Title:  Pulmonary insulin crystals

United States Patent:  6,310,038

Assignee:  Novo Nordisk A/S (Bagsvaerd, DK)

Filed:  March 20, 1998

Foreign Application Priority Data:  Mar 20, 1997[DK]  (0317/97)

The present invention relates to zinc free insulin crystals having a diameter below 10 .mu.m and to therapeutic powder formulations suitable for pulmonary administration containing such insulin crystals. The crystals of the present invention exhibit a better stability profile than powders of essentially the same composition prepared by spray drying, freeze-drying, vacuum drying and open drying. The therapeutic powder formulations elucidate better flowing properties than corresponding amorphous powder formulations.

PREFERRED EMBODIMENTS

The zinc free insulin crystals of the invention are advantageously provided in a crystal structure belonging to the cubic crystal system, preferably in the octadecahedral or dodecahedral crystal forms, since these crystal forms result in readily soluble product having excellent flowing properties.

The diameter of the insulin crystals is advantageously kept in the range of 0.2 to 5 .mu.m, preferably in the range of 0.2 to 2 .mu.m, more preferably in the range of 0.5 and 1 .mu.m, to ensure high bioavailability and suitable profile of action, see PCT application No. WO 95/24183 and PCT application No. WO 96/32149.

In a preferred embodiment the insulin used is selected from the group consisting of human insulin, bovine insulin or porcine insulin, preferably human insulin.

In another preferred embodiment the insulin used is selected from the group consisting of rapid-acting insulins, preferably des(B30) human insulin, Asp^B28 human insulin or Lys^B28 Pro^B29 human insulin.

In another preferred embodiment the insulin used is an insulin derivative, preferably selected from the group consisting of B29-N^.epsilon. -myristoyl-des(B30) human insulin, B29-N^.epsilon. -palmitoyl-des(B30) human insulin, B29-N^.epsilon. -myristoyl human insulin, B29-N^.epsilon. -palmitoyl human insulin, B28-N^.epsilon. -myristoyl LyS^B28 Pro^B29 human insulin, B28-N^.epsilon. -palmitoyl Lys^B28 Pro^B29 human insulin, B30-N^.epsilon. -myristoyl-Thr^B29 Lys^B30 human insulin, B30-N^.epsilon. -palmitoyl-Thr^B29 Lys^B30 human insulin, B29-N^.epsilon. -(N-palmitoyl-.gamma.-glutamyl)-des(B30) human insulin, B29-N^.epsilon. -(N-lithocholyl-.gamma.-glutamil)-des(B30) human insulin, B29-N^.epsilon. -(.omega.-carboxyheptadecanoyl)des(B30) human insulin and B29-N^.epsilon. -(.omega.-carboxyheptadecanoyl) human insulin, more preferably Lys^B29 (N-.epsilon. acylated) des(B30) human insulin.

The insulin derivatives have a protracted onset of action and may thus compensate the very rapid increase in plasma insulin normally associated with pulmonary delivery. By carefully selecting the type of insulin, the present invention enables adjustment of the timing and to obtain the desired biological response within a defined time span.

In order to avoid irritation of the lungs and to eliminate immunological reactions, the employed insulin is preferably insulin which has been purified by chromatography, such as MC insulin (Novo), Single Peak insulin (E. Lilly) and RI insulin (Nordisk).

In a preferred embodiment the zinc free insulin crystals according to the invention further comprise a stabilizing amount of a phenolic compound, preferably m-cresol or phenol, or a mixture of these compounds.

The present invention is furthermore concerned with a therapeutic powder formulation suitable for pulmonary administration comprising the zinc free crystals described above.

In a preferred embodiment this therapeutic powder formulation further comprises an enhancer which enhances the absorption of insulin in the lower respiratory tract.

The enhancer is advantageously a surfactant, preferably selected from the group consisting of salts of fatty acids, bile salts or phospholipids, more preferably a bile salt.

Preferred fatty acids salts are salts of C.sub.10-14 fatty acids, such as sodium caprate, sodium laurate and sodium myristate.

Lysophosphatidylcholine is a preferred phospholipid.

Preferred bile salts are salts of ursodeoxycholate, taurocholate, glycocholate and taurodihydrofusidate. Still more preferred are powder formulations according to the invention wherein the enhancer is a salt of taurocholate, preferably sodium taurocholate.

The molar ratio of insulin to enhancer in the powder formulation of the present invention is preferably 9:1 to 1:9, more preferably between 5:1 to 1:5, and still more preferably between 3:1 to 1:3.

The powder formulations of the present invention may optionally be combined with a carrier or excipient generally accepted as suitable for pulmonary administration. The purpose of adding a carrier or excipient may be as a bulking agent, stabilizing agent or an agent improving the flowing properties.

Suitable carrier agents include 1) carbohydrates, e.g. monosaccharides such as fructose, galactose, glucose, sorbose, and the like; 2) disaccharides, such as lactose, trehalose and the like; 3) polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; 4) alditols, such as mannitol, xylitol, and the like; 5) inorganic salts, such as sodium chloride, and the like; 6) organic salts, such as sodium citrate, sodium ascorbate, and the like. A preferred group of carriers includes trehalose, raffinose, mannitol, sorbitol, xylitol, inositol, sucrose, sodium chloride and sodium citrate.

The crystals of the present invention are advantageously produced according to the following procedure:

a) providing a solution of insulin having a pH between 7.0 and 9.5;

b) mixing said solution with a solution of a salt of an alkali metal or an ammonium salt; and

c) recovering the formed crystals.

The salt of an alkali metal or ammonium is preferably selected from the group consisting of the hydrochloride or acetate of sodium, potassium, lithium or ammonia, or mixtures thereof, more preferably sodium acetate.

In order to suppress the solubility of the crystals formed, the solution of insulin and/or the solution of a salt of an alkali metal or an ammonium salt preferably comprises a water miscible organic solvent in an amount which corresponds to 5 to 25% (v/v) in the solution obtained after mixing.

The water miscible organic solvent is preferably selected from the group consisting of ethanol, methanol, acetone and 2-propanol, more preferably ethanol.

A very uniform distribution of crystal sizes and crystals of the same crystallographic form are obtained when the two solutions are mixed within a period of less than 2 hours, preferably less than 1 hour, more preferably less than 15 minutes, still more preferably less than 5 minutes.

The crystallization process by which uniformly sized, small, zinc free crystals is obtained directly, without the use of milling, micronizing, sieving and other dust generating steps, is much to be preferred from the present state of the art in the manufacture of insulin powders for inhalation.

The concentration of insulin after mixing is preferably between 0.5% and 10%, more preferably between 0.5% and 5%, still more preferably between 0.5% and 2%.

The concentration of salt after mixing is preferably between 0.2 M and 2 M, more preferably about 1 M.

The method according to the present invention may further comprise a washing step, in which the crystals obtained are washed with a solution comprising auxiliary substances to be included in the final dry powder, preferably an enhancer and/or a carbohydrate, and optionally comprising 5-25% of an alcohol, preferably ethanol, 5-50 mM of a preservative preferably phenol, and 0.1-2 M of a salt such as sodium acetate.

Claim 1 of 18 Claims

What is claimed is:

1. Zinc free insulin crystals having a diameter below 10 .mu.m, wherein said insulin is selected from the group consisting of human insulin and Lys^B29 (N-.epsilon.-acylated) des(B30) human insulin.

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