MXPA00003955A - Insoluble insulin compositions - Google Patents

Abstract

The present invention relates to insoluble compositions containing acylated proteins selected from the group consisting of acylated insulin, acylated insulin analog, and acylated proinsulin, and formulations thereof. The formulations are suitable for parenteral delivery or other means of delivery, to a patient for extended control of blood glucose levels. More particularly, the present invention relates to compositions comprised of an acylated protein complexed with zinc, protamine, and a phenolic compound such that the resulting microcrystal is analogous to the neutral protamine Hagedorn (NPH) insulin crystal form. Surprisingly, it has been discovered that compositions of such acylated proteins have therapeutically superior subcutaneous release pharmacokinetics, and more extended and flatter glucodynamics, than presently available commercial preparations of NPH insulin. Yet, the present crystals retain certain advantageous properties of NPH crystals, being readily able to be resuspended and also mixable with soluble insulins.

Description

Insoluble INSULIN COMPOSITIONS BACKGROUND OF THE INVENTION 1. Field of the invention. This invention relates to the field of human medicine. More particularly, this invention relates to the field of pharmaceutical treatment of diabetes diseases and hyperglycemia. 2. Description of the related art. For a long time an objective of insulin therapy has been to mimic the pattern of endogenous insulin secretion in normal individuals. The daily physiological demand for insulin fluctuates and can be separated into two phases: (a) the absorptive phase, which requires an insulin pulse for the disposition of blood glucose discharge related to food, and (b) the post-absorptive phase to record a sustained supply of insulin to regulate the expenditure of hepatic glucose to maintain optimal blood glucose during fasting. Consequently, effective therapy for people with diabetes generally involves the combined use of two types of exogenous insulin formulations: a fast-acting insulin at the time of feeding provided by bolus injections, and the so-called basal insulin, of action REF .: 33009 prolonged, administered by injection once or twice a day to control blood glucose levels between foods. An ideal basal insulin will provide a prolonged and "flat" time of action - that is, it will control blood glucose levels for at least 12 hours and preferably for 24 hours or more, without significant risk of hypoglycemia. In addition, an ideal basal insulin should be mixed with a soluble insulin at the time of feeding, and should not cause irritation or reaction at the site of administration. Finally, basal insulin preparations that are suspension formulations must be capable of being easily and uniformly resuspended by the patient before administration. As has been well understood by those skilled in the art, long-acting insulin formulations that have been obtained by formulating normal insulin as microcrystalline suspensions for subcutaneous injection. Examples of commercial basal insulin preparations include NTH insulin (Hagedorn neutral protamine), protarrtine and zinc insulin (P I) and ulcer (UL). NPH insulin is the most widely used insulin preparation, accounting for 50 to 70 percent of the insulin used throughout the world. It is a suspension of a microcrystalline complex of insulin, zinc, protamine and one or more phenolic preservatives. The NPH insulin preparations are commercially available and incorporate human insulin, porcine insulin, bovine insulin or mixtures thereof. In addition, preparations similar to NPH of a monomeric insulin analog, LysB298, ProB29-human insulin analog [abbreviated herein as "NPL": De Felippis, M.R., U.S. Pat. No. 5,461,031, published October 24, 1995; De Felippis, M.R., U.S. No. 5,650,486, published July 22, 1997; and De Felippis, M.R. U.S. Patent No. 5,747,642, published May 5, 1998]. The NPH insulin microcrystals have a distinctive rod-shaped morphology of typical dimensions approximately 5 micrometers long by 1 micrometer thick and 1 micrometer wide. The prolonged duration of action of the NPH insulin microcrystals results from their slow absorption of the subcutaneous injection site. Currently available therapy using NPH insulin preparations does not provide the ideal "flat" pharmacokinetics needed to maintain an optimal fasting blood glucose for a prolonged period between meals. As a result, treatment with NPH insulin can result in undesirably high levels of insulin in the blood, which can lead to life-threatening hypoglycemia. In addition to not providing an ideal flat pharmacokinetic profile, the duration of NPH insulin is also not ideal.

In particular, a major problem with NPH therapy is the "dusk" phenomenon, which is hyperglycemia that results from the loss of effective glucose control during the night while the patient sleeps. These deficiencies in glycemic control contribute to serious long-term medical complications in diabetes and impose considerable disadvantages and disadvantages on the quality of life for the patient. Protamine zinc insulin (PZI) has a composition similar to NPH, but contains high concentrations of protamine and zinc compared to NPH. The PZI preparations can be made as immediate action amorphous precipitates or long-acting crystalline material. However, PZI is not an ideal basal insulin pharmaceutical substance because it can not be mixed with a soluble insulin for feeding time, and the high concentration of zinc and protamine can cause irritation or reaction at the site of administration. Ultralow human insulin is a microcrystalline preparation of insulin that has higher concentrations of zinc compared to NPH, and does not have protamine or a phenolic preservative incorporated within the microcrystal. Ultralente human preparations provide a moderate time of action that is not adequately flat and do not form stable mixtures with insulin. In addition, they are difficult to resuspend. There have been attempts to resolve the perceived inadequacies of known insulin suspensions. Insulins acylated with fatty acid have been investigated for basal blood glucose control [Havelund, S., et al. , WIPO publication O95 / 07931, March 23, 1995]. Its action in prolonged time is caused by the binding of the fatty acyl portion of these molecules to serum albumin. The fatty acyl chain lengths of these molecules are such that they take advantage of the fatty acid binding capacity of the serum albumin. The fatty acid chains used in the fatty acid-acylated insulins are typically larger than about ten carbon atoms, and the chain lengths of fourteen to sixteen carbon atoms are optimal for binding to serum albumin and a long time of action. Unlike NPH insulin, which is insoluble, the fatty acid-acylated insulins mentioned above are soluble in usual therapeutic insulin concentrations. However, the time of action of these preparations may not be long enough, or sufficiently flat to provide ideal basal control, and they are less potent than insulin, requiring the administration of larger amounts of the drug agent [Radziu, J. ., et al., Diabetologia 41: 116-120, 489-490 (1998)].

Whittingham, J. L., et al. [Biochemistry 36: 2826-283l (1997)] crystallized B29-Ne-tetradecanoyl-des (B30) -human insulin analogue as a hexamer that forms a complex with zinc and phenol for the purpose of structural studies by X-ray crystallography. It is found that the hexamer is in conformation R6 and has certain properties different from human insulin hexamers. Whittingham et al. they do not disclose any pharmaceutical pharmacological property of the crystal that is formed nor do they suggest that such a crystal may have any advantageous property to treat diabetes or hyperglycemia. It is not possible to predict from Whittingham et al. if the protamine-containing crystals of the NPH type can be formed with derivatized insulins and insulin analog or what could be the pharmacokinetic or pharmacodynamic response of such crystals. Therefore, the need remains to identify insulin preparations that have a flatter and longer action time than NPH insulin, which can be mixed with soluble insulins, for feeding time, that can be resuspended easily and that can not generate risks of irritation or reaction at the administration site.

BRIEF DESCRIPTION OF THE INVENTION We have unexpectedly observed that when insulin becomes less soluble by derivatization of one or more of its reactive side groups, derivatized insulin can be incorporated into NPH-like crystals with protamine. When the derivatized protein is precipitated or crystallized, the rate at which the insulin derivatives of the solid form are dissolved is greatly reduced, compared to the rate at which a similar solid form consisting of non-derivatized protein dissolves. We have also discovered that derivatized protein crystals provide a flatter and more prolonged action time compared to crystals composed of a non-derivatized protein. Additionally, we have surprisingly discovered that the benefits of a more flat and longer action time can be obtained even from amorphous precipitates comprising derivatized protein. Accordingly, in its broadest aspect, the present invention provides insoluble compositions comprising a derivatized protein that is selected from the group consisting of insulin derivatives, insulin analog derivatives and proinsulin derivatives, wherein the derivatives are less soluble than the non-derivatized insulin, the insulin analog or the proinsulin. The insoluble compositions are also comprised of a complexing compound, a hexamer stabilizing compound and a divalent metal cation. These insoluble compositions are useful for treating diabetes and hyperglycemia and provide the advantages of having a more flat and prolonged action in time compared to NPH insulin. In addition, they can be mixed in a formulation with soluble protein and soluble derivatized protein. The insoluble compositions of the present invention are in the form of amorphous precipitates and also more preferably in the form of microcxistals. More specifically, the present invention provides microcrystallization forms of acylated fatty acid proteins that are useful for treating diabetes and hyperglycemia. These microcrystals comprise an acylated fatty acid protein which is selected from the group consisting of acylated fatty acid insulin, acylated insulin analogue of fatty acid and acylated proinsulin with fatty acid, protamine, a phenolic preservative and zinc. Such microcrystals will provide a flatter and more prolonged action time than NPH insulin and can be mixed with soluble proteins and soluble derivatized proteins. The invention provides aqueous suspension formulations comprising the insoluble composition and an aqueous solvent. Such suspension formulations may optionally contain a soluble protein, such as human insulin, or a soluble analogue of human insulin, such as a monomeric insulin analog, which controls blood glucose immediately following a food. The microcrystalline formulations of insulins acylated with fatty acid have a superior pharmacodynamics compared to the "human" NPH insulin The present invention is different from the anterior fatty acid acylated insulin technology in that the extension of the action time of the present invention is not it is necessarily based on albumin binding, although binding to albumin may further prolong the time of action of certain compositions of the present invention The invention also pertains to a process for preparing insoluble compositions, and a method for treating diabetes or hyperglycemia which comprises administering a formulation containing an insoluble composition to a patient in need thereof in an amount sufficient to regulate blood glucose levels in the patient.Furthermore, part of the present invention are amorphous precipitates comprising, in its broader aspect, a derivatized protein which is selected from the group consisting of derivatized insulin, derivatized insulin analog and derivatized proinsulin, protamine, a phenolic preservative and zinc, wherein the derivatized protein is less soluble than the non-derivatized protein.

SHORT DESCRIPTION OF THE DRAWING Figure 1 compares the rate of dissolution of porcine insulin NPH (- - -) and that of the microcrystals of human B29-Ne-octanoyl-insulin of this invention ().

DESCRIPTION OF THE INVENTION The term "insoluble composition" refers to the material either in a microcrystalline state or in a state of amorphous precipitate. The presence of microcrystals or an amorphous precipitate can be determined by visual and microscopic examination. The solubility depends on the solvent and a particular composition may be insoluble in one solvent, but soluble in another. The term "microcrystal" means a solid that is constituted primarily of material in a crystalline state, wherein the individual crystals are predominantly of a simple crystallographic composition and are of a microscopic size, typically of a larger dimension within the range of 1 micrometer to 100 micrometers The term "microcrystalline" refers to the state in which the microcrystal is located. The term "amorphous precipitate" refers to insoluble material that is not in crystalline form. A habitually skilled person can distinguish crystals of amorphous precipitate. The precipitates. Amorphous of the present invention have advantageous pharmacological properties by themselves, and are also intermediates in the formation of the microcrystals of the present invention. The term "derivatized protein" refers to a protein that is selected from the group consisting of derivatized insulin, derivatized insulin analogs and derivatized proinsulin that is derivatized (derivatives are produced) by a functional group so that the derivatized protein is less soluble. in an aqueous solvent that what is the non-derivatized protein. Many examples of such derivatized proteins are known in the art, and the determination of solubility of the derivatized proteins and proteins is well known to those skilled in the art. Examples of insulin and derivatized insulin analogs include the benzoyl, p-tolyl-sulfonamide carbonyl and indolyl insulin derivatives and insulin analogs [Havelund, S., et al., WO95 / 07931, published March 23, 1995 ]; alkyloxycarbonyl derivatives of ineulin [Geiger, R., et al. , patent U.S. No. 3,684,791, published August 15, 1972; Brandenberg, D., et al. , US. 3,907,763, published September 23, 1975]; insulin aryloxycarbonyl derivatives [Brandenberg, D., et al. , U.S. 3,907,763, published September 23, 1975]; alkylcarbamyl derivatives [Smyth, D.

G., U.S. Patent No. 3,864,325, published February 4, 1975; Lindsay, D. G. et al. , U.S. Patent No., 3,950,517, published April 13, 1976]; carbamyl, o-acetyl insulin derivatives [Smyth, D. G., U.S. Patent No. 3,864,325 published February 4, 1975]; crosslinked alkyldicarboxylic derivatives [Brandenberg, D., et al. , U.S. Patent No. 3,907,763, published September 23, 1975]; N-carbamyl, O-acetylated insulin derivatives [Smyth, D.G., U.S. Pat. No. 3,868,356, published February 25, 1975]; various O-alkyl esters [Markussen, J., U.S. No. 4,343,898, published August 10, 1982; Morihara, K., et al. , U.S. Patent No. 4,400,465, published on August 23, 1983; Morihara, K., et al. , U.S. Patent No. 4,401,757, published August 30, 1983; Markussen, J., U.S. Patent No. 4,489,159, published December 18, 1984; Obermeier, R., et al. , U.S. Patent No. 4,601,852, published July 22, 1986; and Andresen, F. H., et al. , U.S. Patent No. 4,601,979, published July 22, 1986]; insulin alkylamide derivatives [Balschmidt, P., et al. , U.S. Patent No. 5,430,016, published July 4, 1995]; other additional insulin derivatives [Linsday, D. G., U.S. Pat. No. 3,869,437, published March 4, 1975]; and the acylated fatty acid proteins described herein.

As used herein, the term "acylated protein" refers to a derivatized protein that is selected from the group consisting of insulin, insulin analogs and proinsulin that is acetylated with a portion of organic acid that binds to the protein through an amide bond formed between the acid group of an organic acid compound and an amino group of the protein. In general, the amino group can be an a-amino group of an N-terminal amino acid of the protein, or it can be the e-amino group of a Lye residue of the protein. An acylated protein can be acylated in one or more of the three amino groups that are present in insulin and in most insulin analogues. The monoacylated proteins are acylated in a single amino group. The diacylated proteins are acylated in two amino groups. The triacylated proteins are acylated in three amino groups. The organic acid compounds may be, for example, a fatty acid, an aromatic acid or any other organic compound having a carboxylic acid group which will form an amide bond with an amino group of a protein, and which will cause the aqueous solubility of the derivatized protein is less than the solubility of the non-derivatized protein. The term "acylated protein with fatty acid" refers to an acylated protein that is selected from the group consisting of insulin, insulin analog and proinsulinae that are acylated with a fatty acid that is bound to the protein through an amide bond formed between the acid group of the fatty acid and the amino group of the protein. In general, the amino group can be the a-amino group of the N-terminal amino acid of the protein, or it can be the e-amino group of a Lys residue of the protein. A protein acylated with fatty acid can be acylated in one or more of the three amino groups that are present in insulin and in most insulin analogues. The monoacylated proteins are acylated in a single amino group. The diacylated proteins are acylated in doe amino groups. The triacylated proteins are acylated in three amino groups. Insulin acylated with fatty acid is described in Japanese Patent Publication 1-254,699. See also, Hashimoto, M., et al. , Pharmaceutical Research, 6: 171-176 (1989), and Lindsay, D. G., et al. , Biochemical J. 121-737-745 (1971). A further description of acylated insulins with fatty acid and acylated fatty acid analogues, and methods for their synthesis is found in Baker, J. C, et al. , U.S. 08 / 342,931, filed November 17, 1994 and published as U.S. patent. No. 5,693,609, December 2, 1997; Havelund, S., et al. , WO95 / 07931, published March 23, 1995 and U.S. corresponding No. 5,750,497, May 12, 1998; and Jonassen, I., et al. , W096 / 29342, published September 26, 1996. These descriptions are expressly incorporated herein by reference to describe acylated fatty acid insulins and acylated insulin analogs of fatty acid to allow preparation thereof. The term "acylated fatty acid protein" includes pharmaceutically acceptable salts and complexes of proteins acylated with fatty acid. The term "protein acylated with fatty acid" also includes preparations of acylated proteins wherein the population of acylated protein molecules is homogeneous with respect to the acylation site or sites. For example, the Ne-monoacylated protein, the Bl-Na-monoacylated protein, the Al-Na-monoacylated protein, the A1 protein, B1-Na-diacylated, the Ne protein, Al-N-diacylated, the Ne protein, Bl -Na, diacylated and the Ne, l, Bl-Na, triacylated protein are all included within the term "protein acylated with fatty acid" for the purpose of the present invention. The term also refers to preparations wherein the population of acylated protein molecules have heterogeneous acylation. In the latter case, the term "protein acylated with fatty acid" includes mixtures of monoacylated and diacylated proteins, mixtures of monoacylated and triacylated proteins, mixtures of diacylated and triacylated proteins, and mixtures of monoacylated, diacylated and triaciladae proteins. The term "ineulin", as used herein, refers to human insulin, whose amino acid sequence and spatial structure is well known. Human insulin is made up of an A chain of twenty one amino acids and a B chain of thirty amino acids which are crosslinked or linked by disulfide bridges. An appropriately cross-linked insulin contains three disulfide bridges: one between position 7 of chain A and position 7 of chain B, the second between position 20 of chain A and position 19 of chain B and the third between positions positions 6 and 11 of the A chain. The term "insulin analogue" means proteins having an A chain and a B chain having substantially the same amino acid sequences as the A chain and the B chain of human insulin, respectively, but which differ from the A chain and the B chain of human insulin in that they have one or more amino acid deletions, one or more amino acid substitutions and / or one or more amino acid additions that do not destroy the activity of the insulin or analog of insulin. The "animal insulins" are insulin analogues. Four such insulins are rabbit, porcine, bovine and ovine insulin. The amino acid substitutions that differentiate these animal insulins from human insulin are presented below for the convenience of the reader.

Position of amino acids A8 A9 AlO B30 Insulin from human Thr Ser He Thr Insulin rabbit Thr Ser He Ser Serine porcine insulin Thr Ser He Ala bovine insulin Ala Ser Val Ala ovine insulin Ala Gly Val Ala Another type of insulin analogue, the "monomeric insulin analogue" is well known in the art. Monomeric insulin analogs are structurally very similar to human insulin, and have activity similar to or equal to human insulin, but have one or more deletions, substitutions or additions of amino acids that tend to break the contacts involved in dimerization and hexamerization, which results in a greater tendency to dissociate less aggregated states. Monomeric insulin analogs are fast-acting analogs of human insulin, and are described, for example, in Chance, R.E., et al., U.S. Pat. No. 5,514,646, May 7, 1996; Brems, D. N. et al., Protein Engineering, 5: 527-533 (1992); Brange, J.J. V., et al. , EPO publication No. 214,826, published March 18, 1987; Brange, J.J. V., et al., Patent No. U.S. 5,618,913, April 8, 1997; and Brange, J., et al. Current Opinion in Structural Biology 1: 934-940 (1991). An example of monomeric insulin analogs is described as human insulin wherein Pro at position B28 is substituted with Asp, Lys, Leu, Val or Ala, and wherein Lys at position B29 is Lys or is substituted with Pro, and also AlaB26-human insulin, des (B28-B30) and des (B27) -human insulin. Monomeric insulin analogs used as derivatives in current crystals, or used without derivatizing in solution phase or suepeneion formulations are properly crosslinked in the same positions as in human insulin. Another group of insulin analogs for use in the present invention are those wherein the isoelectric point of the insulin analogue is between about 7.0 and about 8.0. These analogues are referred to as "insulin analogs with pl-displaced". Examples of such insulin analogues include ArgB31, ArgB32 -human insulin, GlyA21, rgB31, ArgB32 - human insulin, ArgAO, rgB31, ArgB32 - human ineulin and ArgAO, GlyA21, ArgB31, rgB32 - human insulin. Another group of insulin analogues consists of insulin analogs having one or more amino acid deletions that do not significantly disrupt the activity of the molecule. This group of insulin analogs is referred to herein as "analogs by deletion". For example, insulin analogues with eupreation of one or more amino acids at exposures B1-B3 are active. Similarly, insulin analogs with deletion of one or more amino acids at positions B28-B30 are active. Examples of "analogs by deletion" include des (B30) -human insulin, desPhe (Bl) -human insulin, des (B27) -human insulin, des (B28-B30) -human insulin, and des (Bl-B3) -human insulin Deletion analogs used as derivatives in the present crystals, or used without derivatization in solution phase or suspension formulations, are appropriately crosslinked in the same positions as human insulin. Optionally, an insulin analog may have substitutions of one or more of its amino acids amidated with other amino acids for chemical stability purposes. For example, Asn and Gln can be substituted with Gly, Ser, Thr, Asp or Glu. In particular, AsnA18, AsnA21 or AsnB3 or any combination of these residues, can be substituted by Gly, Asp, or Glu, for example. In addition, GlnA15 or GlnB4, or both, may be substituted for Asp or Glu. Preferred substitutions are Asp in B21 and Asp in B3. The term "proinsulin" means a single chain peptide molecule that is an insulin precursor. Proinsulin can be converted to insulin or an insulin analog by chemical reactions or preferably, catalyzed by enzymes. In proinsulin, appropriate disulfide linkages are formed as described herein. Proinsulin comprises insulin or an insulin analog and a connecting link or a connecting peptide. A connecting peptide has between 1 and about 35 amino acids. The connecting junction or the connecting peptide are connected to a terminal amino acid of the A chain and to a terminal amino acid of the B chain by an α-amide bond or by two α-amide bonds respectively. Preferably, none of the amino acids in the connecting peptide is cysteine. Preferably, the C-terminal amino acid of the connecting peptide is Lys or Arg. Proinsulin may have the formula XBCAY or may have the formula XACBY, wherein X is hydrogen or is a peptide of 1 to about 100 amino acids having Lys or Arg in its C-terminal amino acid, Y is hydroxy or is a peptide of 1 to about 100 amino acids that has either Lys or Arg at its N-terminal amino acid, A is the A chain of insulin or the A chain of an insulin analog, C is a peptide of one or about 35 amino acids, none of which is cysteine, wherein the C-terminal amino acid is Lys or Arg and B is the B chain of insulin or the B chain of an insulin analogue. A "pharmaceutically acceptable salt" means a salt formed between any one or more charged groups in a protein and any one or more non-toxic and pharmaceutically acceptable cations or anions. Organic and inorganic organic salts include, for example, those prepared from acids such as hydrochloric, sulfuric, sulfonic, tartaric, fumaric, hydrobromic, glycolic, citric, maleic, phosphoric, succinic, acetic, nitric, benzoic, ascorbic, p- toluenesulfonic, benzenesulfonic, naphthalenesulfonic, propionic, carbonic and the like, or for example ammonium, sodium, potassium, calcium or magnesium. The verb "acilar" means forming the amide bond between a fatty acid and an amino group of a protein. A protein is "acylated" when one or more of its amino groups is combined in an amide bond with the acid group of a fatty acid. The term "fatty acid" means a straight chain or branched chain saturated or unsaturated fatty acid having from 1 to 18 carbon atoms. The term "Cl to C18 fatty acid" refers to a straight chain or branched chain, saturated fatty acid having from 1 to 18 carbon atoms. The term "divalent metal cation" refers to the ion or ions that participate to form a complex with a multiplicity of protein molecules. The transition metals, alkali metals and alkaline earth metals are examples of metals that are known and form complexes with insulin. Transition metals are preferred. Zinc is particularly preferred. Other transition metals that can be pharmaceutically acceptable to form complexes with insulin proteins include copper, cobalt and iron. The term "complex" has two meanings in the present invention. First, the term refers to a complex formed between one or more atoms in the proteins that form the complex and one or more divalent metal cations. The atoms in the proteins serve as electrophoretic ligands. The proteins typically form a hexameric complex with divalent transition metal cations. The second meaning of "complex" in the present invention is the association between a compound that forms complexes and the hexamers. The "complex forming compound" is an organic molecule that typically has a multiplicity of positive charges that bind to, or complex with, the hexamers in the insoluble composition, thereby stabilizing it by preventing its dissolution. Examples of suitable complexing compounds in the present invention include protamine, surfen, various globin proteins [Brange, J., Galenics of Insulin, Springer-Verlag, Berlin Heidelberg (1987)] and various polycationic polymer compounds which are known to form complexes with insulin. The term "protamine" refers to a mixture of strongly basic proteins obtained from fish sperm. The average molecular weight of lae proteine in protamine is approximately 4,200 [Hoffmann, J.A., et al., Protein Expression and Purification, 1: 127-133 (1990)]. The term "protamine" can refer to a relatively salt-free protein preparation, often referred to as "protamine base". Protamine also refers to preparations consisting of salts of the proteins. Commercial preparations vary widely in their salt content. Protamines are well known to those skilled in the insulin art and are currently incorporated into the NPH insulin products. A pure fraction of insulin is operable in the present invention, as well as mixtures of proteins. However, commercial preparations of protamine are typically not homogeneous with respect to the proteins presentee. However, they work well in the present invention. The protamine consisting of protamine base is useful in the present invention, to the extent that the protamine preparations are comprised of protamine salts, and those which are mixtures of protamine base and protamine salts. Protamine sulfate is a frequently used protamine salt. The term "suspension" refers to a mixture of a liquid phase and a solid phase consisting of insoluble or sparingly soluble particles that are larger than a colloidal size. Mixtures of NPH microcrystals and an aqueous solvent of suspensions. The mixtures of an amorphous precipitate and an aqueous solvent also form a suspension. The term "suspeneion formulation" means a pharmaceutical composition wherein the active agent is present in a solid phase, for example, a microcrystalline solid, an amorphous precipitate, or both, which is finely dispersed in an aqueous solvent. The finely dispersed solid is such that it can be suspended in a very uniform manner through the aqueous solvent by gentle agitation of the mixture, whereby a reasonably uniform suspension is provided from which a metering volume can be extracted. . Examples of commercially available insulin formulations and suspensions include, for example, NPH, PZI and ultralenta. A small portion of solid material in a microcrystalline suspension formulation can be amorphous. Preferably, the portion of amorphous material is less than 10%, and more preferably, less than 1% solid material in the microcrystalline suspension. Likewise, a small portion of the solid material in an amorphous precipitate suspension can be microcrystalline. "Insulin NPH" refers to the preparation of insulin "Hagedorn neutral protamine". The significance of such a term, and the methods for preparing this insulin preparation, are familiar to those ordinarily skilled in the art of insulin formulation. The term "aqueous solvent" refers to a liquid solvent containing water. An aqueous solvent system may consist solely of water, may be comprised of water plus one or more visible solvents and may contain solutes. Visible solvents are most commonly used with short chain organic alcohols such as methanol, ethanol, propanol, short chain ketones such as acetone, and polyacohols such as glycerol. An "isotonicity agent" is a compound that is physiologically tolerated that imparts adequate tonicity to a formulation to prevent the net flow of water through the membranes of the cells that are in contact with the formulation administered. Glycerol, which is also known as glycerin, is commonly used as an isotonicity agent. Other isotonicity agents include salts, for example, eodium chloride and monosaccharides eg dextrose and lactose. The insoluble compositions of the present invention contain a hexamer stabilizing compound. The term "hexamer stabilizer compound" refers to a small, proteinaceous molecular weight compound that stabilizes the derivatized protein in a state of hexameric aggregation. "Phenolic compounds, particularly phenolic preservatives, are the best known stabilizing compounds for insulin and insulin derivatives, Such hexamer stabilizing compounds stabilize the insulin hexamer by binding it through specific intermolecular contacts." Examples of such agents Hexamer stabilizers include: various phenolic compounds, phenolic preservatives, resorcinol, 4 '-hydroxyacetanilide (tilenol), 4-hydroxybenzamide and 2,7-dihydroxynaphthalene The multi-use formulations of the insoluble compositions of the present invention will contain a preservative, in addition to a hexamer stabilizer compound. The preservative used in the formulations of the present invention may be a phenolic preservative. The term "conservative" refers to a compound added to a pharmaceutical formulation to act as an antimicrobial agent. A parenteral formulation must satisfy the guide lines for conservative effectiveness so that it is commercially available as a multipurpose product. Among the preservatives known in the art as effective and acceptable in parenteral formulations are benzalkonium chloride, benzethonium, chlorhexidine, phenol, m-cresol, benzyl alcohol, methyl paraben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal , benzoic acid and mixtae mixtures thereof. See for example Wallháuser, K. -H. , Develop. Bio. Standard, 24: 9-28 (1974) (S. Krager, Basel). The term "phenolic preservative" includes the compounds phenol, m-cresol, o-cresol, p-cresol, chlorocresol, methyl paraben and mixtures thereof. It is known that certain phenolic preservatives, such as phenol and m-cresol, bind to molecules similar to ineulin and thus induce conformational changes that increase their physical or chemical stability, or both [Birnbaum, D. T., et al. , Pharmaceutical Res. 14: 25-36 (1997); Rahuel-Clermont, S., et al. , Biochemistry? 36: 5837-5845 (1997)]. The term "buffer" or "pharmaceutically acceptable buffer" refers to a compound known to be safe for use in insulin formulations and which has the effect of controlling the pH of the formulation at the desired pH for the formulation. The pH of the formulations of the present invention is from about 6.0 to about 8.0. Preferably, the formulations of the present invention have a pH between about 6.8 and about 7.8. Pharmaceutically acceptable buffers for controlling pH at a moderately acidic pH at a moderately basic pH include compounds such as phosphate, acetate, citrate, arginine, TRIS and histidine. The term "TRIS" refers to 2-amino-2-hydroxymethyl-1,3-propanediol, and to any pharmacologically acceptable salt thereof. The free base and hydrochloride forms are two common forms of TRIS. TRIS is also known in the art as trimethylolaminomethane, tromethamine and tris (hydroxymethyl) aminomethane. Other buffers which are pharmaceutically acceptable and which are suitable for controlling the pH at the desired level are known to chemists with usual skill.

The term "administer" means to introduce a formulation of the present invention into the body of a patient in need thereof to treat a disease or condition. The term "treat" refers to the management and care of the patient having diabetes or hyperglycemia, or another condition for which administration of insulin is indicated for the purpose of combating or alleviating the symptoms and complications of these conditions. Treating includes administering a formulation of the present invention to prevent the onset of symptoms or complications, alleviating symptoms or complications, or eliminating the disease, condition or disorder. As mentioned above, the present invention provides insoluble compositions that have similar properties to NPH insulin in certain aspects, and superior to NPH insulin in others. They are similar to NPH insulin in their physical properties. An optical microscope equipped with an oil immersion objective and with a crossed polarizer is used to examine the microcrystals comprised of B29-Ne-octanoyl-human insulin, zinc, protamine and phenol, prepared according to the method of Preparation 18. Examination at a magnification of 100 × shows that the micro-batch of B29-Ne-octanoyl-human insulin are unique and bar-like, and show a uniform crystalline morphology. The sizes of these microcrystals generally fall within the range of about 2 micrometers long to 8 micrometers long. A direct comparison using this microscope shows that the morphology of these microcrystals appears to be similar to that of the commercially manufactured porcine NPH microcrystals, which have been described elsewhere, as bar-like. The size range of these B29-Ne-s-tannoyl-human insulin microcrystals is also similar to that of the commercially manufactured NPH microcrystals, which generally has an average length of about 5 microns. The commercial manufacturing specification for the average length of NPH microcrystals is from 1 micrometer to 40 micrometers. The microcrystals of the present invention, however, are unexpectedly and unpredictably different from the NPH insulin crystals in their dissolution properties and in their time of action. In particular, the microcrystals of the present invention dissolve much more slowly under conditions that simulate physiological conditions compared to the NPH insulin crystals, and provide a longer and flatter blood glucose control profile compared to NPH insulin. This is demonstrated by the following experiments. It has been found that certain proteins derivatized in soluble form, have time actions not significantly different from regular human insulin.

Three groups of animalee were used. Each animal in the first group receives a dose (0.75 n ol / kg) of Humulin ™ R (soluble human inulin), each animal in the second group receives a dose (0.75 nmol / kg) of B29-Ne-octanoyl-soluble human insulin ("C8-hI") and each animal in the third group receives a dose (0.75 nmol / kg) of B29-Ne-decanoyl-insoluble human insulin ("ClO-hl"). The experiments were carried out essentially as described in Example 5 with five dogs per group.

The proteins are administered subcutaneously. The concentrations of blood groups are determined and are presented in the table below.

Table 1. Blood glucose concentrations before and after the administration of Humulin ™, B29-Ne-octanoyl-soluble human insulin ("C8-hl") or soluble B29-Ne-decanoyl-human insulin ("ClO-hl") in normal dogs simultaneously administering somatostatin to create a transient diabetic state. The values are means ± standard error.

This data clearly shows that B29-Ne-octanoyl-soluble human insulin and human B29-Ne-decanoyl-insulin, administered subcutaneously to normal dogs in a transient diabetic state, provide a general glucose decrease comparable to that obtained with soluble human insulin. Most notably, soluble human B29-Ne-octanoyl-insulin shows a shorter onset and a shorter time of action compared to human insulin. In a second experiment, it is found that the dissolution rate of the B29-Ne-octanoyl-insulin human crystals prepared according to the present invention is markedly longer than that of the commercially manufactured porcine NPH-insulin. This is unexpected in view of the above data. The dissolution rate of NPH porcine insulin is measured by placing 5 microliters of U100 porcine insulin NPH in 3 ml of Dulbecco's phosphate buffered saline solution (without calcium or magnesium) in a square quartz cuvette with a length of 1 cm. a temperature of 22 ° C. Eeta eolution is stirred at constant speed using a magnetic cup agitator. Absorbance measurements at 320 nm are taken at 1 minute intervals. The absorbance at 320 nm corresponds to light scattered by the insoluble particles present in the aqueous suspension. Consequently, as the crystals dissolve, the absorbance approaches zero. The data generated from this experiment is presented in Figure 1 as a dashed line, and show that the porcine NPH microcrystals dissolve completely after about 1 hour. An analogous procedure is followed to measure the dissolution rate of the microcrystals of human B29-Ne-octanoyl-insulin. A volume of 12 microliters of a suspension of human B29-Ne-octanoyl-insulin microcrystals (containing no more than 50 U / ml) prepared according to the preparation procedure 18, is placed in 3 ml of phosphate buffered saline of Dulbecco (without calcium or magnesium) in a square calcium tub 1 cm long. This solution is stirred at the same constant speed at the same temperature of 22 ° C. The data generated from this experiment are presented in Figure 1 as a solid line and show that microcrystals of human B29-Ne-octanoyl-ineulin require much more than 5 hours to dissolve. These experiments establish that, in Dulbecco phosphate-buffered saline solution (without calcium and magnesium), a solution that imitates interstitial fluid in certain aspects, the dissolution speed of the B29-Ne-octanoyl-human insulin microcrystals is significantly more slower than the microcrystals of porcine NPH. Again, this finding is very surprising in light of the previous findings of B29-Ne-octanoyl-soluble human insulin has an action time currently slightly shorter than that of human insulin. The subcutaneous interstitial fluid contains 0.3 mM human serum albumin. Therefore, another experiment is designed to compare the dissolution rates of approximately equal amounts of human B29-Ne-octanoyl-insulin microcrystals and porcine NPH microcrystals in Dulbecco's phosphate-buffered saline containing 0.3 mM human serum albumin. This experiment is performed by placing 25 microliters of NPH porcine ineulin (approximately 3.5 mg insulin / ml) in 2 ml of Dulbecco's phosphate buffered saline solution. (without calcium and magnesium) containing human serum albumin 0. 3 mM. The resulting suspension is gently stirred manually until it is observed that the microcrystals dissolve after approximately 3 to 5 minutes. The rate of dissolution of microcrystals of human B29-Ne-octanoyl-insulin is observed by placing 50 microliters of a microcrystalline formulation of human B29-Ne-octanoyl-insulin (approximately 1.8 mg / ml), prepared essentially as described in the preparation 18 in the present, in 2 ml of Dulbecco's phosphate-buffered saline (without calcium and magnesium) containing 0.3 mM human serum albumin. The resulting suspension is gently stirred manually for about 3 to 5 minutes after which it is observed that minimal dissolution of the suspended microcrystals occurs. The gentle agitation of this solution is continued using a magnetic stirrer which results in a complete dissolution of the suspended B29-Ne-octanoyl-human ineulin microcrystals after about 2 hours.

These experiments establish that the dissolution rate of the microcrystals of human B29-Ne-octanoyl-insulin is significantly lower than the dissolution rate of commercially manufactured porcine NPH microcrystals in Dulbecco's phosphate-buffered saline solution (without calcium and magnesium ) containing 0.3 mM human serum albumin. Because the time-course profile of NPH insulin preparations is strongly related to the rate of dissolution of the microcrystals in the subcutaneous interstitial fluid, it is concluded from these experiments that the microcrystalline suspension formulation of B29- Ne-octanoyl-human insulin has a longer duration of action when administered subcutaneously to diabetic patients than that which exists in commercial preparations of NPH insulin. The insoluble compositions of the present invention may be crystals with bar-like morphology or with an irregular morphology, or they may be amorphous precipitates. Preferred insoluble compositions consist of acylated insulin or acylated insulin analogs, zinc ions, which are present from about 0.3 to about 0.7 moles per mole of derivatized protein, a phenolic preservative which is selected from the group of phenol, m-cresol, o-cresol, p-cresol, chlorocresol, methylparaben and mixtures thereof and is present in sufficient proportions with respect to the derivatized protein to facilitate formation of the conformation of hexamer R6 and protamine, which is present from about 0.15 to about 0.7 moles per mole of derivatized protein. The preferred derivatized proteins are acylated proteins, and the preferred acylated proteins for the microcrystals and the formulations of the present invention are insulin acylated with fatty acid and insulin analogs adhered with fatty acid. Human insulin acylated with fatty acid is greatly preferred. Equally highly preferred are insulin analogs acylated with fatty acid. A preferred group of insulin analogues for preparing acylated insulin analogs used to form the microcrystals of the present invention consists of insulin analogs wherein the amino acid residue at position B28 is Asp, Lys, Leu, Val or Ala, the amino acid residue in position B29 is Lys or Pro, the amino acid residue in the BIO position is His or Asp, the amino acid residue in position Bl is Phe, Aep or deleted alone or in combination with a deletion of the residue in position B2, the residue amino acid at position B30 is Thr, Ala, Ser or is deleted, and the amino acid residue at position B29 is Ser or Asp; with the proviso that in any position B28 or B29 is Lys.

Another preferred group of insulin analogues for use in the present invention consists of those in which the isoelectric point of the insulin analogue is between about 7.0 and about 8.0. These analogues are referred to as "insulin analogues with displaced pH". Examples of insulin analogues with displaced pH include, for example, ArgB3l, ArgB32 - human insulin, GlyA21, rgB31, ArgB32 - human insulin, ArgAO, rgB31, rgB32 - human insulin, and ArgAO, GlyA21, rgB31, ArgB32 - human insulin. Another preferred group of insulin analogues consists of LysB28, ProB29 -human insulin, (B28 is Lys; B29 is Pro); AspB28 -human insulin, (B28 is Asp), human AspBl-insulin, ArgB31, ArgB32 -human insulin, human ArgAO-insulin, AspBl, human GlyB13 -induulin, human B26-ineulin, human GlyA21-insulin, des (ThrB30) - Human insulin and GlyA21, ArgB31, rgB32 - human insulin. Especially preferred insulin analogs include LysB28, ProB29-human inulin, des (ThrB30) -human insulin, AspB28-human insulin and AlaB26-human insulin. Another particularly preferred insulin analogue is GlyA2l, ArgB31, ArgB32-human insulin [Dórechug, M., U.S. No. 5,656,722, Aug. 12, 1997]. The most preferred insulin analog is LysB28, ProB29-human insulin. A preferred group of acylating portions consists of fatty acids that are straight chain and saturated. This group consists of methanoic acid (Cl), ethanoic acid (C2), propanoic acid (C3), n-butanoic acid (C4), n-pentanoic acid (C5), n-hexanoic acid (C6), n-heptanoic acid (C7), n-octanoic acid (C8), n-nonanoic acid (C9), n-decanoic acid (CIO), n-undecanoic acid (Cll), n-dodecanoic acid (C12), n-tridecanoic acid (C13) ), n-tetradecanoic acid (C14), n-pentadecanoic acid (C15), n-hexadecanoic acid (C16), n-heptadecanoic acid (C17) and n-octadecanoic acid (C18). The adjectival forms are formyl (Cl), acetyl (C2), propionyl (C3), butyryl (C4), pentanoyl (C5),. hexanoyl (C6), heptanoyl (C7), octanoyl (C8), nonanoyl (C9), decanoyl (CIO), undecanoyl (Cll), dodecanoil (C12), tridecanoil (C13), tetradecanoyl (C14) or myristoyl, pentadecanoyl (C15) ), hexadecanoyl (C16) or palmitic, heptadecanoyl (C17) and octadecanoyl (C18). A preferred group of fatty acids for forming the acylase proteins with fatty acid used in the microcrystals of the present invention consists of fatty acids having an even number of carbon atoms - that is, saturated fatty acids of C2, C4, C6, C8 , CIO, C12, C14, C16 and C18. Another preferred group of fatty acids for forming the acylated fatty acid proteins used in the microcrystals of the present invention consists of fatty acids having an odd number of carbon atoms - that is, saturated fatty acids of Cl, C3, C5, C7 , C9, 11, C13, C15 and C17. Another preferred group of fatty acids for forming the fatty acid acylated proteins used in the microcrystals of the present invention consists of fatty acids having more than 5 carbon atoms - that is, saturated fatty acids of C6, C7, C8, C9, CIO, Cll, C12, C13, C14, C15, C16, C17 and C18. Another preferred group of fatty acids for forming the fatty acid acylated proteins used in the microcrystals of the present invention consists of fatty acids having less than 9 carbon atoms - that is, saturated fatty acids of Cl, C2, C3, C4, C5, C6, C7 and C8. Another preferred group of fatty acids for forming the fatty acid acylated proteins used in the microcrystals of the present invention consists of fatty acids having between 6 and 8 carbon atoms - that is, saturated fatty acids of C6, C7 and C8. Another preferred group of fatty acids for forming the acylase proteins with fatty acid used in the microcrystals of the present invention consists of fatty acids having more than 4 to 6 carbon atoms - that is, saturated fatty acids of C4, C5 and C6 . Another preferred group of fatty acids for forming the fatty acid acylated proteins used in the microcrystals of the present invention consists of fatty acids having more than 2 to 4 carbon atoms - that is, saturated fatty acids of C2, C3 and C4 . Another preferred group of fatty acids to form fatty acid acylated proteins used in the microcrystals of the present invention consists of fatty acids having 6 carbon atoms - that is, saturated fatty acids of Cl, C2, C3, C4 and C5 Another preferred group of fatty acids for forming the acylated fatty acid proteins used in the microcrystals of the present invention consists of fatty acids having less than 4 carbon atoms - that is,, saturated fatty acids of Cl, C2 and C3. Another preferred group of fatty acids for forming the acylated fatty acid proteins used in the microcrystals of the present invention consists of fatty acids having more than 9 carbon atoms - that is, saturated fatty acids of CIO, Cll, C12, C13, C14, C15, C16, C17 and C18. Another preferred group of fatty acids for forming the fatty acid acylated proteins used in the microcrystals of the present invention consists of fatty acids having an even number of carbon atoms and more than 9 carbon atoms - that is, saturated fatty acids of CIO, C12, C14, C16 and C18.

Another preferred group of fatty acids for forming fatty acid-adsorbed proteins used in the microcrystals of the present invention consists of fatty acids having 12, 14 or 16 carbon atoms - that is, saturated C 12, C 14 and C 16 fatty acids. Another preferred group of fatty acids for forming the acylated fatty acid proteins used in the microcrystals of the present invention consists of fatty acids having 14 or 16 carbon atoms, that is, C 14 and C 16 saturated fatty acids. Acids with 14 carbons are particularly preferred. Fatty acids with 16 carbons are also particularly preferred. Another preferred group of fatty acids for forming the fatty acid acylated proteins used in the microcrystals of the present invention consist of saturated fatty acids having between 4 and 10 carbon atoms, that is, saturated fatty acids C4, C5, C6, C7 , C8, C9 and CIO. Another preferred group of fatty acids to form the proteins acylated with fatty acid used in. the microcrystals of the present invention consist of saturated fatty acids having an even number of carbon atoms between 4 and 10 carbon atoms, that is, C4, C6, C8 and CIO fatty acids. Another preferred group of fatty acids to form the fatty acid acylated proteins used in the microcrystals of the present invention consist of fatty acids having between 6, 8 or 10 carbon atoms. Fatty acids with 6 carbon atoms are particularly preferred. Also particularly preferred are fatty acids with 8 carbons. Fatty acids with 10 carbons are particularly preferred. The skilled artisan will appreciate that the narrower preferred groups are produced by combining the preferred fatty acid groups described above. Another preferred group of acylating portions consists of saturated fatty acids which are branched. A branched fatty acid has at least two branches. The length of a "branch" of a branched fatty acid can be described by the number of carbon atoms in the branch, beginning with the acid carbon. For example, the branched fatty acid, 3-ethyl-5-methylhexanoic acid has three branches that are five, six and six carbons in length. In this case, the "longest" branch is six carbons. As another example, 2,3,4,5-tetraethyloctanoic acid has five branches that are 4, 5, 6, 7 and 8 carbons long. The "longest" branch is 8 carbons. A preferred group of branched fatty acids are those having from three to ten carbon atoms in the longest branch. A representative number of such branched saturated fatty acids will be mentioned to ensure the reader's understanding of the scope of such fatty acids which can be used as acylating portions of the proteins in the present invention: 2-methyl-propionic acid, 3-acid methyl-butyric, 2,2-dimethyl-propionic acid, 2-methyl-pentanoic acid, 3-methyl-pentanoic acid, 4-methyl-pentanoic acid, 2,2-dimethyl-butyric acid, 2,3-dimethyl- butyric, 3, 3-dimethyl-butyric acid, 2-ethyl-butyric acid, 2-methyl-hexanoic acid, 5-methyl-hexanoic acid, 2,2-dimethyl-pentanoic acid, 2,4-dimethyl-pentanoic acid, 2-ethyl-3-methyl-butyric acid, 2-ethyl-pentanoic acid, 3-ethyl-pentanoic acid, 2,2-dimethyl-3-methyl-butyric acid, 2-methyl-heptanoic acid, 3-methyl- heptanoic, 4-methyl-heptanoic acid, 5-methyl-heptanoic acid, 6-methyl-heptanoic acid, 2,2-dimethyl-hexanoic acid, 2,3-dime acid til-hexanoic, 2,4-dimethyl-hexanoic acid, 2,5-dimethyl-hexanoic acid, 3,3-dimethyl-hexanoic acid, 3,4-dimethyl-hexanoic acid, 3,5-dimethyl-hexanoic acid, acid 4, 4-dimethyl-hexanoic acid, 2-ethylhexanoic acid, 3-ethylhexanoic acid, 4-ethylhexanoic acid, 2-propylpentanoic acid, 2-ethylhexanoic acid, 3-ethylhexanoic acid, 2- (1-propyl) pentanoic acid, 2- (2-propyl) pentanoic acid, 2,2-diethyl-butyric acid, 2, 3, 4-trimethyl-pentanoic acid, 2-methyl-octanoic acid, 4-methyl-octanoic acid, 7-methyl-octanoic acid, 2,2-dimethyl-heptanoic acid, 2,6-dimethyl-heptanoic acid, 2-ethyl-2-methyl-hexanoic acid, 3-ethyl-5-methyl-hexanoic acid, 3- (1-propyl) -hexanoic acid, 2- (2-butyl) acid -pntanoic, 2- (2- (2-methylpropyl)) pentanoic acid, 2-methyl-nonanoic acid, 8-methyl-nonanoic acid, 6-ethyl-octanoic acid, 4- (1-propyl) -heptanoic acid, acid 5- (2-propyl) -heptanoic acid, 3-methyl-undecanoic acid, 2-pentyl-heptanoic acid, 2, 3, 4, 5, 6-pentam-he-tanoic acid, 2,6-diethyl-octanoic acid, 2-hexyl-octanoic acid, 2,3,4,5,6,7-hexamethyl-octanoic acid, 3, 3-diethyl-4, 4-diethyl-hexanoic acid, 2-heptyl-nonanoic acid, acid 2, 3, 4, 5-tetrahethyl-octanoic, 2-octyl-decanoic acid and 2- (1-propyl) -3- (1-propyl) -4,5-diethyl-6-methyl-heptanoic acid. Another additional preferred group of acylating portions consists of cyclic alkyl acids having from 5 to 24 carbon atoms, wherein the cyclic alkyl portion or portions have 5 to 7 carbon atoms. A representative number of such cyclic alkyl acids can be mentioned to ensure the reader's understanding in the range of such acids which can be used as acylating portions of the proteins e in the present invention: cyclopentemalmic acid, cyclohexylformamic acid, 1-cyclopentyl acetic acid, 2-cyclohexylacetic acid, 1,2-dicyclopentylacetic acid and the like. A preferred group of derivatized proteins for use in the microcrystals of the present invention consists of monoacylated proteins. Monoacylation in the e-amino group is most preferred. For the ineulin, monoacylation is preferred in LysB29. Similarly, for certain analogous of ineulin, such as LyeB28, ProB29-human insulin analogue, monoacylation in the e-amino group of LysB28 is most preferred. Monoacylation in the a-amino group of the B chain (Bl) is also preferred. Monoacylation of the a-amino group of the A (Al) chain is also preferred. Another preferred group of adiated proteins for use in the microcrystals of the present invention consists of diacylated proteins. The diacylation can be, for example, in the e-amino group of Lys and in the a-amino group of the B chain, or it can be in the e-amino group of Lys and in the a-amino group of the A chain or it can be in the A-amino group of the A chain and in the a-amino group of the B chain. Another preferred group of acylated proteins for use in the microcrystals of the present invention consists of triacylated proteins. Triacylated proteins are those that are acylated in the e-amino group of Lys, in the a-amino group of the B chain and in the a-amino group of the A chain. It is also preferred to use acylated proteins that are a mixture of proteins monoacylated and diacylated. It is also preferred to use acylated proteins which are a mixture of monoacylated and triacylated proteins. Another preferred group of acylated proteins consists of a mixture of diacylated and triacylated proteins.

It is also preferred to use acylated proteins which are a mixture of monoacylated, diacylated and triacylated proteins. Some proteins acylated with fatty acid in the present microcrystals will be mentioned to ensure the reader's understanding with respect to the scope of the present invention. The list is illustrative, and the fact that a protein acylated with a particular fatty acid is not mentioned does not mean that a microcrystal containing it is not within the scope of the present invention. N29 -Ne-formyl -human insulin. Bl-Na-formyl-human insulin. Al-ETa-formyl-human insulin. B29 -Ne-formyl-, human Bl-Na-formyl-insulin. B29-Ne-formyl-, human l -Na- formyl -insulin. Human α-Na-formyl-, Bl-Na-formyl-insulin. B29 -Ne-formyl-, Na-formyl-, human Bl-Na-formyl -insulin.

B29, human TSTe-acetyl -insulin. Bl-N-acetyl-human insulin. Al-Na-acetyl-human insulin. B29 -Ne-acetyl-, human Bl-? A-acetyl -insulin. B29 -Ñe-acetyl-, human Na-acetyl-quinoline. Al-Na-acetyl-, human Bl-Na-acetyl-ineulin. B29-Ne-acetyl-, Na-acetyl-, human Bl-Na-acetyl-insulin. B29-Ne-propionyl-human insulin.

Bl-Ne-propionyl-human insulin. Human Na-propionyl-insulin. B29-Ne-propionyl-, human Bl-Na-propionyl -insulin. B29-Ne-propionyl-, human l-Na-propionyl-insulin. Al-Na-propionyl-, human Bl-Na-propionyl-insulin. B29-Ne -propionyl-, Al-Na-propionyl-, human Bl-Na-propionyl-insulin. B29 = Ne-butyryl-human insulin. Bl-Na-butyryl-human insulin. Al-Na-butyryl-human insulin. B29-Ne-butyryl-, human Bl-Na-butyryl -insulin. B29-Ne-butyryl-, human Na-butyryl-insulin. Al-Na-bu iryl-, human Bl-Na-butyryl-insulin. B29-Ne-bu iryl-, Al-Na-butyryl-, human Bl-N -butyryl-insulin. B29-Ne-pentanoyl-human insulin. Bl-Na-pentanoyl-human insulin. Human al-Na-pentanoyl -insulin. B29 -Ne-pentanoyl-, human Bl-Na-pentanoyl -insulin. B29-Ne-pentanoyl-, human Na-pentanoyl-insulin. Al -Na-pentanoyl-, human Bl-Na-pentanoyl -insulin. B29 -Ne-pentanoyl-, Al-Na-pentanoyl-, human Bl-Na-pentanoyl -insulin. B29-Ne-hexanoyl- human insulin. Bl-Na-hexanoyl-human insulin. Al-Na-hexanoyl-human insulin.

B29-Ne-hexanoyl-, human Bl-Na-hexanoyl-insulin. B29-Ne-hexanoyl-, human Na-hexa.noyl-insulin. Al-N-hexanoyl-, human Bl-Na-hexanoyl-insulin. B29-Ne-hexanoyl-Al-Na-hexanoyl-, human Bl-Na-hexanoyl-insulin. B29-Ne -heptanoyl-human insulin. Human Bl-Na-heptanoyl -insulin. Human al-Na-heptanoyl -insulin. B29 -Ne- eptanoyl-, human Bl-Na-heptanoyl-insulin. B29-Ne-heptanoyl-, human Bl-Na-heptanoyl -insulin. Al-Na-heptanoyl-, human Bl-Na-heptanoyl-insulin. B29-Ne-heptanoyl-, Na-heptanoyl -Bl-Na-heptanoyl-human insulin. B29 -Ne-octanoyl-human insulin. Bl-N -octanoyl -human insulin. Al-Na-octanoyl-human insulin. B29-Ne -octanoyl-, human Bl-Na-octanoyl -insulin. B29-Ne -octanoyl-, human N-octanoyl-insulin. Al-Na-octanoyl-, human Bl-Na-octanoyl-insulin. B29 -Ne -octanoyl-, Na-octanoyl-, Bl-Na-oc anoyl -insulin human. B29-Ne-nonanoil-human insulin. Bl-Na-nonanoil- human insulin. Al-Na-nonanoil-human insulin. B29-Ne-nonanoil-, human Bl-Na-nonanoyl -insulin. B29-Ne-nonanoil-, human Na-nonanoyl-insulin.

Al -Na-nonanoyl-, human Bl-Na-nonanoyl -insulin. B29 -Ne -nonanoyl-, Al -Na -nonanoyl-, Bl -Na -nonanoyl-insul ina human. B29 -Ne-decanoyl-human insulin. Bl-Na-decanoyl-human insulin. Human Na-decanoyl-insulin. B29-Ne -decanoil-, human Bl-Na-decanoyl-insulin. B29 -Ne-decanoyl-, human Na-decanoyl-insulin. Al-Na-decanoyl-, human Bl-Na-decanoyl-insulin. B29 -Ne -decanoyl-, Na-decanoyl-, Bl -Na-decanoyl- human insulin. B28-Ne - formyl-LysB28, ProB29 - human insulin analog. Bl-Na-formyl-LysB28, ProB29 -human insulin analog. Al-Na-formyl-LysB28, ProB29 - human insulin analog. B28-Neoformil-, Bl-Na-formyl-LyeB28, ProB29-human inulin analog. B28 -Ne-formyl-, Al-Na-formyl-LysB28, roB29 -human insulin analogue. Al-Na-formyl-, Bl-Na-formyl-LysB28, ProB29 -human insulin analog. B28 -N? -formyl-, Al-Na-formyl, Bl-Na-formyl-LysB28, ProB29-human insulin analog. B28-Ne-acetyl-LysB28, ProB29-human insulin analog. Bl-Na-acetyl-LyeB28, ProB29 - human insulin analog. Al-Na-acetyl-LysB28, ProB29 - human insulin analog.

B28-Ne-acetyl-, Bl-Na-acetyl-LyeB28, ProB29 - human inulin analog. B28-Ne-acetyl-, Al-Na-acetyl-LyeB28, ProB29-human insulin analog. Al-Na-acetyl-, Bl-Na-acetyl-LysB28, ProB29 -human insulin analog. * B28-Ne-acetyl-, Na-acetyl, Bl-Na-acetyl-LysB28, ProB29-human insulin analog. B28-Ne-propionyl-LyeB28, roB29 - human inulin analog. Bl-Na-propionyl-LysB28, ProB29-human insulin analog.

Al-Na-propionyl-LysB28, ProB29-human insulin analog.

B28-Ne-propionyl-, Bl-Na-propionyl-LysB28, ProB29 - human inulin analog. B28-Ne -propionyl-, Al-Na-propionyl-LysB28, ProB29 -human insulin analog. Al-Na-propionyl-, Bl-Na-propionyl-LysB28, ProB29 -human insulin analog. B28 -Ne -propionyl-, Al -Na -propionyl, Bl -Na -propionyl-LyeB28, ProB29 -analog of human inulin. B28-Ne-butyryl-LysB28, ProB29 - human insulin analog. Bl-Na-butyryl-LysB28, ProB29 - human insulin analog. Al-Na-butyryl-LysB28, ProB29 -human insulin analog. B28-Ne-butyryl-, Bl-Na-butyryl-LysB28, ProB29-human insulin analog.

B28-Ne-butyryl-, Al-Na-butyryl-LyeB28, ProB29-human insulin analog. Al-Na-butyryl-, Bl-Na-butyryl-LysB28, roB29 -human insulin analog. B28-Ne-butyryl-, Na-butyryl, Bl-Na-butyryl-LysB28, ProB29-human insulin analog. B28-Ne-pentanoyl-LysB28, ProB29-human insulin analog.

Bl-Na-pentanoyl-LysB28, ProB29 - Jaumana insulin analog.

Al-Na-pentanoyl-LysB28, ProB29-human insulin analog. B28 -Ne-pentanoyl-, Bl-Na-pentanoyl-LysB28, ProB29 -human insulin analogue. B28-Ne-pentanoyl-, Al-Na-pentanoyl-LyeB28, ProB29 - human inulin analog. Al-Na-pentanoyl-, Bl-Na-pentanoyl-LysB28, ProB29 -human insulin analog. B28-Ne-pentanoyl-, Al-Na-pentanoyl, Bl-Na-pentanoyl Lys? TZ, ProB29-human inulin analogue. B28-Ne-hexanoyl-LysB28, ProB29 -human insulin analog.

Bl-Na-hexanoyl-LysB28, ProB29 - human insulin analog. Al-Na-hexanoyl-LysB28, ProB29 - human inulin analog. B28-Ne-hexanoyl-, Bl-Na-hexanoyl-LysB28, ProB29 -human insulin analog. B28 -Ne -hexanoyl-, Al -Na-hexanoyl-LysB28, ProB29 - human inulin analog.

Al-Na-hexanoyl-, Bl -Na-hexanoyl-LysB28, ProB29 -human insulin analog. B28-Ne -hexanoyl-, Al-Na-hexanoyl, Bl-Na-hexanoyl-LysB28, ProB29-human insulin analog. B28-Ne-heptanoil-LyeB28, ProB29 - human insulin analog.

Bl-Na-heptanoil-LysB28, ProB29 - human insulin analog.

Al-Na-heptanoyl-LysB28, ProB29 - human insulin analog.

B28-Ne-heptanoyl-, Bl-Na-heptanoyl-LysB28, ProB29 -human insulin analog. B28 -Ne-heptanoyl-, Al-Na-heptanoyl-LysB28, ProB29 -human insulin analogue. Al-Na-heptanoyl-, Bl-Na-heptanoyl-LysB28, ProB29 - human insulin analog. B28-Ne-heptanoyl-, Al-Na-heptanoyl, Bl-Na-heptanoyl-LysB28, roB29 - human insulin analog. B 8 -Ne-octanoyl-LysB28, ProB29 - human insulin analog.

Bl -Na- octanoyl-LysB28, ProB29 -human insulin analog. Al-Na-octanoyl-LysB28, ProB29 - human insulin analog. B28 -Ne-octanoyl-, Bl -Na- octanoyl-LysB28, roB29 -human insulin analogue. B28 -Ne-octanoyl-, Al-Na-octanoyl-LysB28, ProB29 -human insulin analog. Al-Na-octanoyl-, Bl -Na- octanoyl-LysB28, ProB29 -human insulin analogue.

B28-Ne-octanoyl-, Al-Na-octanoyl, Bl-Na-octanoyl-LysB28, ProB29-human insulin analog. B28-Ne-nonanoil-LyeB28, ProB29-human insulin analog.

Bl-Na-nonanoil-LyeB28, ProB29 - human insulin analog. Al-Na-nonanoil-LyeB28, ProB29 -human insulin analog. B28-Ne -nonanoyl-, Bl-Na-nonanoyl-LysB28, ProB29-human insulin analog. B28 -Ne-nonanoyl-, l -Na-nonanoyl-LysB28, ProB29 -human insulin analog. Al-Na-nonanoyl-, Bl-Na-nonanoyl-LysB28, ProB29 -human insulin analog. B28 -Ne-nonanoyl-, Al-Na-nonanoyl, Bl-Na-nonanoyl-LysB28, ProB29-human insulin analogue. B28-Ne -decanoil-LysB28, ProB29 - human insulin analog. Bl-Na-decanoyl-LysB28, roB29 - human insulin analog. Al-Na-decanoyl-LysB28, ProB29-human insulin analog. B28 -Ne-decanoyl-, Bl-Na-decanoyl-LysB28, ProB29 -human insulin analogue. B28 -Ne-decanoyl-, Al -Na-decanoyl-LysB28, ProB29 -human insulin analogue. Al-Na-decanoyl-, Bl -Na-decanoyl-LysB28, ProB29 - human inulin analogue. B28-Ne-decanoyl-, l-Na-decanoyl, Bl-Na-decanoyl-LyeB28, ProB29-human insulin analogue. B29 -Ne-pentanoyl-GlyA21, ArgB31, ArgB32 -humaninein. - §4 - _. Bl-Na-hexanoyl-GlyA21, rgB31, rgB32 -human insulin. Al-Na-heptanoyl-GlyA21, ArgB31, rgB32 -human insulin. B29-Ne-octanoyl-, Bl-Na-octanoyl-GlyA21, ArgB31, ArgB32 -human insulin. B29-Ne-propionyl-, Al-Na-propionyl-GlyA21, ArgB31, rgB32 -human insulin. Al-Na-acetyl-, Bl-Na-acetyl-GlyA21, ArgB31, ArgB32 - human insulin. B29 -Ne-formyl-, Na-formyl, Bl-Na-formyl-GlyA21, ArgB31, ArgB32-human insulin. B29-Ne-formyl-des (TyrB26) -human insulin. Bl-Na-acetyl-aspB28 - human inulin B29-Ne -propionyl-, Al, -Na-propionyl-, Bl-Na-propionyl-AspBl, spB3, AspB21-human insulin. B29-Ne-pentanoyl-GlyA21-human insulin. Bl-Na-hexanoyl-GlyA21- human insulin. Al-Na-heptanoyl-GlyA21-human inulin. B29 -Ne-octanoyl-, Bl-Na-octanoyl-GlyA21- human insulin. B29-Ne -propionyl-, Al-Na-propionyl-GlyA21-human inulin. Al-Na-acetyl, Bl-Na-acetyl-GlyA21-human inulin. B29-Ne-formyl-, Na-formyl-, Bl -Na- formyl -GlyA21- human insulin. B29-Ne-butyryl-des (ThrB30) -human insulin. Bl-Na-butyryl-des (ThrB30) -human insulin. Al-Na-butyryl-des (ThrB30) -humaninein.

- E5 -B29-Ne-butyryl-, Bl-Na-butyryl-des (ThrB30) -humaninein. B29-Ne-butyryl-, l-Na-butyryl-dee (ThrB30) -human insulin. Al-Na-butyryl-, Bl-Na-butyryl-des (ThrB30) -human insulin. B29-Ne -butyryl-, Al-Na-butyryl-, Bl-Na-butyryl-des (ThrB30) -human insulin. Aqueous compositions containing water as the main solvent are preferred. Aqueous suspensions in which water is the solvent are highly preferred. The compositions of the present invention are used to treat patients who have diabetes or hyperglycemia. The formulations of the present invention will typically provide derivatized protein at concentrations of about 1 mg / ml to about 10 mg / ml. The present insulin product formulations are typically characterized in terms of the concentration of insulin activity units (units / ml) such as U40, U50, UlOO and so on, which generally corresponds to approximately 1.4, 1.75 preparations. and 3.5 mg / ml, respectively. The dose, route of administration and the number of administrations per route will be determined by the doctor who considers factors such as the therapeutic objectives, the nature and cause of the patient's illness, the gender and weight of the patient, exercise level, habits of feeding, the method of administration and other factors known to medical experts. Over a wide range, a daily dose will be in the range of about 1 nmol / kg of body weight to about 6 nmol / kg of body weight (6 nml is considered equivalent to about 1 unit of insulin activity). A dose of between about 2 and about 3 nmol / kg is typical of the present insulin therapy. The physician with habitual skill in treating diabetes will be able to select the therapeutically most advantageous means for administering the formulations of the present invention. Parenteral routes of administration are preferred. Typical routes of parenteral administration of insulin suspension formulations are the subcutaneous and intramuscular routes. The compositions and formulations of the present invention can also be administered nasally, buccally, pulmonarily or ocularly. Glycerol is preferred at a concentration of 12 mg / ml to 25 mg / ml as an isotonicity agent. The use of glycerol at a concentration of about 15 mg / ml to about 17 mg / ml is additionally preferred for isotonicity. M-cresol and phenol, or mixtures thereof, are the preferred preservatives in formulations of the present invention. The insulin, insulin analogs or proinsulinae used to prepare derivatized proteins can be prepared in any of a variety of recognized techniques for the synthesis of peptides including classical (solution) methods, solid phase methods, semi-synthetic methods and more recently recombinant DNA methods. For example, see Chance, R. E., et al. , U.S. Patent No. 5,514,646, May 7, 1996; EPO publication number 383,472, February 7, 1996; Brange, J.J. V., et al. EPO publication number 214-, 826, March 18, 1987; and Belagaje, R. M. et al. , U.S. Patent No. 5,304,473, April 19, 1994, which describes the preparation of various proinsulin and insulin analogues. These references are expressly incorporated herein by reference. Generally, derivatized proteins are prepared using methods known in the art. The above-mentioned publications for the derivatized protein formulation contain suitable methods for preparing derived proteins. Eetae publications are expressly incorporated as a reference for methods for preparing derivatized proteins. To prepare acylated proteins, the protein is reacted with an activated organic acid, such as an activated fatty acid. The activated fatty acids are derived from commonly used acylating agents, and include activated fatty acid esters, fatty acid halides, activated fatty acid amides such as activated azolyl derivatives [Hansen, LB, WIPO Publication No. 98/02460, 22 January 1998], and fatty acid anhydrides. The use of activated esters, especially esters of N-hydroxysuccinimide of fatty acids, is a particularly advantageous means for acylating a free amino acid with a fatty acid. Lapidot et al. describes the preparation of ester of N-hydroxysuccinimide and its use in the preparation of N-lauroyl-glycine, N-lauroyl-L-serine and N-lauroyl-L-glutamic acid. The term "activated fatty acid esters" means a fatty acid which has been activated using general techniques known in the art [Riordan, J. F. and Vallee, BL, Methods in Enzymology, XXV: 494-499 (1972); Lapidot, Y., et al. , J. Lipid Res. 8: 142-145 (1967)]. Hydroxybenzotriazide (HOBT), N-hydroxyceuccinimide and derivatives thereof are particularly well-known for forming peptide-activated acids. To selectively acylate the e-amino group, various protecting groups can be used to block the a-amino groups during coupling. The selection of a suitable protecting group is known to one skilled in the art and includes p-methoxybenzoxycarbonyl (pmZ). Preferably, the e-amino group is acylated in a one-step eynthesis without the use of amino protecting groups. A process for selective acylation in the Ne -amine group of Lys is described and claimed by Baker, J. C, et al., U.S. Pat. No. 5,646,242, July 8, 1997, the complete description of which is expressly incorporated by reference. A process for preparing a dry powder of an acylated protein is described and claimed by Baker, J. C. et al. Patent No. 5,700,904, December 23, 1997, the entire disclosure of which is hereby expressly incorporated by reference. The main role of zinc in the present invention is to facilitate the formation of Zn (II) hexamers of the derivatized protein. It is known that zinc facilitates the formation of hexamers of insulin and insulin analogues. Likewise, zinc promotes the formation of derivatized insulin hexamers and insulin analogues. The hexamer formation is conveniently obtained by bringing the pH of a solution comprising protein derivatized in the neutral region in the presence of Zn (II) ions, or by adding Zn (II) after the pH has been adjusted in the neutral region . For an efficient yield of microcrystals or an amorphous precipitate, the ratio of zinc to protein derivatized in the microcrystal and the amorphous precipitate of the present invention is bound at the lower limit by about 0.33, that is, two zinc atoms per hexamer of Derivatized protein which is needed for efficient hexamerization. The microcrystal and amorphous precipitate compositions will suitably be formed with about 2 to about 4-6 zinc atoms present. Even more zinc can be used during the process if there is a compound that competes with the protein for zinc binding, such as citrate or phosphate. Excess zinc above the amount needed for hexamerization may be desirable to drive hexamerization more strongly. In addition, excess zinc above the amount needed for hexamerization may be present in a formulation of the present invention, and may be desirable to improve chemical and physical stability, to improve suspeneion capacity and possibly to extend the action in -the time additionally. Consequently, there is a very wide range of zinc: protein ratios allowable in the formulations of the present invention. According to the present invention, zinc is present in the formulation in an amount of from about 0.3 moles to about 7 moles per mole of derivatized protein, and more preferably from about 0.3 moles to about 1.0 moles of derivatized protein. Still highly preferred is the ratio of zinc to derivatized protein from about 0.3 to about 0.7 moles of zinc atoms per mole of derivatized protein. More preferably, the ratio of zinc to derivatized protein is from about 0.30 to about 0.55 moles of zinc atoms per derivatized protein template. For higher zinc formulations that are similar to PZI preparations, the zinc ratio is from about 5 to about 7 moles of zinc per mole of derivatized protein. The zinc compound that provides zinc for the present invention can be any pharmaceutically acceptable zinc compound. The addition of zinc to "insulin preparations is known in the art, and are pharmaceutically acceptable sources of zinc." The zinc compounds preferred for zinc delivery by the present invention include zinc chloride, zinc acetate, zinc citrate, zinc oxide, and zinc oxide. Zinc and zinc nitrate A complexing compound is required for the microcrystals and precipitates of the present invention The complexing compound must be present in sufficient amounts to cause substantial precipitation and hexameric crystallization of the derivatized protein. The amounts can be easily determined for a particular preparation of a particular complexing compound by simple titration experiments Ideally, the concentration of the complexing compound is adjusted so that there is a negligible amount of the complexing compound remaining in the complex. the faee eoluble despuée de complet ar precipitation and crystallization. This requires combining the complexing compound based on an experimentally determined "isophane" ratio. It is expected that this - 2 - relationship is very similar to that of NPH and NPL. However, it may be slightly different because acylation can alter the nature of the protein-protamine interaction. When the protamine is the complexing compound, it is present in the microcrystal in an amount of about 0.15 mg to about 0.5 mg per 3.5 mg of the derivatized protein. The ratio of protamine to protein is preferably from about 0.25 to about 0.40 (mg / mg). Most preferably, the ratio is from about 0.25 to about 0.38 (mg / mg). Preferably, the protamine is in an amount of 0.05 mg to about 0.2 mg per mg of derivatized protein and much more preferably, of about 0.05 to about 0.15 milligrams of protamine per milligram of derivatized protein. Protamine sulfate is the preferred salt form of protamine for use in the present invention. In order to further extend the action time of the compositions of the present invention or to improve their sleepiness, additional protamine and zinc can be added after crystallization. Thus, formulations which have protamine at higher isophane ratios are also within the present invention. For these formulations, the ratio of protamine is from 0.25 mg to approximately 0.5 mg of protamine per mg of the derivatized protein. A required component of the microcrystals and precipitates of the present invention is a hexamer stabilizing compound. Lae structures of three hexameric conformations have been characterized in the literature and are referred to as T6, T3R3 and R6. In the presence of a hexamer stabilizing compound, such as the various phenolic compounds, the R6 conformation is stabilized. Therefore, it is highly probable that the hexamers are in an R6 conformation, or the T3R3 conformation in the crystals and precipitates produced in the presence of a hexamer stabilizing compound, such as phenol. A wide range of hexamer stabilizer compounds are suitable. At least two moles of hexamer stabilizer compound per hexamer or derivatized protein are required for effective stabilization of the hexamers. It is preferred that at least three moles of hexamer stabilizer compound per hexamer or derivatized protein be present in the microcrystals and precipitates of the present invention. The presence of higher ratios of hexamer stabilizer compound, at least up to 25 to 50 times higher, in the solution from which the microcrystals and precipitates are prepared, does not adversely affect the stabilization of the hexamers.

In the formulations of the present invention, a preservative may be present, especially if the formulation is designed to be sampled at multiple times. As mentioned before, a wide range of conservatives is known. Preferably, the preservative is present in the solution in a suitable amount to provide a sufficient antimicrobial effect to meet the requirements of the pharmacopoeia. Preferred preservatives are phenolic preservatives which are listed above. The preferred concentrations for the phenolic preservative are from about 2 mg to about 5 mg per milliliter of the aqueous suspension formulation. These concentrations refer to the total mass of phenolic preservatives because mixtures of individual phenolic preservatives are contemplated. Suitable phenolic preservatives include, for example, phenol, m-cresol and methyl paraben The preferred phenolic compounds are phenol and m-cresol Mixtures of phenolic compounds, such as phenol and m-cresol, are also contemplated and highly preferred. Examples of mixtures of "phenolic compounds are 0.6 mg / ml phenol and 1.6 mg / ml m-cresol, and 0.7 mg / ml phenol and 1.8 mg / ml m-cresol. The microcrystals of the present invention are preferably oblong in shape, and the crystals alone are composed of derivatized protein complexes which complex with a divalent cation, and which include a complexing compound and a hexamer stabilizer compound. The average length of the microcrystals of the present invention is preferably within the range of 1 micrometer to 40 micrometers, and more preferably within the size range of 3 micrometers to 15 micrometers. A preferred composition comprises from about 3 mg to about 6 mg of protamine sulfate per 35 mg of derivatized protein and from about 0.1 to about 0.4 mg of zinc per 35 mg of derivatized protein. Another preferred composition comprises from about 10 mg to about 17 mg of protamine sulfate per 35 mg of derivatized protein and from about 2.0 to about 2.5 mg of zinc per 35 mg of derivatized protein. Another preferred composition comprises, per ml, protamine sulfate 0.34-0.38 mg; zinc, 0.01-0.4 mg; and derivatized protein 3.2-3.8 mg. The use of lae present insoluble compositions to prepare a medication for the treatment of diabetes or hyperglycemia is also contemplated. The amorphous precipitates and crystals of the present invention can be prepared for use in medicaments, or others can be used by many different processes. In summary, the appropriate processes will generally follow the sequence: solubilization, hexamerization, complex formation, precipitation, crietalization and optionally formulation. Solubilization means the derivatized protein solution enough to allow it to form hexamers. Hexamerization refers to the process in which the molecules of the derivatized protein bind with zinc (II) atoms to form hexamers. The formation of complexes indicates the formation of insoluble complexes between the hexamers and the protamine. Precipitation typically results from the formation of insoluble complexes. Crystallization involves the conversion of precipitated hexamer / protamine complexes into crystals, typically bar-like crystals. The solubilization is carried out by dissolving the derivatized protein in an aqueous solvent. The aqueous solvent can be, for example, an acid solution, a neutral solution or a basic solution. The aqueous solvent may be partially constituted by a miscible organic solvent, such as ethanol, acetonitrile, dimethyl sulfoxide and the like. Acidic solutions may be, for example, HCl solutions, advantageously from about 0.01 N HCl to about 1.0 N HCl. Other acids that are pharmaceutically acceptable are also used. The basic solutions can be, for example, NaOH solutions, advantageously from about 0.01N NaOH to about 1.0 NaOH or greater. Other bases that are pharmaceutically acceptable can also be used. In order to stabilize the protein, the concentration of acid or base is preferably as low as possible while still effective to adequately dissolve the derivatized protein. Many derivatized proteins can be dissolved at neutral pH. Solutions for dissolving the derivatized proteins at neutral pH may contain a buffer and optionally, salt, a compound or phenolic compound, zinc or an iaotonicity agent. The solution conditions required for hexamerization are those that allow the formation of zinc-derivatized protein hexamers in solution. Typically, hexamerization requires zinc and neutral pH. The presence of a hexamer stabilizing compound advantageously affects the conformation of the hexamer-derivatized protein and promotes the formation of R6 or T3R3 hexamers. The complex formation step should involve the combination of protamine with hexamer under solution conditions where each is initially soluble. This can be done by combining separate solutions of derivatized hexamer protein and protamine, or by forming a solution of derivatized protein and protamine at acidic or basic pH, and then shifting the pH to the neutral range. During crystallization, the solution conditions must stabilize the crietalizing species. Therefore, the solution conditions will determine the speed and the re-emergence of the crietalization. Likewise, crystallization occurs through an equilibrium involving non-crystalline precipitated derivatized protamine complexes, dissolved derivatized protein-protamine complexes, and crystallized derivatized protein-protamine. The "conditions chosen for crystallization drive the equilibrium towards the formation of crystals." Furthermore, in light of the equilibrium that is established as hypothesis, it is expected that the solubility of the derivatized protein profoundly alters the speed and size due to a lower solubility The net conversion of the precipitate to the solution to the crystal will decrease, and it is well recognized that the decrease in the crystallization rate often results in larger crystals, hence the crystallisation rate and the size of the crystal depend on the size and nature of the derivatizing portion in the derivatized protein The crystallization parameters that affect the crystallization rate and the size of the crystals of the present invention are: size and nature of the acyl group; temperature; the presence and concentration of compounds that compete with the zinc derivatized protein, such as citrate, phosphate and the like; the nature and concentration of the compound or phenolic compound; the concentration of zinc; the presence and concentration of a miscible organic solvent; the time allowed for crystallization; the pH and ionic strength; the identity and concentration of the shock absorber; the concentration-of the precipitants; the presence of planting materials; the shape and material of the container; the agitation speed and the total protein concentration. It is considered "that the temperature and concentration of the competing compounds are of particular importance, competing compounds, such as citrate, alter the speed at which the crystals form, and indirectly, the size and quality of the crystals. The compounds can exert their effect by forming complexes of coordination with zinc in solution, thus competing with relatively weak zinc binding sites on the surface of the zinc-derivatized protein hexamer.The occupation of these sites of weak surface binding probably prevents In addition, many derivatized proteins are partially insoluble in the presence of a little more than 0.333 zinc per mole of derivatized protein, and the presence of competing compounds restores solubility and allows crystallization.The exact concentration of the competing compound is needed optimize for each derivatized protein. erior, of course, is the concentration at which zinc precipitates by the competing compound or the concentration at which the competing residual compound may be pharmaceutically unacceptable, for example, - when it may cause pain or irritation at the site of administration. Next follows an example of a process for preparing the precipitates and crystals of the present invention. A measured amount of a powder of the derivatized protein is dissolved in an aqueous solvent containing a phenolic preservative. To this solution is added a zinc solution as one of its soluble saltse, for example Zn (II) Cl2, to provide about 0.3 moles of zinc per mole of derivatized insulin to about 0.7 moles, or at most 1.0 moles of zinc per mol of derivatized ineulin. Optionally, abeolute ethanol or other suitable organic solvent may be added to this solution in an amount to produce the solution from about 5% to about 10% by volume of organic solvent. This solution can then be filtered through a low protein binding filter of 0.22 microns. A second solution is prepared by dissolving a measured amount of protamine in water equal to one tenth of the concentration by weight of the derivatized insulin solution mentioned above. This solution is filtered through a low protein binding filter of 0.22 microns. The derivatized insulin solution and the protamine solutions are combined in equal volumes, and the resulting solution is then stirred slowly at room temperature (typically about 20-25 ° C) where the microcrystals of the derivatized protein are formed within a period of approximately 12 hours to approximately 10 days. The microcrystals are then separated from the mother liquor and introduced into a different solvent, for storage and administration to a patient. Examples of suitable aqueous solvents are as follows: water for injection containing 25 mM TRIS, 5 mg / ml phenol and 16 mg / ml glycerol; water for injection containing __2 mg / ml of dibasic sodium phosphate, 1.6 mg / ml of m-cresol, 0.65 mg / ml of phenol and 16 mg / ml of glycerol; and water for injection containing 25 mM TRIS, 5 mg / ml phenol, 0.1 M trisodium citrate and 16 mg / ml glycerol. In a preferred embodiment, the crystals are prepared in a manner that eliminates the need to separate the crystals from the mother liquor. Thus, it is desired that the mother liquors themselves be suitable for administration to the patient, or that the mother liquor may be rendered suitable for administration by dilution with a suitable diluent. The term "diluent" will be understood to mean a solution comprised of an aqueous solvent in which the various pharmaceutically acceptable excipients, including without limitation, a buffer, an isotonicity agent, zinc, a preservative, protamine and the like are dissolved. In addition to the derivatized insulin, the divalent cation, the complexing compound and the hexamer stabilizer compound, the pharmaceutical compositions adapted for parenteral administration according to the present invention can utilize additional excipients and carriers such as water miscible organic solvents such as glycerol, sesame oil, aqueous propylene glycol and the like. When present, such agents are usually used in an amount ranging from about 0.5% to about 2.0% by weight based on the final formulation. Examples of such pharmaceutical compositions include aqueous, isotonic, and sterile saline solutions of the derivatized insulin derivative buffered with a pharmaceutically acceptable, pyrogen-free buffer. For additional information regarding the variety of techniques using conventional excipients or carriers for parenteral products, please see Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, PA, USA (1985), which is incorporated herein by reference. reference. In the broad practice of the present invention, it is also contemplated that the formulation may contain a mixture of the microcrystalline formulation and a soluble fraction of the derivatized insulin or a soluble fraction of the normal insulin, or a rapid-acting insulin analogue. such as LysB28, ProB29 -human insulin. Such mixtures are designed to provide a combination of control of glucose levels during feeding time, which is provided by soluble insulin, and a basal control of glucose levels, which is provided by insoluble insulin. The following preparations and examples illustrate and explain the invention. The scope of the invention is not limited by these preparations and examples. The reference to "part" for solid is equivalent to parts by weight. The reference to "parts" for liquids means parts by volume. Percentages, when used to express concentration, mean mass by volume. All temperatures are in degrees centigrade (° C). The term "TRIS" refers to 2-amino-2-hydroxymethyl-1,3-propanediol. The zinc solution of 1-000 parts per million (ppm) is prepared by diluting 1.00 ml of a standard atomic absorption solution of 10,000 ppm zinc [Ricca Chemical Company, zinc in dilute nitric acid] with water to a final volume of 10.00 ml. In many of the preparations described below, the performance of the precipitates and crystals is estimated. The performance estimate is based on the determination of the amount of derivatized insulin or derivatized insulin analogue in the precipitate or crystal, and an estimate of the amount of insulin initially in solution. To determine the amount of derivatized protein, the resolved precipitate or crystal samples are analyzed, and the supernatant prior to the precipitate or the crystals are analyzed by reverse phase CLAP as described below. Briefly, the analytical system is based on a C8 reverse phase column, at 23 ° C. The flow rate is 1.0 ml / min and UV detection at 214 nm is used. Solvent A is 0.1% trifluoroacetic acid (vol: vol) in 10:90 (vol: vol) of acetonitrile: water. Solvent B is 0.1% trifluoroacetic acid (vol: vol) in 90:10 (vol: vol) of acetonitrile: water. The development program is (minutes,% B): (0.1.0); (45.1,75); (50.1,100); (55,100); (57.0); (72.0). All changes are linear. Other analytical systems can be designed by the experts to obtain the same objective. For the preparation for the CLAP analysis, aliquots of the well-mixed suspensions are dissolved when diluted with either 0.01 N HCl or 0.03 N HCL. The CLAP analysis of these solutions gives the total mg / ml of derivatized protein. The aliquots of the suspensions are centrifuged for approximately 5 minutes in an Eppendorf 5415C microcentrifuge at 14,000 rpm.The decanted supernatant is diluted either with 0.01 N HCl or 0. IN and analyzed by CLAP.The precipitate is washed by resuspension in solution , Dulbecco's phosphate-buffered salt (without calcium or magnesium) and re-sedimented by centrifugation The buffer is decanted and the solid is redissolved in 0.01 N HCl The precipitate that has been dissolved again is analyzed by CLAP. uses CLAP to confirm the presence of the expected proteins in the acidified suspension, the redissolved precipitate and the supernatant, and the protein concentrations are also determined.The retention times of the peaks in the chromatograms of the redissolved precipitates are compared with the observed retention times for protamine and the insulin compounds used to produce the formulations. The retention times are always good, which shows that the protamine and the derivatized proteins used to make the formulations are actually incorporated into the crystals. The concentrations of the derivatized proteins are determined by comparing the appropriate peak areas with the areas of a standard. Protamine concentrations are not quantified. A "0.22 mg / ml" derivatized insulin solution is used as standard. A standard containing protamine is carried out, but only for the purpose of determining the retention time. The concentration of protamine is not quantified. In many of the preparations described below, a standard spectrophotometric assay is used to determine how quickly the crystals dissolve in Dulbecco's buffered saline solution (pH 7.4) at room temperature. immediately below are indicated, where appropriate, in the descriptions of the preparations For all dissolution tests a spectrophotometer suitable for measuring in the ultraviolet range is used, and equipped with a 1 cm cuvette and a magnetic tray stirrer. The cuvette containing a small stir bar and 3.00 ml of phosphate buffered solution (PBS) is placed in the cell compartment of the spectrophotometer, the instrument is adjusted to 320 nm and calibrated to zero against the same buffer. add to the cuvette 4.0 microliters of a well-suspended formulation, which usually has a total concentration approximately equivalent to a U50 formulation, or approximately 1.6 to 1.8 mg / ml. After waiting 1.0 minute for mixing, the optical density is recorded at 320 nm. Since the proteins involved in this work do not absorb light at 320 nm, the decrease in optical density is due to a reduction in scred light such as dissolved crystals. The time for the optical density to decrease to half its initial value is typically (tl / 2). As a control, 2.0 microliters of Humulin ™ N UlOO (ie, human insulin NPH, which is also known as human NPH insulin) is added to 3.00 ml of PBS buffer and the optical density is monitored at 320 nm, as in the above . The mean dissolution time (tl / 2) for Humulin ™ N is approximately 6 minutes.

Preparation 1 Gly (A21), Arg (B31), Arg (B32) - human insulin analogue Gly (A21) Arg (B31) Arg (B32) -human insulin is obtained from a fermentation of E. coli in which the precursor molecule of Gly (A21) -human proinsulin is overexpressed in inclusion bodies. A portion of 94.7 g of the inclusion bodies is solubilized in 500 ml of 6 M guanidine hydrochloride containing 0.1 M TRIS, 0.27 M sodium sulfite and 0.1 M sodium tetrathionate, pH 10.5 at room temperature. The pH is rapidly reduced to 8.8 with 12 N HCl. After vigorous stirring in an open container for 45 minutes, the pH is decreased to 2.1 with phosphoric acid and the sample is centrifuged overnight at 4 ° C. The supernatant is decanted and stored at 4 ° C for further processing. The sediment is reextracted with 200 ml of an additional solution of pH 10.5 (see above) and then centrifuged for 3 hours at 4 ° C. This and the previously obtained supernatant where each 4X diluted with 100 mM sodium phosphate, pH 4, precipitates the product and other acid components. After allowing the precipitate to settle, most of the supernatant is decanted and discarded. The resulting suspension is centrifuged, followed by decanting and discarding the additional supernatant, which leaves wet grains of the crude human Gly (S21) S-sulfonate precursor-proinsulin. The granules are solubilized in 1.5 liters of 7 M deionized urea, adjusting the pH to 8 with 5 N NaOH and stirring for several hours at 4 ° C. Salt (NaCl) is then added to obtain a 1 M concentration and the sample is loaded onto a XAD-7 column (14 cm X 20 cm, Toso-Haas, Montgomeryville, PA), previously purged with 50% acetonitrile / ammonium bicarbonate. 50 mM, 50%, 10% acetonitrile / 50 mM ammonium bicarbonate 90%, and finally with 7 M deionized urea / 1 M NaCl / 20 mM TRIS, pH 8. Once loaded, the column is pumped with 4.5 liters of urea 7 M deionized / 1 M NaCl / 20 mM TRIS, pH 8 in solution, followed by 2.8 liters of 50 mM ammonium bicarbonate / 1 M NaCl and 6.5 liters of 50 mM ammonium bicarbonate. The column is eluted with a linear gradient of acetonitrile in 50 mM ammonium bicarbonate, while the eluent is monitored by UV at 280 nm. The peak of interest, the S-sulfonate precursor of partially purified Gly (A21) -proinsulin human, is harvested, lyophilized and subjected to a renaturation / disulfide process as follows. An amount of 5.4 g of the precursor is dissolved in 3 liters of 20 mM glycine, pH 10.5, 4 ° C. Then 15 ml of 240 mM cysteine hydrochloride are added with stirring, while maintaining the pH at 10.5 and the temperature at 4 ° C. The reaction solution is gently stirred at 4 ° C for 27 hours and then suspended by lowering the pH to 3.1 with phosphoric acid. 155 ml of acetonitrile are added, and the solution is loaded on a 5 x 25 cm C4 reverse phase column previously pumped with 60% acetonitrile / 40% water / 0.1% TFA and equilibrated in 10% acetonitrile / 90% water. / TFA 0.1%. Once loaded, the column is pumped with 1 liter of 17.5% acetonitrile / 82.5% water / 0.1% TFA and then eluted with a linear gradient of acetonitrile in 0.1% TFA while monitoring at 280 nm. The selected fractions are accumulated and lyophilized with a recovery of 714 mg. For the conversion of the proinsulin precursor to the desired insulin analogue, 697 mg of Gly (A21) human proinsulin are dissolved in 70 ml of 50 mM ammonium bicarbonate, and then cooled to 4 ° C, pH 8.3. A volume of 0.14 ml of a 1 mg / ml porcine trypsin solution (Sigma Chemical Company, St. Louis, Mo) in 0.01 N HCl is added to the sample solution, which is gently stirred at 4 ° C for approximately 24 hours. hours. An additional 0.14 ml of trypsin solution is added to the reaction solution, which is then stirred for an additional 21 hours and 45 minutes. The reaction is suspended by lowering the pH to 3.2 with 0.7 ml of glacial acetic acid and 0.3 ml of phosphoric acid. The sample solution of Gly (A21) Arg (B31) Arg (B32) - human insulin suspended from the tryptic separation reaction is diluted 4 X with 30% acetonitrile / 50 mM acetic acid 70%, pH 3.1, and loaded onto a 1 x 30 cm S Hyper DF cation exchange column (Biosepra, Marlborough, MA) previously pumped with 30% acetonitrile / 50 mM acetic acid 70% / 500 mM NaCl, pH 3.3, and it is equilibrated in acetonitrile 30% / acetic acid 50_mM 70%. Once loaded, the column is pumped with approximately 50 ml of acetonitrile 30% / acetic acid 50 mM 70%, and then eluted with a linear gradient of NaCl in acetonitrile 30% / acetic acid 50 mM while monitoring the eluent to 276 nm. The selected fractions containing Gly (A21) Arg (B31) Arg (B32) -human insulin are accumulated, diluted 3X with purified water and loaded onto a C4 reverse phase column of 2.2 x 25 cm (Vydac, Hesperia, CA ) pre-pumped with 60% acetonitrile / 40% water / 0.1% TFA, then with 10% acetonitrile / 90% water / 0.1% TFA. Once loaded, the column is pumped with approximately 200 ml of 10% acetonitrile / 90% water / 0.1% TFA and then eluted with a linear gradient of acetonitrile in 0.1% TFA. The selected fractions accumulate and lyophilize which provides a recovery of 101 mg. Analytical CLAP shows a purity greater than 95% of the main peak. Electrosuppression mass spectroscopy (ESMS) analysis of the purified protein provides a molecular pee of 6062.9 (theoretical 6063.0).

Preparation 2 Des (B30) -human insulin Des (B30) -human insulin is prepared from human proinsulin by controlled cryptic hydrolysis. A mass of 2 g of human proinsulin is biosynthesized in recombinant E. coli and purified by conventional methods [Frank, B. H., et al. , in PEPTIDES: Synthesis -Structure -Function. Proceedings of the Seventh Ameri can Peptide Symposium, Rich, D. H. and Gross, E. (Eds.), Pierce Chemical Company, Rockford, p. 729-738, 1981; also, Frank, B.H., U.S. Pat. No. 4,430,266, issued on February 7, 1984, each of which is incorporated as a reference] that is dissolved in 400 ml_ of buffer HEPES 0.1 M, pH 7.5. After the addition of 8 ml of 1 M CaCl 2 (in water) and adjustment of pH to 7.5 with 5 N NaOH, 2 ml of a 10 mg / ml solution of porcine trypsin (Sigma) in 0.01 N HCl are transferred to the Sample solution, while stirring gently. The reaction solution is allowed to stir at room temperature for 2 hours and 42 minutes, at which time it is transferred to an environment at 37 ° C while stirring occasionally. After 1 hour and 45 minutes at 37 ° C, the enzymatic reaction is suspended by lowering the pH to 3.0 with phosphoric acid and the temperature at 4 ° C for storage. Subsequently, the solution is brought to room temperature and diluted with 50 ml of acetonitrile, and then to a final volume of 500 ml with purified water, then loaded onto a column of 2.5 x 58 cm CG-161 (Toso-Haas) previously pumped with 1 hp (column volume) of 40% acetonitrile / 0.1 M 60% ammonium sulfate, pH 2.5, and 2 c.v. of 10% acetonitrile / 0.1 M 90% ammonium sulfate, pH 2.5. Once loaded, the column is pumped with 1 c.v. of 10% acetonitrile / 0.1 M 90% ammonium sulfate, pH 2.5. The column is eluted with a linear gradient of acetonitrile in 0.1 M ammonium sulfate, pH 2.5, while monitoring the eluent at 276 nm. The peak of interest, partially purified of des (B30) -human insulin is collected by accumulating the selected fractions. This cumulative sample of des (B30) - partially purified human insulin is diluted to 1.28 liters with purified water, pH 3.5 and is applied to a cation exchange column of 1 x 29 cm S HyperD F (Bioeepra) previously pumped with 1 c.v. of acetonitrile 30% / TFA 0.1% 70% / NaCl 0.5 M, pH 1.9, and 2 c.v. of acetonitrile 3_0% / TFA 0 ^ 1% 70%, pH 2.3. Once loaded, the column is pumped with 1 c.v. of acetonitrile 30% / TFA 0.1% 70%, pH 2.3, and then eluted with a linear gradient of NaCl in acetonitrile 30% / TFA 0.1% 70%, pH 1.9 to 2.3, while monitoring the eluent at 276 nm. The selected fractions containing des (B30) -purified human insulin are accumulated, diluted with 2.5X with purified water and loaded in a SepPak C8 package of 35 ce. (Waters, Milford, MA) previously cleaned and primed with 2 c.v. of 60% acetonitrile / 0.1% TFA 40% and 2 c.v. of acetonitrile 10% / TFA 0.1% 90%. Once the SepPak is loaded, it is purged with 3 c.v. of 10% acetonitrile / 0.1% TFA 90% and then eluted with 2 c.v. of 60% acetonitrile / 0.1% TFA 40%. The lyophilized eluent provides 500 mg. An assay by analytical CLAP suggests more than 95% of the main peak. Electrospray mass spectroscopy (ESMS) analysis of the purified protein gives a molecular weight of 5706.5 (5707, theoretical).

Preparation 3 Rabbit insulin Rabbit insulin is prepared as described in Chance, R. E., et al. [Proinsulin, Insulin, C-Peptide, Baba, S., et al. (Eds.), Excerpta Medica, Amsterdam-Oxford, pp. 99-105 (1979)].

Preparation 4 Asp (B28) - Human insulin analogue Asp (B28) -humanine is prepared and purified essentially in accordance with the teachings of "Examples 31. and 32 of Chance, RE et al. (US Patent No. 5,700,662, issued December 23, 1997) which it is expressly incorporated herein by reference, des (B23 -30) -human insulin [Bromer, WW and Chance, RE, Biochim, Biophys. Acta, 133: 219-223 (1967), which is incorporated in the present as reference] and the synthetic octapeptide Gly-Phe-Phe-Tyr-Thr-Asp-Lys (Tfa) -Thr, using trypsin-assisted semi-synthesis, purified by gel filtration and reverse phase CLAP, is treated with 15% Ammonium hydroxide (v / v) for 4 hours at room temperature to remove the trifluoroacetate (Tfa) blocking group from Lys (B29), purified by reverse phase CLAP, and lyophilized.

Preparation 5 Synthesis of derivatized proteins The following is a generality with respect to the synthesis of certain derivatized proteins used to prepare the precipitates and crystals of the present invention. The generalities should be read together with the data in Table 2 below. A measured mass of insulin purified from an insulin analog is dissolved in a volume of dimethyl sulfoxide (DMSO) with stirring. Then a measured volume of tetramethylguanidine hydrochloride (TMG) is added and the solution mixed thoroughly. In a separate container, a metric measure of N-acyl-euccinimide (ÑAS) is dissolved in a measured volume of DMSO. A measured volume of the second solution is added to the first solution. The reaction is carried out at room temperature, and the progress of the reaction is monitored by analysis of samples of the reaction mixture using CLAP. The reaction is suspended by adding a measured volume of ethanolamine and then acidifying to pH 2-3, then the reaction mixture is subjected to purification using reverse phase chromatography alone, or using a combination of cation exchange chromatography followed by chromatography. in reversed phase Reverse phase purification is carried out using a FPMA FPVHR (Pharmacia) with UV detection at 214 nm or 280 nm, a fraction collector, a C18 column of 2.2 x 25 cm or 5 x 30 cm, 2.5 or 5 ml / min flow rate, at room temperature The liquid phases are mixtures of a solution A [0.1% trifluoroacetic acid (TFA) in 10:90 acetonitrile: water (vol: ol)] and solution B [0.1% trifluoroacetic acid (TFA) in 70:30 acetonitrile: water (vol: ol)] appropriate to elute and separate the species of interest.The column typically equilibrates and charges while in 100% solution. , a gradi is used linear entity of a certain proportion of solution B to properly separate the reaction products. Fractions that contain the product accumulate. The development of purification methods is within the skill of the "experts." Table 2 below provides experimental data, in accordance with the general description above, for the synthesis of the derivatized proteins which are used to prepare the various embodiments of the present invention. invention The initiate proteins are prepared as described above, or according to conventional methods.The additional purification is used to provide highly purified initial proteins for the synthesis described below.The synthesis of insulin, insulin analogs and proineulin is within the scope of the invention. skill of the art, and can be carried out using recombinant expression, semi-synthesis or solid phase synthesis followed by chain combination The purification of proteins synthesized to a purity suitable for preparing the derivatives used in the present invention is carried out by conventional technique of purific The molecular weight of the purified derivatives is confirmed by mass spectrometry via mass analysis by electrospray (ESMS). The allocation of the acylation site is based on a chromatographic analysis ("CLAP"), or an N-terminal analysis ("N-terminal"), or both.

Table 2. Summary of synthesis of various derivatized proteins * the purification involves a first CLAP in phase inverea, after a CLAP of cation exchange and after a CLAP of faee inverse.

The following are generalities regarding the synthesis of additional derivatized proteins. The generalities should be read together with the data in Table 3, below, to provide complete synteis schemes. A measured mass of insulin purified from an insulin analogue is dissolved by adding it to a measured volume of 50 mM boric acid, pH 2.57. Then a measured volume of acetonitrile, equal to the volume of the boric acid solution, is slowly added with stirring. The volume of "solvent" is the sum of the volumes of boric acid and acetonitrile. The pH of the solution is adjusted between 10.2 and 10.5 using NaOH. -In a "separate container", a measured mass of N-acyl-euccinimide ("ÑAS") dissolved in a measured volume of DMSO is dissolved. A measured volume of the second solution is added to the first solution. The reaction is carried out at room temperature, the pH is maintained above 10.2 as necessary, and the progress of the reaction is monitored by analysis of the samples of the reaction mixture using CLAP. The reaction is suspended by acidifying to pH 2-3. The reaction mixture is then subjected to purified using a "reversed phase chromatography system, as described above." Table 3 provides experimental data, according to the foregoing general, for the synthesis of the derivatized proteins that were used to prepare the various embodiments of the present invention The molecular weight of the purified derivatives is confirmed by mass spectrometry via mass analysis by electrospray (ESMS) The allocation of the acylation site is based either on chromatographic analysis ("CLAP") or in an analysis of the N-terminal part ("N-terminal"), or both.

Table 3. Summary of the synthesis of various deri-atomized proteins Initial protein GlyA21, ArgB31, human insulin des (B27) -analog ArgB32-human insulin analog human insulin protein mass (mg) 86.2 134.8 44.8 solvent (ml) 10 20 acyl chain ÑAS n-octanoyl n-methyl-hexaiioyl n-octanoyl Mass of N-acyl succinimide (mg) 22.4 749 5.6 Volume of DMSO (ml) 0.5 4.93 * 1.0 Volume of solution ÑAS 0.1 15 0.052 0.993 added (ml) Reaction time (min) 40 45 40 Total return (%) 45 45 53 Molecular weight (theoretical) 6189.2 5919.9 5832.8 Molecular Weight (ESMS) 6189.2 5919.9 5832.9 Purity by CLAP (7c) 97 96 Acrylation site (CLAP) Ne Ne Ne * Dieted in acetonitrile instead of DMSO.

* Dissolved in acetonitrile instead of DMSO. ** Performance of Al-Na, 29-Ne-diacyl-derivative of human insulin *** Determined by matrix-assisted laser desorption ionization (MALDI) of mass spectroscopy, instead of "electrospray mass spectroscopy.

* Dissolved in acetonitrile, instead of DMSO. ** Performance of Al-Na, B29-Ne-diacyl-derived human insulin Preparation 6 Microcrystals of B29-Ne-octanoyl-LysB29 human insulin A dry powder of B29-Ne-octanoyl-LysB29-human insulin (7 parts by mass) is dissolved in 1000 parts by volume in an aqueous solvent containing 25 mM TRIS, 0.1 M trisodium citrate and 10 mg / ml phenol at pH 7.6. To this solution is added 150 parts of a 15.3 mM solution of zinc chloride. The pH is adjusted to 7.6 with 1 N HCl and / or 1 N NaOH. Then 120 parts by volume of ethanol are added. This solution is filtered through a low protein binding filter of 0.22 micrometers. A second solution is prepared by dissolving 6 parts by mass of protamine sulfate in 10,000 parts by volume of water and then filtering through a 0.22 micron low protein binding filter. Equal volumes of the acylated insulin solution and the protamine sulfate solution are combined. An amorphous precipitate forms. This suspension is gently stirred for 24 hours at room temperature (typically at a temperature of about 22 ° C). The amorphous precipitate becomes a microcrystalline solid.

Preparation 7 Formulation of microcrystals of B29-Ne-octanoyl-LysB29 human insulin The microcrystals are prepared by the preparation method 6 and separated from the mother liquors and recovered by conventional solid / liquid separation methods such as filtration, centrifugation or decantation. The microcrystals recovered are then suspended in a solution containing 25 mM TRIS, 5 mg / ml phenol and 16 mg / ml glycerol, pH 7.4, such that the final concentration of acylated ineulin corresponds to the equivalent of a solution of 100 mg / ml. U / ml of insulin.

Preparation 8 Microcrystals of B29-Ne-hexanoil-LysB29 human insulin A dry powder of B29-Ne-hexanoil-LysB29 is dissolved (7 parts by mass) in 1000 volume parts of an aqueous solvent consisting of 25 mM TRIS, 0.1 M trisodium citrate, 10 mg / ml phenol and 16 mg / ml glycerol at pH 7.6. To this solution is added 150 parts of a 15.3 mM solution of zinc chloride. The pH 7.6 is mixed with 1 N HCl and / or 1 N NaOH. Then 120 parts by volume of ethanol are added. This solution is filtered through a 0.22 micron low protein binding filter. A second solution is separated by dissolving 6 parts by mass of protamine sulfate in 10,000 parts by volume of water and then filtering through a 0.22 micron low protein binding filter. Equal volumes of the acylated ineulin solution and the protamine eulfate solution are combined. An amorphous precipitate forms. This suspension is stirred slowly for 24 hours at room temperature (typically at a temperature of about 22 ° C). The amorphous precipitate becomes a microcrystalline solid.

Preparation 9 Formulation of B29 -Ne-hexanoyl-LysB29 microcrystals of human insulin The microcrystals prepared by the method of preparation 8 are separated from the mother liquors and recovered by conventional solid / liquid separation methods. The recovered microcrystals are then suspended in a solution - ioo - consisting of 2 mg / ml of sodium dibasic phosphate, 1.6 mg / ml of m-cresol, 0.65 mg / ml of phenol and 16 mg / ml of glycerol, pH 6.8 , so that the final concentration of the acylated insulin corresponds approximately to the equivalent concentration of a 100 U / ml insulin solution.

Preparation 10 Microcrystals of B28-Ne-octanoyl-LysB28, ProB29-human insulin A dry powder of B28-Ne-octanoyl-LysB28-LysB28, ProB29-human insulin (7 parts by mass) is dissolved in 1000 parts by volume of an aqueous solvent consisting of 25 mM TRIS, 0.1 M trisodium citrate and 10 mg / ml. ml of phenol at pH 7.6. To this solution is added 150 parts of a 15.3 mM solution of zinc chloride. The pH is adjusted to 7.6 with 1 N HCl and / or 1 N NaOH. Then 120 parts by volume of ethanol are added. Eeta eolución is filtered through a low-protein binding filter of 0.22 micrometer. A second solution is prepared by dissolving 6 parts by mass of protamine sulfate in 10,000 parts by volume of water and then filtering through a 0.22 micron low protein binding filter. Equal volumes of the acylated insulin solution and the protamine sulfate solution are combined. An amorphous precipitate forms. After 24 hours at room temperature (typically at a temperature of about 22 ° C), the amorphous precipitate becomes a microcrystalline solid.

Preparation 11 Formulation of microcrystals of B28-Ne-octanoyl-LysB28, ProB29 -human insulin The microcrystals prepared by the method of preparation 10 are separated from the master water and recovered by conventional solid / liquid separation methods. The recovered microcrystals are then suspended in a solution consisting of 25 M TRIS, 5 mg / ml phenol, 0.1 M trisodium citrate and 16 mg / ml glycerol, pH 7.6, so that the final concentration of acylated insulin corresponds approximately to the equivalent concentration of a 100 U / ml insulin solution.

Preparation 12 Microcrystals of B28-Ne-butyryl-LysB28, ProB29 -human insulin -I02- A dry powder of B29-Ne-butyryl-LysB29 is dissolved in human insulin (7 parts by mass) in 100 parts by volume of an aqueous solvent consisting of 25 mM TRIS, 0.1 M trisodium citrate and 10 mg / ml phenol at pH 7.6. To this solution is added 150 parts of a 15.3 mM solution of zinc chloride. The pH 7.6 is adjusted with 1 N HCl and / or 1 N NaOH. Then 120 parts by volume of ethanol are added. This solution is filtered through a 0.22 micron low protein binding filter. A second solution is prepared by dissolving 6 parts by mass of protamine sulfate in 10,000 parts by volume of water and then filtering through a 0.22 micron low protein binding filter. Equal volumes of the acylated insulin solution and the protamine sulfate solution are combined. An amorphous precipitate forms. After 24 hours at room temperature (typically at a temperature of about 22 ° C), the amorphous precipitate is converted to a microcrystalline solid.

Preparation 13 Formulation of microcrystals of B28-Ne-butyryl-LysB28, ProB29 -human insulin The microcrystals are prepared by the preparation method 7 and are separated from the mother liquor and recovered by conventional solid / liquid separation methods. Loe / miicrocrystals recovered are then suspended in a solution consisting of 25 mM TRIS, 2.5 mg / ml m-cresol and 16 mg / ml glycerol, pH 7.8, so that the final concentration of acylated insulin corresponds approximately to the equivalent concentration of a 100 U / ml insulin solution.

Preparation 14 Microcrystals of B29-Ne-butyryl-human insulin Prepare B29 -Ne-butyryl-human insulin as described in preparation 5. Dissolve a sample of 16.21 mg of human B29-Ne-butyryl-insulin in 0.8 ml of 0.1 N HCl. After shaking for 5-10 minutes, a volume of 0.32 ml of a zinc nitrate solution containing 1000 parts per million (ppm) of zinc (II) is added, and the resulting solution is carefully mixed by stirring. Deepuée, 3.2 ml of crystallization diluent is added, and the resulting mixture is stirred until it is completely mixed. The crystallization diluent is prepared by dissolving in water, with stirring, 0.603 g of TRIS, 1,007 g of phenol, 1582 g of glycerol and 2,947 g of trisodium citrate. Additional water is added to bring the solution to 100 ml. After mixing the zinc-insulin derivative solution with the crystallization diluent, adjust the pH of the resulting solution to 7.59 using aliquots of 1 N HCl and 1 N NaOH as needed. The pH adjusted solution is filtered through a 0.22 micron low protein binding filter. To a volume of the filtered solution is added an equal volume of an aqueous solution of protamine, prepared by dissolving 18.59 mg of protamine sulfate in water to a final volume of 50 ml. The mixture of the two volumes is subjected to a gentle centrifugation to complete the mixing and then allowed to stand at 25 ° C. They form . bar-like crystals, with a very high performance (greater than 90%). In the spectrophotometric dissolution test described above, the crystals of B29-Ne -butyryl-human insulin have tl / 2 of 32-33 minutes, compared to about 6 minutes for Humulin ™ "N in the same assay.

Preparation 15 Microcrystals of B29 -Ne -pentanoyl-human insulin Human B29-Ne-pentanoyl-insulin is prepared as described in preparation 5. A sample of 16.4 mg of human B29-Ne-pentanoyl-insulin in 0.8 ml of 0.1 N HCl is dissolved. The procedure described in the preparation is followed. 14, except that the pH is adjusted to 7.60, and the protamine solution contains 18.64 mg of protamine sulfate in a total volume of 50 ml. Crystal-like bars are formed with a high yield (greater than 80%). In the spectrophotometric dissolution test described above, the crietalee of human B29-Ne-pentanoyl-ineulin have a tl / 2 of 33-34 min, compared to about 6 min for Humulin ™ in the same assay.

Preparation 16 Microcrystals of B29 -Ne -hexanoyl-human insulin Human B29 -Ne -hexanoyl -insulin is prepared as described in preparation 5. A sample of 15.87 mg of human B29-Ne -hexanoyl -insulin is dissolved in 0.8 ml of 0.1 N HCl. The procedure described in the preparation is followed. 14, except that the pH is adjusted to 7.58. Crystal like bars are formed, with a very high yield (greater than 90%). In the spectrophotometric dissolution test described above, the B29 -Ne -hexanoyl -insulin human crystals have a tl / 2 of 69-70 minutes, compared to about 6 minutes for Humulin ™ N in the same assay.

Preparation 17 Microcrystals of human B29-Ne- (2-methylhexanoyl) -insulin Human B29-Ne- (2-methylhexanoyl) -insulin is prepared as described in preparation 5. A mass of 8.19 mq of human B29-Ne- (2-methylhexanoyl) -insulin is dissolved in 0.400 ml of 0.1 N HCl. After stirring for 5-10 minutes, 0.160 ml of a zinc nitrate solution containing 1000 parts per million (ppm) of zinc (II) is added and the resulting solution is mixed thoroughly. Then 160 ml of the diluent are added and mixed (in 100 ml: 0.604 g of TRIS, 1,003 g of phenol, 3.218 g of glycerol and 3.069 g of trisodium citrate). The pH of this solution is adjusted to 7.61 with small amounts of 1.0 N HCl and 1.0 N NaOH, and then the solution is filtered through a 0.22 micron low protein binding filter. To 2 ml of this filtered solution is added 2 ml of a protamine solution "(in 100 ml, 37.41 mg of protamine) .The mixture is subjected to gentle centrifugation.A precipitate is formed, the mixture is left without alterations to 25 ° C. Bar-like crystals are formed in good yield (greater than 65%) In the spectrophotometric dissolution test described above, the crystals of B29-Ne-2-methylhexanoyl-human insulin have a tl / 2 of 23 minutes, compared to approximately 6 minutes for Humulin ™ in the same day.

Preparation 18 Microcrystals of B29-Ne-octanoyl-human insulin 4.17 mg of B29 -Ne-octanoyl-LysB29-human insulin is dissolved in 1 ml of a solvent consisting of 25 mM TRIS, 0.1 M trisodium citrate and 10 mg / ml phenol at pH 7.6. To this solution is added 7.15 ml of a 15.3 mM solution of zinc chloride. The resulting solution is adjusted to a pH of 7.6 with 1 N NaOH. To this solution is added 0.12 ml of ethanol. The resulting solution is filtered through a 0.22 micron low protein binding filter. A second solution is prepared by dissolving 3.23 mg of protamine sulfate in 10 ml of water and then filtering through a low-protein binding filter of 0.22 micrometer. A volume of 1 ml of the solution of B29-Ne-octanoyl-Lys29-human insulin and 1 ml of protamine sulfate solution combine, resulting in an immediate appearance of an amorphous precipitate. This solution is divided into two 1 ml portions which are transferred to jars and then shaken gently for 19 hours at room temperature (approximately 22 ° C) using an automatic oscillating action stirrer. This procedure results in the formation of a white to off-white microcrystalline solid. The analysis by CLAP of the crystals that have been removed from the mother liquor "and that have been washed carefully demonstrate the presence of protamine inside the crystalline material.

Preparation 19 Formulation of crystalline suspension comprising Ne-octanoyl-human insulin An acid solution of human Ne-octanoyl-insulin is prepared by dissolving 39.7 mg of human Ne-octanoyl-insulin in 1 ml of 0.1 N HCl. This solution is stirred for about 15 minutes. To this solution is added 0.4 ml of a zinc nitrate solution, containing 1000 parts per million (ppm) of zinc (II) with stirring. The zinc nitrate solution is a 1:10 dilution of 10,000 ppm Zn (II) of an atomic absorption standard. To the human Ne-octanoyl-insulin plus the zinc solution are added 4 ml crystallization diluent (40 mg / ml glycerol, TRIS 50 mM, 4 mg / ml m-cresol, 1625 mg / ml phenol, 100 mM trisodium citrate, pH 7.4). The pH of the resulting solution is adjusted to 7.61. The pH adjusted solution is filtered through a 0.22 micron low protein binding filter. 5 milliliters (5 ml) of protamine solution (37.6 mg of protamine sulfate and 50 ml of water) are added to 5 ml of human Ne-octanoyl-insulin in filtered solution. The solution is allowed to stand undisturbed for 63 hours at a controlled temperature of 25 ° C. Microscopic inspection (at 63 o'clock) shows that crystallization has occurred and that the preparation has similar, similarly shaped, bar-like crystals having approximate average lengths of about 10 microns. The crystals settle out by allowing the formulation to rest without being altered. Then 8 milliliters (8 ml) of the supernatant are removed and replaced with 8 ml of a diluent [16 mg / ml glycerol, 20 mM TRIS, 1.6 mg / ml m-cresol, 0.65 mg / ml phenol, trisodium citrate 40 mM, pH 7.4]. The crystals are then resumed. This procedure is carried out in the same way three times, except that on the third occasion the 8 ml of supernatant is replaced with 7 ml of diluent. The amount of ineulinae in the formulation is analyzed by CLAP to quantify the total potency. Total potency refers to the total concentration of human insulin and human Ne-hexanoyl-insulin. An aliquot of 0.050 ml of the completely resuspended formulation is dissolved in 0.950 ml of 0.01 N HCl, and subjected to analysis by CLAP, as described below. For analysis by CLAP, the following conditions were used: a C8 reversed phase column, - 23 ° C constant; 1.0 ml / min, detection at 214 nm; solvent A = acetonitrile 10% - not - ____ (vol / vol) in trifluoroacetic acid aqueous 0.1; solvent B = 90% acetonitrile (vol / vol) in 0.1% aqueous trifluoroacetic acid; linear gradients (0.1 min, 0% B, 45.1 min, 75% B, 50.1 min, 100% B, 55 min 100% B, 57 min, 0% B, 72 min, 0% B). The standards are prepared by dissolving insulin in volume and insulin acylated in volume in 0.01 N HCl. The concentration of each standard is determined by UV spectroscopy. The assumption is made that a 1000 mg / ml solution of human insulin in a 1 cm cuvette has an absorbance of 1.05 units of optical density at the maximum wavelength (approximately 276 nm). This corresponds to a molar extinction coefficient of 6098. The assumption is made that insulin aciladae have the same molar extinction coefficient as human insulin. The solutions are calibrated by UV and then diluted to obtain standards at 0.220, 0.147, 0.073 and 0.022 mg / ml. The standards are run in CLAP and a standard curve of area versus concentration is obtained. The total potency of human Ne-octanoyl-insulin in the crystal formulation of 3.76 mg / ml. The concentration of soluble human Ne-octanoyl insulin is determined at 0.01 mg / ml. By means of the CLAP analysis, no non-acylated human ineulin is found. The rate of dissolution of the crystals is measured by placing 0.005 ml of the formulation suspended uniformly in 3 ml of Dulbecco's phosphate-buffered saline solution - (___) (without calcium or magnesium) in a square quartz cuvette with a path of 1 cm of length, at a trajectory of 22 ° C. This solution is stirred at a constant rate using a magnetic cup agitator. Absorbance measurements are taken at 320 nm at 1 minute intervals. The absorbance at 320 nm corresponds to light scattered by the insoluble particles present in the aqueous suspension. Consequently, to the extent that the microcrystals dissolve, the absorbance approaches zero. The approximate time necessary for 0.005 ml of this formulation to dissolve is greater than 400 minutes. The time necessary for a 0.005 ml sample of commercial Humulin ™ NUO to dissolve under the same ee conditions of about 10 minutes. A particle size measurement is also carried out on a sample of the formulation using an instrument to determine particle size (Multisizer Model HE, Coulter Corp., Miami, FL 33116-9O15). To carry out this measurement, 0.25 ml of the crystal formulation is added to 100 ml of diluent that consists of 14 mM dibasic edetic foefate, 16 mM glycerol, 1.6 mg / ml of m-cresol, 0.65 mg / ml of phenol, pH 7.4 . The size of the orifice of the opening tube of the instrument is 50 micrometers. Particle size data is collected for 50 seconds. The average particle diameter of the crystals is about 6 microns, with an approximately normal distribution spanning a range of particle size from about 2 microns to about 12 microns. This result is similar to the particle size distribution of commercial NPH, as reported in DeFeiippis, M.R., et al. , J. Pharmaceut. Sci. 87: 170-176 (1998).

Preparation 20 Microcrystals of B29-Ne -nonanoyl-human insulin Human Ne -nonanoyl -insulin is prepared as described in preparation 5. A sample of 16.16 mg of human Ne -nonanoyl -insulin is dissolved in 0.8 ml of 0.1 N HCl. The procedure described in preparation 14 is followed except that the protamine solution contains 18.64 mg of protamine sulphate in a total volume of 50 ml. Bar-like crystals are formed, with a high yield (greater than 80%). In the spectrophotometric dissolution test described above, the crystals of human B29 -Ne -nonanoyl -insulin have a tl / 2 of 83 minutes, compared to about 6 minutes for HumulinHR N in the same assay.

Preparation 21 - H3 - __ _ Microbuctals of B29-Ne-decanoyl-human insulin Human B29-Ne-decanoyl-insulin is prepared essentially as described in preparation 5. A sample of 60.7 mg of B29 -Ne-decanoyl-human insulin is dissolved in 1.5 ml of 0.1 N HCl. A volume of 0.6 ml is added. of a zinc nitrate solution containing 1000 parts per million (ppm) of zinc (II) and mixed thoroughly. To 0.7 ml of the resulting solution in "bottle A" is added 2.0 ml of "A" diluent (50 mM citrate, 5 mg / ml phenol, 16 mg / ml glycerol, 25 mM TRIS, pH 7.6). To another 0.7 ml portion of the zinc-derivatized protein solution is added 2.0 ml of "B" diluent (100 mM citrate, 2 mg / ml phenol, 50 mM TRIS, pH 7.6). The pH is adjusted in the bottles to 7.62 and 7.61, respectively, and each is filtered through a 0.22 micron low protein binding filter. One volume of the contents of bottle A (2.5 ml) is mixed with 2.5 ml of a solution of protamine sulfate (7.4 mg of protamine sulphate dissolved in 10. ml of diluent A). A cloudy precipitate immediately develops. The preparation is allowed to stand undisturbed at 25 ° C. Similarly, a volume of the contents of bottle B (2.5 ml) is mixed with 2.5 ml of a solution of protamine sulphate (37.8 mg of protamine sulfate dissolved in 50 ml of water). A cloudy precipitate immediately develops. The preparation is allowed to stand undisturbed at 25 ° C. Microecological examination of the contents of both bottles after 60 hours shows that they have formed in both small crystals. A wall-like morphology is clearly evident for the crystals in flask B. The performance of the crystals in flask B is determined by CLAP, and is 80%. In the spectrophotometric dissolution test described above, the B29-Ne-decanoyl-human insulin crystals in vial B have a tl / 2 of 70 minutes, compared to approximately 6 minutes for Humulin ™ N in the same assay.

Preparation 22 Microcrystals of B29-Ne-dodecanoil-human insulin B29-Ne-dodecanoil-insulin is prepared as described in preparation 5. A sample of 17.0 mg of B29 -Ne-dodecanoyl-human insulin is dissolved in 4.0 ml of diluent (containing in an aqueous solution, per ml of solution , 10 mg of phenol, 32 mg of glycerol, 30 mg of trisodium citrate dihydrate and 6.1 mg of TRIS, pH 8.47). The pH of the insulin analog derivative solution is adjusted to 8.57 using small aliquots of 1 N NaOH. The pH adjusted solution is added to 0.320 ml of a zinc nitrate solution containing 1000 parts per million (ppm) of zinc (II).

The pH of the zinc-derivative derivative of the ineulin analogue is adjusted to 7.59 using small aliquots of 1 N HCl and 1 N NaOH. The adjusted pH solution is filtered through a 0.22 micron low protein binding filter. . To 2.0 ml of the resulting solution in "bottle A" is added 0.25 ml of ethanol. The mixture combines smoothly. To another 2.0 ml of volume of the resulting solution in "bottle B" is added 0.6 ml of ethanol. The mixture combines smoothly. The contents of both bottle A and bottle B are added 2.0 ml of protamine solution (containing, dissolved in water, 0.376 mg of protamine per ml). After adding the protamine solution, each vial contains a cloudy suepeneion. Each fraeco is swirled gently to complete mixing, and then allowed to sit at 25 ° C. Irregular and small crystals are formed, with a very high yield (greater than 90%). In the spectrophotometric dissolution test described above, the B29-Ne-human dodecanoyl-insulin crystals have a tl / 2 greater than 300 minutes, compared to about 6 minutes for Humulin ™ N in the same assay.

Preparation 23 Microcrystals of B29 -Ne-human ededecanoil-insulin Human B29 -Ne-tetradecanoyl-insulin is prepared as described in preparation 5. A sample of 16.42 mg of B29-Ne-tetradecanoyl-human insulin is dissolved in 0.5 ml of 0.1 HCl N. Deepuée to shake for 5-10 minutes, add a volume of 0.32 ml of a zinc nitrate solution containing 1000 parts per million (ppm) of zinc (II) and the resulting solution is mixed thoroughly by stirring. Then, 3.2 ml of the diluent (containing in an aqueous solution, per ml of solution, 10 mg of phenol, 32 mg of glycerol, 30 mg of trisodium citrate dihydrate and 6.1 mg of TRIS, pH 7.58), and the resulting mixture They shake until they are completely mixed. After mixing the zinc-ineulin derivative with the diluent, adjust the pH of the resulting solution to 7.9 and then back to 7.59 using small aliquots of 1 N HCl and 1 N NaOH, as needed. The pH adjusted solution is filtered through a 0.22 micron low protein binding filter. To 1.97 ml of the resulting solution in "fraeco A" is added 0.246 ml of ethanol. _ The mixture is combined gently. To another volume of 1.97 ml of the re-emerging solution in "fraeco B" is added 0.591 ml of ethanol, which results in the formation of a nebulosity in the bottle. The mixture combines smoothly. The contents of both bottle A and bottle B are added 1.97 ml of a protamine solution (containing, dissolved in water, 0.376 mg of protamine per ml). After adding the protamine solution, each bottle contains a cloudy suspension. Each bottle is swirled gently, to complete mixing and then allowed to sit at 25 ° C. Small irregular crystals are formed with a very high yield (greater than 90%). In the spectrophotometric dissolution test described above, the crystals of human B29-Ne-tetradecanoyl-insulin have a tl / 2 greater than 300 minutes, compared to about 6 minutes for Humulin ™ N in the same assay.

Preparation 24 Microcrystals of B29-Ne-hexadecanoil-human insulin B29 -Ne -hexadecanoyl-human ineulin is prepared as described in preparation 5. A sample of 16. 29 mg of B29-Ne -hexadecanoyl -human insulin in 0.5 ml of 0.1 N HCl After stirring for 5-10 minutes, a volume of 0.32 ml of a zinc nitrate solution containing 1000 parts per million (ppm) of zinc (II) is added, and the resulting solution is carefully mixed by agitation.

Then, 3.2 ml of the diluent (containing in an aqueous solution, per ml of solution, 10 mg of phenol, 32 mg of glycerol, mg of trisodium citrate dihydrate and 6.1 mg of TRIS, pH 7. 58), and the resulting mixture is stirred until completely mixed. After mixing the zinc-derivative insulin solution with the diluent, adjust the pH of the resulting solution first to 8.0 and then back to 7.61, using small aliquots of 1 N HCl and 1 N NaOH, as needed. The pH adjusted solution is filtered through a 0.22 micron low protein binding filter. To 2.0 ml of the resulting solution in "fraeco A", 0.25 ml of ethanol are added. The mixture combines smoothly. To another volume of 2.0 ml of the reelectant eolution in "fraeco B" is added 0.6 ml of ethanol, which results in the formation of a nebulosity in the bottle. The mixture combines smoothly. The contents of both bottle A and bottle B ee add 2.0 ml of a protamine solution (containing, dissolved in water, 0.376 mg of protamine per ml). After adding the protamine solution, each bottle contains a cloudy suepeneion. Each fraeco is agitated by gentle centrifugation, to complete the mixing and then allowed to stand at 25 ° C. In both bottles, crystals form. Irregular and small crystals are formed with a very high yield (greater than 90%). In the spectrophotometric dissolution test described above, the crietals of human B29-Ne-hexadecanol-ineulin have a tl / 2 greater than 300 minutes, compared to about 6 minutes for HumulinHR N in the test group.

Preparation 25 Microcrystals of Al-Na-octanoyl-B29-Ne-octanoyl-human insulin A mass of 8.13 mg of Al-Na-octanoyl-B29-Ne-octanoyl-human insulin analogue is dissolved in 1.60 ml of diluent (in 100 ml: 0.604 g of TRIS, 1,003 g of phenol, 3.218 g of glycerol and 3.069 g of trisodium citrate). The pH of this solution is adjusted to 7.61 with small amounts of 1.0 N HCl and 1.0 N NaOH. After stirring for 5-10 minutes, 0.160 ml of a zinc nitrate solution containing 1000 parts per million (ppm) is added. of zinc (II) and the resulting solution is mixed again carefully. The pH is adjusted again to 7.62, and then the solution is filtered through a 0.22 micron low protein binding filter. To 2 ml of this filtered solution is added 2 ml of a protamine solution (in 100 ml, 37.41 mg of protamine). The mixture is stirred gently. A precipitate forms. The mixture is left undisturbed at 25 ° C. Small irregular crystals form.

Preparation 26 Microcrystals of Al-Na-octanoyl-B29-Ne-octanoyl-deB30-human insulin The process of preparation 25 is essentially followed, except that 8.08 mg of Al-Na-octanoyl-B29-Ne-octandl-deB30-insulin is used. human Irregular small crystals form.

Preparation 27 Microcrystals of Al-Na-nonanoil-B29-Ne-nonanoil-desB30- human insulin The process of preparation 25 is essentially followed, except that 8.07 mg of Al-Na-nonanoyl-B29-Ne -nonanoyl-deB30-human insulin is used. Small irregular crystals form.

Preparation 28 Microcrystals of Al-Na-decanoyl-B29-Ne-decanoyl-deB30- human insulin The process of preparation 25 is essentially followed, except that 8.22 mg of Al-Na-decanoyl-B29-Ne-decanoyl-deB30-human insulin is used. Small irregular crystals form.

Preparation 29 Microcrystals of B29-Ne-octanoyl-Gly (A21), Arg (B31), Arg (B32) - human insulin analog S epr ep ara B 2 9 - N e -octanoyl -Gly (A21), Arg (B31), rg (B32) -human insulin analogue as described in preparation 5. Dissolve a maea of 8.6 mg of B29- Ne-octanoyl-Gly (A21), Arg (B31), Arg (B32) -human insulin analogue in 0.4 ml of 0.1 N HCl. After stirring for 5-10 minutes, 0.160 ml of a nitrate solution of zinc, which contains 1000 parts per million (ppm) of zinc (II) and the resulting solution is mixed again carefully. Then, 1.60 ml of diluent (in 100 ml: 0.604 g of TRIS, 1,003 g of phenol, 3.218 g of glycerol and 3.069 g of trisodium citrate) are added and mixed by further stirring. The pH of this solution is adjusted to 7.59 with small amounts of 1.0 N HCl and 1.0 N NaOH, and then the solution is filtered through a 0.22 micron low protein-binding filter. To 2 ml of this filtered solution is added 2 ml of a protamine solution (in 100 ml, 37.41 mg of protamine). The mixture is stirred by gentle centrifugation. A precipitate forms. The mixture is left undisturbed at 25"C. Small irregular crietalee are formed.

Preparation 30 Microcrystals of B29-Ne-octanoyl-des (ThrB30) -human insulin Human B29-Ne-octanoyl-de (ThrB3.0) -mineulin is prepared as described in preparation 5. A sample of 16.21 mg of B29-Ne-octanoyl-de (ThrB30) -human insulin is dissolved in 0.5 ml of 0.1 N HCl. After stirring for 5-10 minutes, a volume of 0.32 ml of a zinc nitrate solution containing 1000 parts per million (ppm) of zinc (II) is added and the mixing solution carefully mixed by stirring . Then, 3.2 ml of the diluent (containing in an aqueous solution, per ml of solution, 10 mg of phenol, 32 mg of glycerol, 30 mg of trieodium citrate dihydrate and 6.1 mg of TRIS, pH 7.58) and the resulting mixture Stir until it is completely mixed. After mixing the zinc-derivative insulin solution with the diluent, adjust the pH of the resulting solution to 8.45 first using small aliquots of 1 N NaOH, as needed. This fails to fully clarify the solution. Then adjust the pH to 7.61, using small aliquots of 1 N HCl, as needed. The pH adjusted solution is filtered through a 0.22 micron low protein binding filter. A volume of the filtered solution is added to an equal volume of an aqueous solution of protamine (containing, dissolved in water, 0.376 mg of protamine per ml). After adding the protamine solution, a cloudy suspension develops. The suspension is gently stirred by centrifugation to complete mixing, and then allowed to settle at 25 ° C. Bar-like crystals are formed with high yield (greater than 80%). In the spectrophotometric dissolution test described above, the crystals of B29-Ne-octanoyl-de (ThrB30) -human insulin have a tl / 2 of 94 minutes, compared approximately 6 minutes for Humulin ™ N in the same assay.

Preparation 31 Microcrystals of B29-Ne-octanoyl-des (B30) - human insulin analogue B29-Ne-octanoyl-de (ThrB30) -human insulin is prepared as described in preparation 5. A meal of 8.09 mg of B29-Ne-octanoyl-de (ThrB30) -human insulin in 0.400 ml of 0.1 HCl is prepared. N. After stirring for 5-10 minutes, add a volume of 0.16 ml of zinc nitrate solution containing 1000 parts per million (ppm) of zinc (II), and the resulting solution is mixed thoroughly by shaking. After stirring for 5-10 minutes, 0.160 ml of a zinc nitrate solution containing 1000 parts per million (ppm) of zinc (II) is added and the resulting solution is mixed thoroughly. Then add 1.60 ml of the diluent (in 100 ml: 0.604 g of TRIS, 1,003 g of phenol, 3.218 g of glycerol and 3.069 g of trisodium citrate) and mix. The pH of this solution is adjusted to 7.61 with a small amount of 1.0 N HCl and 1.0 N NaOH, and then the solution is filtered through a 0.22 micron low protein binding filter. To 2 ml of this filtered solution is added 2 ml of a protamine solution (in 100 ml, 37.41 mg of protamine). The mixture is stirred by rotation gently. A precipitate forms, the mixture is left undisturbed at 25 ° C.

Preparation 32 Microcrystals of B29 -Ne -hexanoyl-bovine insulin B29-Ne -hexanoyl-bovine insulin is prepared as described in preparation 5. A sample of 16.14 mg of B29-Ne -hexanoyl-bovine insulin is dissolved in 0.8 ml of 0.1 N HCl. After shaking for 5-10 minutes , a volume of 0.32 ml of a zinc nitrate solution containing 1000 parts per million (ppm) of zinc (II) is added, and the resulting solution is mixed carefully by agitation. Deepuée is added with 3.2 ml of crietalization diluent (containing, per ml, 10 mg of phenol, 16 of glycerol, 30 mg of trisodium citrate dihydrate and 6.0 mg of TRIS, in water, unadjusted pH), and the resulting mixture shake haeta that ee mixes completely. After mixing the zinc-derivatized insulin solution with the crystallization diluent, adjust the pH of the resulting solution to 7.58 using small aliquots of 1 N HCl and 1 N NaOH, as needed. The pH adjusted solution is filtered through a 0.22 micron low protein binding filter. To a volume of the filtered solution is added an equal volume of an aqueous solution of protamine (0.375 mg of protamine sulfate / ml of solution, in water, unadjusted pH). The mixture of the two volumes is stirred gently to complete mixing. A cloudy suepeneion is formed, which is shaken gently to complete the mixing, and afterwards allowed to stand at 25 ° C. Eimilar crystals are formed to bars, with a very high yield (greater than 90%). In the spectrophotometric die lysis assay described above, the B29-Ne-hexanoyl bovine insulin crystals have a tl / 2 greater than 300 minutes, compared to about 6 minutes for Humulin ™ N in the honeymoon.

Preparation 33 Microcrystals of B29 -Ne-ovine hexanoyl-insulin B29-Ne -hexaneyl-ovine insulin is prepared as described in preparation 5. A 16.15 mg of B29 -Ne-hexanoyl-ovine inulin is dissolved in 0.8 ml of 0.1 HCl. N. Deepuée to stir for 5-10 minutes, add a volume of 0.32 ml of a zinc nitrate solution containing 1000 parts per million (ppm) of zinc (II), and the resulting solution is mixed thoroughly by stirring. The solution is hazy, and the addition of 0.1 ml of 0.1 N HCl does not cause the nebulosity to completely disperse. The procedure of preparation 32 is then followed, except that the pH is adjusted to 7.61 instead of 7.58. Small irregular crystals are formed with a high yield (greater than 80%). In the spectrophotometric dissolution test described above, the B29 -Ne-hexanoyl-ovine insulin crystals have a tl / 2 of 184 minutes, compared to about 6 minutes for Humulin ™ N in the same assay.

Preparation 34 Microcrystals of B29-Ne-octanoilinsulina porcine Porcine B29-Ne-octanoyl insulin is prepared as described in preparation 5. A sample of 16.78 mg of porcine B29-Ne-octanoyl-insulin is dissolved in 0.5 ml of 0.1 N HCl. After stirring for 5-10 minutes , a volume of 0.32 ml of a zinc nitrate solution containing 1000 parts per million (ppm) of zinc (II) is added, and the resulting solution is mixed thoroughly by stirring. Then, 3.2 ml of the diluent (containing in an aqueous solution per ml of solution, 10 mg of phenol, 32 mg of glycerol, 30 mg of trisodium citrate dihydrate and 6.1 mg of TRIS, pH 8.5), and the resulting mixture is stirred until it is mixed completely. After mixing the zinc-derivatized insulin solution with the diluent, adjust the pH of the resulting solution to 8.4 first using aliquots of 1 N NaOH as needed. The pH is then adjusted to 7.60 using small aliquots of 1 N HCl, as needed. After this, the procedure of preparation 32 is followed. Small irregular crystals are formed with a high yield (greater than 80%). In the spectrophotometric solution described above, the porcine B29-Ne-octanoylinsulin crystals have a tl / 2 greater than 300 minutes, compared to approximately 6 minutes for Humulin ™ N in the same assay.

Preparation 35 B29-Ne-octanoyl-insulin microcrystals from rabbit Rabbit B29-Ne-octanoyl-insulin is prepared as described in Preparation 5. A sample of 8.10 mg of rabbit B29-Ne-octanoyl-insulin is dissolved in 0.25 ml of 0.1 N HCl. After shaking for 5- 10 minutes, a volume of 0.16 ml of a zinc nitrate solution containing 1000 parts per million (ppm) of zinc (II) is added and the resulting solution is mixed thoroughly by shaking. Then, 1.6 ml of diluent (containing in an aqueous solution, per ml of solution, 10 mg of phenol, 32 mg of glycerol, 30 mg of trisodium citrate dihydrate and 6.1 mg of TRIS, pH 8.5), and the resulting mixture Shake until it is completely mixed. After mixing the zinc-derivatized insulin solution with the diluent, the pH of the re-emerging solution is first adjusted to 8.34 using small aliquots of 1 N NaOH, as needed. Then adjust the pH to 7.57, using small aliquots of 1 N HCl, as needed. After this, the preparation procedure is followed 32. _ Small irregular crystals are formed, with high yield (greater than 80%). In the spectrophotometric dissolution test described above, the rabbit B29-Ne-octanoyl-ineulin crystals have a tl / 2 of 119 minutes, compared to about 6 minutes for Humulin® N in the same assay.

Preparation 36 Micro-crystals of B29-Ne-octanoyl-des (B27) -human insulin analog B29-Ne-octanoyl-dee (B27) -human insulin analogue is prepared as described in preparation 5. Dissolve a mass of 8.02 mg of B29-Ne-octanoyl-dee (B27) -human inulin analogue in 0.400 ml of 0.1 N HCl. After stirring for 5-10 minutes, 0.160 ml of a zinc nitrate solution containing 1000 parts per million is added ( ppm) of zinc (II) and the resulting solution is mixed thoroughly. Then, 1.60 ml of diluent (in 100 ml: 0.604 g of TRIS, 1,003 g of phenol, 3.218 g of glycerol and 3.069 g of trisodium citrate) are added and mixed. The pH of this solution is adjusted to 7.61 with small amounts of 1.0 N HCl and 1.0 N NaOH, and then the solution is filtered through a 0.22 micron low protein binding filter. To 2 ml of this filtered solution is added 2 ml of a protamine solution (in 100 ml, 37.41 mg of protamine). The mixture is stirred gently. A precipitate forms. The mixture is left undisturbed at 25 ° C. After that day, "they form crietals in the shape of a bar, alone, well formed.

Preparation 37 Microcrystals of B29-Ne-octanoyl-Asp (B28) -human insulin analog A mass of 8.16 g of B29-Ne-octanoyl -Asp (B28) -human insulin analog is dissolved in 1.60 ml of diluent (in 100 ml: 0.604 g of TRIS, 1,003 g of phenol, 3,218 g of glycerol, and 3,069 g of trisodium citrate). The pH of this solution is adjusted to 7.61 with a small amount of 1.0 N HCl and 1.0 N NaOH. After stirring for 5-10 minutes, 0.160 ml of a zinc nitrate solution containing 1000 parts per million (ppm) is added. of zinc (II) and the resulting solution is mixed again carefully. The pH is adjusted back to 7.62 and after the eolution is filtered through a 0.22 micron low protein binding filter. To 2 ml of this filtered solution is added 2 ml of a protamine solution (in 100 ml, 37.41 mg of protamine). The mixture is stirred gently. A precipitate forms. The mixture is left undisturbed at 25 ° C. Small irregular crystals are formed, with a high yield (greater than 80%). In the spectrophotometric dissolution test described above, the crystals of B29-Nc-octanoyl-Asp (B28) -human insulin analog have a tl / 2 of 15 minutes, compared to about 6 minutes for Humulin ™ N in the same assay.

Preparation 38 Microcrystals of B28-Ne-butyryl-LysB28, ProB29 -human insulin B28-Ne-butyryl-LyeB28, ProB29-human ineulin is prepared as described in preparation 5. A mixture of 16.09 mg of? 28-Ne-butyryl is dissolved -LyeB28, ProB29- human insulin in 0.8 ml of 0.1 N HCl. After stirring for 5-10 minutes, add a volume of 0.32 ml of zinc nitrate containing 1000 parts per million (ppm) of zinc (II), and the resulting solution is carefully mixed by stirring. Then 3.2 ml of crystallization diluent is added and the resulting mixture is stirred and thoroughly mixed. (The crietalization diluent is prepared by dissolving in water, with agitation, 0.603 g of TRIS, 1.07 g of phenol, 1582 g of glycerol and 2,947 g of trisodium citrate, adding additional water to bring the solution to 100 ml). After mixing the zinc-derivative insulin derivative with the crystallization diluent, the pH of the resulting solution is adjusted to 7.60 using small aliquots of 1 N HCl and 1 N NaOH, as needed. The pH adjusted solution is filtered through a 0.22 micron low protein binding filter. To a volume of the filtered solution is added an equal volume of an aqueous solution of protamine, prepared by dissolving 18.64 mg of protamine sulphate in water to a final volume of 50 ml. The mixture of the two volumes is stirred gently to complete mixing and then allowed to stand at 25 ° C. "Preparation 39 Microcrystals of B28-Ne-hexanoil-LysB28, ProB29 -human insulin B28 -Ne -hexanoyl-LysB28, ProB29 -human insulin is prepared as described in preparation 5. A sample of 15.95 mg of B28-Ne-hexanoyl-LyeB28, ProB29-human insulin is dissolved in 0.8 ml of 0.1 N HCl. After the preparation procedure 38. Small irregular crietalee with high yield (more than 80%) are formed. In the spectrophotometric dissolution test described above, the crystals of B28-Ne-hexanoyl-LyeB28, ProB29-human insulin have a tl / 2 of 5-6 minutes, compared to about 6 minutes for Humulin ™ in the same assay.

Preparation 40 _ Microcrystals of B28-Ne-hexanoyl-LysB28, ProB29- human insulin Prepare B28-Ne-hexanoyl-LysB28, ProB29-human insulin as described in preparation 5. Dissolve a sample of 16.8 mg of B28-Ne-hexanoyl-LysB28, ProB29-human insulin in 4.0 ml of diluent (containing in an aqueous solution, per ml of solution, 10 mg of phenol, 32 mg of glycerol, 30 mg of triedic citrate dihydrate and 6.1 mg of TRIS, pH 7.58). The pH of the solution of the insulin analogue derivative is adjusted to 8.4 using small aliquots of 1N NaOH. A_ the pH adjusted solution is added 0.320 ml of a zinc nitrate solution containing 1000 parts per million (ppm) of zinc (II). The pH of the zinc analog derivative-insulin solution is adjusted to 7.61 using small aliquots of IN HCl and IN NaOH. The pH adjusted solution is filtered through a low protein binding filter of 0.22 microns. To 2.0 ml of the resulting solution in the §? A bottle, 0.25 ml of ethanol is added. The mixture is combined gently, and the solution becomes turbid. To another volume of 2.0 ml of the resulting solution in the flask B ^ is added 0.J6 ml of ethanol. The mixture is combined gently, and the solution becomes opalescent. To the contents of bottle A and bottle B ee add 2.0 ml of the protamine solution (containing 0.376 mg of protamine per ml dissolved in water). After adding the protamine solution, each vial contains a cloudy suspension. Each bottle is shaken gently to complete mixing and then allowed to sit at 25 ° C. The cracks are formed in both jars. The composition of the solution in vial A is analyzed for the remaining concentration of the insulin analogue derivative, and the crietalee are subjected to dissolution tests.

Preparation 41 Microcrystals of B28-Ne-octanoyl-LysB28, ProB29 -human insulin Prepare B28-Ne-octanoyl-LysB28, ProB29-human insulin as described in preparation 5. A sample of 16.02 mg of B28-Ne-octanoyl-LysB28, ProB29-human insulin in 0.8 ml of 0.1 N HCl is prepared. Subsequently, the process of preparation 38 is essentially followed. Small irregular crystals with high yield (more than 80%) are formed. In the spectrophotometric dissolution test described above, the crystals of B28-Ne-octanoyl-LysB28, ProB29 -human insulin have a tl / 2 of 7-8 minutes, compared to about 6 minutes of HumulinRN in the same assay.

Preparation 42 _ Microcrystals of B28-Ne-octanoyl-LysB28, ProB29 -human insulin Prepare B28-Ne-octanoyl-LyeB28, ProB29 -humaninein as described in preparation 5. Dissolve a sample of 16.35 mg of B28-Ne-octanoyl-LyeB28, ProB29-human ineulin in 4.0 ml of diluent (containing in an aqueous solution, per ml of solution, 10 mg of phenol, 32 mg of glycerol, 30 mg of trisodium citrate dihydrate and 6.1 mg of TRIS, pH 7.58). The pH of the solution of the insulin analogue derivative is adjusted to 8.4 using small aliquots of NaOH IN. To the solution with adjusted pH, 0.320 ml of a zinc nitrate solution containing 1000 parts per million (ppm) of zinc (II) is added. The pH of the zinc analog derivative-insulin solution is adjusted to 7.62 using small aliquots of IN HCl and IN NaOH. The pH adjusted solution is filtered through a low protein binding filter of 0.22 microns. To 2.0 ml of the resulting solution in the ^ bottle ^ 0.25 ml of ethanol are added. The mixture combines smoothly, and the solution becomes cloudy. To another volume of 2.0 ml of the resulting solution in the flask B ^ ^ is added 0.6 ml of ethanol. The mixture combines smoothly, and the solution becomes cloudy. To the content of both bottle A and bottle B, 2.0 ml of the protamine solution (containing 0.376 mg of protamine per ml dissolved in water) is added. After adding the protamine solution, each fraeco contains a cloudy euepeneion. Each bottle is shaken gently to complete mixing and then allowed to sit at 25 ° C. The crystals are formed in both bottles. The composition of the solution in vial A is analyzed for the remaining concentration of the insulin analogue derivative, and the crystals are subjected to dissolution tests. Small irregular crystals are formed with high yield (more than 80%). In the spectrophotometric dissolution test described above, the crystals of rabbit B28-Ne-octanoyl-insulin have a tl / 2 of 15 minutes, compared to about 6 minutes of Humulin ™ in the same assay.

Preparation 43 Amorphous suspension of B29-Ne -octanoyl-human insulin Human B29 -Ne-octanoyl -ineulin is prepared as described in preparation 5. A human B29-Ne-octanoyl-insulin insulin (20.31 mg of solid, having 16.95 mg of protein) is dissolved in 0.5 ml of 0.1 HCl. N. After adding 200 microliters of a zinc nitrate solution containing 1000 parts per million (ppm) of zinc (II), followed by 2.0 ml of a diluent containing, per ml: 1.625 mg of phenol, 4 mg of m- cresol, 40 mg of glycerol, 5 mg of anhydrous sodium dibasic phosphate, and 7.5 mg of triedic citrate dihydrate, with a final pH of 7.6. After adding the diluent, adjust the pH of the resulting solution to 7.58 with 0.090 ml of 1N NaOH. The solution is then passed through a sterile 0.22 micron low protein binding filter and refrigerated overnight. At this point, the concentration of the insulin derivative is 6.074 mg / ml. The next morning, the solution does not have a visible precipitate. 2.50 ml volume of the solution is mixed with 2.875 ml of a solution of protamine sulfate containing, per ml, 0.75 mg of solid protamine sulfate in water, and in the immediate form amorphous precipitate. The concentration of human B29 -Ne-octanoyl-ineulin ee of 2.825 mg / ml after adding protamine. Suspeneion is injected into two dogs approximately 1 hour and 40 minutes after mixing the ineulin derivative with protamine.

Preparation 44 Amorphous suspension of B28-Ne-ministoil-B28, Pro29 human insulin analog B28-Ne-myrietoyl-LyeB28, Pro29 human Iululin is prepared essentially as described in preparation 5. A "sample of B28-Ne-myrietoyl-LyeB28, human ProB29 -inhuman (20.43 mg of eolide, 18.53 mg of protein) ee It is dissolved in 5 ml of 0.1 N HCl, then 200 microliters of a zinc nitrate solution containing 1000 parts per million (ppm) of zinc (II) and 2.0 ml of formulation diluent are added. ml: 1.6 mg of phenol, 4 mg of m-cresol, 40 mg of glycerol, 5 mg of anhydrous sodium dibasic phosphate and 7.5 mg of trisodium phosphate dihydrate, with a final pH of 7.6. 5.9 to 8.7 with 100 microliters of IN NaOH The formulation is clear, then the pH is reduced to 7.59 by adding 20 microliters of 1N HCl At this point, the protein concentration is 6.57 mg / ml. through a sterile 0.22 micron low protein binding filter and it frigid during the night. The next morning, the formulation does not show a visible precipitate. A 2.50 ml portion of the solution is mixed with 2.875 ml of the protamine solution (0.75 mg / ml of solid protamine sulfate dissolved in water) and an amorphous solution is formed. The concentration of B28-Ne-myristoyl-LysB28, ProB29-human insulin ee should be reduced to 3.056 mg / ml by the addition of the protamine solution. Samples are prepared for CLAP analysis expeditiously after protamine has been added. Based on the known peak retention times, the CLAP analysis shows that the insoluble material contains protamine and B28-Ne-myristoyl-LyeB28, ProB29-human quinulin. It is found that the concentration of B28-Ne-myrietoyl-LyeB28, ProB29-human ineulin ee of 0.005 mg / ml, and in a sample of the precipitate is redissolved to the original volume, the concentration is 3.13 mg / ml. The concentration of B28-Ne-myristoyl-LysB28, ProB29-human insulin in an acidified sweating sample is 3.34 mg / ml.

Preparation 45 Microcrystals of B29 -Ne-octanoyl-human insulin A dry powder of human B29 -Ne-octanoyl-insulin (7 parts by mass) is dissolved in 175 parts by volume of 0. ICI HCl and then a solution of zinc chloride (60 parts in arolumen, prepared by dissolving the oxide) is added. of zinc in HCl to provide a 15.3 mM concentration of zinc). To this solution is added 800 parts by volume of an aqueous solvent comprising 25 mM TRIS, 10 g / ml phenol, 0.1 M citrate, 40 mg / ml glycerol, in water, at a pH value of 7.6. The resulting solution is adjusted to a pH value of 7.6 and is then filtered through a low protein binding filter of 0.22 micrometers. An additional solution is prepared by dissolving 7 parts by mass of protamine sulphate in 10,000 parts by volume of water. The protamine solution is filtered through a 0.22 micron low protein binding filter. Equal volumes of the derivatized protein solution and the protamine solution are combined by adding the protamine solution to the acylated ineulin solution. An amorphous precipitate forms. It is allowed that eetapeneion repoee ein alterations, for 48 hours at a temperature of 25 ° C. The microcrystals of the resulting preparation will provide an extended and flatter action in time compared to an equal dose of NPH human insulin.

Preparation 46 Microcrystals of B29-Ne-octanoyl-human insulin The process of preparation 45 is followed. The suspension is allowed to stand undisturbed for 48 hours at a temperature of 30 ° C. Similar results are obtained.

Preparation 47 Microcrystals of B29-Ne-octanoyl-human insulin A dry powder of human B29-Ne-octanoyl-insulin (7 parts by mass) is dissolved in 175 parts by volume of 0. IN HCl, and then a solution of zinc chloride (60 parts by volume) is added., prepares by dissolving zinc oxide in HCl to provide a concentration of 15.3 mM zinc. To this solution is added 1000 parts by mass of an aqueous solvent comprising 35 mM dibasic sodium phosphate, 4 mg / ml m-cresol, 1.6 mg / ml phenol, 25 mM citrate and 40 mg / ml glycerol, water, pH 7.6. The resulting solution is adjusted to pH 7.6, and then filtered through a 0.22 micron low protein binding filter. An additional solution is prepared by dissolving 6 parts by mass of protamine eulfate in 10,000 parts by volume of water and then by filtering through a 0.22 micron low-protein binding filter. Equal volumes of the derivatized insulin solution and the protamine solution are combined by adding the protamine solution to a solution of acylated protein. An amorphous precipitate forms. This suspension is allowed to remain undisturbed for 1 week at a temperature of 25 ° C. The microcrystals in the resulting preparation will provide an extended and flatter time of action compared to an equal dose of NPH human insulin.

Preparation 48 Microcrystals of B29-Ne-octanoyl-human insulin A dry powder of human B29-Ne-octanoyl-insulin (7 parts by mass) is dissolved in 175 parts by volume of 0.1N HCl, and then a solution of zinc chloride (60 parts in volume, prepared by dissolving) is added. zinc oxide in HCl to provide a concentration of 15.3 mM zinc). To this solution is added 1000 parts by mass of a solvent comprising 35 mM dibasic sodium phosphate, 4 mg / ml m-cresol, 1.6 mg / ml phenol, 10 mM citrate, 40 mg / ml glycerol, in water , pH 7.6. The resulting solution is adjusted to pH 7.6 and then filtered through a 0.22 micron low protein binding filter. An additional solution is prepared to dieolve 6 partse in protamine sulfate maea in 10,000 parts by volume of water and then by filtering through a 0.22 micron low protein binding filter. Equal volumes of the acylated insulin solution and the protamine solution are combined by adding the protamine solution to a solution of acylated ineulin. An amorphous precipitate forms. It is allowed that eeta suspeneion rest without alterations for 1 week at a temperature of 25 ° C. The microcrystals in the resulting preparation will provide an extended and flatter time of action compared to an equal dose of NPH human insulin.

Preparation 49 Microcrystals of B29-Ne-octanoyl-human insulin The process of preparation 47 is followed. The suspension is allowed to stand undisturbed for 60 hours at a temperature of 30 ° C. The microcrystals in the resulting preparation will provide an extended and flatter time of action compared to an equal dose of NPH human insulin.

Preparation 50 Microcrystals of B29-Ne-octanoyl-human insulin A solution is prepared by adding to water for injection (WFI, 1000 parts by volume): phenol (0.65 parts by mass), m-cresol (1.6 parts by mass) and glycerin (16 parts in maea). Deepuée dissolves in this solution protamine sulphate powder (0.6 parts by mass). A solution of zinc chloride (6 parts by volume) is prepared by dissolving zinc oxide in HCl to provide a 15.3 mM concentration of zinc in 0. IN HCl which is subsequently added. Dry powder of human B29-Ne-octanoyl-insulin (7 parts by mass) is added and dissolved with stirring. The pH is adjusted to approximately 3 to help the solution, if necessary, with small amounts of IN HCl and IN NaOH. The pH is then adjusted within the range of 3-3.6, with small amounts of IN HCl and 1N NaOH. Eeta eolution is filtered through a low-protein binding filter of 0.22 micrometers. A second solution is prepared by dissolving sodium dibasic phosphate (7.56 parts by mass), phenol (0.65 parts by mass), m-cresol (1.6 parts by mass) and glycerin (16 parts by mass) in water for injection (1000 parts) in volume). The pH of this solution is adjusted to a value such that the combination of a volume of this solution with an equal volume of the B29-Ne-octanoyl-human insulin solution results in a pH value of about 7.5 to about 7.7. After properly adjusting the pH of this buffer solution, it is filtered through a 0.22 micron low protein binding filter. Equal volumes of the buffer solution and the human B29 -Ne-octanoyl-insulin solution are combined. An amorphous precipitate is formed immediately, which becomes crystalline upon standing for 60 hours without alterations at a controlled temperature of 25 ° C. The microcrystals in the resulting preparation will provide an extended and flatter action time compared to an equal dose of NPH human Iululin.

Preparation 51 Microcrystals of B29-Ne-octanoyl-human insulin A solution is prepared by adding to water for injection (1000 parts by volume) sodium phosphate (3.78 parts by mass), phenol (0.65 parts by mass), m-cresol (1.6 parts in maea) and glycerin (16 parts in mass). A solution of zinc chloride (6 parts by volume) prepared by dissolving zinc oxide in HCl is then added to provide a concentration of 153 mM zinc in 0.1 N HCl. A dry powder of human B29 -Ne-octanoyl-insulin (7 parts by mass) is added and dissolved with stirring. The pH is adjusted to approximately 3 with the help of dissolution if necessary, with small amounts of IN HCl and IN NaOH. After adjusting the pH to 7.6 with 10% HCl and 10% NaOH. Eeta eolution is filtered through a low-protein binding filter of 0.22 micrometers. A second solution is prepared by dissolving sodium dibasic phosphate (3.78 parts by mass), phenol (0.65 parts by mass), m-cresol (1.6 parts by mass) and glycerin (16 parts in maea) in water for injection (1000 parts). in volume). Then powdered protamine sulfate solution (0.6 parts by mass) is dissolved in this solution. The pH of this solution is adjusted to 7.6. This solution is filtered through a 0.22 micron low protein binding filter. Equal volumes of this protamine solution and the B29-Ne-octanoyl-human insulin solution are combined. An amorphous precipitate is formed immediately, which becomes crietaline upon standing for 60 hours without alterations at a controlled temperature of 25 ° C. The microcrystals in the resulting preparation will provide an extended and flatter time of action compared to an equal dose of NPH human insulin. ~ Preparation 52 Microcrystals of human B29-Ne- (2-ethylhexanoyl) -insulin Human B29-Ne- (2-ethylhexanoyl) -insulin is prepared as described in preparation 5. A mass of 8.00 mg of human B29-Ne- (2-ethylhexanoyl) -insulin is dissolved in 0.400 ml of 0.1N HCl . Subsequently, the process of preparation 17 is followed essentially. A precipitate is formed. The mixture is allowed to stand undisturbed at 25 ° C. They form cristalee eimilaree a barrae, with high yield (greater than 80%). In the epectrophotometric solution assay described above, the crystals of human B29-Ne- (2-ethylhexanoyl) -insulin have a tl / 2 of 34-35 minutes, compared to about 6 minutes for Humulin® N in the same assay.

Example 1 In vivo test in diabetic dogs The protracted action of a suspension formulation containing microcrystals prepared as described in any of the preparations herein is tested in diabetic dogs by comparing their ability to control hyperglycemia with that of the control compounds. A doeie once a day of approximately 0.2 unit / kg body weight is used. Eeta doeie will be equivalent to approximately 1.2 nmol / kg. On the test days, the blood glucose is monitored for 24 hours after the subcutaneous injection of the suepeneion formulation. The control compounds are human ineulin and human NPH ineulin. The microcrystalline suspension formulations of the present invention will reduce blood glucose levels and will have a prolonged action time compared to human NPH insulin. when it tests a comparable doeie.

Example 2 Action in the time of crystals in rats To 1898 ml of the crystal formulation prepared according to Preparation 19 is added 3,102 ml of diluent (16 mg / ml glycerol, 20 mM TRIS, 1.6 mg / ml m-cresol, 0.65 mg / ml phenol, 40 mM trisodium citrate, pH 7.4). This provides 5 ml of a U40 formulation, which is tested in BBDP / Wor rats, a genetically characterized, maintained and available animal model from University of Massachusetts Medical Center (Worchester, MA) in connection with Biomedical Research Models, Inc. (Rutland , MA). The rattae line DPBB / Wor is susceptible to diabetes, and shows insulin-dependent diabetes mellitus (autoimmune). 40 BBDP / Wor rats are randomly assigned [20 males / 20 females, aged 4-5 months, maintained with long-acting insulin (PZI)], by gender, to 8 experimental groups, A, B, C, D, E, F, G, and H. The groups A (5 males) and B (5 females) are treated for 2 days with the ultra-fast composition of human insulin U40 having 2.5 mg / ml zinc. Groups C (5 males) and D (5 females) are treated for 2 days with the ultra-fast composition of U40 human insulin having 1.25 mg / ml zinc. Groups E (5 males) and F (5 females) are treated for 2 days with insulin Pork-pork PZI U40 (PZI). The groups G (5 males) and H (5 females) are treated for 2 days with a crystalline formulation according to the present invention, as described in this example. Each rat is given daily injections of its group formulation during the 2 days before the blood glucose is determined, and on the day on which the blood glucose is determined. A sample is obtained half an hour before administering the sample formulation. The animals were injected with either 0.8 U / 100 g of body weight (males) or 1.1 U / 100 g of body weight (females) at 11:30 A.M. Blood is obtained by cutting the tail (without anesthesia), briefly stored on ice, centrifuged and glucose is determined using a Beckman II glucose analyzer. The blood samples are obtained just before the administration of the test formulations, and at 2, 4, 6, 8, 12, 16, 20 and 24 hours after the administration. The crystal formulations of the present invention control blood glucose for a time comparable to that obtained with the long-acting ineulin preparations.

Example 3 Action in the time of crystals, in rats The test procedure described above in the example is repeated, with a second 5 ml sample of a U40 formulation of a suspension, prepared as described above. 35 BBDP / Wor rats are maintained [18 males / 17 females, age 4-5 months, maintained with long-acting insulin (PZI)], randomly assigned by gender to 6 experimental groups I, J, K, L, M, and N. Groups I (8. males) and J (8 females) are treated for 3 days with the crietalee formulation according to the present invention, as described in this example, above. The groups K (5 males) and L (4 females) are treated for 3 days with an ultra-fast human insulin composition U40 having 2.5 mg / ml zinc.

The groups M (5 males) and N (5 females) are treated for 3 days with PZI cow-pork U40 (PZI) insulin. Each rat is given daily injections of its group formulation during the 3 days before the blood glucose is determined, and on the day in which the blood glucose is determined. The blood is obtained half an hour before administering the test formulations. The animals were injected subcutaneously with either 0.9 U / 100 g of body weight (males) or l.l U / 100 g of corporal peeo (female) at 11:30 A.M. Blood is obtained by a cut in the tail (in anesthesia), and stored briefly on ice, centrifuged and glucose is determined using a Beckman II glucose analyzer. The blood samples are obtained just before the administration of the test formulations, and at 2, 4, 6, 8, 12, 16, 20 and 24 hours after the administration, the crystal formulations of the present invention control blood glucose for a time comparable to that obtained with long-acting insulin preparations.

Example 4 Action in the time of crystals, in rats 26 rats are randomly assigned by gender_ BBDP / Wor [13 males / 13 female, with ages of 4-6 months, maintained with long-lasting protamine zinc insulin (PZI)], a 4 experimental groups, O, P, Q, and R. Loe groups O (8 males) and P (8 female) are treated for 3 days with the crystal formulation according to the present invention, as described in example 2 Groups Q (5 males) and R (4 females) are treated for 3 days with PZI cow-pork U40 (PZI) insulin. Each rat is given daily injections of its group formulation during the 3 days before the glycoea is determined in blood, and on the day in which the blood glucose is determined. The blood is obtained half an hour before administering the test formulations. The animals are administered subcutaneously with either 0.9 U / 100 g of body weight (males) or 1.1 U / 100 g of body weight (females) at 11:30 A.M. Blood is obtained by a cut in the tail (without anesthesia), briefly stored on ice, centrifuged and glucose determined using a Beckman II glucose analyzer. The blood samples are obtained just before the administration of the test formulations, and at 2 o'clock, 4, 6, 8, 12, 16, 20 and 24 hours after administration.

The crystalline formulations of the present invention control blood glucose for a time comparable to that obtained with long-acting insulin preparations.

Example 5 Amorphous precipitate of human B29-Ne-octanoyl-insulin tested in dogs The action over time of a formulation containing an amorphous precipitate of human protamine and B29 -Ne-octanoyl-ineulin, prepared as described in preparation 43, is determined in 2 normal dogs (2 nmol / kg, subcutaneous). The dogs received a consistent infusion of somatostatin to create a transient diabetic state. The data were compared with those observed in the same model after administration of ultralow human insulin (3 nmol / kg, n = 5) and with saline (n = 6). Experiments were carried out in chronically calculated, chronically calculated male and female mongrel dogs, fasted overnight with a weight of 10-17 kg.

(Marschall Farms, North Rose, NY). At least 10 days before the study, the animals were anaesthetized with isoflurane (Anaquest, Madieon, Wl) and silicone catheters were attached to the vascular access ports (V-A-PMR, Access Technologies, Norfolk Medical, Skokie, IL) inserted into the femoral artery and the femoral vein. The catheters were filled with a glycerol / heparin solution (3: 1, v / v; final heparin concentration of 250 klU / ml; glycerol from Sigma Chemical Co., St. Louis, MO, and Elkins-Sinn heparin, Inc., Cherry Hill, NJ) to avoid occlusion of the catheter, and the wounds were closed. Kefzol (Eli Lilly &Co., Indianapolis, IN) (20 mg / kg, IV and 20 mg / kg, IM) was administered preoperatively, and Keflex was administered postoperatively (250 mg, po once a day for 7 days). days) to avoid infections. Torbugesic (1.5 mg / kg, I.M.) was administered postoperatively to control pain. Blood is drawn just before the day of the study to determine the health of the animal. Only animals with hematocrit greater than 38% and leukocyte counts less than 16,000 / mm3 were used (hematology analyzer: Cell-Dyn 900, Sequoia-Turner, Mountain View, CA). The morning of the experiment, the orifices were accessed (Accese Technologiee, Norfolk Medical, Skokie, IL); the content of the catheters was aspirated; the catheters were purged with ealine elation (Baxter Healthcare Corp., Deerfield, IL); The dog is placed in a cage, and the extension line (protected by a stainless steel cover and attached to an oscillating system [Instech Laboratories, Plymouth Meeting, PA]) is attached to the access lines to the holes.

The dogs were allowed to acclimate for 10 minutes to the environment of the cage before an arterial blood sample was taken for determination of the fasting concentrations of insulin and blood glucose (time = -30 minutes). At this time, a continuous IV infusion of cyclic eomatoetatin (0.65 μg / kg / min), BACHEM California, Torrance, CA) was initiated and continued for the next 30.5 hours 30 minutes after the start of the infusion (time = 0 minutes), an arterial blood sample is extracted, and a subcutaneous bolus of test substance, or vehicle, is injected into the dorsal aspect of the neck, arterial blood samples are taken every 3 hours thereafter to determine the concentrations of Glucose and Insulin in Plaema Blood samples are collected in vacuum blood collection tubes containing disodium EDTA (Terumo Medical Corp., Elkton, MD) and placed immediately on ice.The samples are centrifuged and the plasma The resulting glucose is transferred to polypropylene test tubes and stored: on ice for the duration of the study Plasma glucose concentrations were determined on the day of the study using the gluco method sa oxidase in a Beckman glucoea analyzer (Beckman Inetruments, Inc., Brea, CA). The samples for the other tests were stored at -80 ° C until the time of analysis.

Insulin concentrations were determined using the double antibody radioimmunoassay. At the conclusion of the experiment, the catheters were purged with fresh saline, treated with Kefzol (20 mg / kg), filled with the glycerol / heparin mixture; antibiotic (Keflex, 250 mg) p.o. To minimize the number of animals that were used and allow the matching of the data set when possible, the animals were studied several times. The experiments in the animals that were re-studied were carried out with a minimum difference of one week. The amorphous precipitate formulation of human B29-Ne-octanoyl-ineulin, prepared as described above, provides effective control of blood glucose for almost 27 hours, compared to only about 21 hours for ultra-slow human insulin. The precipitate provides significantly more flat and more widespread control of glucose levels compared to ultra-slow human insulin. For example, the nadir of the blood glucose concentration is obtained after 1.5 hours for the precipitate, and then the glucose level increases to a nearly constant level. In comparison, the nadir for ultralow human insulin is reached after 9 hours, and then the blood glucose level increases relatively rapidly. The glucose level in the nadir is 72 mg / dl for the formulation of the derivatized insulin precipitate, while it is 56 mg / dl for the ultraleast formulation. Finally, plasma insulin levels corroborate these observations, which correlate well with the greater planarity and extension of time action of the amorphous precipitate of B29 -Ne-octanoyl-human insulin in comparison with ultra-slow human insulin.

Example 6 Micro-crystals of B29 -Ne -octanoyl-human insulin tested in dogs The glucodynamics of the two crystal-containing formulations of human B29-Ne-octanoyl-human ineulin, prepared as described in Preparation 19, or essentially as described in Preparation 19, were determined in normal dogs, essentially using the protocol described in FIG. Example 5. Each dog was given one of two microcrystal preparations at a dose of 2 nmol / kg, subcutaneously. The experiments were carried out on 3 different occasions. The data from these 3 experiments were combined. A total of 10 dogs received each of the doses of 2 nmol / kg of one of the 2 preparations. In a separate experiment, a microcrystalline formulation of B29-Ne-octanoyl-human insulin is administered subcutaneously, essentially as described in Preparation 19, to each of 5 dogs at a dose of 3 nmol / kg. As controls, NPH human insulin (2 nmol / kg, n = 5) and saline vehicle (n = 5) were used. In each experiment, the dogs received a concomitant infusion of eomatoetatin to create a transient diabetic state. The formulation of microcrystals comprising B29-Ne -octanoyl-human insulin, administered at 2 nmol / kg and having an effective action time of 27 hours, compared to 21 hours for human insulin NPH at the same dose. The glucodynamic profile shows a hypoglycemically lower tendency compared to human NPH inulin, which is an advantageous quality. At 3 nmol / kg, microcrystals comprising B29-Ne-octanoyl-human insulin effectively control the level of glycoea for at least 30 hours. In the nadir of glucose, approximately the same glucose level is obtained as that obtained after the administration of human NPH inulin (specifically, approximately 65 mg / dl). However, the duration of such levels of glucose suppressed is much shorter for micro-diets comprising B29-Ne-human octanil-insulin (approximately 3 hours) compared to NPH human insulin (approximately 7.5 hours). After nadir, the blood glucose levels for the group receiving microcrystals comprising B29-Ne-octanoyl-human insulin vary only between 91 and 115 mg / dL for up to 30 hours, after which there is no additional data available. In contrast, glucose levels in the group receiving human insulin NPH at 2 nmol / kg vary from 89 to 145 after the nadir is reached.

Example 7 Micro-crystals of B29-Ne-hexanoil-human insulin tested in dogs The glucodynamics of formulations containing microcrystals of B29 -Ne -hexanoyl-human insulin were prepared in normal dogs, prepared as described in Preparation 16, essentially using the protocol described in Example 5. The formulation of microcrystals comprising B29-Ne -hexanoyl-human ineulin, administered at 2 nmol / kg, has an action effective in time, for 24 hours, compared to 24 hours for human NPH inulin to the myeloma. The glucodynamic profile is flatter, showing a lower hypoglycemic tendency than human insulin NPH. In the glucose nadir, about the same level of glucoea is obtained as that obtained after the administration of human insulin NPH (specifically, approximately 67 mg / dl, vereue 64 mg / dl for human NPH ineulin). However, the duration of such suppressed glucose levels is much shorter for microcrystals comprising B29 -Ne -hexanoyl-human ineulin (approximately 3 hours) compared to NPH human insulin (approximately 7.5 hours). The principles, preferred embodiments and modes of operation of the present invention have been described in the above specification. The invention which is intended to protect in the present, however, is not considered to be limited to the particular forms described, but which should be considered as illustrative rather than restrictive. Variations and changes can be made by those skilled in the art without departing from the spirit of the invention. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Claims (84)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A microcrystal, characterized in that it comprises: a) a derivatized protein that is selected from the group consisting of derivatized insulin, derivatized insulin analogs and derivatized proinsulins, - b) a complexing compound; c) a hexamer reactant compound; and d) a divalent metal cation.

2. The microcrystal according to claim 1, characterized in that the complexing compound is protamine which is present from about 0.15 mg to about 0.5 mg per 3.5 mg of derivatized protein.

3. The microcrystalline according to claim 2, characterized in that the zinc divalent metal cation, which is present from about 0.3 mol to about 0.7 mol per mol of derivatized protein.

4. The microcrystal according to claim 3, characterized in that the hexamer stabilizing compound is a phenolic preservative selected from the group consisting of phenol, m-cresol, o-cresol, p-cresol, chlorocresol, methylparaben and mixtures thereof , and is present in sufficient proportions with respect to the derivatized protein to facilitate formation of the conformation of the R6 hexamer.

5. The microcrystal according to claim 4, characterized in that the derivatized protein is an acylated protein that is selected from the group consisting of acylated insulin and acylated insulin analogues.

6. The microcrystal according to claim 5, characterized in that the derivatized protein is an acylated fatty acid insulin.

7. The microcrystal according to claim 6, characterized in that the derivatized protein is insulin that is acylated with a saturated straight-chain fatty acid.

8. The microcrystal according to claim 7, characterized in that the derivatized protein is insulin that is monoacylated in the LysB29-Ne-amino group of ineulin.

9. The microcrystalline according to claim 8, characterized in that the derivatized protein is acylated with a fatty acid which is selected from the group consisting of n-hexanoic acid, n-heptanoic acid, n-octanoic acid, n-nonanoic acid and n-nonanoic acid. -decanoic.

10. The microcrystal according to claim 9, characterized in that the derivatized protein is selected from the group consisting of B29 -Ne -hexanoyl - human insulin, B29-Ne -octanoyl-human insulin and B29-Ne-decanoyl-human insulin.

11. The microcrystal according to claim 8, characterized in that the derivatized protein is acylated with a fatty acid selected from the group consisting of n-dodecanoic acid, n-tetradecanoic acid and n-hexadecanoic acid.

12. The microcrystal according to claim 7, characterized in that the insulin acylated with fatty acid is a diacylated insulin that is acylated in the LysB29-Ne-amino group and also is acylated in a N-terminal Na-amino group, wherein the acid fatty acid is selected from the group consisting of n-hexanoic acid, n-heptanoic acid, n-octanoic acid, n-nonanoic acid and n-decanoic acid.

13. The microcrystal according to claim 6, characterized in that the derivatized protein is insulin that is acylated with a branched chain saturated fatty acid.

14. The microcrystal according to claim 13, characterized in that the branched, saturated fatty acid has from 3 to 10 carbon atoms in its longest branch.

15. The microcrystal according to claim 5, characterized in that the derivatized protein is an insulin analog acylated with fatty acid.

16. The microcrystal according to claim 15, characterized in that the derivatized protein is an insulin analog that is acylated with a saturated, straight-chain fatty acid.

17. The microcrystal according to claim 16, characterized in that the derivatized protein is monoacylated in the Ne-amino group.

18. The microcrystal according to claim 17, characterized in that the derivatized protein is acylated with a fatty acid that is selected from the group consisting of n-hexanoic acid, n-heptanoic acid, n-octanoic acid, n-nonanoic acid and n-nonanoic acid. -decanoic.

19. The microcrystal according to claim 18, characterized in that the derivatized protein is selected from the group consisting of animal insulins acylated with fatty acid, monomeric insulin analogs adhered with "fatty acid, suppression analogs acylated with _, fatty acid and analogue of Ineulin with embedded pl, acylated with fatty acid.

"20. The microcrystalline according to claim 19, characterized in that the derivatized protein ee des (B30) analog of human insulin acylated with fatty acid, LysB28, Pro29- human insulin analog acylated with fatty acid or AspB28 analog of acylated human insulin with. fatty acid.

21. The microcrystal according to claim 20, characterized in that the derivatized protein is des (B30) analogue of human Iululin acylated with fatty acid.

22. The microcrystal according to claim 17, characterized in that the derivatized protein is acylated with a fatty acid selected from the group consisting of n-dodecanoic acid, n-tetradecanoic acid and n-hexadecanoic acid.

23. The microcrystalline according to claim 22, characterized in that the derivatized protein is selected from the group consisting of animal ineulins acylated with fatty acid, analogue of monomeric ineulin acylated with fatty acid, analogs of suppression "acylated with fatty acid, and insulin analogues. with displaced pl, acylated with fatty acid.

24. The microcrystal according to claim 23, characterized in that the derivatized protein is des (B30) analogs of human insulin acylated with fatty acid, LysB28, ProB29 analogous of human inulin acylated with fatty acid or AspB28 analog of human insulin acylated with fatty acid.

25. The microcrystal according to claim 24, characterized in that the derivatized protein is des (B30) analogue of human inulin acylated with fatty acid.

26. The microcrystal according to claim 25, characterized in that the derivatized protein is B29-Ne-myristoyl-des (B30) -human insulin analogue.

27. The microcrystal according to claim 26, characterized in that the derivatized protein is B28-Ne-myristoyl-LysB28, ProB29 -human insulin analogue.

28. The microcrystal according to claim 15, characterized in that the derivatized protein is an insulin analog that is acylated with saturated fatty acid, branched chain.

29. The microcrystal according to claim 28, characterized in that the branched chain saturated fatty acid has from 3 to 10 carbon atoms in its longest branch.

30. The microcrystalline according to claim 1, characterized in that the microcrystalline has rod-like morphology.

31. The microcrystal according to claim 1, characterized in that the microcrystal has irregular morphology.

32. A formulation in suepeneion, comprising an unsolvable fae and a fae in solution, wherein the insoluble phase is comprised of the microcrystal according to claim 1, and the phase in solution is comprised of water.

33. The suspension formulation, according to claim 32, characterized in that the insoluble phase is comprised of the microcrystal according to claim 2.

34. The suspension formulation, according to claim 33, characterized in that the solution phase is further comprised of a phenolic preservative at a concentration of about 0.5 mg per ml to about 6 mg per ml of solution, a pharmaceutically acceptable buffer and an agent of isotonicity.

35. The euspension formulation, according to claim 34, characterized in that the eroding factor is also comprised of insulin, an insulin analogue, an acylated insulin or an acylated insulin analogue.

36. The suspension formulation, according to claim 35, characterized in that the phase in solution is comprised of insulin.

37. The suspension formulation, according to claim 35, characterized in that the phase in solution is comprised of an insulin analogue.

38. The suspension formulation, according to claim 37, characterized in that the insulin analog is a monomeric insulin analogue.

39. The suspension formulation, according to claim 38, characterized in that insulin analog ee LyeB28, ProB29 - human insulin analogue.

40. The suspension formulation, according to claim 32, characterized in that the solution phase is further comprised of zinc and protamine, wherein the ratio of zinc to the protein derivatized in the euspension formulation is from about 5 to about 7 moles of zinc atoms per mole of derivatized protein, and the ratio of protamine to the protein derivatized in the euepeneion formulation ee from about 0.25 mg haeta about 0.5 mg per mg of «Derivatized protein.

41. A process for preparing the microcrystal according to claim 1, characterized in that it comprises: a) dissolving a derivatized protein, a hexamer stabilizing compound and a divalent metal cation in an aqueous solvent having a pH that will allow the formation of hexamer of the derivatized protein; and b) add a complexing compound.

42. A process for preparing the microcrystal according to claim 1, characterized in that it comprises: a) dissolving the derivatized protein, a hexamer stabilizing compound and a divalent metal cation in an aqueous solvent having a pH that will not allow the formation of hexamers of the derivatized protein, and b) adjusting the pH between approximately 6.8 and approximately 7.8; and c) adding a complexing compound.

43. A method for treating diabetes, characterized in that it comprises administering the formulation according to claim 32 to a patient in need thereof, in an amount sufficient to regulate blood glucose levels in the patient.

44. An amorphous precipitate, characterized in that it comprises: a) a derivatized protein, which is selected from the group that you derived from derivatized insulin, derivatized insulin analogs, and derivatized proinsulinae; b) a complex forming compoteto; c) a hexamer reactant compound; and d) a divalent metal cation.

45. The amorphous precipitate according to claim 44, characterized in that the complexing compound is protamine, which is presented from about 0.15 mg to about 0.5 mg per 3.5 mg of derivatized protein.

46. The amorphous precipitate according to claim 45, characterized in that the zinc divalent metal cation, which is present from about 0.3 moles to about 0.7 moles per mole of derivatized protein.

47. The amorphous precipitate according to claim 46, characterized in that the hexamer stabilizing compound is a phenolic preservative selected from the group consisting of phenol, m-cresol, o-cresol, p-cresol, chlorocresol, methylparaben and mixtures thereof. the same, and is present in sufficient proportions with respect to the derivatized protein to facilitate formation of the conformation of the R6 hexamer.

48. The amorphous precipitate according to claim 47, characterized in that the derivatized protein is an acylated protein selected from the group consisting of acylated insulin and acylated insulin analogues.

49. The amorphous precipitate according to claim 48, characterized in that the derivatized protein is an acylated fatty acid insulin.

50. The amorphous precipitate according to claim 49, characterized in that the derivatized protein is insulin which is acylated with a saturated straight-chain fatty acid.

51. The amorphous precipitate according to claim 50, characterized in that the derivatized protein is insulin that is monoacylated in the LysB29-Ne-amino group of ineulin.

52. The amorphous precipitate according to claim 51, characterized in that the derivatized protein is acylated with a fatty acid which is selected from the group consisting of n-hexanoic acid, n-heptanoic acid, n-octanoic acid, n-nonanoic acid and n-decanoic acid.

53. The amorphous precipitate according to claim 52, characterized in that the derivatized protein is selected from the group consisting of B29-Ne-hexanoil-human insulin, B29 -Ne-octanoyl-human insulin and B29 -Ne-decanoyl-human insulin.

54. The amorphous precipitate according to claim 51, characterized in that the derivatized protein is acylated with a fatty acid which is selected from the group that connects n-dodecanoic acid, n-tetradecanoic acid and n-hexadecanoic acid.

55. The amorphous precipitate according to claim 50, characterized in that the insulin acylated with fatty acid is a diacylated ineulin which is acylated in the group LysB29-Ne-amino and is also acylated in a N-terminal group Na-amino, and in wherein the fatty acid is selected from the group consisting of n-hexanoic acid, n-heptanoic acid, n-octanoic acid, n-nonanoic acid and n-decanoic acid.

56. The amorphous precipitate according to claim 49, characterized in that the derivatized protein is ineulin which is acylated with a saturated, straight chain fatty acid.

57. The amorphous precipitate according to claim 56, characterized in that the saturated and branched fatty acid has from 3 to 10 carbon atoms in the longest branch.

58. The amorphous precipitate according to claim 48, characterized in that the protein derivatized with an insulin analog acylated with fatty acid.

59. The amorphous precipitate according to claim 58, characterized in that the derivatized process is an analogue of ineulin which is acylated with saturated fatty acid, straight chain.

60. The amorphous precipitate according to claim 59, characterized in that the derivatized protein is monoacylated in the Ne-amino group.

61. The amorphous precipitate according to claim 60, characterized in that the derivatized protein is acylated with a fatty acid which is selected from the group consisting of n-hexanoic acid, n-heptanoic acid, n-octanoic acid, n-nonanoic acid and n-decanoic acid.

62. The amorphous precipitate according to claim 61, characterized in that the derivatized protein is selected from the group that you connected of animal insulins acylated with fatty acid, monomeric insulin analogs acylated with fatty acid, analogs of suppression acylated with fatty acid, and analogues of Insulin with displaced pl, acylated with fatty acid.

63. The amorphous precipitate according to claim 62, characterized in that the derivatized protein is des (B30) analogue of human insulin acylated with fatty acid, LysB28, ProB29 analog of human insulin acylated with fatty acid or AspB28 -analog of acylated human inulin with fatty acid.

64. The amorphous precipitate according to claim 63, characterized in that the derivatized protein is des (B30) analog of human insulin acylated with fatty acid.

65. The amorphous precipitate, according to claim 60, characterized in that the derivatized protein is acylated with a fatty acid selected from the group consisting of n-dodecanoic acid, n-tetradecanoic acid and n-hexadecanoic acid.

66. The amorphous precipitate according to claim 65, characterized in that the derivatized protein is selected from the group consisting of animal insulins acylated with fatty acid, monomeric insulin analogs acylated with fatty acid, analogs by supreeion, acylated with fatty acid, and the like of insulin with displaced pl, acylated with fatty acid.

67. The amorphous precipitate according to claim 66, characterized in that the derivatized protein is des (B30) analog of human insulin acylated with fatty acid, LysB28, ProB29 analogue of human inulin, acylated with fatty acid or AspB28 - acylated human inulin analogue with fatty acid.

68. The amorphous precipitate according to claim 67, characterized in that the derivatized protein ee des (B30) analog of human insulin, acylated with fatty acid.

69. The amorphous precipitate according to claim 68, characterized in that the derivatized protein is B29-Ne -myristoyl-des (B30) -human inulin analogue.

70. The amorphous precipitate according to claim 69, characterized in that the derivatized protein ee B28-Ne-myrietoyl-LyeB28, Pro29-human inulin analogue.

71. The amorphous precipitate according to claim 58, characterized in that the derivatized protein is an insulin analogue which is acylated with branched chain saturated fatty acid.

72. The amorphous precipitate according to claim 71, characterized in that the branched chain saturated fatty acid has from 3 to 10 carbon atoms and its longest branch.

73. A suspension formulation, characterized in that it comprises a phase insoluble in a phase in solution, wherein the insoluble phase is comprised of the amorphous precipitate according to claim 44, the phase in solution is comprised of water.

74. The suspension formulation, according to claim 45, characterized in that the insoluble phase is comprised of the amorphous precipitate according to claim 2.

75. The suspension formulation, according to claim 41, characterized in that the fae in solution. it is further comprised of a phenolic preservative at a concentration of about 0.5 mg per ml to about 6 mg per ml of solution, a pharmaceutically acceptable buffer, and an isotonicity agent.

76. The euspension formulation, according to claim 75, characterized in that the solution phase is additionally comprised of insulin, an insulin analogue, an acylated ineulin or an acylated insulin analogue.

77. The suspeneion formulation, according to claim 76, characterized in that the phase in solution is comprised of insulin.

78. The suspension formulation, according to claim 76, characterized in that the phase in solution is comprised of an analogue of ineulin.

79. The euspension formulation, according to claim 78, characterized in that the insulin analog is a monomeric insulin analogue.

80. The suepeneion formulation, according to claim 79, characterized in that the insulin analogue is LysB28, ProB29 - human inulin analogue.

81. The euepeneion formulation, according to claim 73, characterized in that the solution phase is additionally comprised of zinc and protamine, wherein the ratio of zinc to the protein derivatized in the suspeneion formulation is from about 5 to about 7 moles of zinc atoms per mole of derivatized protein, and the ratio of protamine to protein derivatized in the euepeneion formulation ee from about 0.25 mg to about 0.5 mg per mg of derivatized protein.

82. A process for preparing the amorphous precipitate according to claim 45, characterized in that it comprises: a) dyeing a derivatized protein, a hexamer stabilizer composition and a divalent metal cation, in an aqueous solvent having a pH that will allow the formation of hexamers of the derivatized protein, and b) adding a complexing compound.

83. A process for preparing the amorphous precipitate according to claim 45, characterized in that it comprises: a) dissolving the derivatized protein, a hexamer stabilizing compound and a divalent metal cation in an aqueous solvent having a pH that will not allow the formation of hexamers of the derivatized protein, and b) adjusting the pH of between about 6.8 and about 7.8; and c) adding a complexing compound.

84. A method for treating diabetes, characterized by comprising administering the formulation according to claim 73 to a patient in need thereof, in an amount sufficient to regulate blood glucose levels in the patient.