JP2002543092A - Insulin crystals for pulmonary administration - Google Patents

Abstract

  (57) [Summary] The present invention relates to a crystal comprising a derivatized insulin or a derivatized insulin analog, a method for producing such a crystal, and a method for treating diabetes comprising administering the crystal to a patient in need of the treatment of diabetes to regulate blood glucose. About. Obtain crystals having the desired average size. By adjusting the average crystal size and conveniently administering the crystals by inhalation via the mouth and allowing them to accumulate in the deep lung where they are absorbed over time, blood glucose during meals and overnight can be controlled.

Description

DETAILED DESCRIPTION OF THE INVENTION

CROSS REFERENCE This application claims the benefit of US Provisional Application No. 60 / 131,170, filed April 27, 1999, the contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the invention. The invention belongs to the field of human medicine. More particularly, the invention belongs to the field of treatment of diabetes and hyperglycemia. 2. Description of related technology. Simulating the pattern of endogenous insulin secretion in normal individuals has long been a goal of insulin therapy. The daily physiological demands for insulin fluctuate, and (a) the absorption phase (absorptive) which requires the application of insulin (pulse) to eliminate sudden fluctuations in dietary blood glucose (blood glucose)
post-absorptive ph), which requires a continuous supply of insulin to regulate the release of hepatic glucose to maintain optimal fasting blood glucose.
ase). Thus, in general, effective treatments for people with diabetes include mealtime fast-acting insulin given as a bolus injection,
A combination of two long-acting exogenous insulin preparations, so-called basal insulins, administered by injection once or twice a day to regulate blood sugar levels between meals is required.

[0003] Conventional insulin therapy involves only two injections per day. More intensive insulin therapy, injecting insulin three or more times a day, is available from Diabetes Con
As shown by the trol and Complications Trial (DCCT) test, it results in reduced complications. Unfortunately, many diabetics reluctant to receive intensive therapy because the many injections required to maintain tight regulation of glucose levels are uncomfortable. Non-injectable insulin is desirable to increase patient compliance with intensive insulin therapy and reduce the risk of complications.

[0004] Many investigators have studied non-injectable insulin, such as the oral, rectal, transdermal, and nasal routes. To date, these types of administration have been ineffective due to poor absorption of insulin, low serum insulin levels, stimulation of the supply site or no significant reduction in serum glucose levels.

[0005] Another well-studied route for administering non-injectable insulin is from the lungs. Due to its relatively low molecular weight (5,800 daltons), insulin appears to be an ideal candidate for pulmonary administration. Administration of insulin by inhalation and its absorption from the lungs was first reported in 1925. However, after administration by inhalation, small proteins such as insulin are rapidly absorbed from the lungs due to the very large surface area of the lungs and the relatively porous membrane. Plasma concentrations rapidly peak and fall rapidly. These pharmacokinetics may be suitable for regulating blood glucose in the absorption phase, but are completely inappropriate in the post absorption phase. Therefore, a method of administering long-acting insulin by inhalation remains a challenge.

The following is a review of the inhalation of insulin and other proteins: "Aeroso
l Insulin-A Brief Review, Patton, JS and Platz, RM, Respiratory
Drug Delivery IV, edited by P. Byron, Interpharm Press (1994); `` Delivery of B
iotherapeutics by Inhalation Aerosol ", Niven, R., Crit. Rev. in Therape
utic Drug Carrier Systems 12: 151 231 (1995); and `` Drug Delivery via th
e Respiratory Tract, Byron, PR and Patton, JS, J. Aerosol Medic
ine 7: 49-75 (1994).

[0007] The efficiency of supply and accumulation of particles on the surface of the deep lung, the alveoli, depends on the aerodynamics of the particles.
There is a strong relationship with (target) median diameter (MMAD). The optimal MMAD is about 2-3 microns. Above and below this range, the substance will not accumulate much at the alveoli surface.

[0008] Phagocytosis of insoluble or slowly dissolving particles is the most important clearance mechanism in the deep lung [Rudt, SH, et al., J. et al.
Controlled Release 25: 123-138 (1993); Tabata, Y., et al., J. Biomedical
Material Res. 22: 837-842 (1988); Wang, JA, et al., In: Respiratory D
rug Delivery. Buffalo Grove, IL; Interpharm 1997, vol. VI, pp. 187-192;
Edwards, DA, et al., Science 276: 1868-1871 (1997); Gehr, PM, Micro
sc. Res. Tech. 26: 423-436 (1993)]. Once accumulated on the alveoli surface, particles with actual sizes in the range of about 1 to about 10 microns are phagocytosed by lung macrophages (phagocy
tose). It is known that very large particles, such as those having a diameter of about 10 microns or more, are not efficiently phagocytosed by lung macrophages. Small particles whose actual size is in the nanometer size range appear to escape macrophage uptake. Unfortunately, particles with optimal properties for delivery and accumulation often have actual particle sizes in the range where macrophage attack is expected.

Have described the long term release of insulin from macroporous particles administered to the deep lung by inhalation [J. Appl. Physiol. 85: 379-385 (1998); Sc
ience 276: 1868-1871 (1997)]. The persistence of the administered particles is due to the large actual size of the particles (> 10 microns), but the rate of accumulation in the deep lung is relatively small, despite their large physical size. Due to the low density of the particles giving rise to size (approximately 3-4 micrometers MMAD). These particles include insulin encapsulated in a biodegradable copolymer (poly-lactic-co-glycolic acid). Insulin is released over a period of at least 4 days due to the slow degradation of the copolymer.

[0010] Crystallization of insulin, for example using NPH-insulin and various Lente products, is a well-known means of providing long-term glycemic control to people with diabetes. Insulin crystals are uniformly administered by the parenteral route of administration, usually the subcutaneous route. Acceptable high biodegradability of insulin-containing crystals was not achieved when delivered to the deep lung by inhalation. This is a complex defense mechanism involving the biology, immunology and chemistry of the lung surface for the removal of particles in the air with particle properties that avoid the upper airway entrapment mechanism, especially microorganisms and aerosols containing microorganisms It seems to be.

[0011] Hughes, BL et al. [PCT / US98 / 23040, filed Oct. 29, 1998] describe a method and pharmaceutical composition for administering fatty acylated insulin and insulin analogues by inhalation to treat diabetes. Was described. The composition is a solution or a powder of an amorphous substance and is not crystalline. Hughes et al. Derivatized insulin B28-Nε-myristoyl-LysB28, ProB29 human insulin analog, and B29-Nε-palmitoyl-
It was demonstrated that human insulin was adsorbed in an amount effective to lower glucose levels. The pharmacokinetics of fatty acylated insulin administered via the lung were delayed compared to non-acylated insulin, but the length of the delay was shorter than that of NPH-insulin supplied subcutaneously.

Havelund, S. [W098 / 42749, published October 1, 1998] is resistant to aggregation and clumping, which is said to be suitable for administration by inhalation. And zinc-free crystalline powders of insulin, insulin analogs and insulin derivatives.

[0013] Whittingham, JL et al. [Biochemistry 36: 2826-2831 (1997)] describe B29-Nε-tetradecanoyl-des (B30) -human insulin analog and zinc for structural studies by X-ray crystallography. Very large crystals consisting of These crystals were too large for efficient accumulation in the deep lung when administered by inhalation. These crystals would have to be milled to produce a substance of a suitable particle size to achieve efficient accumulation when administered by inhalation.

Brader, M. [US Patent Application No. 09/177685, filed October 22, 1998; PCT / US98 / 22
434; EP 0911035, published April 28, 1999, herein referred to as Brader
I], and Brader, M. [US Patent Application No. 09/217275, filed December 21, 1998; PCT / US98 / 27299, referred to herein as Brader II]
Microcrystals containing derivatized insulin and derivatized proteins containing derivatized insulin analogs, methods of making the crystals, and methods of administering them to treat (treat) diabetes are described. The crystals are rod-shaped, commercially available rod-shaped
It has the size of NPH insulin crystals and is about 5 microns long. Such crystals would be too large to obtain optimal accumulation in the deep lung when administered by mouth inhalation.

[0015] Brader II provides a long list of parameters that are believed to affect crystallization rate and size, in which the temperature and concentration of competing compounds, such as citrate, were mentioned. However, Brader II does not specify a relationship between crystal size and temperature, and only infers the effect of competing compounds on size, which would slow the rate of crystallization. In addition, Brader II does not mention chloride anions among many parameters that are thought to affect the crystal size of the derivatized protein.

SUMMARY OF THE INVENTION The present invention provides a stable population of crystals consisting of derivatized insulin or a derivatized insulin analog and a divalent metal cation, characterized by an average diameter in the range of 1-3 microns. Including. More specifically, the invention relates to a crystal having a uni-modal symmetric particle distribution, comprising: a) a compound of the formula: B29-Nε-X-human insulin, wherein X is butyryl, pentanoyl, A volume-averaged spherical equivalent diameter comprising a derivatized insulin selected from the group consisting of hexanoyl, heptanoyl, octanoyl, nonanyl, and decanoyl), b) zinc, c) a phenolic preservative, and d) protamine. spherical equiva
lent diameter) of 1 to 3 microns.

The present invention relates to a co-crystal comprising insulin or an insulin analog, and a derivatized insulin or a derivatized insulin analog and a divalent metal cation, the average diameter of which is in the range of 1-3 microns. Including stable population. That is, more particularly, the present invention relates to a co-crystal having a uni-modal symmetric particle distribution, comprising: insulin, and a) a compound of the formula: B29-Nε-X-human insulin (where X Is selected from the group consisting of butyryl, pentanoyl, hexanoyl, heptanoyl, octanoyl, nonanyl, and decanoyl), b) zinc, c) a phenolic preservative, and d) protamine. It also includes co-crystals characterized by an equivalent diameter of 1-3 microns (where the ratio of derivatized insulin to total protein is at least 50%).

The invention also includes a pharmaceutical composition comprising the crystal or co-crystal together with one or more pharmaceutically acceptable excipients, carriers, or aqueous solvents in which the crystals are stable. The pharmaceutical composition may be for parenteral administration, more preferably for administration by inhalation through the mouth of a patient for accumulation in the deep lung (alveoli).

In the parenteral pharmaceutical composition, the crystals may optionally contain isotonic agents, buffers, preservatives,
And insulin or insulin analog are suspended in a pharmaceutically acceptable aqueous solvent. In a pharmaceutical composition for pulmonary administration, the crystals may be provided in a dry powder (eg, by a dry powder inhaler), optionally together with other dry excipients, a dry powder of insulin or an insulin analog, and carrier particles. )). The crystals may also be formulated for administration by inhalation by mouth to a patient in liquid form by suspending the crystals in a pharmaceutically acceptable aqueous solvent containing insulin and an insulin analog, if desired. Good.

The present invention provides a method for producing crystals and co-crystals of a size that increases the absorption efficiency in the deep lung when administered by inhalation through the mouth. The method comprises producing a suspending agent having a neutral pH by performing the following steps a) -f) in any order in the absence of citrate salt, provided that step f) comprises step a): Followed by step f) prior to step e), steps b) and c) prior to step f): a) dissolving the derivatized insulin in an acidic solvent at acidic pH, b) adding the phenolic preservative In addition, c) zinc is added; d) chloride anion is brought to a final concentration of about 100 mM to about 150 mM above that introduced by pH adjustment.
E) add protamine, f) adjust to neutral pH, then raise the temperature of the neutral pH suspension from about 25 ° C. to 12 hours to about 96 hours.
Keep at about 37 ° C.

The present invention also includes the use of crystals in the manufacture of a medicament for treating diabetes or hyperglycemia by inhalation, wherein the treatment is performed using an inhalation device such that the medicament accumulates in the patient's lungs. Administering an effective amount of a medicament to a patient in need thereof). The present invention also includes the use of a co-crystal in the manufacture of a medicament for treating diabetes or hyperglycemia by inhalation, wherein the treatment comprises administering an effective amount of the medicament using an inhalation device such that the medicament builds up in the lungs of the patient. Administering the medicament to a patient in need thereof).

The present invention also provides a method of administering a crystal or co-crystal by inhalation. The present invention also provides crystals and co-crystals which are physically stable and more readily resuspendable than the crystals and co-crystals prepared by the methods described in Brader I and Brader II. Methods for producing crystals and co-crystals having improved properties include producing a suspension having a neutral pH by performing the following steps a) -g) in the absence of citrate. (However, when step g) follows step a) and step g) precedes step f), steps b) and c) precede step g)): a) converting the derivatized insulin to an aqueous solvent at acidic pH B) Add phenolic preservative, c) Add zinc, d) Add chloride anion to a final concentration of about 15 mM to 150 mM chloride ion above that introduced by pH adjustment, e) The acid salt is added to a concentration of 1 mM to 10 mM, f) protamine is added, g) adjusted to neutral pH, and then for 12 hours to about 96 hours, the temperature of the neutral pH suspension is about 20 ° C. to about
Keep at 37 ° C. In co-crystals, insulin is also dissolved using the method described above, and the ratio of derivatized insulin to total protein is at least 50%. Crystals produced by this method are physically more stable than crystals produced according to the disclosures of Brader I and Brader II and are more easily resuspended. These are convenient for the patient.

The present invention is the discovery of the dramatic and unexpected effect of chloride ions on crystal and co-crystal particle size, and physical stability and resuspension. Ability to produce crystals and co-crystals with a volume average sphere equivalent diameter in the range of about 1 micron to about 3 microns, ideally suited to accumulate particles in the lung with high efficiency without further sorting or reducing size. Derive from the further discovery of the combined effects of chloride ions, higher temperatures, and the absence or low levels of citrate.

The present invention solves two problems not currently addressed in the art. First, previous pulmonary methods for delivering insulin did not provide adequate time-effects to regulate blood glucose between meals and overnight (overnight). Second, currently known soluble, long-acting insulins and insulin analogs are supplied by painful subcutaneous injection of a needle, which is inconvenient for preparing samples for injection. According to the present invention, patients in need of insulin to regulate blood glucose levels can benefit from a slow and convenient uptake of insulin compared to inhalation of underivatized insulin, and inconvenience compared to subcutaneous administration. You will benefit from less pain. The crystals and co-crystals may be prepared using any of a variety of devices suitable for administration by inhalation, for example as a solution or suspension in a carrier,
Or it can be supplied as a dry powder. The acylated insulin can be administered using an inhalation device, such as a nebulizer, a metered dose inhaler, a dry powder inhaler, a sprayer and the like.

The present invention also provides a method of administering a pharmaceutical composition comprising a crystal or co-crystal and either insulin or an insulin analog to a patient in need thereof by inhalation. Administration of such a combination results in both postprandial and basal regulation of blood glucose levels. The method avoids injections, thus improving patient comfort and
Patient compliance increased compared to normal insulin delivery.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows blood glucose levels after administration of crystals and co-crystals in F344 rats: 100% C8-
Intratracheal instillation of BHI 1 mg / kg (solid square, solid line); 75% C8-BHI: 25% BHI Intratracheal instillation of 1 mg / kg (open square, solid line); 75% C8-BHI: 25% BHI 1 mg / kg Subcutaneous administration (filled circles, broken line);
And subcutaneous administration of NPH insulin 1 mg / kg as control (solid triangle, solid line).

DETAILED DESCRIPTION OF THE INVENTION As used herein, the term “crystal” refers to a microparticle that includes derivatized insulin or a derivatized insulin analog, divalent metal cations, complexing compounds, and hexamer-stabilizing compounds. Means crystal. This type of crystal is described in Brader, M. [US patent application Ser. No. 09/17768.
No. 5, filed Oct. 22, 1998, PCT / US98 / 22434, European Patent Publication No. 0911035, 19
Published April 28, 1999]. As used herein, the term "co-crystal" refers to microcrystals including insulin or insulin analogs, derivatized insulin or derivatized insulin analogs, divalent metal cations, complex-forming compounds, and hexamer-stabilizing compounds. Means This type of co-crystal is disclosed in Brader, M. [US Patent Application Serial No. 09/217275, December 21, 1998.
, Filed in PCT / US98 / 27299].

The term “crystallite” refers to a solid that has a microscopic size, typically the longest dimension, in the range of 1 micron to 100 microns, mainly including the crystalline material. The term "microcrystalline" refers to a state that is microcrystalline.

The term “rod” refers to a unique microcrystalline morphology, also described as a tetragonal rod with a pyramid tip. The morphology of the microcrystals of the invention is easily determined by microscopic examination.

The term “protein” may have its usual meaning (ie, a polymer of amino acids). As used herein, the term “protein” has a narrower meaning (
That is, it also has a protein selected from the group consisting of insulin, insulin analogs, and proinsulin). The term “underivatized protein” also refers to insulin,
Represents a protein selected from the group consisting of insulin analogs and proinsulin.

The term “total protein” as used in context in the claims and elsewhere refers to proteins (insulin, insulin analogs, or proinsulin) and derivatized proteins (derivatized insulin, derivatized insulin analog, or derivatized) Proinsulin). Protamine and other known complexing compounds are also proteins in the broadest sense of the term, but the term "total protein"
Does not include them.

The term “derivatized protein” means that the derivatized protein is less soluble in an aqueous solvent than the underivatized protein, more liposoluble than underivatized insulin, or the corresponding of the underivatized protein. A protein selected from the group consisting of a derivatized insulin and a derivatized insulin analog derivatized with a functional group to form a complex with zinc and protamine that is less soluble than the complex. Determining whether proteins and derivatized proteins are soluble or lipophilic is well known to those skilled in the art. Derivatized proteins and their solubility in complexes with zinc and protamine are well known in the art [Graham and Pomeroy
, J. Pharm. Pharmacol. 36: 427-430 (1983), DeFelippis, MR and Frank,
B., EP735,048), or the method used herein.

Benzoyl, p-toluyl-sulfonamidocarbonyl and indolyl derivatives of insulin and insulin analogues [Havelund, S. et al., W095 / 07931, March 1995
Published on Aug. 23], an alkyloxycarbonyl derivative of insulin [Geiger, R. et al., US Pat. No. 3,684,791, issued Aug. 15, 1972, Brandenberg, D. et al., US Pat.
07,763, issued September 23, 1975], aryloxycarbonyl derivative of insulin
[Brandenberg, D. et al., US 3,907,763, issued September 23, 1975], alkylcarbamyl derivative [Smyth, DG, U.S. Patent No. 3,864,325, issued February 4, 1975, Lindsay,
DG et al., US Patent No. 3,950,517, issued April 13, 1976], carbamyl and O-acetyl derivatives of insulin [Smyth, DG, US Patent No. 3,864,325, issued February 4, 1975], cross-linked alkyl dicarboxylic Derivatives [Brandenberg, D. et al., U.S. Pat.
07,763, issued September 23, 1975], N-carbamyl, O-acetylated insulin derivative
[Smyth, DG, U.S. Patent No. 3,868,356, issued February 25, 1975], various O-alkyl esters [Markussen, J., U.S. Patent No.4,343,898, issued August 10, 1982, Mori
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. 1
No. 59, issued December 18, 1984, Obermeier, R. et al., U.S. Pat.No. 4,601,852, 1986
Published July 22, and Andresen, FH et al., U.S. Pat.No. 4,601,979, July 22, 1986
Published in the United States], alkyl amide derivatives of insulin [Balschmidt, P. et al., US Pat.
No. 430,016, issued on July 4, 1995], and various other insulin derivatives [Lindsay, DG
And U.S. Pat. No. 3,869,437, issued Mar. 4, 1975], and many examples of such derivatized proteins are known in the art, including the fatty acid acylated proteins described herein.

As used herein, the term “acylated protein” refers to an organic acid that binds to a protein via an amide bond formed between the acid group of the organic acid compound and the amino group of the protein. 1 represents a derivatized protein selected from the group consisting of partially acylated insulin and insulin analogs. Generally, the amino group is the α-amino group of the N-terminal amino acid of the protein or the L-amino acid of the protein.
It may be the ε-amino group of the ys residue. Acylated proteins may be acylated at one or more of the three amino groups present on insulin and most insulin analogs. Mono-acylated proteins have one amino group acylated. The di-acylated protein has two amino groups acylated. Tri-acylated proteins have three amino groups acylated. The organic acid compound forms, for example, an amide bond with a fatty acid, an aromatic acid, or an amino group of the protein, and the zinc / protamine complex of the derivatized protein has reduced solubility or water solubility as compared with the underivatized protein. Other organic compounds having a carboxylic acid group that decreases or increases lipophilicity may be used.

The term “fatty acid-acylated protein” consists of insulin and insulin analogs that are acylated with a fatty acid that binds the protein via an amide bond formed between the acid group of the fatty acid and an amino acid of the protein. Represents an acylated protein selected from the group. Generally, the amino acid is α of the N-terminal amino acid of the protein.
-It may be an amino group or the ε-amino acid of a Lys residue of the protein. Fatty acid-acylated proteins may be acylated on one or more of the three amino acids present in insulin and most insulin analogs. Mono-acylated proteins have one amino group acylated. The di-acylated protein has two amino groups acylated. Tri-acylated proteins have three amino groups acylated. Fatty acid-acylated insulins are disclosed in Japanese Patent Application No. 1-254,699. Hashimoto, M. et al., Pharmaceutical Resea
rch, 6: 171-176 (1989), and Lindsay, DG et al., Biochemical J. 121: 737-7.
See also 45 (1971). Further disclosure of fatty acylated insulin and fatty acylated insulin analogs, and methods of synthesizing them, is provided by Baker, JC et al., U.
S.08 / 342,931 (filed Nov. 17, 1994, issued on Dec. 2, 1997 as U.S. Pat. No. 5,693,609), Havelund, S. et al., W095 / 07931 (published Mar. 23, 1995), and No. 5,750,497 (issued May 12, 1998) and Jonassen, I. et al., W096 / 2
9342 (released September 26, 1996).

The term “fatty acid-acylated protein” includes pharmaceutically acceptable salts and complexes of fatty acid-acylated protein. The term "fatty acid-acylated protein" also includes preparations of acylated proteins in which the population of the acylated protein molecule is uniform with respect to the acylation site or sites. For example, Nε-mono-acylated protein, B1-Nα-mono-acylated protein, A1-N
α-mono-acylated protein, A1, B1-Nα-di-acylated protein, Ne, A1-Nα,
Di-acylated protein, Nε, B1-Nα, di-acylated protein, and Nε, A1, B1
-Nα, tri-acylated proteins are all termed “fatty acid-
Acylated protein ". The term also describes preparations in which the acylated protein molecule population has heterologous acylation. In the latter case, the term "fatty acid acylated protein" includes a mixture of monoacylated protein and diacylated protein, a mixture of monoacylated protein and triacylated protein, and a mixture of monoacylated protein, diacylated protein, and triacylated protein. Is included.

[0037] As used herein, the term "insulin" refers to human insulin whose amino acid sequence and unique structure are well known. Human insulin consists of a 21 amino acid A chain and a 30 amino acid B chain cross-linked by disulfide bonds. Properly crosslinked insulin contains three disulfide bridges, the first between position 7 of the A chain and the position 7 of the B chain,
The second is between position 20 of the A chain and position 19 of the B chain, and the third is between positions 6 and 11 of the A chain.

The term “insulin analog” refers to one or more amino acids that have A and B chains that are substantially the same as the A and B chains of human insulin, respectively, but that do not disrupt the insulin activity of the insulin analog. A protein that differs from the A and B chains of human insulin by having deletions, one or more amino acid substitutions, and / or one or more amino acid additions.

“Animal insulin” is an analog of insulin as defined herein because it is an analog of human insulin. Four such animal insulins are rabbit, pig, bovine, and sheep insulin. Amino acid substitutions that distinguish these animal insulins from human insulin are provided below for the convenience of the reader.

[0040]

Another type of insulin analog, a “monomer insulin analog”, is well known in the art. Monomeric insulin analogs are structurally very similar to human insulin and have similar or the same activity as human insulin, but dimerization and hexamerization with a reduced tendency to lead to higher aggregation states Have one or more amino acid deletions, substitutions or additions that tend to interfere with the necessary contacts. Monomeric insulin analogues are fast acting human insulin analogues, such as Chan
ce, RE et al., US Patent No. 5,514,646 (May 7, 1996); Brems, DN et al., Protein.
Engineering, 5: 527-533 (1992); Brange, JJV et al., EPO Publication No. 214,826 (
Brange, JJV et al., U.S. Patent No. 5,618,913 (April 8, 1997).
And Brange, J. et al., Current Opinion in Structural Biology 1: 934-94.
0 (1991). Examples of monomeric insulin analogs include Pro at B28, Asp,
Human insulin substituted with Lys, Leu, Val, or Ala, and Lys at position B29 is Lys or substituted with Pro, and AlaB26-human insulin, death (B28-B30)
Human insulin, and des (B27) -human insulin. Monomeric insulin analogs used as derivatives in the present crystals or used without derivatization in the solution phase of a suspended formulation are suitably cross-linked at the same locations as human insulin.

Another group of insulin analogs for use in the present invention are insulin analogs having an isoelectric point of about 7.0 to about 8.0. These analogs are referred to as “pI shifted insulin analogs. Examples of such insulin analogs include ArgB31, ArgB32-human insulin, GlyA21, ArgB31, ArgB32-human insulin, ArgAO, ArgB31, ArgB32-human insulin. And ArgAO, GlyA21, ArgB31, ArgB32-human insulin.

Another group of insulin analogs consists of insulin analogs having one or more amino acid deletions that do not significantly interfere with the activity of the molecule. This group of insulin analogs is designated herein as "deletion analogs." For example, 1 at B1-B3
Insulin analogs having a deletion of or more amino acids are active. Similarly, insulin analogs having a deletion of one or more amino acids at positions B28-B30 are active. Examples of "deletion analogs" include des (B30) -human insulin, desPhe (B1
) -Human insulin, des (B27) -human insulin, des (B28-B30) -human insulin, and des (B1-B3) -human insulin. Deletion analogs used as derivatives in the present crystals or used without derivatization in the solution phase of a suspension formulation are:
Properly cross-linked at the same position as human insulin.

Amidated amino acids, particularly asparagine residues, in insulin are known to be chemically unstable [Jorgensen, KH et al., US Pat. No. 5,008,241 (1991)
Dorschug, M., U.S. Patent No. 5,656,722 (August 12, 1997). In particular, they are capable of deamidation and various reactions under certain well-known conditions. Thus, if desired, the insulin analog may be insulin or an insulin analog in which one or more amidated residues have been replaced with another amino acid to achieve chemical stability. For example, Asn or Gln may be substituted with a non-amidated amino acid, with preferred amino acid substitutions for Asn or Gln being Gly, Ser, Thr, Asp, or Glu. In particular, AsnAl8, AsnA21, or AsnB3, or any combination of these residues, may be replaced, for example, with Gly, Asp, or Glu.
GlnA15 or GlnB4, or both, may be replaced with either Asp or Glu. Preferred substitutions are Asp at position B21 and Asp at B3. Substitutions that do not alter the charge of the protein molecule are also preferred, as are substitutions with neutral amino acids of Asn or Gln.

The term “proinsulin” refers to a single-chain peptide molecule that is a precursor of insulin. Proinsulin may be converted to insulin or an insulin analog by a chemical reaction, or preferably by an enzyme-catalyzed reaction. Proinsulin forms the appropriate disulfide bond as described below. Proinsulin includes insulin or an insulin analog, and a linking or linking peptide.
The linking peptide has 1 to about 35 amino acids. The linking bond or the linking peptide bonds to the terminal amino acid of the A chain and the terminal amino acid of the B chain through an α-amide bond and two α-amide bonds, respectively. Preferably, the amino acids in the linking peptide are not cysteines. Preferably, the C-terminal amino acid of the connecting peptide is L
ys or Arg. Proinsulin may have the formula: XBCAY or the formula: XACBY (where X is hydrogen or a peptide of 1 to about 100 amino acids where the C-terminal amino acid is either Lys or Arg. Y is hydroxy or N
-A peptide of 1 to about 100 amino acids whose terminal amino acid is either Lys or Arg. A is the A chain of insulin or the A chain of an insulin analog, and C is 1 to about 3
A peptide of 5 amino acids, none of which is cysteine, the C-terminal amino acid is Lys or Arg, and B is the B chain of insulin or the B chain of an insulin analog. ).

“Pharmaceutically acceptable salt” refers to one or more charged groups in a protein,
A salt formed between any one or more pharmaceutically acceptable non-toxic cations or anions is meant. Organic and inorganic salts include, for example, acids such as hydrochloric acid, sulfuric acid, sulfonic acid, tartaric acid, fumaric acid, hydrobromic acid, glycolic acid, citric acid, maleic acid, phosphoric acid, succinic acid, acetic acid, nitric acid, benzoic acid Includes acids, ascorbic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, propionic acid, carboxylic acids, and the like, or salts prepared from, for example, ammonium, sodium, potassium, calcium, or magnesium.

The term “acylate” means forming an amide bond between an amino group of a protein and a fatty acid. A protein is "acylated" when one or more amino groups of the protein is linked to an acid group of a fatty acid via an amide bond. The term "fatty acid" means a saturated or unsaturated, straight or branched chain fatty acid having 1 to 18 carbon atoms. The term "C1-C18 fatty acid" refers to a saturated straight or branched chain fatty acid having 1 to 18 carbon atoms.

The term “divalent metal cation” refers to an ion or ions that participate in forming a complex with various protein molecules. Transition metals, alkali metals, and alkaline earth metals are examples of metals known to complex with insulin. Transition metals are preferred. Zinc is particularly preferred. Other transition metals that are pharmaceutically acceptable to complex with the insulin protein are copper, cobalt, and iron.

The term “complex” has two meanings in the present invention. First, the term describes a complex formed between one or more atoms of a complexing protein and one or more divalent metal cations. The atoms in the protein serve as electron donating ligands. The protein typically forms a hexameric complex with a divalent transition metal cation. The second meaning of "complex" in the present invention is a bond between a complex-forming compound and a hexamer. A “complexing compound” is typically an organic compound that binds to hexamers in an insoluble composition, or stabilizes against dissolution by having various positive charges that form a complex. Is a molecule. Examples of stable complex-forming compounds in the present invention include protamine, sulfen, various globin proteins [Brange, J.,
Galenics of Insulin, Springer-Verlag, Berlin Heidelberg (1987)], and various polycationic polymer compounds known to form complexes with insulin.

The term “protamine” refers to a mixture of strongly basic proteins from fish sperm. The average molecular weight of the protein in protamine is about 4,200 [Hoffmann, JA et al., P.
rotein Expression and Purification, 1: 127-133 (1990)]. "Protamine"
It can represent a relatively salt-free preparation of the protein, often called "protamine base". Protamine also refers to a preparation consisting of a salt of the protein. Commercial preparations differ greatly in salt content.

Protamine is well known to those skilled in the insulin art and is currently included in the NPH insulin product. Pure fractions of protamine as well as protamine mixtures can be used in the present invention. However, commercial protamine preparations are typically not homogeneous with respect to the proteins present. Nevertheless, they are effective in the present invention. Protamine comprising a protamine base is effective in the present invention, as are protamine preparations comprising a salt of protamine and protamine preparations comprising a mixture of a protamine base and a protamine salt. Protamine sulfate is a frequently used protamine salt. All mass ratios for protamine are obtained for protamine free base. One skilled in the art can determine the amount of other protamine preparations that meet a particular mass ratio for protamine.

The term “suspension” refers to a mixture of solid and liquid phases consisting of insoluble or sparingly soluble particles larger than the colloid size. A mixture of NPH crystallites and an aqueous solvent forms a suspension. The term “suspension (suspension) formulation” refers to a pharmaceutical composition in which the active substance is present in a solid phase, which is finely dispersed in an aqueous solvent, for example, a microcrystalline solid, an amorphous precipitate, or both. . The finely dispersed solid is suspended completely gently throughout the aqueous solvent by gently agitating the mixture to provide a suspension with suitable uniformity for extracting the dosage volume. Examples of commercially available insulin suspension formulations include, for example, NPH, PZI
, And Ultralente. The low percentage of solid material in the microcrystalline suspension formulation may be amorphous. Preferably, the proportion of amorphous material is no more than 10% of the solid material in the microcrystalline suspension, most preferably no more than 1%. Similarly, the low percentage of solid material in the amorphous precipitate suspension may be microcrystalline.

“NPH insulin” refers to a “Neutral Protamine Hagedorn” formulation of insulin. Synonyms include human insulin NPH and insulin NPH, among many others. Humulins® N is a commercial formulation of NPH insulin. A related term is "NPL" for LysB28, ProB29-human insulin analog. The meaning of these terms, and how to make them, will be well known to those skilled in the art of insulin formulation.

The term “aqueous solvent” refers to an aqueous solvent containing water. The aqueous solvent system may consist only of water, may consist of water and one or more miscible solvents, and may contain solutes. More commonly used miscible solvents are short-chain organic alcohols such as methanol, ethanol, propanol, short-chain ketones such as acetone, and polyalcohols such as glycerol.

“Isotonic substances” are compounds that are physiologically acceptable and give the formulation adequate tonicity to prevent net flow of water across cell membranes in contact with the administered formulation. . Glycerol, also known as glycerin, is commonly used as an isotonic substance. Other isotonic substances include salts such as sodium chloride, and monosaccharides such as dextrose and lactose.

The crystals and co-crystals of the present invention include a hexamer stabilizing compound. The term “hexamer stabilizing compound” refers to a non-proteinaceous, low molecular weight compound that stabilizes a protein bound to a hexamer or a derivatized protein. Phenolic compounds, especially phenolic preservatives, are the best known stabilizing compounds for insulin and insulin derivatives. The hexamer stabilizing compound stabilizes the hexamer by binding to the hexamer by specific intermolecular contact. Examples of hexamer stabilizers include various phenolic compounds, phenolic preservatives, resorcinol, 4'-hydroxyacetanilide, 4-hydroxybenzamide, and 2,7-dihydroxynaphthalene. Multi-use formulations of the insoluble compositions of the present invention will include a preservative in addition to the hexamer stabilizing compound. The preservative used in the formulations of the present invention may be a phenolic preservative and may be the same or different from the hexamer stabilizing compound.

The term “preservative” refers to a compound that is added to a pharmaceutical formulation to act as an antimicrobial. Parenteral formulations must meet guidelines for the effectiveness of preservatives to be a commercially available multi-use product. Effective and acceptable preservatives known in the art for parenteral formulations include benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methyl paraben, chlorobutanol, o-cresol, p-cresol , Chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. For example, Wa
llhaeusser, K.-H., Develop.Biol.Standard, 24: 9-28 (1974) (S. Krager, Ba
sel).

The term “phenolic preservative” includes the compounds phenol, m-cresol, o-cresol, p-cresol, chlorocresol, methylparaben, and mixtures thereof. Certain phenolic preservatives, such as phenol and m-cresol, are known to bind to insulin-like molecules and induce structural changes that increase physical or chemical stability or both [ Birnbaum, DT,
et al., Pharmaceutical. Res. 14: 25-36 (1997); Rahuel-Clermont, S., et a.
l., Biochemistry 36: 5837-5845 (1997)].

The term “buffering agent” or “pharmaceutically acceptable buffer” is known to be safe for use in insulin formulations and has the effect of adjusting the pH of the formulation to the desired pH for the formulation. Represents a compound having The pH of the formulation of the present invention is from about 6.0 to about 8.0. Preferably, the formulations of the present invention have a pH of about 6.8 to about 7.8. pH from mild acidic pH to mild basic
Pharmaceutically acceptable buffers for adjusting pH include compounds such as phosphate, acetate, citrate, arginine, Tris, and histidine. "Tris" refers to 2-amino-2-hydroxymethyl-1,3, -propanediol and all pharmaceutically acceptable salts thereof. The free base and hydrochloric acid forms are the two common forms of Tris. Tris is also known in the art as trimethylolaminomethane, tromethamine, and tris (hydroxymethyl) aminomethane. Other pharmaceutically acceptable buffers suitable for adjusting the pH to the desired level are known to those of ordinary skill in the art.

The term “administering” means introducing a formulation of the present invention into a patient in need thereof to treat a disease or condition. The term "treating" refers to treating patients with diabetes or hyperglycemia or other conditions that require insulin administration to address and alleviate the symptoms or complications of the condition. management) and care.
Treating includes administering a formulation of the present invention to prevent the onset of symptoms or complications, reduce the symptoms or complications, or eliminate the disease, condition, or disorder.

The terms “MMAD” and “MMEAD” are well known in the art, and refer to “mass median aerodynamic diameter” and “mass median equivalent aerodynamic diameter,” respectively. Represents The terms are substantially equivalent. The "aerodynamic equivalent" size of a particle is the diameter of a unit density sphere that exhibits the same aerodynamic (aerodynamic) behavior as the particle, regardless of its actual density or shape. The MMAD is measured using a cascade impactor to determine the particle size as a function of the aerodynamic behavior of the particles in a high velocity airflow. The median (50%) particle size is obtained from a linear regression analysis of the cumulative distribution data. The term "VMSED" stands for "volume average spherical equivalent diameter". This term is well known in the field of particle sizing.

[0062] The crystals and co-crystals of the present invention have a rod-like or irregular morphology. Preferably, the crystals and co-crystals are acylated insulin or an acylated insulin analog, zinc ion (present at about 0.3 to about 0.7 mole per mole of total protein),
Sufficient to stabilize the total protein T3R3 or R6 hexamer structure, selected from the group consisting of phenol, m-cresol, o-cresol, p-cresol, chlorocresol, and mixtures thereof Phenolic preservatives, and protamine (present at about 0.15 to about 0.7 mg / 3.5 mg total protein).

[0063] A preferred group of insulin analogs for making derivatized insulin analogs for use in forming crystals and co-crystals are animal insulin, deletion analogs, and
Consists of a pI-shifted analog. A more preferred group consists of animal insulin, and deletion analogs. Deletion analogs are more preferred.

Another preferred group of insulin analogs for use in the crystals and co-crystals of the present invention consists of monomeric insulin analogs. The amino acid residue at position B28 is Asp, Lys, Leu, Val,
Or Ala, the amino acid residue at position B29 is Lys or Pro, the amino acid residue at position B10 is His or Asp, and the amino acid residue at position B1 is Phe, Asp, or B3 (alone or with deletion of the residue at position B2)
The amino acid residue at position 0 is Thr, Ala, Ser or is deleted, and the amino acid residue at position B9 is Ser or Asp (provided that either B28 or B29 is Lys) Monomeric insulin analogues are particularly preferred.

Another preferred group of insulin analogues for use in the present invention are those wherein the isoelectric point of the insulin analogue is from about 7.0 to about 8.0. These analogs are referred to as "pI-shifted insulin analogs." pI-shifted insulin analogues include, for example, ArgB31, Ar
gB32-human insulin, GlyA21, ArgB31, ArgB32-human insulin, ArgAO, ArgB31, A
rgB32-human insulin, and ArgAO, GlyA21, ArgB31, ArgB32-human insulin.

Another preferred group of insulin analogs is LysB28, ProB29-human insulin (where B28 is
Lys, B29 is Pro), AspB28-human insulin (B28 is Asp), AspB1
-Human insulin, ArgB31, ArgB32-Human insulin, ArgAO-Human insulin, Asp
B1, GluB13-human insulin, AlaB26-human insulin, GlyA21-human insulin,
It consists of death (ThrB30) -human insulin, and GlyA21, ArgB31, ArgB32-human insulin.

Particularly preferred insulin analogs include LysB28, ProB29-human insulin, Des (T
hrB30) -human insulin, AspB28-human insulin, and AlaB26-human insulin. Another particularly preferred insulin analog is GlyA21, ArgB31, ArgB32-
Human insulin [Doerschug, M., U.S. Patent No. 5,656,722, 1997, August 12,
Day]. The most preferred insulin analog is LysB28, ProB29-human insulin. Preferred derivatized proteins are acylated proteins, and preferred acylated proteins for the formulations and microcrystals of the invention are fatty acylated insulin and fatty acylated insulin analogs. Fatty acid-acylated human insulin is also highly preferred. Fatty acid-acylated insulin analogues are also highly preferred.

Certain groups used to derivatize insulin or insulin analogs (collectively proteins) do not significantly reduce the biological activity of the protein, are non-toxic when bound to the protein, and Most importantly, any chemical moiety that reduces the solubility of the zinc / protamine complex of the derivatized protein, reduces aqueous solubility, or increases lipophilicity.

One preferred group of acylating moieties consists of linear, saturated fatty acids.
This group includes methanic acid (C1), 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 (C10), n-undecanoic acid (Cll), n-dodecanoic acid (C12), n
-Consisting of tridecanoic acid (C13), n-tetradecanoic acid (C14), n-pentadecanoic acid (C15), n-hexadecanoic acid (C16), n-heptadecanoic acid (C17), and n-octadecanoic acid (C18). Adjective forms are formyl (C1), acetyl (C2), propionyl (C3), butyryl
(C4), pentanoyl (C5), hexanoyl (C6), heptanoyl (C7), octanoyl
(C8), nonanoyl (C9), decanoyl (C10), undecanoyl (Cll), dodecanoyl
(C12), tridecanoyl (C13), tetradecanoyl (C14) or myristoyl, pentadecanoyl (C15), hexadecanoyl (C16) or palmitic, heptadecanoyl (C17), and octadecanoyl (C18) or stearic. is there.

A preferred group of fatty acids for forming the fatty acid-acylated proteins used in the microcrystals of the present invention are fatty acids having an even number of carbon atoms, ie, C2, C4, C6, C8, C10, C12.
, C14, C16, and C18 saturated fatty acids. Another preferred group of fatty acids for forming the acylated proteins used in the microcrystals of the present invention are fatty acids having an odd number of carbon atoms, i.e., C1, C3, C5, C7, C9,
Consists of C11, C13, C15, and C17 saturated fatty acids.

Another preferred group of fatty acids for forming fatty acid-acylated proteins for use in the microcrystals of the present invention are fatty acids having more than 5 carbon atoms, ie, C6, C7, C8, C9
, C10, C11, C12, C13, C14, C15, C16, C17, and C18 saturated fatty acids. Another preferred group of fatty acids for forming fatty acid-acylated proteins for use in the microcrystals of the present invention are fatty acids having less than 9 carbon atoms, i.e., C1, C2, C3, C4.
, C5, C6, C7, and C8 saturated fatty acids. Another preferred group of fatty acids for forming fatty acid-acylated proteins for use in the microcrystals of the present invention consists of fatty acids having 6-8 carbon atoms, ie, C6, C7, and C8 saturated fatty acids.

Another preferred group of fatty acids for forming fatty acid-acylated proteins for use in the microcrystals of the present invention consists of fatty acids having 4 to 6 carbon atoms, ie, C4, C5, and C6 saturated fatty acids. Another preferred group of fatty acids for forming fatty acid-acylated proteins for use in the microcrystals of the present invention consists of fatty acids having 2 to 4 carbon atoms, ie, C2, C3, and C4 saturated fatty acids. Another preferred group of fatty acids for forming fatty acid-acylated proteins for use in the microcrystals of the present invention are fatty acids having less than 6 carbon atoms, i.e., C1, C2, C3, C4.
, And C5 saturated fatty acids.

Another preferred group of fatty acids for forming fatty acid-acylated proteins for use in the microcrystals of the present invention are fatty acids having less than 4 carbon atoms, ie, C1, C2, and
Consists of C3 saturated fatty acids. Another preferred group of fatty acids for forming fatty acid-acylated proteins for use in the microcrystals of the invention are fatty acids having more than 9 carbon atoms, ie, C10, Cll, C12.
, C13, C14, C15, C16, C17, and C18 saturated fatty acids. Another preferred group of fatty acids for forming fatty acid-acylated proteins for use in the microcrystals of the present invention is the even fatty acids having more than 9 carbon atoms, ie, C10, C12.
, C14, C16, and C18 saturated fatty acids.

Another preferred group of fatty acids for forming fatty acid-acylated proteins for use in the microcrystals of the present invention are fatty acids having 12, 14, or 16 carbon atoms, ie, C12, C14,
And C16 saturated fatty acids. Another preferred group of fatty acids for forming fatty acid-acylated proteins for use in the microcrystals of the present invention consists of C14 or C16 fatty acids, ie, C14 and C16 saturated fatty acids. C14 fatty acids are particularly preferred. C16 fatty acids 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 are fatty acids having 4 to 10 carbon atoms, ie, C4, C5, C6, C7, C8, C
Consists of 9, and C10 saturated fatty acids. Another preferred group of fatty acids for forming fatty acid-acylated proteins for use in the microcrystals of the present invention consists of even-numbered fatty acids having 4 to 10 carbon atoms, ie, C4, C6, C8, and C10 saturated fatty acids. Another preferred group of fatty acids for forming fatty acid-acylated proteins for use in the microcrystals of the present invention consists of fatty acids having 6, 8, or 10 carbon atoms. Particularly preferred are fatty acids having 6 carbon atoms. C8 fatty acids are also particularly preferred. Particularly preferred are fatty acids having 10 carbon atoms. Those skilled in the art will appreciate that the narrower preferred group is constituted by a combination of the preferred groups of the above fatty acids.

[0076] Another preferred group of acylating moieties consists of branched saturated fatty acids. Branched fatty acids have at least two branches. The length of the "branch" of a branched fatty acid could be expressed in terms of 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 with 5, 6, and 6 carbons in length. In this case, the “longest” branch has 6 carbon atoms. As another example, 2,3,4,5-tetraethyloctanoic acid has five branches with 4, 5, 6, 7, and 8 carbons in length. A preferred group of branched fatty acids are those with the longest branch having 3 to 10 carbon atoms.

[0077] Representative numbers of such branched saturated fatty acids are provided to ensure that the reader will understand the range of such fatty acids that may be used as the acylating moiety of the proteins of the present invention:
2-methyl-propionic acid, 2-methylbutyric acid, 3-methyl-butyric acid, 2
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 acid, 3,3-dimethyl -Butyric acid, 2-ethylbutyric 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 acid, 4-methyl-heptanoic acid, 5-methyl-heptanoic acid, 6
-Methylheptanoic acid, 2,2-dimethyl-hexanoic acid, 2,3-dimethylhexanoic acid, 2,4-
Dimethyl-hexanoic acid, 2,5-dimethylhexanoic acid, 3,3-dimethyl-hexanoic acid, 3
, 4-dimethylhexanoic acid, 3,5-dimethyl-hexanoic acid, 4,4-dimethyl-hexanoic acid, 2-ethyl-hexanoic acid, 3-ethyl-hexanoic acid, 4-ethyl-hexanoic acid, 2-propyl- Pentanoic acid, 2-ethyl-hexanoic acid, 3-ethyl-hexanoic acid, 4-ethyl-hexanoic 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-methyloctanoic acid, 2,2-dimethyl-heptanoic acid, 2,6-dimethyl-heptane Acid, 2-ethyl-2-methyl-hexanoic acid, 3-ethyl-5-methyl-hexanoic acid, 3- (1-propyl) -hexanoic acid, 2- (2-butyl) -pentanoic acid, 2- (2 -(2-methylpropyl)) pentanoic acid, 2-methyl-nonanoic acid, 8-methyl-nonanoic acid, 6-ethyl-octanoic acid, 4- (
1-propyl) -heptanoic acid, 5- (2-propyl) -heptanoic acid, 3-methyl-undecanoic acid, 2-pentyl-heptanoic acid, 2,3,4,5,6-pentamethyl-heptanoic 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, 2,3,4,5-tetraethyl-octanoic acid, 2-octyl-decanoic acid, and 2- (1-propyl) -3- (1-propyl) -4,5-diethyl-6-methyl-heptanoic acid.

Yet another preferred group of acylated moieties consists of 5-24 carbon cyclic alkyl acids wherein the cyclic alkyl moiety or moieties have 5-7 carbon atoms. Representative numbers of such cyclic alkyl acids are provided to assure the reader of the range of such acids that may be used as the acylating moiety of the proteins of the invention: cyclopentyl-formic acid, cyclohexyl-formic acid, 1 -Cyclopentyl-acetic acid, 2-
Cyclohexyl-acetic acid, 1,2-dicyclopentyl-acetic acid and the like.

A preferred group of derivatized proteins consists of monoacylated proteins. Most preferred is monoacylation of the ε-amino group. For insulin, monoacylation of LysB29 is preferred. Similarly, for certain insulin analogs, such as LysB28, ProB29-human insulin analog, monoacylation of the ε-amino group of LysB28 is most preferred. B
Monoacylation of the α-amino group of the chain (B1) is also preferred. Monoacylation of the α-amino group of the A chain (A1) is also preferred.

Another group of acylated proteins consists of diacylated proteins. The diacylation can be, for example, the ε-amino group of Lys and the α-amino group of the B chain, or the α-amino group of the A chain and the α-amino group of the B chain. Another group of acylated proteins consists of triacylated proteins. A triacylated protein is one in which the ε-amino group of Lys, the α-amino group of the B chain, and the α-amino group of the A chain are acylated. 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 diabetic or hyperglycemic patients. The formulations of the invention will typically provide a concentration of about 1 mg / mL to about 10 mg / mL of the derivatized protein. The insulin product formulations of the present invention typically have a concentration of units of insulin activity (units / mL), such as U40, U50, U100 (approximately about 1.4, 1.75, respectively).
, And 3.5 mg / mL preparation). Dose, route of administration,
And the number of doses per day may depend on the purpose of treatment, the nature and cause of the patient's disease, the gender and weight of the patient, exercise level, eating habits, dosing regimen, and other factors known to the skilled physician. The physician will decide upon consideration. Broadly, the daily dose is about
1 nmol / kg body weight to about 6 nmol / kg body weight (6 nmol is considered equal to about 1 insulin activity unit). A dose of about 2 to about 3 nmol / kg is typical for the present insulin therapy.

A physician treating diabetes will be able to select the most therapeutically advantageous method for administering the formulation of the present invention. Parenteral routes of administration are preferred. Typical parenteral routes of administration of insulin suspensions are subcutaneous and intramuscular. The compositions and formulations of the present invention could also be administered by the nasal, buccal, pulmonary, or ocular route. The pulmonary route is particularly advantageous in reducing pain and inconvenience. The crystals and co-crystals of the present invention are particularly well suited for pulmonary administration.

Glycerol at a concentration of 12 mg / mL to 25 mg / mL is preferred as the isotonic substance. It is even more highly preferred to use glycerol at a concentration of about 15 mg / mL to about 17 mg / mL for isotonicity. M-cresol and phenol, or mixtures thereof, are preferred preservatives in the formulations of the present invention.

Insulin or insulin analogs used to produce derivatized proteins can be prepared by any of a variety of known methods, including traditional (solution) methods, solid-phase methods, semi-synthetic methods, and more recently, recombinant DNA methods. It can be produced by peptide synthesis technology. For example, Ch. Discloses methods for making various proinsulins and insulin analogs.
ance, RE et al., U.S. Patent No. 5,514,646 (May 7, 1996); EPO Publication No. 383,47.
No. 2 (February 7, 1996); Brange, JJV et al., EPO Publication No. 214,826 (March 1987)
And Belagaje, RM et al., U.S. Patent No. 5,304,473 (April 19, 1994). These references form part of the present specification.

Generally, derivatized proteins are produced using methods known in the art. The above publications describing derivatized proteins include suitable methods for producing derivatized proteins. These publications form a part of the present specification as a method for producing a derivatized protein. To produce an acylated protein, the protein is reacted with an activated organic acid, for example, an acylated fatty acid. Activated fatty acids are derivatives of commonly used acylating substances, including activated esters of fatty acids, fatty acid halides, activated amides of fatty acids, such as activated azolide derivatives [H
ansen, LB, WIPO Publication No. 98/02460 (January 22, 1998)], and fatty acid anhydrides. Activated esters, especially N-hydroxysuccinimide esters of fatty acids, are a particularly advantageous method for acylating free amino acids with fatty acids. Lapidot et al. Reported the production of N-hydroxysuccinimide ester and N-lauroyl-
Glycine, N-lauroyl-L-serine, and its use in the production of N-lauroyl-L-glutamic acid are described. The term “activated fatty acid ester” means a fatty acid that has been activated using common techniques known in the art [Riordan, JF
And Vallee, BL, Methods in Enzymology, XXV: 494-499 (1972); Lapidot,
Y. et al., J. Lipid Res. 8: 142-145 (1967)]. Hydroxybenzotriazide (HOBT)
, N-hydroxysuccinimide and its derivatives are particularly well known for forming activated acids for peptide synthesis.

For selective acylation of the ε-amino group, various protecting groups could be used to block the α-amino group during coupling. The selection of a suitable protecting group is known to those skilled in the art and includes p-methoxybenzoxycarbonyl (pmz). Preferably, the ε-amino acids are acylated by a one-step synthesis without using an amino protecting group. A method for the selective acylation of Lys' Nε-amino group is disclosed and claimed in Baker, JC et al., US Pat. No. 5,646,242 (July 8, 1997), the contents of which are incorporated herein by reference. Make up). A method for producing a dry powder of an acylated protein
Baker, JC et al., U.S. Pat. No. 5,700,904, issued Dec. 23, 1997, which is hereby incorporated by reference.

The main role of the zinc according to the invention is that the protein and the derivatized protein Zn (II)
To facilitate the formation of the hexamers either separately as mixed hexamers or together as hybrid hexamers. Zinc promotes the formation of hexamers of insulin and insulin analogs. Similarly, zinc promotes the formation of hexamers of derivatized insulin and insulin analogs. Hexamer formation can be achieved by adjusting the pH of the solution containing the protein or derivatized protein, or both, to a neutral region in the presence of Zn (II) ions, or to adjusting the pH to a neutral region after the pH is adjusted to the neutral region. ) Is conveniently achieved.

For efficient recovery of crystals and co-crystals, limit the molar ratio of zinc to total protein to a lower limit of about 0.33 (ie, about 2% zinc required for efficient hexamerization).
Atom / hexamer). In the absence of compounds that compete with insulin when binding to zinc, crystals and co-crystals are suitably formed when about 2 to about 4-6 zinc atoms are present. If there are compounds that compete with insulin when binding to zinc, such as compounds containing citric acid or phosphoric acid, even more zinc may be used during the treatment. For more potent hexamerization, an excess of zinc over the minimum required for efficient hexamerization may be desirable. Also, an excess of zinc over a minimal amount can be present in the formulations of the present invention and is desirable to improve chemical and physical stability, improve suspension, and possibly further extend time-effects May. Thus, the range of acceptable zinc: protein ratios in the insoluble compositions, methods, and formulations of the present invention is fairly broad.

According to the present invention, zinc is present in the formulation from about 0.3 mol to about 7 mol / mol total protein, more preferably, from about 0.3 mol to about 1.0 mol / mol total protein. Even more preferably, the zinc / derivatized protein ratio is from about 0.3 to about 0.7 mol / mol total protein of zinc atoms. Most preferably, the zinc / derivatized protein ratio is from about 0.30 to about 0.55 mol / mol total protein of zinc atom. In the same high zinc formulation as the PZI preparation, the zinc ratio is from about 5 to about 7 mol zinc / mol total protein.

The zinc compound that provides zinc for the present invention can be any pharmaceutically acceptable zinc compound. Adding zinc to insulin preparations is known in the art, as are sources of pharmaceutically acceptable zinc. Preferred zinc compounds that supply zinc for the present invention include zinc chloride, zinc acetate, zinc citrate, zinc oxide, and zinc nitrate.

The microcrystals and precipitates of the present invention require a complex-forming compound. The complex-forming compound must be present in an amount sufficient to cause substantial precipitation and crystallization of the hexamer. Such an amount of a particular complex forming compound for a particular preparation can be readily determined by simple titration experiments. Ideally, the concentration of the complex forming compound is adjusted so that only a small amount of the complex forming agent remains in the soluble phase after precipitation and crystallization are complete. This requires mixing the complexing agent based on the experimentally determined "isophene" ratio. This ratio is expected to be very similar to that of NPH and NPL. However, derivatization may be slightly different as it affects the nature of the protein-protamine interaction.

When protamine is the complex forming compound, protamine is present in the crystals and co-crystals in an amount from about 0.15 mg to about 0.5 mg / 3.5 mg total protein. The protamine / total protein ratio is preferably between about 0.25 and about 0.40 (mg / 3.5 mg). More preferably, the ratio is from about 0.25 to about 0.38 (mg / 3.5 mg). Preferably, the amount of protamine is
0.05 mg to about 0.2 mg / mg total protein, more preferably about 0.05 to about 0.15 mg / mg total protein. Protamine sulfate is a more preferred salt of protamine used in the present invention. If protamine sulfate or other protamine salts are used, the amount used will need to be adjusted for the amount of protamine free base and used in the same application at a rate equal to the ratio of the molecular weight of the salt to protamine.

[0093] In order to further extend the chronological action of the composition of the invention or to improve its suspendability, additional protamine and zinc may be added after crystallization. That is, formulations having protamine higher than the isophane ratio are also within the scope of the invention. In these formulations, the protamine ratio is 0.25 mg protamine to about 0.5 mg / mg total protein.

A required component of the crystals and co-crystals of the present invention is a hexamer-stabilizing compound. The structures of the three hexameric conformations have been characterized in the literature and include T6, T3R3, and
Represented by R6. In the presence of a hexamer-stabilizing compound, such as various phenolic compounds, the R6 conformation is stabilized. Thus, the hexamer is likely to be in the R6 or T3R3 conformation in crystals and co-crystals formed in the presence of a hexamer stabilizing compound, such as phenol or m-cresol. A wide range of hexamer-stabilizing compounds are suitable. The compound must be present in a sufficient ratio to total protein to stabilize the R6 hexamer conformation. To achieve this, at least 3 moles of hexamer stabilizing compound per hexamer is required to effectively stabilize the hexamer. The crystals and co-crystals of the present invention preferably contain at least 3 moles of a hexamer stabilizing compound per hexamer. The presence of higher ratios of hexamer stabilizing compounds of at least 25-50 fold in the solution producing the microcrystals and precipitates will not adversely affect hexamer stabilization.

[0095] Preservatives may be present in the formulations of the present invention, particularly if the formulation is intended to be sampled multiple times. As noted above, a wide variety of suitable preservatives are known. Preferably, the preservative is present in the solution in an appropriate amount to provide an antimicrobial effect sufficient to meet the requirements of the Pharmacopoeia.

Preferred preservatives are the phenolic preservatives listed above. The preferred concentration of the phenolic preservative is from about 2 mg to about 5 mg / ml of the aqueous suspension formulation. These concentrations represent the total amount of phenolic preservative, as a mixture of individual phenolic preservatives is expected. Suitable phenolic preservatives include, for example, phenol, m-cresol, and methyl paraben. Preferred phenolic preservatives are phenol and m-cresol. Mixtures of phenolic compounds such as phenol and m-cresol are also envisioned 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 m / mL
g / mL phenol and 1.8 mg / mL m-cresol.

The crystals and co-crystals of the present invention have an oblong shape, also known as a “rod”, containing a complex-forming compound and a hexamer-stabilizing compound consisting of a derivatized protein, a divalent metal cation. It is preferably a single crystal. A preferred composition comprises about 3 mg to about 6 mg / 35 mg total protein of protamine sulfate, and about 0.1 to about 0.4 mg / 35 mg total zinc. Another preferred composition comprises about 10 mg to about 17 mg / 35 mg protamine sulfate total protein, and about 2.0 to about 2.5 mg / 35 mg zinc.
Includes total protein. Another preferred composition is protamine sulfate 0.34 to 0.3 per mL.
Contains 8 mg, zinc 0.01-0.04 mg, and 3.2-3.8 mg total protein.

[0098] Both underivatized and derivatized proteins are required for the co-crystals of the present invention. The ratio between the total amounts of these proteins determines the degree of time extension of the preparation. Preferred ratio of moles of derivatized protein to moles of derivatized protein (
The ratio of the number of moles of the protein to the number of moles of the derivatized protein) is about 1: 100 and about 1
00: 1. A more preferred ratio of moles of derivatized protein to moles of derivatized protein is from about 1: 1 to about 100: 1. Another preferred ratio of moles of derivatized protein to moles of derivatized protein is from about 1: 1 to about 20: 1. Yet another preferred ratio of moles of derivatized protein to moles of derivatized protein is about
2: 1 to about 20: 1, about 2: 1 to about 10: 1, about 2: 1 to about 5: 1, about 3: 1 to about 5: 1, about 1: 1 to about 1:20,
About 1: 1 to about 1:10, about 1: 2 to about 1:20, about 1: 2 to about 1:10, about 1: 2 to about 1: 5, about 1: 3 to about 1: 5
, About 10: 1 to about 1:10, about 9: 1 to about 1: 9, about 5: 1 to about 1: 5, about 3: 1 to about 1: 3.

The present invention provides a method for producing crystals and co-crystals. Briefly, a suitable method generally involves one step of the following procedure: solubilization (when starting with a dry substance), hexamerization, homogenization, complex formation, precipitation, crystallization, Formulation as desired; or solubilization (when starting with a dry substance), homogenization, hexamerization, complex formation, precipitation, crystallization, and, optionally, formulation.

[0100] Solubilization means that the derivatized proteins and proteins dissolve them sufficiently to form hexamers. Hexamerization refers to the process by which proteins and derivatized protein molecules combine with zinc (II) atoms to form hexamers.
Complex formation means forming an insoluble complex between the hexamer and protamine. For crystallization, precipitated hexamer / protaminecon crystals,
Typically involves conversion to rods.

[0101] Solubilization is achieved by dissolving the derivatized protein and the protein in an aqueous solvent. The aqueous protein can be, for example, an acidic, neutral, or basic solution. Aqueous solvents may include, in part, miscible organic solvents such as ethanol, acetonitrile, dimethylsulfoxide, and the like. The acidic solvent may be, for example, an HCl solution, conveniently from about 0.01 N HCl to about 1.0 N HCl. Other pharmaceutically acceptable acids may also be used. The basic solution is, for example, a NaOH solution, conveniently about 0.01N.
NaOH to about 1.0 N NaOH or higher. Other pharmaceutically acceptable bases may also be used. For protein stability, it is preferred that the acid or base concentration be as low as possible while remaining effective to properly dissolve the protein and derivatized protein.

[0102] Most proteins (insulin and insulin analogs) and many derivatized proteins may be dissolved at an appropriate concentration at neutral pH. Solutions for dissolving derivatized proteins at neutral pH may include buffers and, optionally, one or more additional solutes, such as salts, phenolic compounds, zinc, and isotonic agents.

If hexamerization occurs before homogenization, two populations of homogeneous hexamers are formed first, and then the populations are mixed to form a mixed hexamer. If homogenization occurs first, the hexamer will yield a hybrid hexamer. To produce an insoluble composition comprising a hybrid hexamer as described above, proteins and derivatized proteins are separated into monomer or dimer aggregation stages before hexamerizing with divalent metal cations. Homogenize under preferred conditions. To achieve the required separation, the protein and the derivatized protein may be mixed under strongly acidic or strongly alkaline conditions. The degree of separation, and thus homogenization, is affected by the solution conditions chosen for this step. Insulin and related proteins readily self-associate in a series of reactions that produce dimers, hexamers, and other conjugates. The distribution of these bound forms at equilibrium depends on many parameters, including pH. These coupling reactions are usually predominantly monomer-dimer-
It is thought to contain hexameric assemblies. Therefore, depending on the solution conditions chosen, mixing of the monomers, dimers, or mixtures thereof should be achieved by homogenization. For example, homogenization in 1N HCl is 0.1N HCl
The fraction of monomer blend may be higher than in the middle (possibly containing more dimer blend). A homogenization step is effective for producing a composition containing a hybrid hexamer, but the homogenization conditions used may result in very little fractionation of a homogeneous hexamer of the protein or derivatized protein. Or there are very few.

A composition consisting of a mixed hexamer contains mainly two types of hexamers, a protein hexamer and a derivatized protein hexamer. In this case, the homogenization step occurs after the hexamerization step and homogenization of the hexamer is achieved before complex formation with the complex-forming compound. Thus, the homogenization step is performed under solution conditions that stabilize the Zn (II) -insulin hexamer. Solution conditions for stabilizing insulin hexamers are well known in the literature.

The solution conditions required for hexamerization are conditions for forming a hybrid hexamer or a mixed hexamer in a solution. This condition may be the same as or very similar to the condition that causes the insulin or insulin analog to hexamerize. Typically, hexamerization requires zinc and a neutral to slightly basic pH (about pH 6.8 to about 8.4). The presence of a hexamer stabilizer favorably affects hexamers by promoting the derivatized protein (and in some cases also the protein) R6 or T3R3 conformation. For certain monomeric insulin analogs, hexamer stabilizing compounds are required for hexamer formation.

For compositions consisting of hybrid hexamers, seven hexamers: P ₆ , P ₅ D ₁ , P ₄ D ₂ , P ₃ D ₃ , P ₂ D ₄ , P ₁ D ₅ , and D ₆ is expected (wherein, P is a protein monomer, D is represents the derivatized protein monomer). The statistical distribution of the hexamer is expected to fit the Poisson distribution and will be influenced by the relative ratio of protein and derivatized protein, and the degree of separation (dissociation) before hexamerization. For example, the four major hybrid hexamers from a homogenized solution composed primarily of dimers are: P ₆ ,
_{P 4 D 2, P 2 D} 4, and is expected to D _6. For compositions consisting of mixed hexamers, only two hexamers P6 and D6 are expected to predominate.

The complex formation step must involve mixing the complex forming compound and the hexamer under solution conditions in which each is initially soluble. This can be done by mixing separate solutions of hexamer and protamine, or by first adding an acidic or basic pH
Form a solution of protein, derivatized protein, and protamine at
This could be achieved by changing the pH to the neutral range.

Solution conditions during crystallization must stabilize what crystallizes and convert the precipitate to a solute to facilitate crystallization. That is, solution conditions will determine the rate and result of crystallization. Crystallization appears to involve a complex equilibrium involving amorphous precipitates, dissolved hexamer-protamine complex, and crystals. To obtain microcrystals, the conditions chosen for crystallization must shift the equilibrium towards crystal formation. In addition, the solubility of the derivatized protein greatly affects the rate and size of the crystallization, since the assumed equilibrium suggests that low solubility results in a slow net conversion of the precipitate to solutes and crystals. It is expected to do. Further, when the crystallization speed is reduced, the crystal often becomes larger. That is, it is considered that the crystallization rate and the crystal size depend on the size and properties of the derivatized portion of the derivatized protein.

Crystallization parameters previously thought to affect the crystallization rate and crystal size of the present invention (see Brader I and Brader II) include the size and nature of the acyl group,
"Competition compounds" that compete with proteins and derivatized proteins for temperature, zinc, such as the presence and concentration of citrate, phosphate, etc., the nature and concentration of phenolic compounds, zinc concentration, the presence and concentration of miscible organic solvents. , Time allowed for crystallization, pH
Ionic strength, buffer identity and concentration, precipitant concentration, seed material presence, vessel shape and material, agitation speed, and total protein concentration. The concentration and temperature of the competitor compounds were considered particularly important.

Quite surprisingly, it has now been found that chloride ions, apart from their effect on ionic strength, play an important role in determining the size of crystals and co-crystals. The exact nature of the effect of chlorine on crystals and co-crystals is not known. No specific link between chloride ion concentration and crystallization rate has been previously described for these crystals and co-crystals.

A competing compound, such as citrate, could affect the rate of crystal formation, and indirectly, the size and quality of the crystal. These compounds could form a coordination complex with zinc in solution and affect by competing with zinc for relatively weak zinc binding sites on the hexamer surface. Occupation of this weak surface binding site probably prevents crystallization. Furthermore, many derivatized proteins are only partially insoluble in the presence of only 0.333 zinc / mole derivatized protein, and the presence of competing compounds restores solubility and leads to crystallization. The optimal concentration of the competitor compound can be determined using conventional techniques for any combination of protein and derivatized protein. Of course, the upper limit is the concentration at which zinc is precipitated by the competitor compound, or the concentration at which residual competitor compound is not pharmaceutically acceptable, causing pain or irritation at the site of administration.

Examples of the method for producing the precipitates and crystals of the present invention are shown below. The measured amount of the derivatized protein and the measured amount of the protein are dissolved in a suitable solvent containing the hexamer-stabilizing compound, eg, a phenolic compound, or mixed to form a solution in the solvent. To this solution, one of the soluble salt (e.g. Zn (II) Cl ₂₎ zinc solution as added, zinc about 0.3 mol / mol derivatized insulin-zinc about 0.7 mole or about 1.0 mole / mole of total protein (protein + Derivatized protein). If desired, anhydrous ethanol or another miscible organic solvent may be added to the solution in an amount that results in a solution of about 5% to about 10% (by volume) of the organic solvent. This solution may then be filtered through a 0.22 micron low protein binding filter. The protamine solution is prepared by dissolving the measured amount of protamine in an aqueous solvent. This solution may be filtered through a 0.22 micron protein low binding filter. The solution of the protein and the derivatized protein is mixed with the protamine solution, where a precipitate first forms. The resulting suspension is stirred slowly at room temperature (typically about 20-25 ° C.) and then for about 4 hours to about
Microcrystals form within a period of 10 days.

Next, the microcrystals are separated from the mother solution and placed in a different solvent for storage and administration to the patient. Examples of suitable aqueous solvents are: water for injection containing 25 mM Tris, 5 mg / mL phenol, and 16 mg / mL glycerol, 2 mg / mL sodium diphosphate, 1.6 mg / mL m-cresol, 0.65 mg / mL phenol, and water for injection containing 16 mg / mL glycerol, and 25 mM Tris, 5 mg / mL phenol, 0.1 M citric acid 3
Water for injection containing sodium and 16 mg / mL glycerol.

In another method of preparing the insoluble composition of the present invention, for example, a measured amount of dry derivatized protein and a measured amount of dry protein are combined together in an acidic aqueous solvent, such as
Dissolve in 0.1N-1.0N HCl. The solution is agitated to ensure thorough mixing of the derivatized protein and protein. By predefining the ratio of derivatized protein powder to protein powder in this mixture, a similar ratio of derivatized protein to protein in the insoluble composition to be produced is obtained. A separately prepared aqueous solution of a phenolic preservative and, optionally, a pharmaceutically acceptable buffer is mixed with an acidic solution of the protein. Next, the pH of the resulting solution was adjusted to about 6.8.
To about 8.4, preferably about 6.8 to about 8.0, or pH about 7.2 to about 7.8, and most preferably about 7.4 to about 7.8. This solution contains the soluble salt (eg, Zn (II) Cl ₂ )
A solution of zinc, one of
Mol / mol total insulin. The solution may be adjusted to the above pH, preferably about 7.4 to 7.6, and then filtered through a 0.22 micron protein low binding filter. A protamine solution is prepared by dissolving the measured amount of protamine in an aqueous solvent. The protamine solution may be filtered through a 0.22 micron low protein binding filter. The solution of the protein and the derivatized protein is mixed, where a precipitate first forms. The resulting suspension is stirred slowly at room temperature (typically about 20-25 ° C.) and then
Microcrystals form within a period of about 4 hours to about 10 days.

In another method of making the insoluble composition of the present invention, an initially measured amount of the derivatized protein is dissolved in an aqueous solvent containing a phenolic preservative. To this solution was added which is one zinc solution of the soluble salts (for example Zn (II) Cl _2), and zinc about 0.3 mol / mol derivatized protein-zinc about 4 mol / mol-derivatized protein. The pH of the resulting solution is about 6.8 to about 8.4, preferably about 6.8 to about 8.0, or pH about 7.2 to about 7.8,
And most preferably from about 7.4 to about 7.8. A second solution is separately prepared by dissolving a measured amount of a protein selected from the group consisting of insulin, insulin analogs, and proinsulin in an aqueous solvent containing a phenolic preservative. To this solution is added a solution of zinc, one of its soluble salts (eg, Zn (II) Cl ₂ ) to make about 0.3 mol / mol protein zinc to about 4 mol / mol zinc. Then
The pH of the resulting solution is about 6.8 to about 8.4, preferably about 6.8 to about 8.0, or preferably
Adjust the pH to about 7.2 to about 7.8, and most preferably about 7.4 to about 7.8 or 7.4 to 7.6. The derivatized protein solution and portions of the protein solution are mixed in a predefined ratio to achieve a similar derivatized protein / protein ratio as the insoluble composition. The solution is agitated to ensure thorough mixing of the derivatized protein solution with the protein. The solution may then be adjusted to a pH of about 7.6 and then filtered through a 0.22 micron low protein binding filter. The measured amount of protamine may be dissolved in an aqueous solvent to prepare a protamine solution. The protamine solution may be filtered through a 0.22 micron low protein binding filter. The solution of the protein and the derivatized protein is mixed, where a precipitate first forms. The resulting suspension is stirred slowly at room temperature (typically about 20-25 ° C.), then microcrystals form within a period of about 4 hours to about 10 days.

While not exhaustively describing the vast number of types of methods for producing the insoluble compositions of the present invention, the following are additional methods of the present invention: protein, derivatized protein, hexamer stable Dissolving the derivatized compound and the divalent metal cation in an aqueous solvent having a pH that allows hexamer formation, and then adding the complex-forming compound; a protein, a derivatized protein, a hexamer-stabilizing compound, and a divalent metal The cation is dissolved in an aqueous solvent having a pH that does not allow the formation of hexamers and the pH is adjusted to about 6.8 to about 7
.8, and then add the complex forming compound. The protein, hexamer stabilizing compound, and divalent metal cation are dissolved in an aqueous solvent having a pH that allows hexamer formation, and the derivatized protein, hexamer The dimer stabilizing compound, and the divalent metal cation are separately dissolved in an aqueous solvent having a pH that allows hexamer formation, the two solutions are mixed thoroughly, and then the complex-forming compound is added. The hexamer stabilizing compound, divalent metal cation, and complex forming compound are dissolved in an aqueous solvent (the resulting solution has a pH that does not cause precipitation), and the derivatized protein, hexamer stabilizing compound, divalent The metal cation and the complex forming compound are separately dissolved in an aqueous solvent (the resulting solution is at a pH that does not precipitate) and the two solutions are mixed thoroughly. Adjusted to pH value occurs precipitation of the solution, protein, derivatized protein, a hexamer stabilizing compound, a divalent metal cation,
And dissolving the complex-forming compound in an aqueous solvent (the resulting solution is at a pH that does not precipitate) and adjusting the pH of the solution to a value that causes precipitation, proteins, derivatized proteins, hexamer-stabilizing compounds, And the divalent metal cations are dissolved in an aqueous solvent (the resulting solution has a pH that does not precipitate when the complexing agent is added), and the complexing compound is added, and the pH of the solution is adjusted to a value that causes precipitation. Dissolve the protein, hexamer-stabilizing compound, and divalent metal cation in an aqueous solvent (the resulting solution should not precipitate when the complex-forming compound is added).
), Separately dissolving the derivatized protein, hexamer-stabilizing compound, and divalent metal cation in an aqueous solvent (the resulting solution is at a pH that does not precipitate when the complex-forming compound is added) ), Add the complex-forming compound to the solution,
Dissolve the protein, derivatized protein, hexamer stabilizing compound, and divalent metal cation in an aqueous solvent by adjusting the pH of the solution to a value that causes precipitation (the resulting solution is PH that does not cause precipitation), adjust the pH of the solution to a value at which precipitation occurs when the complex-forming compound is added, and then add the complex-forming compound to the solution; protein, hexamer-stabilizing compound, and Dissolve the divalent metal cation in an aqueous solvent (the resulting solution has a pH that does not precipitate when the complex-forming compound is added)
), Separately dissolving the derivatized protein, hexamer-stabilizing compound, and divalent metal cation in an aqueous solvent (the resulting solution is at a pH that does not precipitate when the complex-forming compound is added) ), Mix these two solutions thoroughly and mix
The pH is adjusted to a value at which precipitation occurs when the complex forming compound is added, and then the complex forming compound is added to the solution.

[0117] In a preferred embodiment, the microcrystals are produced in advance by a method that does not require the separation of the microcrystals from the mother liquor. That is, it is preferred that the mother liquor is suitable for administration to a patient by itself, or that the mother liquor can be made suitable for administration by diluting it with a suitable diluent. The term diluent refers to a solution consisting of an aqueous solution in which various pharmaceutically acceptable excipients are dissolved, including but not limited to buffers, isotonic agents, zinc, preservatives, protamine, and the like. means.

Protein, derivatized protein, divalent ion, complex forming compound,
Pharmaceutical compositions suitable for parenteral administration in accordance with the present invention, in addition to the hexamer stabilizing compound, and excipients, carriers, such as water-miscible organic solvents such as glycerol, sesame oil, aqueous propylene glycol, and the like. May be used. When present, such materials are typically used in amounts up to about 2.0% by weight of the final formulation. For additional information on various techniques using conventional excipients or carriers for parenteral products, see Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing.
See Company, Easton, PA, USA (1985), which is incorporated herein by reference.

In the general practice of the invention, it is also envisioned that the formulation may comprise a mixture of soluble fractions and microcrystals of a protein selected from insulin, derivatized insulin, insulin derivatives, and derivatized insulin analogs. Is done. Examples of such pharmaceutical compositions include sterile, pyrogen-free, insulin, insulin analogs, derivatized insulin, or derivatized insulin analogs buffered with a pharmaceutically acceptable buffer. Isotonic aqueous saline solutions are included. For the soluble phase, insulin or an acutely acting insulin analogue, such as LysB28, ProB29-human insulin or AspB28-human insulin, is preferred. Such a mixture is designed to provide a combination of prandial regulation of blood glucose provided by soluble insulin and basal regulation of blood glucose provided by insoluble insulin. The ratio of total protein in the insoluble phase (protein + derivatized protein) and total protein in the soluble phase ranges from about 9: 1 to about 1: 9. A preferred range for this ratio is from about 9: 1 to
It is about 1: 1, more preferably about 7: 3. Other ratios are 1: 1 and 3: 7.

An effective amount of a crystal or co-crystal for inhalation will require from about 0.5 μg / kg to about 200 μg / kg total protein. Preferred doses are from about 5 μg / kg to about 100 μg / kg, from about 10 μg / kg to
About 100 μg / kg, about 20 μg / kg to about 100 μg / kg, or about 30 μg / kg to about 100 μg / kg. More preferably, the dose is about 10 μg / kg to about 60 μg / kg, 20 μg / kg to about 60 μg / kg
Or 30 μg / kg to about 60 μg / kg. A therapeutically effective amount is the level of insulin protein, the physical condition of the patient, the condition of the patient's lungs, the efficacy and bioavailability of the protein, the total protein administered with another insulin, such as fast acting or prandial insulin or another therapeutic agent. A skilled physician can decide whether to do so or to consider factors, including other factors known to the physician. Effective initiation therapy includes "titration" of the patient, i.e., starting with a low dose, monitoring blood glucose levels, and increasing the dose required to achieve the desired blood glucose level.

According to the present invention, crystals and co-crystals are supplied by inhalation to achieve a favorable slow uptake of insulin protein compared to both inhalation of underivatized insulin protein and inhalation of soluble derivatized protein. Is done. Inhaled administration results in pharmacokinetics comparable to subcutaneous administration of crystalline insulin.

According to the present invention, crystals and co-crystals are supplied by any of a variety of inhalation devices and methods known in the art for administering insulin or other proteins by inhalation [Rubsamen, US Patent No. 5,364,838 (issued Nov. 15, 1994); Rubsamen, US Pat. No. 5,672,581 (issued September 30, 1997); Platz et al., WIPO
Publication No. W096 / 32149 (published October 17, 1996); Patton et al., WIPO Publication No.
No. 95/24183 (published Sep. 14, 1995); Johnson et al., US Pat. No. 5,654,007 (1997
Goodman et al., US Patent No. 5,404,871 (issued April 11, 1995); R
Ubsamen et al., US Patent No. 5,672,581 (issued September 30, 1997); Gonda et al., US Patent No. 5,743,250 (issued April 28, 1998); Rubsamen, US Patent No. 5,419,315 (199)
Rubsamen et al., US Patent No. 5,558,085 (issued September 24, 1996).
Gonda et al., WIPO Publication No.W098 / 33480 (published August 6, 1998); Rubsamen, U.S. Patent No. 5,364,838 (issued November 15, 1994); Laube et al., U.S. Patent No. 5,320,094 (1994 Issued June 14, Eljamal et al., US Pat. No. 5,780,014 (issued July 14, 1998); Backstrom et al., US Pat. No. 5,658,878 (issued August 19, 1997); Backstrom et al., US Pat. No. 5,518,998. (Issued May 21, 1996); Backstrom et al., 5,506,203 (19
Meezan et al., U.S. Patent No. 5,661,130 (issued August 26, 1997);
Hodson et al., US Pat. No. 5,655,523 (issued Aug. 12, 1997); Schultz et al., US Pat. No. 5,645,051 (issued Jul. 8, 1997); Eisele et al., US Pat. No. 5,622,166 (1997)
Mecikalski et al., US Pat. No. 5,577,497 (November 26, 1996); M.
ecikalski et al., U.S. Patent No. 5,492,112 (issued February 20, 1996); Williams et al., U.S. Patent No. 5,327,883 (issued July 12, 1994); Williams, U.S. Patent No. 5,277,195.
(Issued January 11, 1994)].

Devices used to administer crystals and co-crystals according to the present invention include those well known in the art, such as metered dose inhalers, liquid nebulizers, dry powder inhalers, nebulizers, hot vaporizers and the like. And AERx® pulmonary drug delivery system developed by Aradigm Corporation, dry powder formulation and delivery tool developed by Inhale Therapeutic Systems, Inc., and Sp developed by Dura Pharmaceuticals, Inc.
Devices provided by the developed technology including the iros® dry powder inhaler system are included. Other suitable techniques include an electrohydrodynamic aerosolizer. The inhalation device may be, for example, about 10 μm MMAD or less, preferably about 1 to 5 μm MMAD, more preferably about 1 to about 3 μm MMAD, and most preferably about
Small particles in the range of 2 to about 3 μm MMAD should be provided. In addition, the inhalation device is small enough to be easy to use and convenient to carry,
It must be practical in that it can provide multiple doses and is durable. Specific examples of commercially available inhalation devices suitable for practicing the present invention include Turbohaler (Astra), Rotahal
er (Glaxo), Diskus (Glaxo), Ultravent nebulizer (Mallinckrodt), Acorn II
Nebulizer (Marquest Medical Products), Ventolin metered dose inhaler (Glaxo), Sp
and inhaler powder inhalers (Fisons). Insulin and fatty acid-acylated insulin may conveniently be supplied by a dry powder inhaler or nebulizer. Dry powder inhalers for administering crystals and co-crystals have several desirable features. For example, delivery by such inhalation devices is conveniently reliable, reproducible and accurate.

As the skilled artisan will appreciate, the nature and amount of the pharmaceutical composition, and the duration of a single dose administration, will depend on the type of inhalation device used. For some aerosol delivery systems, such as nebulizers, the frequency of administration and the length of time the system operates will depend primarily on the concentration of crystals and co-crystals in the aerosol.
For example, those with a short administration time can be used with a high concentration of crystals and co-crystals in the nebulizer solution. Devices such as metered dose inhalers can produce high concentrations of aerosols and can be operated to deliver a desired amount of crystals or co-crystals in a short time. Devices such as dry powder inhalers deliver the active substance until a substance with a given medicinal effect is released from the device. In this type of inhaler, the amount of crystals and co-crystals in a given amount of powder determines the dose delivered in a single dose.

[0125] The particle size of the crystals and co-crystals supplied by the inhalation device determines the extent to which the particles are carried to the lower respiratory tract or alveoli, which are most favorable for accumulation due to their large surface area. Preferably at least about 10% of the crystals and co-crystals, preferably about 10%
~ 20% or more accumulate in the lower lung. It is known that the maximum efficiency of lung accumulation in humans breathing in the mouth is obtained with about 2 μm to about 3 μm MMAD. Above about 5 μm MMAD, lung accumulation is substantially reduced. Below about 1 μm MMAD, lung accumulation is reduced, making it difficult to provide a sufficient amount of particles to be therapeutically effective. Preferably, the particle size of the crystals and co-crystals is about 10 μm or less, preferably in the range of about 1 μm to about 5 μm MMAD, more preferably in the range of about 1 to about 3 μm MMAD, and most preferably in the range of about 2 to about 3 μm MMAD.
AD.

For the production of dry powders, methods such as scraper blades or air blasting are typically used to produce particles from solid formulations of fatty acylated insulin proteins. Generally, the particles are generated in a container and then transported into the patient's lungs via a carrier airflow. Typically, in current dry powder inhalers, the power to terminate solids and airflow is provided solely by the patient's inhalation. One suitable dry powder inhaler is the Turbohaler manufactured by Astra.

Delivery of the crystals and co-crystals of the invention by inhalation may be through an inhalation device, such as, but not limited to, a jet nebulizer, a dry powder inhaler, an ultrasonic nebulizer, a piston pump, or a voltage nebulizer. Is achieved by
Liquid solutions for nebulizers may include substances such as, but not limited to, buffers, preservatives, or surfactants. The dry powder formulation may include, but is not limited to, sucrose, lactose, dextrose, mannitol,
Spray-dried powders from solutions of sugars or polyols such as trehalose, starch are included.

Typically, crystalline and co-crystalline formulations for administration from a dry powder inhaler include:
Preferably, it includes fine dry powders of crystals and co-crystals produced without using grinding or other mechanical operations. The powder may also include underivatized insulin or insulin analogs, fillers, buffers, carriers, excipients, other additives, etc., that provide a relatively rapid onset and short duration of action. it can. Crystalline and co-crystalline dry powder formulations provide the necessary dilution of the powder to be delivered from a particular powder inhaler, which facilitates the processing of the formulation, provides favorable powder properties to the formulation, and reduces the amount of powder from the inhalation device. Additives may be included to facilitate dispersion, stabilize the formulation (eg, antioxidants or buffers), or flavor the formulation.

Advantageously, the additives do not adversely affect the patient's respiratory tract. The crystals and co-crystals can be mixed with an additive, and the solid formulation can be coated with particles of the additive or comprise particles of the crystals and co-crystals mixed with the particles. Typical additives include monosaccharides, disaccharides, and polysaccharides, sugar alcohols, and other polyols, such as lactose, glucose, raffinose, melezitose, lactitol,
Includes maltitol, trehalose, sucrose, mannitol, starch, or combinations thereof, surfactants such as sorbitol, diphosphatidylcholine, or lecithin. Typical additives, such as fillers, are present in an amount effective for the purpose, often from about 50% to about 90% by weight of the formulation. Additives known in the art for protein formulations can also be included in the formulation. See, for example, Japanese Patent Application No. J04041421 (published February 12, 1992, Taisho Pharmaceutical).

Conveniently, for administration as a dry powder, the crystals or co-crystals have an NMAD of about 10 μm or less, preferably about 1 μm to about 5 μm MMAD, more preferably about 1 to about 3 μm MMAD.
, Most preferably about 2 to about 3 μm MMAD. The preferred particle size is effective to efficiently supply and accumulate in the alveoli of the patient's lungs. Preferably, the dry powder consists for the most part of particles that have been produced such that the majority of particles have a desired range of sizes.

A spray containing crystals or co-crystals can be produced by forcing the crystals or co-crystals through a nozzle under pressure. Nozzle size and configuration, applied pressure, and liquid feed rate can be selected to achieve the desired output and particle size. The electrospray can be generated by an electric field connecting a capillary or nozzle feed. Conveniently the particle size of the particles supplied by the sprayer is less than or equal to about 10 μm, preferably in the range of about 1 μm to about 5 μm MMAD, more preferably in the range of about 1 to about 3 μm MMAD, most preferably in the range of about 2 to about 3 μm MMAD. Administration as a spray is the preferred method for crystals and co-crystals. Typically, crystals and co-crystal formulations suitable for use with a sprayer are:
The aqueous solution contains crystals or co-crystals at a concentration of about 1 mg to about 20 mg of total protein per mL of solution. The formulation can include substances such as excipients, buffers, isotonic substances, preservatives, surfactants, and zinc. The formulation may also include substances or excipients to stabilize the protein, such as buffers, reducing agents, bulk proteins or carbohydrates. Bulk proteins useful for formulating crystals or co-crystals include albumin, protamine, and the like. Typical carbohydrates useful in spray formulations include sucrose, mannitol, lactose, trehalose, glucose and the like. Spray formulations can also include surfactants that can reduce or prevent agglomeration that occurs on the surface of the crystals or co-crystals that result from spraying the solution in the formation of an aerosol. A variety of conventional surfactants can be used, for example, polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Generally, the amount is from 0.001% by weight of the formulation
4% by weight. Particularly preferred surfactants for the purposes of the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, and the like.
Additional materials known in the art for protein formulations are also included in the formulation.

The crystals or co-crystals can be administered by a nebulizer, such as a jet nebulizer or an ultrasonic nebulizer. Typically, jet nebulizers use a source of compressed air to create a high velocity air jet through an opening. As the gas spreads out of the nozzle, a low pressure area is created and a suspension of crystals or co-crystals is pumped through a capillary connected to the liquid reservoir. The suspension ejected from the capillary exits the capillary and flies like an arrow to form unstable filaments and droplets, thereby producing an aerosol. The desired performance characteristics can be obtained from the jet nebulizer with a range of configurations, flow rates, and adjustment plates. In an ultrasonic nebulizer, high frequency electrical energy, typically a piezo-electric transducer, is used to obtain oscillating mechanical energy.
This energy is transferred to the crystal or co-crystal suspension, either directly or via a coupling solution, producing an aerosol. Conveniently, the particles comprising crystals or co-crystals provided by the nebulizer have a particle size of about 10 μm or less, preferably in the range of about 1 μm to about 5 μm MMAD, more preferably in the range of about 1 to about 3 μm MMAD, most preferably. Is about 2 to about 3 μm MMAD.

Crystal formulations suitable for use with jet or ultrasonic nebulizers include:
Typically, the aqueous solution contains crystals or co-crystals at a concentration of about 1 mg to about 20 mg of total protein per mL of solution. The formulation can include substances such as excipients, buffers, isotonic substances, preservatives, surfactants, and preferably zinc. The formulation may also include substances or excipients to stabilize the protein, such as buffers, reducing agents, bulk proteins or carbohydrates. Bulk proteins useful for formulation include albumin and protamine. Typical carbohydrates useful for formulation include sucrose, mannitol, lactose, trehalose, glucose and the like. The formulation can also include a surfactant that can reduce or prevent aggregation of fatty acid-acylated insulin protein on the surface caused by spraying the solution in the formation of an aerosol. A variety of conventional surfactants can be used, for example, polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbital fatty acid esters. Generally, the amount will be from 0.001% to 4% by weight of the formulation. Particularly preferred surfactants for the purposes of the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, and the like.

In a fixed dose inhaler (MDI), a propellant, a suspension of crystals or co-crystals, and any excipients or other additives are contained in a can as a mixture with a liquefied compressed gas. The metering valve is actuated, preferably in the range of about 10 μm or less, preferably about 1 μm.
The mixture is released as an aerosol in the range of μm to about 5 μm MMAD, more preferably in the range of about 1 to about 3 μm MMAD, and most preferably in the range of about 2 to about 3 μm MMAD. The desired aerosol particle size is determined by jet milling, spray drying, critical point cond
ensation) can be obtained using a preparation of crystals or co-crystals manufactured by various methods known to those skilled in the art, including ensation. It is preferred to avoid mechanical methods by adjusting the crystallization according to the method of the present invention. 3M for preferred metered dose inhaler
Or those using a hydrofluorocarbon propellant manufactured by Glaxo.

Formulations of crystals or co-crystals for use with metered-dose inhalers include crystals or co-crystals as fine powder in suspension in a non-aqueous medium, for example, suspended in a propellant with the aid of a surfactant. There will be. Propellants include any conventional materials used for this purpose, such as chlorofluorocarbon, hydrochlorofluorocarbon, hydrofluorocarbon, or trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane. , HFA-134a (hydrofluoroalkane-134a), and HFA-227 (hydrofluoroalkane-227). Preferably, the propellant is a hydrofluorocarbon. Surfactants can be chosen to stabilize the crystals or co-crystals as a suspension in the propellant, to protect the active against chemical degradation, and the like. Suitable surfactants include sorbitan trioleate, soya lecithin, oleic acid, and the like. The formulation may also contain additional substances and excipients. The present invention will be better understood from the following examples. These examples represent specific embodiments of the present invention and do not limit the scope of the present invention.

The particle size is measured as follows. The particle diameter of the crystals and co-crystals may be measured using the following apparatus: Coulter® Multisizer 646 or equivalent (
Coulter Corporation, Hialeah, FL), Sampling Stand II, Model 999 or equivalent (Coulter Corporation, Hialeah, FL), 50 μm Coulter® aperture tube, pH meter calibrated with a pH buffer sandwiching the desired Ph value. 7.5 g of dibasic sodium phosphate crystals in 1 L of water, 3.2 g of m-cresol, 32 g of glycerin,
And a stock diluent (2X concentration) containing 1.46 g of phenol. 5N HCl or 5
Adjust the pH to 7.35-7.45 with N NaOH and filter through a 0.22 μm or smaller pore size filter. Store at room temperature. One volume of the stock diluent and one volume of water are mixed and filtered again to prepare a working diluent (1X concentration).

Re-suspend the test sample. If in vial, resuspend by inverting 10 times with palm and inverting 10 times. If in a cartridge, use the palm of your hand
Spin and invert 10 times 3 times to resuspend. Pipet 0.25 mL of sample into 100 mL of working diluent. Generally, this gives a particle count in the range of 90,000-300,000 counts with a sampling time of 50 seconds. If the particle count is below this range, the sample may be over-diluted, so it is necessary to use a sample volume of 0.25 mL or more to obtain the appropriate count. Add an appropriate volume of suspension to obtain a particle count in the range of 100,000-300,000. Simultaneous correction should not exceed 15%. For thicker samples, a volume of 0.25 mL or less may need to be used. The beaker containing the diluted sample is placed on a sampling stand to make sure that the external electrode is submerged in water. Measure once per sample. The measurement is performed continuously and gradually with stirring over a sampling time of 50 seconds.

For the instrument, the following parameters are typical. Page 1 Aperture diameter: 50 μm Aperture length: 53 μm Setup: Manual Analysis: Sample Calibration: Recall Kd: 505.00 (default depends on each calibration) Size: 5 Units: μm Page 2 Current and gain: Manual aperture current : 400μA Gain: 4 Polarity: + Device control: Time Time: 50s Channel count: 0 Total count: 0t

Page 3 Channel: 256 Auto Scaling: On Edit: Off Simultaneous Compensation: On Analysis Volume: Particle Relative Density: 1 Diff Value:% End Tone: On Page 4 Communication Setup RS232C Serial Output Baud Rate: 9600 Equipment: Computer Autoplot: None Print / Plot Option Channel Data: None Analog Plot: None Screen Dump: Yes Overlay Mode: None Format: Enhanced Computer Option Send (Send) STX / ETX: None End Field Char: 59 End Line Char: CR loading zero: None

The Coulter® Multisizer is used for particle characterization, ie measuring particle number and size. The device operates on the principle that a change in electrical resistance occurs when particles suspended in a conductive liquid pass through a small opening with electrodes on either side. The change in resistance is related to the particle volume and results in a short electrical pulse that is essentially proportional to the particle volume. By measuring the particle volume, the equivalent volume diameter can be calculated.
A size distribution curve is obtained by electronically sorting a series of pulses. The number and volume statistics and their distribution are obtained by software integrated with the instrument.

The abbreviations used in the following Preparation Examples and Examples are as follows: BHI: biosynthetic human insulin biosynthesized in an organism transformed with recombinant DNA
Zn-BHI: biosynthetic human insulin containing 0.33 or human insulin per molecule zinc atom zinc crystal ^{C6-BHI: B29-N ε} - hexanoyl - human insulin ^{C8-BHI: B29-N ε} - decanoyl - human insulin VMSED: Volume average spherical equivalent diameter of crystal or co-crystal particle size distribution; units in microns SD: Standard deviation of crystal or co-crystal particle size distribution; units in microns

Production Example 1 A stock solution of 40 mg / mL C8-BHI is prepared by dissolving lyophilized C8-BHI powder at pH 1.2. 25 μL of 12.44 mg / mL zinc oxide was added to 1 mL of 40 mg / mL C8-BHI. To 1 mL of this solution, add 4 mL of crystallization buffer containing 40 mg / mL glycerin, 4.4 mg / mL m-cresol, 1.8 mg / mL phenol, 9.375 mg / mL secondary phosphate, and 7.35 mg / mL trisodium citrate. Was. The pH of the resulting solution was adjusted to 7.6 with 5N NaOH. The solution was filtered through a Millipore Millex-GV filter and mixed with an equal volume of 0.64 mg / mL protamine sulfate. A precipitate formed quickly. The sample was stored gently at a temperature adjusted to 25 ° C. After 24 hours, rod-like crystals containing some amorphous material were observed. The particle size (particle size) distribution was broad and the volume average spherical equivalent diameter (VMSED) was 9.7 microns.

Production Example 2 The method of Production Example 1 was carried out except that the crystallization buffer contained 7.3 mg / mL sodium chloride. A precipitate was formed immediately after the addition of protamine sulfate. The sample was stored gently at a temperature adjusted to 25 ° C. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. The particle size distribution was much narrower than without sodium chloride, VMSED 5.8 microns. This also provides a means of producing 100% C8-BHI / protamine crystals.

Production Example 3 In both cases, 40 mg / mL stock solutions of C8-BHI and Zn-BHI were prepared at pH 1.2. 40mg / m
0.85 mL of LC8-BHI was mixed with 0.15 mL of 40 mg / mL Zn-BHI. 12 to this insulin solution
.44 mg / mL zinc oxide 21.25 μL was added. In 1 mL of this solution, add 40 mg / mL glycerin, 4
.4 mg / mL m-cresol, 1.8 mg / mL phenol, 9.375 mg / mL secondary phosphate,
And 4 mL of crystallization buffer containing 7.35 mg / mL trisodium citrate. The pH of the resulting solution was adjusted to 7.6 with 5N NaOH. The solution was filtered through a Millipore Millex-GV filter and mixed with an equal volume of 0.64 mg / mL protamine sulfate. A precipitate formed quickly. The sample was stored gently at a temperature adjusted to 25 ° C. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. VMSED was 7.7 microns.

Production Example 4 The method of Production Example 3 was carried out except that the crystallization buffer contained 7.3 mg / mL sodium chloride. A precipitate was formed immediately after the addition of protamine sulfate. The sample was stored gently at a temperature adjusted to 25 ° C. After 24 hours, the amorphous precipitate turned into rod-like crystalline material.
The effect of adding sodium chloride was clear and significant. Again, the particle size distribution was fairly narrow and VMSED was reduced to 4.8 microns.

Production Example 5 C40-mg / mL C8-BHI and Zn-BHI stock solutions were prepared at pH 1.2. 40mg / m
0.75 mL of LC8-BHI was mixed with 0.25 mL of 40 mg / mL Zn-BHI. 12 to this insulin solution
.44 mg / mL zinc oxide 18.75 μL was added. In 1 mL of this solution, 40 mg / mL glycerin, 4.
4 mg / mL m-cresol, 1.8 mg / mL phenol, 9.375 mg / mL secondary phosphate,
And 4 mL of crystallization buffer containing 7.35 mg / mL trisodium citrate. The pH of the resulting solution was adjusted to 7.6 with 5N NaOH. The solution was filtered through a Millipore Millex-GV filter and mixed with an equal volume of 0.64 mg / mL protamine sulfate. A precipitate formed quickly. The sample was stored gently at a temperature adjusted to 25 ° C. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. VMSED was 6.2 microns.

Production Example 6 The method of Production Example 5 was carried out except that the crystallization buffer contained 7.3 mg / mL sodium chloride. A precipitate was formed immediately after the addition of protamine sulfate. The sample was stored gently at a temperature adjusted to 25 ° C. After 24 hours, the amorphous precipitate turned into rod-like crystalline material.
The effect of adding sodium chloride was clear and significant. Again, the particle size distribution was fairly narrow and the VMSED was reduced to 4.2 microns.

Production Example 7 In both cases, 40 mg / mL C8-BHI and Zn-BHI stock solutions were prepared at pH 1.2. 40mg / m
0.65 mL of LC8-BHI was mixed with 0.35 mL of 40 mg / mL Zn-BHI. 12 to this insulin solution
16.25 mg / mL zinc oxide 16.25 μL was added. In 1 mL of this solution, 40 mg / mL glycerin, 4.
4 mg / mL m-cresol, 1.8 mg / mL phenol, 9.375 mg / mL secondary phosphate,
And 4 mL of crystallization buffer containing 7.35 mg / mL trisodium citrate. The pH of the resulting solution was adjusted to 7.6 with 5N NaOH. The solution was filtered through a Millipore Millex-GV filter and mixed with an equal volume of 0.64 mg / mL protamine sulfate. A precipitate formed quickly. The sample was stored gently at a temperature adjusted to 25 ° C. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. VMSED was 5.0 microns.

Production Example 8 The method of Production Example 7 was carried out except that the crystallization buffer contained 7.3 mg / mL sodium chloride. A precipitate was formed immediately after the addition of protamine sulfate. The sample was stored gently at a temperature adjusted to 25 ° C. After 24 hours, the amorphous precipitate turned into rod-like crystalline material.
The effect of adding sodium chloride was clear and significant. Again, the particle size distribution was fairly narrow and VMSED was reduced to 3.8 microns.

Preparation 9 The procedure of Preparation 1 was followed, except that C8-BHI was first dissolved at pH 2.4 instead of pH 1.2. After 24 hours, the material was still largely amorphous, with some rod-like crystals. The particle size distribution was multi-modal.

Production Example 10 The method of Production Example 9 was carried out except that the crystallization buffer contained 7.3 mg / mL sodium chloride. A precipitate was formed immediately after the addition of protamine sulfate. The sample was stored gently at a temperature adjusted to 25 ° C. After 24 hours, the amorphous precipitate changed to a rod-like crystalline material, unlike the production method without adding sodium chloride. VMSED was 8.4 microns. This shows a controlled example of the production of 100% C8-BHI protamine crystals, where a low average, narrow particle size distribution Gaussian distribution was achieved.

Preparation Example 11 The procedure of Preparation Example 3 was followed except that C8-BHI was first dissolved at pH 2.4 instead of pH 1.2. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. VMSED was 11.0 microns.

Production Example 12 The procedure of Production Example 11 was carried out except that the crystallization buffer contained 7.3 mg / mL sodium chloride. A precipitate was formed immediately after the addition of protamine sulfate. The sample was stored gently at a temperature adjusted to 25 ° C. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. The effect of adding sodium chloride was clear and significant. Again, the particle size distribution was fairly narrow and VMSED was reduced to 6.5 microns.

Preparation Example 13 The procedure of Preparation Example 5 was followed except that C8-BHI was first dissolved at pH 2.4 instead of pH 1.2. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. VMSED was 8.7 microns.

Production Example 14 The method of Production Example 13 was carried out except that the crystallization buffer contained 7.3 mg / mL sodium chloride. A precipitate was formed immediately after the addition of protamine sulfate. The sample was stored gently at a temperature adjusted to 25 ° C. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. The effect of adding sodium chloride was clear and significant. Again, the particle size distribution was fairly narrow and VMSED was reduced to 6.0 microns.

Preparation Example 15 The procedure of Preparation Example 7 was followed except that C8-BHI was first dissolved at pH 2.4 instead of pH 1.2. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. VMSED was 7.0 microns.

Production Example 16 The method of Production Example 15 was carried out except that the crystallization buffer contained 7.3 mg / mL sodium chloride. A precipitate was formed immediately after the addition of protamine sulfate. The sample was stored gently at a temperature adjusted to 25 ° C. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. The effect of adding sodium chloride was clear and significant. Again, the particle size distribution was quite narrow and the VMSED was reduced to 4.7 microns.

Production Example 17 A C8-BHI solution was prepared by dissolving 61.4 mg of lyophilized C8-BHI powder in 1.54 mL of 0.1N HCl. A Zn-BHI solution was prepared by dissolving 20.5 mg of Zn-BHI crystals in 0.51 mL of 0.1 N HCl. Zn-B
1.5 mL of C8-BHI solution was added to the HI solution. To the Zn-BHI and C8-BHI solutions, 49 μL of a 12.44 mg / mL zinc oxide stock solution was added. Add 40 mg / mL glycerin, 4.4 mg / m
L m-cresol, 1.8 mg / mL phenol, 9.375 mg / mL secondary phosphate, and
8 mL of crystallization buffer containing 7.35 mg / mL trisodium citrate was added. The pH of the resulting solution was adjusted to 7.6 with 5N NaOH. The solution was filtered through a Millipore Millex-GV filter and mixed with an equal volume of 0.64 mg / mL protamine sulfate. A precipitate formed quickly. The suspension was aliquoted and stored gently at temperatures adjusted to 20, 25 and 30 ° C. twenty four
After time, the amorphous precipitate turned into rod-like crystalline material. The effect of temperature was clear and significant. VMSED was 7.8, 6.9, and 5.5 microns at 20, 25, and 30 ° C., respectively.

Summary of Production Examples 1-17

The following conclusions are obtained based on Production Examples 1 to 17 described above. First, for the same proportion of C8-BHI, dissolution at pH 1.2 results in a smaller, tighter particle size distribution than dissolution at pH 2.4. Second, the lower the proportion of C8-BHI, the smaller the crystals and the smaller the standard deviation. Third, the addition of sodium chloride results in smaller crystals and smaller standard deviations. This obviously provides a means to adjust the particle size by various possible modifications in the formulation and manufacturing process. Finally, the addition of sodium chloride obviously also provides a means of producing 100% C8-BHI crystals.

Production Example 18 A 50 mL stock solution of about 26.5 mg / mL C8-BHI and 8.9 mg / mL Zn-BHI was prepared at pH 1.2. To this insulin solution was added 12.44 mg / mL zinc oxide (1 mL). In this solution,
40 mg / mL glycerin, 4.4 mg / mL m-cresol, 1.8 mg / mL phenol, 9.375 mg /
200 mL of crystallization buffer containing mL second phosphate and 7.35 mg / mL trisodium citrate was added. The pH of the resulting solution was adjusted to 7.6 with 5N NaOH. Mil solution
It was filtered through a lipore Millex-GV filter and mixed with an equal volume of 0.64 mg / mL protamine sulfate. A precipitate formed quickly. The sample was stored gently at a temperature adjusted to 25 ° C. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. VMSED was 7.8 microns.

Production Example 19 About 120 mL of a stock solution of about 10.5 mg / mL of C8-BHI and about 3.5 mg / mL of Zn-BHI was prepared at pH 2.
Prepared in 4. To this insulin solution was added 12.44 mg / mL zinc oxide (1 mL). To this solution was added 64 mg / mL glycerin, 7.0 mg / mL m-cresol, 2.9 mg / mL phenol,
About 125 mL of crystallization buffer containing mg / mL secondary phosphate and 11.76 mg / mL trisodium citrate was added. The pH of the resulting solution was adjusted to 7.6 with 5N NaOH. Water was added to bring the volume to 250 mL. The solution was filtered through a Millipore Millex-GV filter,
It was mixed with an equal volume of 0.64 mg / mL protamine sulfate. A precipitate formed quickly. twenty five
The sample was stored gently at a temperature adjusted to ° C. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. VMSED was 9.2 microns.

Preparation 20 The procedure of Preparation 19 was followed except that the crystallization buffer also contained 11.69 mg / mL sodium chloride. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. VMSED was 5.3 microns.

Production Example 21 A 25 mL stock solution of approximately 10.5 mg / mL C8-BHI and 3.5 mg / mL Zn-BHI was prepared at pH 2.5. 12.44 mg / mL zinc oxide 0.24 μL was added to 2.5 mL of this insulin solution. To this solution was added 2.5 mL of crystallization buffer containing 64 mg / mL glycerin, 7.0 mg / mL m-cresol, 2.9 mg / mL phenol, 15 mg / mL secondary phosphate, and 11.76 mg / mL trisodium citrate. . The pH of the resulting solution was adjusted to 7.6 with 5N NaOH. The solution was filtered through a Millipore Millex-GV filter and mixed with an equal volume of 0.64 mg / mL protamine sulfate. A precipitate formed quickly. The sample was stored gently at a temperature adjusted to 25 ° C. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. VMSED is 7.0
9 microns.

Production Example 22 The procedure of Production Example 21 was followed except that the crystallization buffer also contained 11.69 mg / mL sodium chloride. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. VMSED was 5.72 microns.

Production Example 23 The procedure of Production Example 21 was followed except that the crystallization buffer also contained 14.91 mg / mL potassium chloride. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. VMSED was 5.39 microns.

Production Example 24 The procedure of Production Example 21 was followed except that the crystallization buffer also contained 16.41 mg / mL sodium acetate. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. VMSED was 7.39 microns.

Preparation 25 The procedure of Preparation 21 was followed except that the crystallization buffer also contained 46.02 mg / mL sodium tartrate. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. VMSED was 8.07 microns.

Production Example 26 The procedure of Production Example 21 was followed except that the crystallization buffer also contained 28.41 mg / mL sodium sulfate. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. VMSED was 7.07 microns.

Production Example 27 The procedure of Production Example 21 was followed except that the crystallization buffer also contained 13.6 mg / mL sodium formate. After 24 hours, the amorphous precipitate turned into rod-like crystalline material. VMSED was 6.90 microns.

Summary of Production Examples 18 to 27

The following conclusions are obtained from Production Examples 18 to 27. First, the scale for crystallization is
Not adding sodium chloride only affects the average particle size. Addition of sodium chloride (i.e., chloride anion) will give larger and more reproducible results using manufacturable methods. The effect of sodium chloride is due to chlorine, not sodium. The other anions tested had no or little effect. These results also indicate that the effect of sodium chloride is not due to an increase in ionic strength, but to a specific effect of chloride anion.

Production Example 28 100% C8-BHI Crystal Preparation for Rat Intratracheal Instillation Test A dry lyophilized powder of C8-BHI (471 mg) was dissolved in 11.77 mL of 0.1 N HCl. To this solution, 3.532 mL of a 1000 ppm zinc nitrate solution was added and stirred. An aqueous solution (pH 7.63) containing 9.5 mg / mL disodium phosphate heptahydrate, 0.375 M NaCl, 4 mg / mL m-cresol, 1.65 mg / mL phenol, and 16 mg / mL glycerol was added to this solution. mL was added. The pH was adjusted to 7.6 with small amounts of 1N HCl and 1N NaOH. This solution was then filtered through a 0.2 micron protein low binding filter. 86.8 protamine sulfate dry powder
mg was dissolved in 115.73 mL of water and then filtered through a 0.2 micron protein low binding filter. 55.95 mL of the C8-BHI solution was mixed with 55.95 mL of the protamine sulfate solution. A white precipitate formed. The suspension was mixed gently to mix thoroughly. This preparation
It was left in a 30 ° C. water bath for 24 hours. Inspection under an optical microscope indicated that microcrystalline solids, including rod-like crystals, were present. VMS by measuring particle size distribution by Coulter technology
The ED was found to be 4.7 microns.

The mother liquor of this preparation was replaced with an aqueous solution containing 0.7 mg / mL phenol, 1.6 mg / mL m-cresol, and 0.3 mg / mL disodium phosphate heptahydrate according to the following procedure. Centrifuge 50 mL of the suspension at 3000 rpm for 12 minutes at 23 ° C., remove 40 mL of the mother liquor without disturbing the solid phase, 0.7 mg / mL phenol, 1.6 mg / mL m-cresol, and 0.3 mg / mL.
The solution was replaced with an aqueous solution containing disodium phosphate heptahydrate. This method was repeated more than once. After replacing the mother liquor in this manner, the microcrystals were again analyzed using a Coulter counter and found to have a VMSED of 3.5 microns.

Analytical characterization by HPLC indicated that almost all insulin and protamine sulfate were present in the solid phase. Intratracheal instillation of rats
) For testing, the required concentration by appropriately diluting these formulations with an aqueous solution containing 0.7 mg / mL phenol, 1.6 mg / mL m-cresol, and 0.3 mg / mL disodium phosphate heptahydrate Diluted.

Production Example 29 75% C8-BHI crystal preparation for rat intratracheal instillation test A dry lyophilized powder of C8-BHI (330 mg) was dissolved in 8.25 mL of 0.1N HCl. A dry powder of human insulin-Zn crystals (116 mg) was dissolved in 2.9 mL of 0.1 HCl. 2.75m of the latter solution
L was mixed with the C8-BHI solution to give a mixture of C8-BHI and human insulin in the appropriate weight ratio of 75:25. This solution was stirred and mixed. 2.866 mL of 1000 ppm zinc nitrate solution
Was added and stirred. To this solution was added 9.5 mg / mL disodium phosphate heptahydrate, 0.375
47 mL of an aqueous solution (pH 7.63) containing M NaCl, 4 mg / mL m-cresol, 1.65 mg / mL phenol, and 16 mg / mL glycerol was added. Adjust the pH with a small amount of 1N HCl and 1N NaOH
Was adjusted to 7.6. This solution was then filtered through a 0.2 micron protein low binding filter. 86.8 mg of dry powder of protamine sulfate was dissolved in 115.73 mL of water, and then dissolved in water.
Filtered through a 2 micron protein low binding filter. 50 mL of a mixed solution of C8-BHI and human insulin was mixed with 50 mL of a protamine sulfate solution. A white precipitate formed. The suspension was mixed gently to mix thoroughly. Put this preparation in a 30 ° C water bath
It was left for 24 hours. Inspection under an optical microscope indicated that microcrystalline solids, including rod-like crystals, were present. Measurement of the particle size distribution by the Coulter technique indicated a VMSED of 4.4 microns.

The mother liquor of this preparation was prepared as described in Example 28 with 0.7 mg / mL phenol, 1.6 mg / m
Replaced with an aqueous solution containing Lm-cresol and 0.3 mg / mL disodium phosphate heptahydrate. After this substitution of the mother liquor, the microcrystals were again analyzed using a Coulter counter and found to have a VMSED of 3.9 microns.

Analytical characterization by HPLC indicated that almost all insulin and protamine sulfate were present in the solid phase. Appropriately dilute these formulations with an aqueous solution containing 0.7 mg / mL phenol, 1.6 mg / mL m-cresol, and 0.3 mg / mL disodium phosphate heptahydrate for intratracheal instillation studies in rats To the required concentration.

The procedures described in Preparative Examples 28 and 29 demonstrate that, with appropriate adjustment of the crystallization conditions, crystals containing derivatized insulin with zinc and protamine have the preferred size for optimizing accumulation in the deep lung (preferably (<5 microns, more preferably <3 microns)
Can be prepared. Important parameters in these preparation examples were the NaCl concentration during crystallization (150 mM) and the temperature during crystallization (30 ° C.).

Production Example 30 The mother liquors of Production Examples 28 and 30 were replaced with water according to the following procedure. 5 mL of suspension 2
Centrifugation was performed at 3 ° C. and 3000 rpm for 12 minutes. 4 mL of the mother liquor was removed without disturbing the solid phase and replaced with water. This procedure was repeated two more times. The resulting suspension was lyophilized. The solid obtained after lyophilization was reconstituted with 5 mL of water. When observed under a light microscope, the resulting suspension contained small microcrystals.

Production Example 31 The mother liquor of Production Examples 28 and 29 was replaced as described in Production Example 30, except that the crystals were washed with a solution of 0.9 g / 100 mL of NaCl. After washing three times, the resulting suspension was lyophilized. The solid obtained after lyophilization was reconstituted with 5 mL of water. When observed under a light microscope, the resulting suspension contained small microcrystals.

The procedures described in Preparations 30 and 31 are based on the method of the invention (ie, for example, Preparation 28
And 29) show that the microcrystals obtained according to (29) can be freeze-dried without impairing the properties of the crystals. Such microcrystalline powder can be used in a dry powder inhaler for lung supply. These results also indicate that these microcrystals can be mixed with water to form a stable aqueous suspension with minimal excipients. Such an aqueous suspension can be used in a nebulizer for lung supply.

Preparation 32 After crystallization according to the procedure described in either Preparation 28 or 29 (determined by light microscopy), the microcrystals are separated from the mother liquor by a conventional solid / liquid separation method and recovered.
Next, the recovered microcrystals are buffered (eg, 0.3 mg / mL disodium phosphate heptahydrate), antimicrobial preservative (eg, 0.65 mg / mL phenol and / or 1.6 mg / mL m-cresol). ), And isotonic substances (e.g., 16 mg / mL glycerol or 9 mg / mL NaCl
) And adjust the pH to 6.8.

Preparation 33 After crystallization according to the procedure described in either Preparation 28 or 29 (determined by light microscopy), the microcrystals are separated from the mother liquor by a conventional solid / liquid separation method and recovered.
Next, the recovered microcrystals are resuspended according to Production Example 32 except that the resuspension solution does not contain an antimicrobial preservative. Such preparations may be more appropriate when it is desired to be free of irritants and unnecessary excipients.

Preparation 34 VMSED Dissolution of 2.1 micron 75% C8-BHI co-crystal A 75% C8-BHI co-crystal was prepared on a 10 mL scale by the same procedure as in Preparation 29.
The VMSED of the crystals produced by this procedure was 2.1 microns. The in vitro dissolution rate of the crystals was tested using a spectrophotometric dissolution assay. 3 mL of phosphate buffered saline was placed in a quartz cuvette containing a small stir bar. The solution was stirred at a constant speed. The absorbance was zeroed. 5 μL of the uniformly suspended formulation was rapidly suspended at the bottom of the cuvette. One minute after adding the formulation,
305 nm absorbance data was obtained as a function of time. The change in absorbance was tracked as a function of time.

The absorbance decreases as the scattering particles dissolve. The time until the absorbance decreases by half of the complete decrease in absorbance is indicated by the parameter t _1/2 . This parameter is useful for comparing the dissolution rates of different crystal preparations. As t _1/2 increases, the dissolution rate decreases. In the above dissolution test, t _1/2 of the cocrystal produced according to Production Example 34 was 50 minutes. Under the same dissolution conditions, the t1 _{/ 2} of NPH-human insulin crystals was typically about 10 minutes or less. That is, the dissolution of the co-crystal of Preparative Example 34 was significantly slower than NPH, although the VMSED was smaller and a concomitant increase in surface area was expected. This result supports the conclusion that the smaller crystallites of the present invention result in a more sustained release compared to NPH-human insulin crystals.

Preparation Examples 35-40 The crystallization buffer for three samples was free of sodium chloride, or contained 75 mM sodium chloride and 150 mM sodium chloride, except that the temperature was adjusted to 30 ° C. According to the procedure, three types of each crystal sample were prepared on a 10 mL scale. When 150 mM NaCl was present during crystallization, both 75% and 100% C8-BHI compositions resulted in very small particles with a mean particle diameter of about 2 microns and a narrow size distribution.

[0188]

Production Examples 41-44 Dry powder (24.0 mg) of C8-BHI was dissolved in 1.20 mL of 0.1 N HCl. Another solution was prepared by dissolving the dried powder of zinc zinc human insulin (8.0 mg) in 0.400 mL of 0.1 N HCl.
These two solutions were mixed to obtain 1.60 mL of a mixed solution of human insulin and C8-BHI. To this solution, 448 μL of a 15.3 mM zinc chloride solution was added with stirring. Add 10 mg / mL disodium phosphate heptahydrate, 7.5 mg / mL trisodium citrate · 2
6.4 mL of an aqueous solvent (pH 7.59) consisting of hydrate, 4 mg / mL m-cresol, 2 mg / mL phenol, and 40 mg / mL glycerol was added. Adjust the pH with small amounts of 1N HCl and 1N NaOH
Adjusted to 7.61. The solution was then filtered through a 0.22 micron low protein binding filter. The second solution was prepared by dissolving 72.1 mg of protamine sulfate in 96 mL of water, and then adding 0.2 ml of 0.2 mL of water.
Prepared by filtration through a 22 micron protein low binding filter. An 8 mL amount of the insulin mixed solution was mixed with 8 mL of the protamine sulfate solution. An amorphous precipitate formed.
The suspension was gently stirred to complete the mixing. The preparation was divided into 4 volumes of 4 mL and allowed to stand at temperatures of 15 ° C, 25 ° C, 30 ° C, and 35 ° C, respectively, for 90 hours. Optical microscope (100
Observation under 0x) indicated that in each case the amorphous precipitate had turned into a microcrystalline solid of uniform appearance containing a single rod. Precipitates corresponding to temperatures of 15 ° C., 25 ° C., 30 ° C. and 35 ° C. are shown in Production Examples 41, 42, 43 and 44, respectively.

Each sample was centrifuged at 3000 rpm for 12 minutes to precipitate crystals. 3.2 mL of supernatant in each production example
Is decanted off, and 4 mg / mL disodium phosphate heptahydrate, 3 mg / mL citric acid 3
Replaced with 3.2 mL of an aqueous diluent (pH 7.61) containing sodium dihydrate, 0.8 mg / mL phenol, and 16 mg / mL glycerol. This diluent exchange step was repeated for the second and third times (however, in the third time, 3.2 mL of the aqueous diluent was replaced with 2.8 mL of the aqueous diluent). The VMSED of this preparation is shown in the table below.

[0191]

Production Example 45-48 A dry powder (24.0 mg) of C8-BHI was dissolved in 0.60 mL of 0.1 N HCl. Another solution was prepared by dissolving dry powder of human insulin zinc crystals (8.2 mg) in 0.20 mL of 0.1 N HCl.
These two solutions were mixed to obtain 0.80 mL of a mixed solution of human insulin and C8-BHI.
To this solution, 300 μL of a 15.3 mM zinc chloride solution was added with stirring. 0.27 of this solution
Divided into 4 volumes of 5 mL. Contains 35 mM disodium phosphate heptahydrate, 4 mg / mL m-cresol, 1.6 mg / mL phenol, and 40 mg / mL glycerol (pH 7.6) with different sodium citrate concentrations (0 mM, 12.5 mM, 37.5 mM, and 8
(7.5 mM) 4 different crystallization buffers were prepared (Preparation Examples 45, 46, 47 and 48).
1.1 mL of crystallization buffer was added to each of 0.275 mL of the four samples of the protein solution. The pH of each solution was adjusted to 7.6 with a small amount of 1N HCl and 1N NaOH. Each solution was then filtered through a 0.22 micron low protein binding filter. To 1 mL of each solution, 37.6 mg of protamine sulfate was dissolved in 50 mL of water, and then 1 mL of a protamine sulfate solution prepared by filtration through a 0.22 micron low protein binding filter was added. In each case, an amorphous was formed. Each preparation was gently agitated to complete mixing. The four preparations were left at 25 ° C. for 23 hours. When observed under an optical microscope (1000x), in each case the amorphous precipitate turned into a microcrystalline solid of uniform appearance containing a single rod, and the average crystal size of each preparation was as shown in the table. Turned out different.

[0193]

Production Example 49 A dry powder of C8-BHI (60.7 mg) was dissolved in 1.50 mL of 0.1N HCl. 600 μL of a 15.3 mM zinc chloride solution was added to this solution with stirring. To 0.70 mL of this solution, 50 mM Tris,
10 mg / mL phenol, 30 mg / mL trisodium citrate dihydrate, and 31 mg / mL
2 mL of an aqueous solvent consisting of glycerol (pH 7.60) was added. The pH was adjusted to 7.61 with small amounts of 1N HCl and 1N NaOH. The solution was then filtered through a 0.22 micron low protein binding filter. A second solution was prepared by dissolving 37.8 mg of protamine sulfate in 50 mL of water and then filtering through a 0.22 micron low protein binding filter.
An amount of 2.5 mL of the C8-BHI mixed solution was mixed with 2.5 mL of the protamine sulfate solution. An amorphous precipitate formed. The suspension was gently stirred to complete the mixing. Preparation at 25 ° C for 60
Let stand for hours. Observation under an optical microscope (1000x) showed that the amorphous precipitate had turned into a microcrystalline solid of uniform appearance, including rod-like crystals with an estimated approximate average particle size of 2 microns.

Production Example 50 A dry powder (39.2 mg) of C8-BHI was dissolved in 1000 parts of 0.1N HCl. To this solution, 400 μL of a 15.3 mM zinc chloride solution was added with stirring. 5 mg / mL disodium phosphate anhydrous, 25 mM trisodium citrate, 1.6 mg / mL phenol, 4 mg / mL m-
Cresol and 4 mL of an aqueous solvent (pH 7.6) consisting of 40 mg / mL glycerol were added. The pH was adjusted to 7.60 with small amounts of 1N HCl and 1N NaOH. Then, add this solution to 0.22
Filtered through a micron protein low binding filter. A second solution was prepared by dissolving 37.3 mg of protamine sulfate in 50 mL of water and then filtering through a 0.22 micron low protein binding filter. An amount of 5 mL of the C8-BHI mixed solution was mixed with 5 mL of the protamine sulfate solution. An amorphous precipitate formed. The suspension was gently stirred to complete the mixing. The preparation was left at 25 ° C. for 47 hours. Observation under an optical microscope (1000x) showed that the amorphous precipitate had turned into a microcrystalline solid of uniform appearance, including a single rod-shaped crystal of approximately average length 2 microns.

Production Examples 51-54 As shown in the table below, 75% C8-BHI was used according to the procedure of Example 1 or Example 19 except that the final citrate and sodium chloride concentrations in the four samples were as follows. Crystals were produced. After 24 hours, in each case the amorphous precipitate turned into rod-like crystalline material. VMESD of each preparation
And SD are shown in the above table.

Production Example 55 Microcrystal Preparation Microcrystals prepared according to any of Production Examples 1 to 51 are separated from the mother liquor by a conventional solid / liquid separation method and recovered. Next, the recovered microcrystals were suspended in a solution (pH 6.8) consisting of 2 mg / mL disodium phosphate, 1.6 mg / mL m-cresol, 0.65 mg / ml phenol, and 16 mg / ml glycerol, and the final insulin activity was measured. Concentration about 100U / m
L.

Production Example 56 Microcrystal Preparation Microcrystals prepared according to any of Production Examples 1 to 51 are separated from the mother liquor by a conventional solid / liquid separation method and recovered. Next, the recovered microcrystals are suspended in a solution consisting of 0.65 mg / ml phenol in water to bring the final concentration of insulin activity to about 100 U / mL. Adjust the pH to about 6.8 with 1N HCl and 1N NaOH.

PREPARATIONS 57-83 All preparations produced 75% C8-BHI co-crystals. 3 In all but the Preparation Examples (57, 62, and 63), the crystallization buffer was adjusted substantially as needed to achieve the indicated final citrate and sodium chloride concentrations. 19 steps were followed. The scale was 32-50 mL. The table shows the pH at which the protein was dissolved. These preparations further support the above conclusions and show that VMSED is inversely proportional to citrate concentrations in the range of at least 0-10 mM.

[0200]

Preparations 84-89 Effect of Zinc All preparations resulted in 75% C8-BHI co-crystals. In all preparations, the crystallization buffer was substantially in accordance with the procedure of Preparation 19 with a final citrate of 10 mM and a sodium chloride concentration of 50 mM. The scale was 20 mL. Perform protein lysis
pH was 2.5. Zinc was added as shown in the table. The zinc concentration is indicated by the weight ratio of the total protein (insulin + insulin derivative). 0.1% or more zinc is required,
There was no consistent effect on VMSED at zinc concentrations between 0.5% and 5.8%.

[0202]

Production Example 90 Production of co-crystal of 85% Nε-octanoyl-LysB29 human insulin and human insulin containing protamine and Zn A dry freeze-dried powder of Nε-octanoyl-LysB29 human insulin (1678.5 mg) was prepared in 0.1%.
Dissolved in 42.5 mL of N HCl. Human insulin-Zn crystal dry powder (286 mg) was added to 0.1N HCl 7
Dissolved in .5 mL. 7.5 mL of the latter solution was mixed with Nε-octanoyl-LysB29 human insulin to obtain a mixture of Nε-octanoyl-LysB29 human insulin and human insulin in an appropriate weight ratio of 75:25. This solution was stirred and mixed. Add 10mg / mL Zn to this solution
1.38 g of the solution were added. 9.5 mg / mL disodium phosphate heptahydrate, 0.375
200 mL of an aqueous solution (pH 7.63) consisting of M NaCl, 4 mg / mL m-cresol, 1.65 mg / mL phenol, and 16 mg / mL glycerol was added. Adjust the pH with small amounts of 5N HCl and 5N
Adjusted to 7.6 with NaOH. This solution was then filtered through a 0.2 micron low protein binding filter. A second solution was prepared by dissolving 262.9 mg of dry protamine sulfate powder in 305.69 g of water, and then filtering through a 0.2 micron protein low binding filter. Nε-octanoyl-LysB29 mixture solution of human insulin and human insulin 2
62.84 g was mixed with 259.6 g of protamine sulfate solution. A white precipitate formed. The suspension was gently stirred to complete the mixing. The preparation was left at 32 ° C. for 24 hours. Observation under an optical microscope indicated that a microcrystalline solid was present. Particle size distribution measurement by Coulter technique indicated an average particle diameter of 2.5 microns.
The mother liquor of this preparation was prepared as follows with a liquid solution consisting of 0.04 mg / mL phenol, 0.11 mg / mL m-cresol, and 0.3 mg / mL disodium phosphate heptahydrate, and 24 mg / mL glycerin. Replaced. Without disturbing the solid phase, 450 mL of the supernatant was removed from the well-settled precipitate and replaced with the aqueous solution. Analytical characterization by HPLC indicated a composition of 85.2% C8-BHI and 14.8% BHI. In canine intratracheal instillation experiments, these formulations were diluted to the required concentration by appropriate dilution with the aqueous solution used for the replacement. The microcrystals of this Production Example had an average particle diameter of 2.5 microns by Coulter multisizing. This preparation was used for intratracheal instillation in dogs.

Production Example 91 Isolation of an 85% co-crystal formulation (from Production Example 90 above) in powder form (approximately 1 g scale) About 400 mL of the crystal suspension obtained from Production Example 90 is applied to a 0.22 micron filtration device under vacuum And filtered. The solid is washed with about 5 mL of pure alcohol and air dried by applying vacuum to the filter. The agglomerated powder was recovered using a clean spatula.

Preparation 92 In Vitro Dissolution Properties of 85% Co-Crystal Formulations (from Preparations 90 and 91 above) The in vitro dissolution rate of the microcrystalline suspension obtained from Preparation 90 was measured at pH 7.4 The measurement was performed at 25 ° C. using acid-buffered saline (PBS). The PBS buffer contained 1 mg / mL bovine serum albumin to minimize the loss due to insulin adsorption. Total insulin
One volume of the suspension containing 1.8 mg was suspended in 200 mL of the buffer solution and stirred at a constant speed of 180 rpm. At regular intervals, aliquots of the solution were filtered through a 0.2 micron protein low binding filter and assayed for total dissolved insulin. An unfiltered control sample was also analyzed to determine total available insulin. Based on this assay, Preparation 90 took about 6 hours to completely dissolve. In contrast, NPH crystals dissolved in about 2 minutes. Further, Production Example 90 shows that as it dissolves, a constant ratio of C8-BH to BHI
I was released, and the co-crystallinity of Production Example 90 was confirmed. The isolated powder (Production Example 91) was prepared in Production Example 9
Retained a slow dissolution profile of 0, suggesting that the method of powder isolation and drying did not affect the dissolution profile. About 50% of the powder dissolved in about 3 hours. During this period, the powder was released at a constant ratio of about 85% C8-BHI and 15% BHI as it dissolved.
In addition, this homogeneous dissolving property confirms that the filtration and air-drying methods do not alter the basic properties of the microcrystals.

[0206] These in vitro dissolution data suggest that this crystallite, with a particle diameter of about 2.5 microns, dissolves very slowly compared to NPH, despite the increased surface area. These data support the possibility that Preparation Example 90 could serve as a sustained release formulation. These data further suggest that the isolation and drying methods do not alter the unique dissolution properties of the suspension.

Example 1 Two types of crystalline insulin, 100% C8-BHI crystal and 75% C8-BHI: 25% BHI co-crystal
Was tested. In both of these cases, intratracheally instilled compounds are higher for longer durations than previous studies performed in rats using normal insulin and immunoreactive insulin comparable to the results of subcutaneously supplied SPH human insulin. Resulted in blood levels. Interestingly, when lung lavage fluid was tested 4 and 8 hours after lung instillation, insulin crystals were observed mainly free and complete in lung lavage fluid, with relatively few crystals being found in alveolar macrophages. there were. The literature indicates that the digestion of the particles by macrophages in the lung is largely complete within a few hours, so most of the crystalline material was expected to have been taken up and digested by alveolar macrophages. This surprising finding is that if crystalline insulin is insoluble enough to prevent too rapid dissolution in the lung fluid, it will remain available to slowly dissolve and be absorbed in the blood. Suggest.
Thus, supplying these two types of crystalline insulin to the lungs may be a non-invasive approach for sustained release of insulin resulting in basal regulation of glucose.

When blood glucose levels were measured in these rat experiments, blood glucose levels decreased in proportion to the level of insulin absorption in the blood. These experiments show that for sustained release of insulin into the blood, pulmonary delivery is a suitable method for delivering insoluble insulin crystals to the lungs. Aerosol inhalation will be a method used in clinical use to deliver crystalline insulin to the patient's lungs to avoid the need for injections and improve patient compliance.

Example 2 Twelve male F344 rats / group were used in this study. The administration groups are as follows. Group 01: Intratracheal instillation of 100% C8-BHI 1 mg / kg produced according to Production Example 28 above. Group 02: 75% C8-BHI produced according to Production Example 29 above: 25% BHI 1 mg / kg intratracheal instillation. Group 03: 75% C8-BHI produced according to the above Production Example 29: 25% BHI 1 mg / kg subcutaneous administration. Group 04: subcutaneous administration of 1 mg / kg NPH insulin. Blood samples were administered and collected at 0 (pre), 0.5, 1, 4, 8, 16, and 24 hours. The blood samples were centrifuged to collect serum, and the blood level and glucose concentration of the test substance were measured. FIG. 1 shows the results of glucose measurement. These preparations, which were instilled into the trachea of rats, reduced blood glucose, resulted in sustained release comparable to subcutaneous administration of NPH-human insulin, and surprisingly high activity. These data indicate that these crystals and co-crystals can be administered by inhalation and particles with properties known to cause them to accumulate in the patient's lungs. Accumulate in the lung, making it reasonable to predict that these crystals or co-crystals will dissolve in the lungs and insulin activity will be absorbed into the patient's blood from the accumulated particles.

Example 3 Physical Stability and Resuspability Test This test evaluated the physical stability of four different 75% C8-BHI co-crystal formulations in a 3.0-mL cartridge under physical stress conditions. . Production Example 51-54 was filled in a 3.0 mL cartridge, and subjected to an accelerated physical stability test. Each cartridge was filled with 44 cartridges. 38 cartridges of each preparation were placed in an insulin shaker system at a constant temperature of 37 ° C. for 14 days. During this period, the material was stirred at 30 rpm for 4 hours a day. The cartridges were examined for fibrosis (aggregation or agglomeration) and showed physical changes on days 0, 2, 5, 7, 9, 11, and 14. 5 ° C without stirring (excluding resuspension) of a set of control cartridges (6 for each production example)
(With the side of the vertical cap raised) and examined every 7 days.

The cartridge did not show any of the following conditions as determined by visual inspection by trained personnel: Separated particles that settle more rapidly from a homogeneous milky suspension with slow sedimentation. Change to suspension showing, film on cartridge wall,
Frost, clamping (large aggregates), failure to resuspend, or any combination of the above reasons. The number of disappointing (failed) cartridges after the 14-day accelerated physical stability test was approximately the same for Production Examples 51 and 54. Production Example 53 shows the minimum number and Production Example 52
Had slightly more failures than Production Example 53 but less than Production Example 52 or 55.

Example 4 Glucose Response to 85% C8 Insulin: 15% BHI Co-Crystal Powder Inhaled into the Lung of F344 Rats To study the effect of powder in air supplied to the lung, rat lung was inhaled using an inhalation method. An experiment was performed in which 85% C8 insulin powder (Preparation Examples 90-92) was blown into the mixture. The dose is 2mg / kg
And Fasted male Fischer 344 rats (200-250 g) were briefly anesthetized with isoflurane and intubated into the trachea. Aliquots of the pre-weighed powder were prepared using inhalation-grade lactose and a geometric dilution method to ensure that the insulin crystals were homogeneously mixed with the carrier lactose at a concentration of 5% by weight. 10 mg of the powder was filled into size 00 capsules which were then attached to a Penn Century inhalation device. The Penn Century device was introduced into the endotracheal cannula and the powder was blown into the lungs by rapidly releasing 3 mL of air from a manual syringe. Dosing was synchronized with the animal's inspiration. 0 (before dosing) and 0 after dosing for glucose measurement
Blood samples were taken at .25, 0.5, 1, 3, 4, 6, 8, and 12 hours.

The figure below shows the glucose response (mean SE) after inhalation of 85% C8 insulin: 15% BHI crystal powder into rat lung. For unknown reasons, the 8-hour data point is likely to be significantly out of the normal distribution because there were two rats in this group with glucose levels above 50% of the baseline level. Ignoring this data point, the glucose response is similar to the 75% C8 insulin obtained in the previous experiment above.
: Extended time exactly as data obtained after inhalation of 25% BHI crystal suspension
-Show the action profile. These data indicate that both powder and liquid suspensions in air introduced into the lung produce similar reactions. Previous rat experiments have shown qualitatively similar glucose responses after inhalation of various types of crystalline insulin. Taken together, these results suggest that the results of the instillation test are excellent predictors of the results obtained from airborne powders introduced into the lungs. As this experiment shows, one crystalline insulin has a long-term effect on the glucose response after introducing air powder into the lung, and similar behavior is expected when other crystalline insulin is introduced into the lung as air powder. .

Glucose changes after intratracheal inhalation of 85% C8-insulin co-crystal and intratracheal instillation of 75% CS-insulin co-crystal in F344 rats

Example 5 Comparison of Three Intratracheal Insulin Crystalline Formulations to Beagle Dogs This study demonstrates the kinetics of NPH crystals after subcutaneous (sc) administration, NPH crystals, MicroUltralente crystals, and 85 It was designed to compare the kinetics after intratracheal (IB) administration of% C8 insulin co-crystal. The survival status was tested at Lilly's Toxicology Research Laboratories. Two male and three female adult Beagle dogs were used in this study. Weight at the start of the test is 7.1 ~
The range was 14.6 kg. Test substances used were 85% C8-insulin co-crystal, MicroUltralente crystal, and NP
It was an H crystal. Each test substance was specially formulated for pulmonary administration using modified crystallization conditions to obtain 2-3 μm particles. As shown in Table 1, a specific vehicle was used for each crystalline insulin preparation.

Table 1. Crystalline insulin formulations and related vehicles

Anesthetized fasted animals received 85% C8 insulin co-crystal, Mi
croUltralente crystals and NPH crystals were administered. As shown in Table 2, a dose of 0.25 mL / kg was instilled into the trachea by a bronchial fiberscope as a dose to the lung. For comparison purposes, each dog received 0.75 U / kg NPH crystals subcutaneously. Each treatment was separated by at least 7 days. Blood samples were pre-treatment and 0.5, 1, 2, 3, 4, 5, 6, 7, 8,
Collected at 9, 10, 12, 16, and 24 hours. For subcutaneous and pulmonary administration of NPH, blood samples were collected before treatment and at 0.5, 1, 2, 4, 6, 8, 10, 12, 16, and 24 hours after administration. The glucose concentration of the serum obtained from these samples was analyzed.

Table 2. Feeding dose

Results and Discussion Intratracheal instillation of all crystalline insulin formulations resulted in significant suppression of serum glucose levels (Table 3, FIG. 1). Mean blood glucose reached the initial low point at 2 hours for all formulations.

Table 3. Blood glucose after intratracheal or subcutaneous administration of a crystalline insulin formulation in Beagle dogs (% baseline)

[0221] Glucose levels remained significantly suppressed in all formulations up to about 10 hours after intratracheal or subcutaneous administration. The time-effect of sustained release was similar in all cases as far as intratracheal instillation of all formulations, as well as at least subcutaneous administration of NPH. N
Two dogs after intratracheal administration of PH and 85% C8 insulin co-crystal showed significant low glucose levels 7-10 hours post-administration, but were assisted by oral or intravenous administration of 50% dextrose. The potential sustained release effect of the group and the overall mean were reduced because of the artificial increase in serum glucose levels in the animals that helped with this event.

Changes in serum glucose levels after subcutaneous administration of NPH or intratracheal administration of NPH, MicroUltralente, and 85% C8-insulin co-crystal (mean ± SE)

Example 6 Aerosol Characterization of Three Insulin Crystalline Formulations A dry powder formulation of three crystalline insulins was aerosolized using a Wright Dust Feed (WDF) generator (operated at 10 LPM). Pass through a cyclone or WDF designed to remove large particles before entering the 12-L head-dome exposure system
Aerosol was generated by WDF directly through the head-dome. Gelman Typ
e Si / Model 218K Cascade Im with A / E glass fiber filter
The mass median aerodynamic diameter (MMAD) was measured using a pactor (cascade impactor). The airflow through the Cascade Impactor was 3 L / min and sampling times ranged from 5 to 50 minutes. Aerosol concentrations were determined by obtaining gravimetric samples during exposure.

Results Table 1 shows the particle size and gravimetric data of the three crystalline insulins. Table 1. Aerosol characterization Average (± SE) test substance MMEAD GSD Aerosol concentration (mg / L) 85% C8-insulin w / cyclone 1.15 3.98 NA w / o cyclone 1.30 4.03 0.069 10% MicroUltralente / 90% lactose w / Cyclone 2.74 1.61 0.280 w / o Cyclone 4.07 1.97 0.104 Spray-dry MicroUltralente w / cyclone 1.79 2.90 0.088 w / o Cyclone 2.31 3.04 0.040

These data indicate that the generation of aerosols of these crystalline insulins results in a very respirable particle size distribution. The data obtained from Examples 1-5 confirms that the instillation test predicts the effect of the inhaled dry powder. Tests were performed in beagle dogs to compare a) aerosol inhalation of a normal insulin solution, b) inhalation of a normal insulin powder, and C) glucose response after instillation of a normal insulin solution. The attached figures show that the time-effect profile of the glucose response is steep in all cases, with similar results for all three types of dose administration. The time to minimum glucose levels is less than 2 hours in all cases, with the reaction returning to near baseline within 4-5 hours. (Laube BL, Georgopoulus, Adams GK. 1992. Aerosolized insulin supplied from the lung is effective in normalizing plasma glucose levels in non-insulin dependent diabetes. J. Biopharm. Sci 3 (1992) 163. -169). Or powder (Patto
n J, Bukar J., and Najarajan S. 1999. Inhaled insulin.Adv.Drug Delive
ry Rev. 35: 235-247). These results confirm that the inhalation test predicts the same results achieved in the instillation test. A similar glucose response in rats after administration of an intratracheal instillation suspension of the same C8 co-crystal compared to inhalation of the C8 co-crystal powder also directly supports this consideration.

The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many modifications and changes may be made within the spirit and scope of the invention. All publications and patent applications in this specification are indicative of the levels of those skilled in the art to which this invention belongs.

[Brief description of the drawings]

FIG. 1 is a graph showing blood glucose levels after administration of crystals and co-crystals to F344 rats.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 3/10 A61K 37/26 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU) , TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, D , DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ , TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Benjamin Lee Hughes United States 46208 Blue Ridge Road, Indianapolis, Indiana No. 402 ( 72) Inventor Shun Li United States 46268 Bancaster Drive, Indianapolis, Indiana No. 7620 (72) Inventor N. Kinman United States 46032 Carmel, Indiana Putnam Reis 10745 (72) Inventor Muppara Skumar Bent Oak Lane, Indianapolis, Indiana 46236 United States 12604 (72) Inventor Ronald Keith Wolf United States 46033 Carmel, Indiana Brookshire Parkway 12329 F term (reference) 4C076 AA29 BB01 BB27 CC21 DD21 DD37 EE41 FF68 GG02 GG11 4C084 AA03 BA44 CA62 DB34 MA43 MA52 MA55 MA56 NA10 ZC35 4H045 AA30 DA37 EA27

Claims (26)

[Claims] 1. A crystal having a uni-modal symmetric particle distribution, comprising: a) a compound of the formula: B29-Nε-X-human insulin, wherein X is butyryl, pentanoyl, hexanoyl, heptanoyl, Volume mean spherical equivalent diameter (derivatized insulin) selected from the group consisting of octanoyl, nonanyl, and decanoyl), b) zinc, c) phenolic preservative, and d) protamine. equiva
a crystal having a lent diameter of 1 to 3 microns. 2. The crystal of claim 1, wherein the ratio of derivatized insulin to total protein is at least about 50%. 3. The crystal according to claim 1, wherein the derivatized protein is B29-Nε-octanoyl-human insulin. 4. The crystal according to claim 2, wherein the ratio of the derivatized insulin is 75%. 5. The crystal of claim 1, wherein zinc is present in about 0.3 mole to about 0.7 mole per mole of total protein. 6. The phenolic preservative is selected from the group consisting of phenol, m-cresol, and a mixture of phenol and m-cresol, and the total protein 1
A crystal according to any of the preceding claims, being present in a ratio of at least 0.5 mole per mole. 7. The crystal of claim 1, wherein protamine is present at about 0.15 mg to about 0.5 mg per 3.5 mg of total protein. 8. The crystal according to claim 1, further comprising a carrier,
Pharmaceutical compositions for administration by inhalation via mouth containing absorption enhancing compounds, excipients, or solvents. 9. The pharmaceutical composition according to claim 8, further comprising insulin, an insulin analog or a derivative, or a pharmaceutically acceptable salt thereof. 10. The method according to claim 1, for producing a medicament for treating diabetes.
Use of the crystal according to any one of the above items 7. 11. The method according to claim 1, comprising performing the following steps a) to f) in any order in the absence of citrate to produce a neutral pH suspension. (However, the step f) follows the step a), and the step f) is the step e).
Prior to step b) and c) prior to step f)): a) dissolving the derivatized insulin in an aqueous solvent at acidic pH, b) adding a phenolic preservative, c) adding zinc, d) Chloride anion is added to a final concentration of about 100 mM to about 150 mM chloride anion above that introduced by pH adjustment, e) Protamine added, f) adjusted to neutral pH, and then 12 hours to about 96 hours, The temperature of the neutral pH suspension is about 25 ° C
Keep at about 37 ° C. 12. The method of claim 11, wherein the insulin is also soluble and the ratio of derivatized insulin to total protein is at least 50%. 13. The derivatized insulin is B29-Nε-octanoyl-human insulin, and the chloride anion is added to a final concentration of about 120 above that introduced by pH adjustment.
13. The method according to claim 11 or 12, wherein the temperature is about 30 ° C in addition to ~ 150mM. 14. The derivatized insulin is B29-Nε-octanoyl-human insulin, wherein the percentage of derivatized insulin is at least 75% of the total protein, and the final concentration above which chloride anions are introduced by pH adjustment. 13. The method according to claim 11 or 12, wherein the temperature is about 30 ° C in addition to about 120-150 mM. 15. A method for treating diabetes, which comprises administering the crystal according to claim 1 parenterally. 16. A method for treating diabetes, which comprises administering the crystal according to claim 1 by inhalation through the mouth. 17. A method for producing crystals, comprising producing a suspension having a neutral pH by performing the following steps a) -g) in any order in the absence of citrate, provided that g) precedes step a) and steps b) and c) precede step g) when step g) precedes step f): a) dissolving the derivatized insulin in an aqueous solvent at acidic pH; ) Add phenolic preservative, c) Add zinc, d) Add chloride anion to a final concentration of about 15 mM to 150 mM chloride ion above that introduced by pH adjustment, e) Add citrate to 1 mM 1010 mM, f) Protamine, g) Adjust to neutral pH, then raise the temperature of the neutral pH suspension from about 20 ° C. to about 12 hours to about 96 hours.
Keep at 37 ° C. 18. The method of claim 17, wherein the insulin is also soluble and the ratio of derivatized insulin to total protein is at least 50%. 19. The method according to claim 18, wherein the proportion of the derivatized insulin is 75%. 20. The method according to claim 19, wherein the derivatized insulin is B29-Nε-octanoyl-human insulin. 21. Zinc is added to a concentration of 0.3 mole to 0.7 mole per mole of total protein and the phenolic preservative is phenol, m-cresol, and phenol and m-cresol.
2. A mixture selected from the group consisting of cresols and added to a concentration such that the ratio of phenolic preservative per mole of total protein is at least 0.5 mole.
21. The method according to any one of 7 to 20. 22. A crystal produced according to any of claims 17 to 20. 23. A pharmaceutical composition comprising the crystal according to claim 22, and further comprising a carrier, an absorption enhancing compound, an excipient or a solvent. 24. Insulin, an insulin analogue or a derivative,
24. The pharmaceutical composition according to claim 23, comprising a pharmaceutically acceptable salt thereof. 25. A method for producing a medicament for treating diabetes.
Use of the described crystals. 26. A method for treating diabetes, which comprises administering the crystal according to claim 22 parenterally.