. A complex prepared by the process of claim 6 or claim 7.

9. A parenteral formulation which comprises the complex of any one of claims 1 to 5 or 8 and which further comprises about 0.2 to about 1.5mg protamine/1001U insulin analogue; about 0.35 o about 0.9% zinc by weight; and a phenolic derivative.

10. A parenteral pharmaceutical insulin analogue-protamine formulation, which comprises the complex of any one of claims 1 to 5 or 8 or the formulation of claim 9 together with a pharmaceutically acceptable diluent or carrier.

11. A parenteral pharmaceutical formulation, which comprises: Lys 2 Pro 929 -human insulin, about 0.3mg protamine/1001U of insulin analogue, about 0.7% zinc by weight, about 1.7mg/mL m- cresol, about 0.7mg/mL phenol, about 16mg/mL glycerin and about 378mg/mL dibasic sodium 30 phosphate,

12. The parenteral pharmaceutical formulation as claimed in any one of claims 9 to 11, which further comprises a soluble human insulin analogue as hereinbefore defined.

13. A parenteral pharmaceutical formulation, which comprises: zinc; a phenolic derivative selected from m-cresol, phenol and mixtures thereof; and a mixture of soluble insulin analogue and insulin analogue-protamine crystals; wherein the ratio by weight of the soluble insulin analogue to the insulin analogue-protamine crystals is about 1:99 to 99:1; said insulin analogue is human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val, or Ala; and Lys at position B29 is Lys or Pro; des(B28-B30)-human insulin; or des(B27)-human insulin; provided that the insulin analogue is not AspB 2 8 -human insulin. 24/08 '99 TUE 18:28 [TX/RX NO 9682] 6021 S24. AUG. 1999 18:30 SPRUSON FERGUSON 61 2 92615486 NO. 5794 P. 22/39 22

14. The parenteral pharmaceutical formulation of claim 13, wherein the ratio by weight of the two components is about 75:25 to 25:75. The parenteral pharmaceutical formulation of claim 14, which comprises: Lyse 2 8 ProS 2 9 human insulin and LysB 28 ProBs2-human insulin-protamine crystals. s 16. The parenteral pharmaceutical formulation of claim 15, wherein the ratio by weight of the two components is 50:50, 75:25, or 25:75.

17. A parenteral pharmaceutical formulation which comprises the formulation of any one of claims 11 to 16 combined with a pharmaceutically acceptable diluent or carrier.

18. A process of preparing a parenteral pharmaceutical formulation as claimed in claim 10 or 1l claim 17, which process comprises: suspending insulin analogue-protamine crystals in a pharmaceutically acceptable diluent or carrier.

19. The process of claim 18, wherein the process is carried out at a temperature from about 13"C to about 17°C.

20. A parenteral pharmaceutical formulation prepared by the process of claim 18 or claim 19, is 21. A process for preparing a LysB 2 8 Pro 29 -human insulin-protamine crystal, which comprises: combining an aqueous solution of Lys 82 8 ProB 29 -human insulin in a hexamer association state, and a protamine solution at a temperature from about 8C to about 22*C; said aqueous solution comprising from about 0.35 to about 0.9% zinc by weight, LysB28Pro 29 -human insulin, and a phenolic derivative at a pH of about 7.1 to about 7.6; said protamine solution comprising protamine at a pH of about 7,1 to about 7.6 such that the final concentration of protamine is about 0.27 to about 0.32mg protamine/1001U of insulin analogue.

22. The process of claim 21, wherein the temperature is 15"C; the zinc concentration is 0.7% to and the protamine concentration is-0.3mg/1001U of insulin -nalogue.

23. A Lys 28 Pro -human insulin-protamine crystal prepared by the process of claim 21 or 25 claim 22.

24. A parenteral pharmaceutical formulation comprising the Lys' 2 8 ProB 29 -human insulin- Sprotamine crystal of claim 23 together with a pharmaceutically acceptable diluent or carrier. A process for preparing an AspB 28 -human insulin protamine crystal, which process comprises: combining an aqueous solution of Asps 28 -human insulin in a hexamer association state, 30 and a protamine solution at a temperature from about 5 0 C to 22"C; said aqueous solution comprising about 0.35 to about 0.9% zinc by weight, AspB 2 8 -human insulin, and a phenolic derivative at a pH of about 7.1 to about 7.6; said protamine solution comprising protamine at a pH of about 7.1 to about 7.6 such that the final concentration of protamine is about 0.27 to about 0.32mg protamine/100 U of insulin analogue.

26. The process of claim 25, wherein the process is carried out at a temperature from about 8"C to about 22"C.

27. The process of claim 26, wherein the process is carried out in less than about 4mg/mL sodium chloride.

28. An AspB 28 -human insulin protamine crystal prepared by the process of any one of claims 25 to 27 24/08 '99 TUE 18:28 [TX/RX NO 9682] Q1022 24, AUG. 1999 18:30 SPRUSON FERGUSON 61 2 92615486 NO, 5794 P. 23/39 23

29. A parenteral pharmaceutical formulation comprising the AspB 2 0 -human insulin protamine crystal of claim 28 together with a pharmaceutically acceptable diluent or carrier. A method of treating a patient suffering from diabetes mellitus, which method comprises administering to said patient an insulin analogue-protamine complex of any one of claims 1 to 5 or 8 or a formulation of any one of claims 9 to 17 or

31. The method of claim 30, wherein the insulin analogue-protamine complex are LysB 2 8 ProBs-human insulin-protamine crystals.

32. A method of treating a patient suffering from diabetes mellitus, which method comprises administering to said patient an insulin analogue-protamine complex, which comprises: human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val, or Ala, and Lys at position B29 is Lys or Pro, des(B28-B30)-human insulin, or des(B27)-human insulin; protamine; zinc; and a phenolic derivative selected from m-cresol, phenol, and mixtures thereof, provided that when the insulin is AspB 2 8 -human insulin the concentration of protamine is less than 10% by weight.

33. The method of claim 32 wherein the complex is combined with a pharmaceutically acceptable diluent or carrier in the form of a formulation.

34. A method of treating a patient suffering from diabetes mellitus, which method comprises administering to said patient a LysB 28 Pro-human insulin of claim 23 or a formulation of claim 24.

35. A method of treating a patient suffering from diabetes mellitus, which method comprises administering to said patient a parenteral pharmaceutical formulation, which comprises: zinc; a phenolic derivative selected from m-cresol, phenol and mixtures thereof; and a mixture of soluble insulin analogue and insulin analogue-protamine crystals; wherein the ratio by weight of the soluble insulin analogue to the insulin analogue-protamine crystals is about 1:99 to 99:1; said insulin analogue is human insulin wherein Pro at-position.B28 is substituted with Asp, Lys, Leu, Val, or Ala; Sand Lys at position B29 is Lys or Pro; des(B28-B30)-human insulin; or des(827)-human insulin, S. 25 provided that when the insulin is Asp28 -human insulin the concentration of protamine is less than 10% by weight.

36. The method of claim 35 wherein the formulation is combined with a pharmaceutically acceptable diluent or carrier.

37. A method of treating a patient suffering from diabetes mellitus, which method comprises a, 3o administering to said patient an AspB 2 8-human insulin-protamine crystal of claim 28 or a formulation of claim 29.

38. Use of an insulin analogue-protamine complex of any one of claims 1 to 5 or 8 for the manufacture of a medicament to treat a patient suffering from diabetes mellitus.

39. An insulin analogue-protamine complex of any one of claims 1 to 5 or 8 when used for the treatment of a patient suffering from diabetes mellitus. Use of an insulin analogue-protamine complex, which comprises: human insulin wherein Pro at position 828 is substituted with Asp, Lys, Leu, Val, or Ala, and Lys at position B29 is Lys or Pro, des(B28-B30)-human insulin, or des(B27)-human insulin: protamine; zinc; and a phenolic derivative selected from m-cresol, phenol, and mixtures thereof, provided that when the insulin is Aspe 2 a-human 24/08 '99 TUE 18:28 [TX/RX NO 9682] &023 24. AUG. 1999 18:31 SPRUSON FERGUSON 61 2 92615486 NO. 5794 P. 24/39 24 insulin the concentration of protamine is less than 10% by weight for the manufacture of a medicament to treat a patient suffering from diabetes mellitus,

41. An insulin analogue-protamine complex, which comprises: human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val, or Ala, and Lys at position B29 is Lys or Pro, s des(B28-B30)-human insulin, or des(B27)-human insulin, protamine; zinc; and a phenolic derivative selected from m-cresol, phenol, and mixtures thereof, provided that when the insulin is AspB 2 8-human insulin the concentration of protamine is less than 10% by weight.when used for the treatment of a patient suffenng from diabetes mellitus.

42. Use of a LysM2ProB-human insulin of claim 23 for the manufacture of a medicament to treat a patient suffering from diabetes mellitus.

43. A LysB 2 BProB 29 -human insulin of claim 23 when used to treat a patient suffering from diabetes mellitus.

44. Use of zinc; a phenolic derivative selected from m-cresol, phenol and mixtures thereof; S. and a mixture of soluble insulin analogue and insulin analogue-protamine crystals; wherein the ratio 15 by weight of the soluble insulin analogue to the insulin analogue-protamine crystals is about 1:99 to 99:1; said insulin analogue is human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val, or Ala; and Lys at position B29 is Lys or Pro; des(B28-B30)-human insulin; or des(B27)- human insulin, provided that when the insulin is Asp2-human insulin the concentration of protamine is less than 10% by weight for the manufacture of a medicament to treat a patient suffering from diabetes mellitus. Zinc; a phenolic derivative selected from m-cresol, phenol and mixtures thereof; and a mixture of soluble insulin analogue and insulin analogue-protamine crystals; wherein the ratio by w eight of the soluble insulin analogue to the insulin analogue-protamine crystals is about 1:99 to 99:1; said insulin analogue is human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, 25 Val, or Ala; and Lys at position B29 is Lys or Pro; des(B28-B30)-human insulin; or des(B27)-human Sinsulin, provided that when the insulin is AspB 2 8-human insulin the concentration of protamine is less than 10% by weight when used for treating a patient suffering from diabetes mellitus.

46. Use of an AspB 2 8 -human insulin-protamine crystal of claim 28 for the manufacture of a Smedicament to treat a patient suffering from diabetes mellitus. 30 47. An Aspe2B-human insulin-protamine crystal of claim 28 when used to treat a patient suffering from diabetes mellitus.

48. A parenteral pharmaceutical insulin analog-protamine formulation, which comprises: a mixture of a soluble human insulin analog solution characterised in that the soluble analog is in a hexamer complex and insulin analog-protamine crystals; wherein the ration by weight of the two components is about 1:99 to 99:1 insulin analog to insulin analog-protamine crystal; wherein said insulin analog is human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val, or Ala; and Lys at position B29 is Lys or Pro; des (B28-B30)-human insulin; or des (B27)-human insulin.

49. The parenteral pharmaceutical insulin analog-protamine formulation of claim 48, which comprises: soluble Asp 2 hexamer complexes and AspBa-protamine crystals, 24/08 '99 TUE 18:28 [TX/RX NO 9682] ~]024 S 24, AUG. 1999 18:31 SPRUSON FERGUSON 61 2 92615486 NO, 5794 P. 25/39 09 9 9 a. 4. 0 4 9@ 4* 9 The parenteral pharmaceutical insulin analog-protamine formulation of claim 48, which comprises: soluble LysB 2 BPro B 29 hexamer complexes and LysB 2 zProB 9 -protamine crystals.

51. The parenteral pharmaceutical insulin analog-protamine formulation of any one of claims 48 to 50, wherein the soluble human insulin analog solution further comprises about 0.35% Zn to about 0.9% Zn by weight.

52. The parenteral pharmaceutical insulin analog-protamine formulation of claim 51, wherein the human insulin analog is AspB 2 -human insulin.

53. The parenteral pharmaceutical insulin analog-protamine formulation of claim 51, wherein the human insulin analog is LyszeProB29-human insulin.

54. An insulin analog-protamine complex, with comprises: human insulin wherein Pro at position 828 is substituted with: Asp, Lys, Leu, Val, or Ala, and Lys at position B29 is Lys or Pro, Des(B28-B30)-human insulin, or des(B27)-human insulin; protamine; zinc; and a phenolic derivative; provided that when the insulin is AspE 2 S-human insulin the concentration of protamine is less than 10% by weight. 15 55. An insulin analog-protamine complex, according to claim 54 which is LysB 2 BPro29-human insulin; protamine; zinc; and a phenolic derivative,

56. The complex of claim 54 which is 0.2 to 1.5mg protamine per 1001U of insulin analog, 0.35 to 0.9% zinc by weight, and a phenolic derivative.

57. The complex of claim 55, which is 0.2 to 0.32mg protamine per 1001U of insulin analog, 0.35 to 0.9% zinc by weight, and a phenolic derivative.

58. The complex of claim 56 which is AspB28-human insulin, 0.2 to 0.35mg protamine/1001U of insulin analog, 0.35 to 0.9% zinc by weight, and a phenolic derivative,

59. The complex of any one of claims 54, 55, 56, or 57 in which the complex is in the form of crystals. 2s 60. A parenteral pharmaceutical insulin analog-protamine formulation, which comprises the complex of claims 54, 55, 56, 57, or 58.

61. A parenteral pharmaceutical formulation which comprises: LyseMPro 529 -human insulin, 0.27 to 0.32mg protamine per 1001U insulin analog, and 0.35 to 0.9% zinc by weight; and a phenolic derivative.

62. A parenteral pharmaceutical formulation which comprises: Asp 92 8-human insulin, 0.27 to 0.35mg protamine per 1001U insulin analog, and 0.35 to 0.9% zinc by weight and a phenolic derivative, 63, The parenteral pharmaceutical formulation as claimed in any one of claims 60 through 62, which further comprises soluble insulin analog.

64. A parenteral pharmaceutical formulation, which comprises: a mixture of soluble insulin analog solution comprising a human insulin analog, zinc and a phenolic derivative; and insulin analog- protamine crystals; wherein the ratio by weight of the two components is 1:99 to 99:1 insulin analog to insulin analog-protamine crystals; said insulin analog is: human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val or Ala and Lys at position B29 is Lys or Pro, des(B28-B30)-human 24/08 '99 TUE 18:28 [TX/RX NO 9682] E025 24. AUG. 1999 18:32 SPRUSON FERGUSON 61 2 92615486 NO, 5794 P. 26/39 26 insulin, or des(B27)-human insulin; provided that when the insulin is Asp 2 8 a-human insulin the concentration of protamine is less than 10% by weight. A parenteral pharmaceutical formulation, which comprises: a mixture of soluble insulin analog solution comprising a human insulin analog, zinc and a phenolic derivative; and insulin analog- s protamine crystals; wherein the ratio by weight of the two components is 1:99 to 99:1 insulin analog to insulin analog-protamine crystals; and wherein said insulin analog is Lysa2AProa 9 -human insulin. 66 A parenteral pharmaceutical formulation of claim 64 or 65 wherein the ratio by weight of the two components is 75:25 to 25:75.

67. A parenteral pharmaceutical formulation of claim 66 wherein the ratio by weight of the two components is 50:50.

68. A parenteral pharmaceutical formulation of claim 66 which comprises: LysB 28 Pro 29 human insulin and Lys 82 8 ProB 2 9 -human insulin-protamine crystals.

69. A parenteral pharmaceutical formulation of claim 68 wherein the ratio by weight of the two components is 50:50, 75:25, or 25.75.

70. A parenteral pharmaceutical insulin analog-protamine formulation, which comprises: a mixture of a soluble human insulin analog solution comprising a human insulin analog, zinc and a phenolic derivative characterized in that substantially all of the soluble analog is in a hexamer complex and an insulin analog-protamine complex; wherein the ratio by weight of the two components is 1:99 to 99:1 soluble insulin analog to insulin analog-protamine complex; said insulin analog is: human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val or Ala, and Lys at position B29 is Lys or Pro, des(B28-B30)-human insulin, or des(B27)-human insulin.

71. The parenteral pharmaceutical insulin analog-protamine formulation of claim 70 wherein the ratio by weight of the two components is 75:25 to 25:75.

72. The parenteral pharmaceutical insulin analog-protamine formulation of claim 70 or 71 S 25 which comprises: soluble Asp 2 8 hexamer complexes and AspB28-protamine complexes.

73. The parenteral pharmaceutical insulin analog-protamine formulation of claim 70 or 71 which comprises: soluble LysB 2 8 ProBZ 2 hexamer complexes and LysBMProB 2 g-protamine complexes.

74. The parenteral pharmaceutical insulin analog-protamine formulations of any one of claims 70, 71, 72, or 73 which contains crystals of insulin analog-protamine complex. 30 75. The parenteral pharmaceutical insulin analog-protamine formulation of any one of claims to 74 wherein the soluble human insulin analog solution comprises 0.35% Zn to 0.9% Zn by weight.

76. The parenteral pharmaceutical insulin analog-protamine formulation of claim 75 wherein the human insulin analog is AspB2-human insulin.

77. The parenteral pharmaceutical insulin analog-protamine formulation of claim 75 wherein the human insulin analog is Lys 2 8 sPro--human insulin.

78. A process for preparing the complex in any one of claims 54 to 59 which comprises: combining a monomeric insulin analog, protamine, zinc and a phenolic derivative in an aqueous solvent and allowing the complex to form. 24/08 '99 TUE 18:28 [TX/RX NO 9682] I026 24, AUG. 1999 18:32 SPRUSON FERGUSON 61 2 92615486 NO, 5794 P. 27/39 27

79. A process for preparing AspB 28 -human insulin protamine crystals, which comprises: combining an aqueous solution of AspB 2 8 -human insulin in a hexamer associated state, and a protamine solution at a temperature from 13° to 17°C; said aqueous solution comprising 0.35 to 0.9% zinc by weight, Asps 2 1-human insulin, and a phenolic derivative at a pH of 7.1 to 7.6; said protamine s solution comprising protamine at a pH of 7.1 to 7.6 such that the final concentration of protamine is 0.27 to 0.32mg protamine/1001U of insulin analog. An insulin analogue-protamine complex of any one of claims 1 to 5 or 8 for use in the treatment of a patient suffering from diabetes mellitus,

81. An insulin analogue-protamine complex, which comprises: human insulin wherein Pro at io position B28 is substituted with Asp, Lys, Leu, Val, or Ala, and Lys at position B29 is Lys or Pro, des(B28-B30)-human insulin, or des(B27)-human insulin; protamine; zinc; and a phenolic derivative selected from m-cresol, phenol, and mixtures thereof, provided that when the insulin is Asp9 28 -human insulin the concentration of protamine is less than 10% by weight for use in the treatment of a patient suffering from diabetes mellitus.

82. A LysB 2 8ProB 29 -human insulin of claim 23 for use in treating a patient suffering from diabetes mellitus.

83. Zinc; a phenolic derivative selected from m-cresol, phenol and mixtures thereof; and a mixture of soluble insulin analogue and insulin analogue-protamine crystals; wherein the ratio by weight of the soluble insulin analogue to the insulin analogue-protamine crystals is about 1:99 to 99:1; said insulin analogue is human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val, or Ala; and Lys at position B29 is Lys or Pro; des(B28-B30)-human insulin; or des(B27)-human insulin, provided that when the insulin is AspB 28 -hunan insulin the concentration of protamine is less than 10% by weight for use in treating a patient suffering from diabetes mellitus.

84. An AspB 2 8-human insulin-protamine crystal of claim 28 for use in treating a patient o 25 suffering from diabetes mellitus

85. A medicament to treat a patient suffering from diabetes mellitus when manufactured by the use according to any one of claims 38, 40, 42, 44 or 46. Dated 24 August 1999 ""ELI LILLY AND COMPANY Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 24/08 '99 TUE 18:28 [TX/R0X NO 9682] 027 24/08 '99 TUE 18:28 [TX/RX NO 9682] Q0j27