the expression “ enhancer ” as used herein refers to a substance enhancing the absorption of insulin , insulin analogue or insulin derivative through the layer of epithelial cells lining the alveoli of the lung into the adjacent pulmonary vasculature , i . e . the amount of insulin absorbed into the systemic system is higher than the amount absorbed in the absence of enhancer . in the present context the expression “ powder ” refers to a collection of essentially dry particles , i . e . the moisture content being below about 10 % by weight , preferably below 6 % by weight , and most preferably below 4 % by weight . the diameter of the crystals is defined as the martin &# 39 ; s diameter . it is measured as the length of the line , parallel to the ocular scale , that divides the randomly oriented crystals into two equal projected areas it is an object of the present invention to provide an insulin powder suitable for pulmonary delivery which has a reduced tendency to associate into aggregates in the dry powder compared to the pulmonary insulin particles described in the prior art . according to the present invention this object has been accomplished by providing zinc free insulin crystals having a diameter below 10 μm . the crystals of the present invention furthermore exhibit a better stability profile than powders of essentially the same composition prepared by spray drying , freeze - drying , vacuum drying and open drying . this is probably due to the amorphous state of powders prepared by the other methods described . by this means it is possible to store the powder formulations based on the crystals of the present invention at room temperature in contrary to human insulin preparations for injections and some amorphous insulin powders without stabilizing agent which have to be stored between 2 □ c to 8 □ c . furthermore , therapeutical powder formulations comprising the insulin crystals of the invention elucidates better flowing properties than corresponding amorphous powder formulations . 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 μm , preferably in the range of 0 . 2 to 2 μm , more preferably in the range of 0 . 5 and 1 μm , 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 ε - myristoyl - des ( b30 ) human insulin , b29 - n ε - palmitoyl - des ( b30 ) human insulin , b29 - n ε - myristoyl human insulin , b29 - n ε - palmitoyl human insulin , b28 - n ε - myristoyl lys b28 pro b29 human insulin , b28 - n ε - palmitoyl lys b28 pro b29 human insulin , b30 - n ε - myristoyl - thr b29 lys b30 human insulin , b30 - n ε - palmitoyl - thr b29 lys b30 human insulin , b29 - n ε -( n - palmitoyl - γ - glutamyl )- des ( b30 ) human insulin , b29 - n ε -( n - lithocholyl - γ - glutamyl )- des ( b30 ) human insulin , b29 - n ε -( ω - carboxyheptadecanoyl )- des ( b30 ) human insulin and b29 - n ε -( ω - carboxyheptadecanoyl ) human insulin , more preferably lys b29 ( n - ε acylated ) des ( b30 ) human insulin . the insulin derivatives has 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 10 - 14 fatty acids , such as sodium caprate , sodium laurate and sodium myristate . 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 : providing a solution of insulin having a ph between 7 . 0 and 9 . 5 ; mixing said solution with a solution of a salt of an alkali metal or an ammonium salt ; and 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 crystallisation 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 . this invention is further illustrated by the following examples which , however , are not to be construed as limiting . 2 g of highly purified human insulin is dissolved in 100 ml 10 mm tris buffer , ph 8 . 0 in 20 % ( v / v ) of ethanol in water . to this solution is added 100 ml 2 m sodium acetate under stirring . a precipitate forms immediately . after 2 days at room temperature microscopy shows small crystals having a diameter between 0 . 5 and 1 μm . the crystals are collected by centrifugation at − 10 □ c , washed once with 20 ml ice cold 10 % ethanol ( v / v ) in water , isolated by centrifugation and dried by lyophilization . the obtained crystals are shown in fig1 . 10 mg of human insulin and 5 mg of taurocholic acid sodium salt are dissolved in 500 μl 10 mm tris buffer , ph 8 . 0 in 20 % ( v / v ) of ethanol in water . to this solution is added 500 μl 2 m sodium acetate . microscopy after 1 hour and after 24 hours shows identically appearance of the crystals , i . e . uniformly sized crystals having diameters between 0 . 5 and 1 μm . the crystals were washed three times with 100 μl 10 % ( v / v ) ethanol in water at − 10 □ c and dried in vacuo . hplc of the crystals showed that the washings had removed the taurocholic acid sodium salt from the insulin crystals . crystallisation in the presence of tween 80 , bis ( 2 - ethylhexyl ) sulfosuccinate sodium salt , chitosan , l - α - lysophosphatidylcholine myristoyl and polyoxyethylene sorbitan monolaurate . crystallisation was performed as described in example 2 except that taurocholic acid sodium salt was replaced by 0 . 6 % ( w / v ) tween 80 , 0 . 56 % ( w / v ) bis ( 2 - ethylhexyl ) sulfosuccinate sodium salt , 0 . 32 % ( w / v ) chitosan , 0 . 52 % ( w / v ) l - αlysophosphtidylcholine myristoyl , and 1 % ( w / v ) polyoxyethylene sorbitan monolaurate , respectively . all five examples resulted in uniformly sized crystals having diameters between 0 . 5 and 1 μm . crystallisation was performed in 10 % ( v / v ) ethanol as described in example 1 , using 4 combinations of ph and concentration of sodium acetate : 4 . 1 : ph 7 . 5 and 1 m sodium acetate 4 . 2 : ph 7 . 5 and 1 . 5 m sodium acetate 4 . 3 : ph 9 . 0 and 1 m sodium acetate 4 . 4 : ph 9 . 0 and 1 . 5 m sodium acetate all 4 combinations yielded uniformly sized crystals having diameters between 0 . 5 and 1 μm . crystallisation was performed in 15 % ( v / v ) ethanol , using 6 combinations of ph and concentration of sodium acetate : 5 . 1 : ph 7 . 5 and 1 m sodium acetate 5 . 2 : ph 7 . 5 and 1 . 5 m sodium acetate 5 . 3 : ph 7 . 5 and 2 m sodium acetate 5 . 4 : ph 9 . 0 and 1 m sodium acetate 5 . 5 : ph 9 . 0 and 1 . 5 m sodium acetate 5 . 6 : ph 9 . 0 and 2 m sodium acetate all 6 combinations yielded uniformly sized crystals having diameters between 0 . 5 and 1 μm . crystallisation was performed in 20 % ( v / v ) ethanol using 4 combinations of ph and concentration of sodium acetate : 6 . 1 : ph 7 . 5 and 1 m sodium acetate 6 . 2 : ph 7 . 5 and 1 . 5 m sodium acetate 6 . 3 : ph 7 . 5 and 2 m sodium acetate 6 . 4 : ph 9 . 0 and 1 m sodium acetate all 4 combinations yielded uniformly sized crystals having diameters between 0 . 5 and 1 μm . crystallisation at ph 7 . 5 , 8 . 0 , 8 . 5 and 9 . 0 in 20 % ethanol ( v / v ) using slow addition of sodium acetate . crystallisation was performed using solutions as described in example 1 , except that the 2 m sodium acetate was dissolved in 20 % ( v / v ) ethanol in water . the ph of the insulin solutions were adjusted to 7 . 5 , 8 . 0 , 8 . 5 and 9 . 0 , respectively . the sodium acetate solution was added in aliquots over a period of 2 hours , using 10 min between additions . at all 4 ph values uniformly sized crystals having diameters between 0 . 5 and 1 μm were obtained . crystallisation of lys b29 ( ε - myristoyl ) des ( b30 ) human insulin in the presence of taurocholic acid sodium salt . 10 mg of lys b29 ( ε - myristoyl ) des ( b30 ) human insulin and 5 mg of taurocholic acid sodium salt are dissolved in 500 μl 10 mm tris buffer , ph 8 . 0 in 20 % ( v / v ) of ethanol in water . to this solution is added 500 μl 2 m sodium acetate . microscopy after 1 hour and after 24 hours shows identically appearance of the crystals , i . e . uniformly sized crystals having diameters between 0 . 5 and 1 μm . the crystals were washed once with 300 μl 10 % ( v / v ) ethanol in water at − 10 □ c and dried in vacuo . hplc of the crystals showed that the washings had removed the palmitoyl - thr b29 lys b30 human insulin , b29 - n ε -( n - palmitoyl - γ - glutamyl )- des ( b30 ) human insulin , b29 - n ε -( n - lithocholyl - γ - glutamyl )- des ( b30 ) human insulin , b29 - n ε -( ω - carboxyheptadecanoyl )- des ( b30 ) human insulin and b29 - n ε -( ω - carboxyheptadecanoyl ) human insulin .
the present invention provides methods and compositions for treating diabetes by administering acylated insulin or an acylated insulin analog via a pulmonary route . the insulin or insulin analog may be in the form of a dry powder or a solution .