Insoluble insulin compositions

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.

BACKGROUND OF THE INVENTION 1. FIELD OF THE INVENTION
 This invention is in the field of human medicine. More particularly, this
 invention is in the field of pharmaceutical treatment of the diseases of
 diabetes and hyperglycemia. 2. Description of Related Art
 It has long been a goal of insulin therapy 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 requiring a pulse of insulin to dispose
 of the meal-related blood glucose surge, and (b) the post-absorptive phase
 requiring a sustained delivery of insulin to regulate hepatic glucose
 output for maintaining optimal fasting blood glucose.
 Accordingly, effective therapy for people with diabetes generally involves
 the combined use of two types of exogenous insulin formulations: a rapid
 acting meal time insulin provided by bolus injections and a long-acting,
 so-called, basal insulin, administered by injection once or twice daily to
 control blood glucose levels between meals. An ideal basal insulin will
 provide an extended and "flat" time 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. Furthermore, an ideal basal
 insulin should be mixable with a soluble meal-time insulin, and should not
 cause irritation or reaction at the site of administration. Finally, basal
 insulin preparations that are suspension formulations should be able to be
 readily, and uniformly resuspended by the patient prior to administration.
 As is well understood by those skilled in this art, long-acting insulin
 formulations have been obtained by formulating normal insulin as
 microcrystalline suspensions for subcutaneous injection. Examples of
 commercial basal insulin preparations include NPH (Neutral Protamine
 Hagedorn) insulin, protamine zinc insulin (PZI), and ultralente (UL). NPH
 insulin is the most widely-used insulin preparation, constituting from 50
 to 70 percent of the insulin used worldwide. It is a suspension of a
 microcrystalline complex of insulin, zinc, protamine, and one or more
 phenolic preservatives. NPH insulin preparations are commercially
 available incorporating human insulin, pork insulin, beef insulin, or
 mixtures thereof. Also, NPH-like preparations of a monomeric insulin
 analog, LysB298,ProB29-human insulin analog, are known in the art
 [abbreviated herein as "NPL": De Felippis, M. R., U.S. Pat. No. 5,461,031,
 issued Oct. 24, 1995; De Felippis, M. R., U.S. Pat. No. 5,650,486, issued
 Jul. 22, 1997; and De Felippis, M. R., U.S. Pat. No. 5,747,642, issued May
 5, 1998].
 NPH insulin microcrystals possess a distinctive rod-shaped morphology of
 typical dimensions about 5 microns long by 1 micron thick and 1 micron
 wide. The extended duration of action of NPH insulin microcrystals results
 from their slow absorption from the subcutaneous injection site.
 Therapy using currently-available NPH insulin preparations fails to provide
 the ideal "flat" pharmacokinetics necessary to maintain optimal fasting
 blood glucose for an extended period of time between meals. Consequently,
 treatment with NPH insulin can result in undesirably high levels of
 insulin in the blood, which may cause life-threatening hypoglycemia.
 In addition to failing to provide an ideal flat pharmacokinetic profile,
 the duration of action of NPH insulin also is not ideal. In particular, a
 major problem with NPH therapy is the "dawn phenomenon" which is
 hyperglycemia that results from the loss of effective glucose control
 overnight while the patient is sleeping. These deficiencies in glycemic
 control contribute to serious long-term medical complications of diabetes
 and impose considerable inconvenience and quality-of-life disadvantages to
 the patient.
 Protamine zinc insulin (PZI) has a composition similar to NPH, but contains
 higher levels of protamine and zinc than NPH. PZI preparations may be made
 as intermediate-acting amorphous precipitates or long-acting crystalline
 material. PZI, however, is not an ideal basal insulin pharmaceutical
 because it is not mixable with a soluble meal-time insulin, and the high
 zinc and protamine can cause irritation or reaction at the site of
 administration.
 Human insulin ultralente is a microcrystalline preparation of insulin
 having higher levels of zinc than NPH, and not having either protamine or
 a phenolic preservative incorporated into the microcrystal. Human
 ultralente preparations provide moderate time action that is not suitably
 flat, and they do not form stable mixtures with insulin. Furthermore, they
 are difficult to resuspend.
 There have been attempts to address the perceived inadequacies of known
 insulin suspensions. Fatty acid-acylated insulins have been investigated
 for basal control of blood glucose [Havelund, S., et al., WIPO publication
 WO95/07931, Mar. 23, 1995]. Their extended time action is caused by
 binding of the fatty acyl portion of these molecules to serum albumin. The
 fatty acyl chain lengths of these molecules is such as to take advantage
 of the fatty acid binding capability of serum albumin. The fatty acid
 chains used in fatty acid-acylated insulins are typically longer than
 about ten carbon atoms, and chain lengths of fourteen and sixteen carbon
 atoms are optimal for binding to serum albumin and extending time action.
 Unlike NPH insulin, which is insoluble, the aforementioned fatty
 acid-acylated insulins are soluble at the usual therapeutic concentrations
 of insulin. However, the time action of these preparations may not be
 sufficiently long enough, or flat enough, to provide ideal basal control,
 and they are less potent than insulin, thereby requiring administration of
 greater amounts of the drug agent [Radziuk, J., et al., Diabetologia
 41:116-120, 489-490 (1998)].
 Whittingham, J. L., et al. [Biochemistry 36:2826-2831 (1997)] crystallized
 B29-N.epsilon.-tetradecanoyl-des(B30)-human insulin analog as a hexamer
 complex with zinc and phenol for the purpose of structural studies by
 X-ray crystallography. The hexamer was found to be in the R6 conformation,
 and to have certain properties different from hexamers of human insulin.
 Whittingham, et al. do not disclose any pharmaceutical or pharmacological
 properties of the crystal that was formed, nor do they suggest that such a
 crystal would have any advantageous properties for treating diabetes or
 hyperglycemia. It is not possible to predict from Whittingham, et al.
 whether protamine-containing crystals of the NPH type could be formed with
 derivatized insulins and insulin analogs, or what the pharmacokinetics or
 pharmacodynamic response of such crystals would be.
 Thus, there remains a need to identify insulin preparations that have
 flatter and longer time action than NPH insulin, that are mixable with
 soluble, meal-time insulins, that can be readily resuspended, and that do
 not pose risk of irritation or reaction at the site of administration.
 SUMMARY OF THE INVENTION
 I have unexpectedly observed that when insulin is made less soluble by
 derivatizing one or more of its reactive side groups, the 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 derivative dissolves from the solid form is greatly reduced
 compared with the rate at which similar solid forms comprised of
 un-derivatized protein dissolve. I have furthermore discovered that
 crystals of derivatized proteins provide flatter and longer time action
 than do crystals comprised of un-derivatized protein. Additionally, I have
 surprisingly discovered that the benefits of flatter and longer time
 action 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 selected from the
 group consisting of insulin derivatives, insulin analog derivatives, and
 proinsulin derivatives, wherein the derivatives are less soluble than the
 underivatized insulin, insulin analog, or proinsulin. The insoluble
 compositions also are 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 flatter and longer time action than NPH
 insulin. Furthermore, they are mixable in a formulation with soluble
 protein and with 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 microcrystals.
 More specifically, the present invention provides microcrystalline forms of
 fatty acid-acylated proteins that are useful for treating diabetes and
 hyperglycemia. These microcrystals comprise a fatty acid-acylated protein
 selected from the group consisting of fatty acid-acylated insulin, fatty
 acid-acylated insulin analog, and fatty acid-acylated proinsulin,
 protamine, a phenolic preservative, and zinc. Such microcrystals will
 provide both flatter and longer time action than NPH insulin, and are
 mixable 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 contain, optionally, a soluble protein, such as human insulin, or a
 soluble analog of human insulin, such as a monomeric insulin analog, that
 control blood glucose immediately following a meal. The microcrystalline
 formulations of fatty acid-acylated insulins have superior
 pharmacodynamics compared with human insulin NPH. The present invention is
 distinct from previous fatty acid-acylated insulin technology in that the
 extension of time action of the present invention does not rely
 necessarily on albumin-binding, though albumin binding may further
 protract the time action of certain of the compositions of the present
 invention.
 The invention also pertains to a process for preparing the insoluble
 compositions, and a method of treating diabetes or hyperglycemia
 comprising administering a formulation containing an insoluble composition
 to a patient in need thereof in a quantity sufficient to regulate blood
 glucose levels in the patient.
 Also part of the present invention are amorphous precipitates, comprising,
 in their broadest aspect, a derivatized protein 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 underivatized
 protein.

DESCRIPTION OF THE INVENTION
 The term "insoluble composition" refers to matter in either a
 microcrystalline state or in an amorphous precipitate state. The presence
 of microcrystals or amorphous precipitate can be ascertained by visual and
 microscopic examination. Solubility depends on solvent, and a particular
 composition may be insoluble in one solvent, but soluble in another.
 The term "microcrystal" means a solid that is comprised primarily of matter
 in a crystalline state, wherein the individual crystals are predominantly
 of a single crystallographic composition and are of a microscopic size,
 typically of longest dimension within the range 1 micron to 100 microns.
 The term "microcrystalline" refers to the state of being a microcrystal.
 The term "amorphous precipitate" refers to insoluble material that is not
 crystalline in form. The person of ordinary skill can distinguish crystals
 from amorphous precipitate. The amorphous precipitates of the present
 invention have advantageous pharmacological properties in their own right,
 and also are intermediates in the formation of the microcrystals of the
 present invention.
 The term "derivatized protein" refers to a protein selected from the group
 consisting of derivatized insulin, derivatized insulin analogs, and
 derivatized proinsulin that is derivatized by a functional group such that
 the derivatized protein is less soluble in an aqueous solvent than is the
 un-derivatized protein. Many examples of such derivatized proteins are
 known in the art, and the determination of solubility of proteins and
 derivatized proteins is well-known to the skilled person. Examples of
 derivatized insulin and insulin analogs include benzoyl,
 p-tolyl-sulfonamide carbonyl, and indolyl derivatives of insulin and
 insulin analogs [Havelund, S., et al., WO95/07931, published Mar. 23,
 1995]; alkyloxycarbonyl derivatives of insulin [Geiger, R., et al., U.S.
 Pat. No. 3,684,791, issued Aug. 15, 1972; Brandenberg, D., et al., U.S.
 Pat. No. 3,907,763, issued Sep. 23, 1975]; aryloxycarbonyl derivatives of
 insulin [Brandenberg, D., et al., U.S. Pat. No. 3,907,763, issued Sep. 23,
 1975]; alkylcarbamyl derivatives [Smyth, D. G., U.S. Pat. No. 3,864,325,
 issued Feb. 4, 1975; Lindsay, D. G., et al., U.S. Pat. No. 3,950,517,
 issued Apr. 13, 1976]; carbamyl, O-acetyl derivatives of insulin [Smyth,
 D. G., U.S. Pat. No. 3,864,325 issued Feb. 4, 1975]; cross-linked, alkyl
 dicarboxyl derivatives [Brandenberg, D., et al., U.S. Pat. No. 3,907,763,
 issued Sep. 23, 1975]; N-carbamyl, O-acetylated insulin derivatives
 [Smyth, D. G., U.S. Pat. No. 3,868,356, issued Feb. 25, 1975]; various
 O-alkyl esters [Markussen, J., U.S. Pat. No. 4,343,898, issued Aug. 10,
 1982; Morihara, K., et al., U.S. Pat. No. 4,400,465, issued Aug. 23, 1983;
 Morihara, K., et al., U.S. Pat. No. 4,401,757, issued Aug. 30, 1983;
 Markussen, J., U.S. Pat. No. 4,489,159, issued Dec. 18, 1984; Obermeier,
 R., et al., U.S. Pat. No. 4,601,852, issued Jul. 22, 1986; and Andresen,
 F. H., et al., U.S. Pat. No. 4,601,979, issued Jul. 22, 1986]; alkylamide
 derivatives of insulin [Balschmidt, P., et al., U.S. Pat. No. 5,430,016,
 issued 4 July 1995]; various other derivatives of insulin [Lindsay, D. G.,
 U.S. Pat. No. 3,869,437, issued Mar. 4, 1975]; and the fatty acid-acylated
 proteins that are described herein.
 The term "acylated protein" as used herein refers to a derivatized protein
 selected from the group consisting of insulin, insulin analogs, and
 proinsulin that is acylated with an organic acid moiety that is bonded 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 may be the .alpha.-amino group of an N-terminal amino acid of
 the protein, or may be the .epsilon.-amino group of a Lys residue of the
 protein. An acylated protein may be acylated at one or more of the three
 amino groups that are present in insulin and in most insulin analogs.
 Mono-acylated proteins are acylated at a single amino group. Di-acylated
 proteins are acylated at two amino groups. Tri-acylated proteins are
 acylated at three amino groups. The organic acid compound may be, for
 example, a fatty acid, an aromatic acid, or any other organic compound
 having a carboxylic acid group that will form an amide bond with an amino
 group of a protein, and that will cause the aqueous solubility of the
 derivatized protein to be lower than the solubility of the un-derivatized
 protein.
 The term "fatty acid-acylated protein" refers to a an acylated protein
 selected from the group consisting of insulin, insulin analogs, and
 proinsulins that is acylated with a fatty acid that is bonded to the
 protein through an amide bond formed between the acid group of the fatty
 acid and an amino group of the protein. In general, the amino group may be
 the .alpha.-amino group of an N-terminal amino acid of the protein, or may
 be the .epsilon.-amino group of a Lys residue of the protein. A fatty
 acid-acylated protein may be acylated at one or more of the three amino
 groups that are present in insulin and in most insulin analogs.
 Mono-acylated proteins are acylated at a single amino group. Di-acylated
 proteins are acylated at two amino groups. Tri-acylated proteins are
 acylated at three amino groups. Fatty acid-acylated insulin is disclosed
 in a Japanese patent application 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). Further disclosure of fatty
 acid-acylated insulins and fatty acylated insulin analogs, and of methods
 for their synthesis, is found in Baker, J. C., et al, U.S. Ser. No.
 08/342,931, filed Nov. 17, 1994 and issued as U.S. Pat. No. 5,693,609,
 Dec. 2, 1997; Havelund, S., et al., WO95/07931, published Mar. 23, 1995,
 and a corresponding U.S. Pat. No. 5,750,497, May 12, 1998; and Jonassen,
 I., et al., WO96/29342, published Sep. 26, 1996. These disclosures are
 expressly incorporated herein by reference for describing fatty
 acid-acylated insulins and fatty acid-acylated insulin analogs and for
 enabling preparation of the same.
 The term "fatty acid-acylated protein" includes pharmaceutically acceptable
 salts and complexes of fatty acid-acylated proteins. The term "fatty
 acid-acylated protein" also includes preparations of acylated proteins
 wherein the population of acylated protein molecules is homogeneous with
 respect to the site or sites of acylation. For example,
 N.epsilon.-mono-acylated protein, B1-N.alpha.-mono-acylated protein,
 Al-N.alpha.-mono-acylated protein, A1,B1-N.alpha.-di-acylated protein,
 N.epsilon., A1-N.alpha., di-acylated protein, N.epsilon.,B1-N.alpha.,
 di-acylated protein, and N.epsilon., A1,B1-N.alpha., tri-acylated protein
 are all encompassed within the term "fatty acid-acylated protein" for the
 purpose of the present invention. The term also refers to preparations
 wherein the population of acylated protein molecules has heterogeneous
 acylation. In the latter case, the term "fatty acid-acylated protein"
 includes mixtures of mono-acylated and di-acylated proteins, mixtures of
 mono-acylated and tri-acylated proteins, mixtures of di-acylated and
 tri-acylated proteins, and mixtures of mono-acylated, di-acylated, and
 tri-acylated proteins.
 The term "insulin" as used herein, refers to human insulin, whose amino
 acid sequence and special structure are well-known. Human insulin is
 comprised of a twenty-one amino acid A-chain and a thirty-amino acid
 B-chain which are cross-linked by disulfide bonds. A properly cross-linked
 insulin contains three disulfide bridges: one between position 7 of the
 A-chain and position 7 of the B-chain, a second between position 20 of the
 A-chain and position 19 of the B-chain, and a third between positions 6
 and 11 of the A-chain.
 The term "insulin analog" means proteins that have an A-chain and a B-chain
 that have substantially the same amino acid sequences as the A-chain and
 B-chain of human insulin, respectively, but differ from the A-chain and
 B-chain of human insulin by having one or more amino acid deletions, one
 or more amino acid replacements, and/or one or more amino acid additions
 that do not destroy the insulin activity of the insulin analog.
 "Animal insulins" are insulin analogs. Four such animal insulins are
 rabbit, pork, beef, and sheep insulin. The amino acid substitutions that
 distinguish these animal insulins from human insulin are presented below
 for the reader's convenience.