FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
PROVISIONAL SPECIFICATION
(See section 10 and rule13)
1. TITLE OF THE INVENTION:
PHARMACEUTICAL COMPOSITIONS COMPRISING INSULIN ANALOGS
2. APPLICANT (S)
(a) NAME: WOCKHARDT LTD.
(b) NATIONALITY: INDIAN
(c) ADDRESS: D4-MIDC Area, Chikalthana, Aurangabad - 431 210,
(M.S.) INDIA.

3. PREAMBLE TO THE DESCRIPTION
The invention relates to an insulin analog protamine crystals comprising monomeric insulin analog, protamine, zinc and one or more ligand selected from group consisting of benzyl alcohol, phenylethanol, phenoxyethanol, benzoic acid, mandelic acid, 2,2,2 trifluro-1-phenyl alcohol, phenylphosphonic acid and derivatives thereof. The invention further provides pharmaceutical composition comprising the same.
The following specification particularly describes the invention and the manner in which it is to be performed.

4. Description
The invention provides an insulin analog protamine crystals comprising monomeric insulin analog, protamine, zinc and one or more ligand selected from group consisting of benzyl alcohol, phenylethanol, phenoxyethanol, benzoic acid, mandelic acid, 2,2,2 trifluro-1-phenyl alcohol, phenylphosphonic acid and derivatives thereof. The invention further provides pharmaceutical composition comprising the same.
insulin lispro, insulin aspart and insulin giulisine are rapid-acting human insulin analog. Insulin lispro is Lys(B28), Pro(B29) human insulin analog, created when the amino acids at positions 28 and 29 on the B -chain of human insulin are reversed. Insulin Lispro is available in the market under the trade name of Humalog or Humalog Mix (in combination with lispro protamine solution).
Insulin aspart is homologous with regular human insulin with the exception of a single substitution of the amino acid proline by aspartic acid in position B28. It is marketed under the trade name of Novolog or Novolog Mix.
Insulin giulisine differs from human insulin in that the amino acid asparagine at position B3 is replaced by lysine and the lysine in position B29 is replaced by glutamic acid. It is available under the trade name of Apidra.
Regular human insulin and insulin analog tend to self associate in solution. Insulin exists in solution (depending on concentration and pH) as monomers or dinners and in rhombohedraf crystals as hexamers. in solution at neutral pH and at physiological concentrations (about 1 ng/ml) insulin exists as a monomer, and it is the monomer which is the active form of the hormone. At higher concentrations at acidic or neutral pH (in the absence of zinc) the insulin monomer self-associates to form dimers and (in the presence of zinc) hexamers. Zinc is added to promote association to hexamers and increase chemical stability.
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Phenolic ligands such as m-cresol, phenol, or a mixture of m-cresol and phenol and non phenolic ligands such as bi- or tricyclic alcohols and purines also affect insulin conformation and association by binding to the insulin hexamer and inducing an allostehc conformational change. The ligand-bound state is known as the R-state, and the apo-form is known as the T-state after the nomenclature of (Monod J, Wyman J, Changeux JP. 1965. On the nature of allostehc transitions: A plausible model. J Mol Biol 12:88-118.). An intermediate conformational stage T3R3 exist between R-state and T-state exist. For insulin, phenolic ligands induce the Te-state hexamer to convert to an R6-state, whereby the eight N-terminal residues of the B-chain convert from an extended conformation to an a-helix. Thus an injection of a solution of R-state insulin hexamers must convert to T-state hexamers, followed by dissociation. The rate-limiting step for the absorption of insulin solutions after subcutaneous injection is considered to be the dissociation of self-associated hexamers to monomers.
Addition of protamine to pharmaceutical preparations containing insulin, insulin analog results in formation of crystaf. Crystalline insufin protamine complex results in retarding the release of insulin and thus prolongs the time of action. In the crystals of the prolonged acting insulin preparations, the insulin is also found to be hexameric (Balschmidt et al.: Acta Chryst. B47, (1991), 975-986).
US Patent No. 5,747,642 and US Patent No. 5,461,031 discloses insulin analog-protamine crystals and pharmaceutical compositions containing the same.
US Patent No. 5,474,978 and US Patent No. 6,489,292 discloses hexameric complex of insulin analog.
US Patent No. 6,960,561 and US Patent No. 6,211,144 disclose a formulation comprising insulin analog.

US Patent No. 6,221,633 discloses insulin analog and depot formulations comprising insulin analog and protamine.
It was observed while working on the insulin analog protamine formulations that addition of protamine to a solution comprising insulin analog, zinc and one or more ligand selected from group consisting of benzyl alcohol, phenylethanol, phenoxyethanol, benzoic acid, mandelic acid, 2,2,2 trifluro-1-phenyl alcohol, phenylphosphonic acid and derivatives thereof results in formation of well defined crystals. The above mentioned ligands induce an allosteric conformational change resulting in formation of stable hexameric complex and when protamine was added to the hexamer, it results in formation of crystals.
One of the embodiments of the invention provides insulin analog-protamine crystals comprising monomeric human insulin analog or derivative, zinc ions, protamine and one or more ligand selected from group consisting of benzyl alcohol, phenylethanol, phenoxyethanol, benzoic acid, mandelic acid, 2,2,2 trifluro-1-phenyl alcohol, phenylphosphonic acid and derivatives thereof.
The term "monomeric insulin analog" as used herein, designates a fast acting insulin analog that is less prone to dimerization or self-association. A monomeric insulin analog of the invention is human insulin comprising one or more of the following modifications: (1) Asparagine at B3 position is substituted with lysine; (2) Pro at position B28 is substituted with Asp, Lys, Leu, Val, or Ala; (2) Lys at position B29 is Lysine, glutamic acid or Proline. In addition to these substitutions, the monomeric insulin analog may also comprise one or more of the following modifications: (1) Asn at position A21 may be substituted with Asp, Gly or Glu; (2) Asn at position B3 may be substituted with Asp and Gin. One skilled in the art would recognize that other modifications to the monomeric insulin analog are possible and widely accepted in the art.
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In another embodiment of the invention, the monomeric human insuiin analogs are not acylated.
In another embodiment of the invention, monomeric insulin analog or derivative thereof is selected from the group consisting of AspB28-human insulin, LysB28ProB29, or LysB3GluB29.
In another embodiment of the invention, the human insulin analog is present in the form of hexameric complex comprising human insulin analog, zinc ions and one or more ligand selected from group consisting of benzyl alcohol, phenylethanol, phenoxyethanol, benzoic acid, mandelic acid, 2,2,2 trifluro-1-phenyl alcohol, phenylphosphonic acid and derivatives thereof.
In another embodiment of the invention, the insulin analog-protamine crystals further comprises crystallizing aids such as urea or methylparaben.
In another embodiment amount of insulin analog in hexameric complex or formulation containing the hexameric complex varies from about 0.5mg/ml to about 20mg/ml.
The amount of insulin lispro in hexameric complex or formulation containing the hexameric complex varies from about 0.5mg/ml to about 20mg/ml.
In another embodiment of the invention the ligand is benzyl alcohol.
In another embodiment of the invention, zinc is added in the form of zinc salts selected from the group consisting of zinc acetate, zinc bromide, zinc chloride, zinc fluoride, zinc iodide, zinc oxide and zinc sulfate.
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The concentration of zinc in insulin analog hexamer or formulation containing the same varies from about 10|ig/ml to about 50p.g/ml. The concentration of benzyl alcohol varies from about 1.0 mg/ml to 2.5 mg/ml.
The amount of protamine present in the insulin analog protamine crystals varies from about 0.2 mg/ml to 1 mg/ml.
One of the embodiments of the invention provides a pharmaceutical composition comprising insulin analog-protamine crystals comprising monomeric human insulin analog or derivative, zinc ions, protamine and one or more ligand selected from group consisting of benzyl alcohol, phenylethanol, phenoxyethanol, benzoic acid, mandelic acid, 2,2,2 trifluro-1-phenyl alcohol, phenylphosphonic acid and derivatives thereof.
In another embodiment of the invention, the pharmaceutical composition further comprises of buffer, complexing agent, surfactant, stabilizers or isotonicity agent.
Suitable buffers are selected from the group consisting of sodium phosphate, TRIS, sodium acetate, and sodium citrate.
In another embodiment of the invention isotonicity agent can be selected from the group consisting of glycerol, mannitol, sorbitol, lactose, dextrose, trehalose, sodium chloride, propylene glycol and dimethyl sulfone.
Suitable surfactants or stabilizers include one or more of polysorbate, a poloxyethylene ether, a polyethylene glycol ether, or urea.
Suitable complexing agents include one or more of ethylenediamine tetraacetic acid, ethyleneglycol tetraacetic acid, and salts thereof.
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In another embodiment of the invention, the pH of the composition is from about 6.5 to about 8.0.
In another embodiment of the invention, the composition is a liquid for parenteral administration.
The composition of the present invention is free of phenol or phenolic derivatives such as cresol or metacresol.
In another embodiment of the invention, crystals comprising monomeric insulin analog, zinc and and one or more ligand selected from group consisting of benzyl alcohol, phenylethanol, phenoxyethanol, benzoic acid, mandelic acid, 2,2,2 trifluro-1-phenyl alcohol, phenylphosphonic acid derivatives thereof can be given in combination with insulin lispro hexamer solution. The ratio of crystals comprising insulin analog protamine to dissolved insulin analog is in range of 1:99 to 99:1.
Vehicles suitable for formulating the invention include but are not limited to water for injection.
Example 1
Table 1: Liquid Composition of Insulin Lispro protamine

Sr. No. Ingredients Units- mg/ml
1 Lispro API 3.5
2 Benzyl alcohol 1.6-2.5
3 Urea 0.65-1.6
4 Dibasic sodium phosphate 3.78
5 Zinc 0.025 ,
6 Protamine sulphate 0.76
7 Glycerol 16
Water for injection
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Procedure - Benzyl alcohol, urea, disodium hydrogen phosphate, and glycerol were added in water for injection. Protamine sulphate solution in water for injection was added to the above solution. Insulin Lispro and zinc oxide were dissolved in an acidic solution which was than added to the above solution by gentle mixing. The final pH of the solution was adjusted 7.2 ± 0.2. The final solution was filtered through a 0.2 u syringe filter.
While the invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the invention.
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We Claim
1. Insulin analog-protamine crystals comprising monomeric human insulin
analog or derivative, zinc ions, protamine and one or more ligand selected
from group consisting of benzyl alcohol, phenylethanol, phenoxyethanol,
benzoic acid, mandelic acid, 2,2,2 trifluro-1-phenyl alcohol,
phenylphosphonic acid and derivatives thereof.
2. The insulin analog protamine crystals of claim 1, wherein the human insulin analog or derivative is a hexameric complex.
3. The insulin analog protamine crystals of claim 1, wherein the monomeric insulin analog or derivative thereof is selected from the group consisting of AspB28-human insulin, LysB28ProB29, and LysB3GluB29.
4. The insulin analog protamine crystals of claim 1, further comprises of crystallizing aid selected from the group consisting of urea and methylparaben.
5. The insulin analog protamine crystals of claim 1, wherein the ligand is benzyl alcohol.
6. The insulin analog protamine crystals of claim 1, wherein the zinc is added in the form of zinc salts selected from the group consisting of zinc acetate, zinc bromide, zinc chloride, zinc fluoride, zinc iodide, zinc oxide and zinc sulfate.
7. A pharmaceutical composition comprising the insulin analog-protamine crystals of claim 1.
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8. The pharmaceutical composition of claim 7, additionally comprises of one or more insulin analog hexamer in ratio of 1:99 to 99:1.
9. The pharmaceutical composition of claim 7, further comprises of buffer, complexing agent, surfactant, stabilizers and isotonicity agent.
10. The pharmaceutical composition of claim 7, wherein the pH of the composition is from about 6.5 to about 8.0.
Dated this day of July, 2008 For Wockhardt Limited
(JLU_
(Mandar kodgule) Authorized Signatory
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