Title:  Complex of human growth hormone and zinc

United States Patent:  6,191,107

Assignee:  Takeda Chemical Industries, Ltd. (Osaka, JP)

Filed:  September 11, 1998

Foreign Application Priority Data:    Sep 26, 1997[JP] (9-261251)

The present invention provides a complex of human growth hormone and zinc containing human growth hormone and zinc at a molar ratio of about 1:1.6 to about 1:2.4, and a sustained-release preparation which comprises the complex of human growth hormone and zinc and a biodegradable polymer and which has a high entrapment ratio of human growth hormone and exhibits a stable sustained-release suppressing the initial burst.

DETAILED DESCRIPTION OF INVENTION

Current sustained-release preparations containing GH have been produced by a method which comprises making GH in water (in water phase) and dispersing the water phase in organic solvent (in oil phase) containing a biodegradable polymer to make a water-in-oil emulsion. But, in this method, GH is remarkably denaturalized in the production process or on the shelves and a sufficient entrapment ratio and release is not obtained. On the one hand, a method which comprises dispersing GH powder into organic solvent (in oil phase) containing a biodegradable polymer to make a solid-in-oil dispersion is not appropriate for producing a sustained-release preparation on a large scale, since it is necessary to maintain stability by spraying the solid-in-oil dispersion into liquid nitrogen. Furthermore, since GH is not in the form of fine particles and is usually used after atomizing, the activity of GH is remarkably lowered by atomization of GH and it is difficult to make a solid-in-oil dispersion containing GH having a high content.

Thus, it is very difficult to maintain stability of GH and micronize GH in the process for producing preparations. Further, it is very difficult to produce quality sustained-release preparations containing a high content of GH on a large-scale without lowering the activity of GH but at the same time maintaining quality and stability.

Therefore, a clinically useful preparation comprising GH which overcomes the above problems and has constant release over a long period of time, and a method for producing the sustained-release preparation on a large scale at high yield, are desired.

The present inventors made extensive and intensive studies and as a result, made a complex of GH and zinc containing GH and zinc at a molar ratio of about 1:1.6 to about 1:2.4 for the first time. Further, they found that the complex is substantially water-soluble and the micronization of the complex is easier than GH itself, without lowering the activity of GH, and when the obtained complex of GH and zinc having a small particle diameter is used to produce a sustained-release preparation, the sustained-release preparation can be produced on a large scale with assured stability and without denaturalizing GH in the process, having also an enhanced entrapment of GH and an improvement in release properties.

Namely, the present invention provides

(1) a complex of human growth hormone and zinc containing human growth hormone and zinc at a molar ratio of about 1:1.6 to about 1:2.4,

(2) a complex of the above (1), which is water-soluble,

(3) a complex of the above (1), wherein the mean particle diameter of the complex is less than about 10 .mu.m,

(4) a sustained-release preparation, which comprises the complex of the above (1) and a biodegradable polymer,

(5) a preparation of the above (4), wherein the biodegradable polymer is an aliphatic polyester,

(6) a preparation of the above (5), wherein the aliphatic polyester is a polymer of lactic acid and glycolic acid,

(7) a preparation of the above (6), wherein the content ratio of a polymer of lactic acid and glycolic acid is 100/0 to 40/60 (mole %),

(8) a preparation of the above (5), wherein the weight-average molecular weight of the aliphatic polyester is about 3,000 to about 20,000,

(9) a preparation of the above (5), wherein the aliphatic polyester is a salt of polyvalent metal,

(10) a preparation of the above (9), wherein the polyvalent metal is zinc,

(11) a preparation of the above (4), wherein the preparation is a microcapsule,

(12) a preparation of the above (11), wherein the microcapsule is for injection,

(13) a preparation of the above (4), wherein the initial burst ratio of GH is less than about 50%,

(14) a method for producing a complex of human growth hormone and zinc, which comprises mixing human growth hormone and zinc salt at a molar ratio of about 1:1.6 to about 1:2.4,

(15) a method for producing micronized human growth hormone, which comprises forming a complex of human growth hormone and zinc containing human growth hormone and zinc at a molar ratio of about 1:1.6 to about 1:2.4 and atomizing them,

(16) use of a complex of human growth hormone and zinc containing human growth hormone and zinc at a molar ratio of about 1:1.6 to about 1:2.4 for producing a sustained-release preparation containing human growth hormone,

(17) a method for producing a sustained-release preparation containing human growth hormone, which comprises dispersing a complex of human growth hormone and zinc containing human growth hormone and zinc at a molar ratio of about 1:1.6 to about 1:2.4 in an oil phase containing a biodegradable polymer to make a solid-in-oil emulsion, adding the solid-in-oil emulsion to water phase to make a solid-in-oil-in-water emulsion, and then in-water drying the solid-in-oil-in-water emulsion,

(18) a pharmaceutical composition which comprises the complex of the above (1), and

(19) a pharmaceutical composition for treating or preventing pituitary drawfism, which comprises the complex of the above (1).

GH used in the present invention may be any type, for example, natural type (extracted products, etc.) or genetic recombinant type GH (Nature Vol.281, page 544 (1979), Vol.293, page 408(1981), Proc. Natl. Acad. Sci. USA, Vol.80, page 397(1983), Biotechnol., Vol.5, page 161(1981), etc.), and genetic recombinant type GH is preferred in terms of its safety and quality. Further, in the present invention, muteins, derivatives, analogous and active fragments of GH may be used as GH (J.Biol.Chem., Vol.253, page 2679 (1978), B.B.R.C., Vol.92, page 511 (1980), Endocrinol., Vol.109, page 1301(1981), Protein Eng. Vol.3, page 49(1989), etc.).

Complexes of GH and zinc according to the present invention may be produced by any method, for example, methods generally used in the production of complexes suiteable for a molar ratio of GH and zinc in the range of from about 1:1.6 to about 1:2.4. The said complex of GH and zinc is usually produced by bringing GH in to contact with a water-soluble zinc salt. This contact reaction is preferably employed in a solvent, for example, aqueous-solvent. The reaction time ranges from 1 minute to 1 hour. The reaction temperature ranges from 4^oC. to 37^oC. Water-soluble zinc salts used in this method, include salts of zinc and inorganic acids, salts of zinc and organic acids and so on. Inorganic acids include hydrochloric acid, sulfuric acid, nitric acid, thiocyanic acid and so on and organic acids include aliphatic carboxylic acids, aromatic acids and so on.

Examples of aliphatic carboxylic acids used as organic acids, are aliphatic monocarboxylic acids, aliphatic dicarboxylic acids, and aliphatic tricarboxylic acids. These aliphatic carboxylic acids may be saturated or unsaturated. The aliphatic carboxylic acid is preferably an aliphatic carboxylic acid having 2 to 9 carbon atoms.

Examples of aliphatic monocarboxylic acids are saturated aliphatic monocarboxylic acids having 2 to 9 carbon atoms (e.g., acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, caprynic acid etc.) and unsaturated aliphatic monocarboxylic acids having 2 to 9 carbon atoms (e.g., acrylic acid, propiolic acid, methacrylic acid, crotonic acid, isocrotonic acid etc.).

Examples of aliphatic dicarboxylic acids are saturated aliphatic dicarboxylic acids having 2 to 9 carbon atoms (e.g., malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid etc.) and unsaturated aliphatic dicarboxylic acids having 2 to 9 carbon atoms (e.g., maleic acid, fumaric acid, citraconic acid, mesaconic acid etc.).

Examples of aliphatic tricarboxylic acids are saturated aliphatic tricarboxylic acids having 2 to 9 carbon atoms (e.g., tricarballylic acid, 1,2,3-butanetricarboxylic acid etc.).

The above-mentioned aliphatic carboxylic acids may have 1 or 2 hydroxyl groups. Such aliphatic carboxylic acids include glycolic acid, lactic acid, glyceric acid, tartronic acid, malic acid, tartaric acid, citric acid and so on.

The aliphatic carboxylic acid is preferably an aliphatic monocarboxylic acid, more preferably an aliphatic monocarboxylic acid having 2 to 9 carbon atoms, and still more preferably a saturated aliphatic monocarboxylic acid having 2 or 3 carbon atoms. Examples of particularly preferable aliphatic carboxylic acids include acetic acid and so on.

Examples of aromatic carboxylic acids used as the above-organic acid are benzoic acid and salicylic acid, with preference given to benzoic acid.

For example, a complex of GH and zinc in the present invention is produced by mixing GH and a water-soluble zinc salt at a mixing ratio (molar ratio) of about 1:1.6 to about 1:2.4, preferably about 1:1.8 to about 1:2.2 in a aqueous solvent (e.g., aqueous solutions containing ethanol, acetonitril or acetone at a concentration (for example, about 1 to about 10% (W/W)) which does not exert an adverse influence on the solubility of GH and a water-soluble zinc salt, preferably water). The said complex may be a compound (complex salt, double salt, salt, and organic metal compound etc.) formed by intermolecular binding between GH and zinc or a mixture of compounds which differ in their binding patterns. The composition ratio (molar ratio) of GH and zinc in the complex of GH and in the present invention is within the scope of about 1:1.6 to about 1:2.4, preferably about 1:1.8 to about 1:2.2, more preferably about 1:2. In complex of GH and zinc of the present invention, as though it is preferred that all of GH and zinc contained at a molar ratio of about 1:1.6 to about 1:2.4 is in the form of a complex, GH and/or zinc which do not form a complex may be present.

The pH of the aqueous solution resulting from the above mixing must be such that the bioactivity of GH is not affected, and such that each solubility of GH and zinc salt is not lowered in excess. Although the mixing procedure is normally conducted in distilled water, it may be conducted in water adjusted to be weakly acidic, neutral, or weakly alkaline pH (pH 6 to 9) as necessary. the concentration of GH and water-soluble zinc salt in the water may range within each solubility.

The thus-obtained complex of GH and zinc in water is substantially water-soluble since no precipitate is visibly found in the water. A substantially water-soluble complex of GH and zinc means that the solubility of the complex in 1ml of water (pH 6 to 8) at normal temperature is more than about 2 mg.

This complex of GH and zinc in water is used for producing a pharmaceutical composition, preferably a sustained-release preparation after being vacuum dried or lyophylized and micronized.

The obtained powder of the complex of GH and zinc is fine-grained and is easier to handle than bulky powder of GH free from zinc, and is very useful for producing a sustained-release preparation on a large scale. For example, a complex of GH and zinc can be obtained as a powder with a mean particle diameter of less than about 10 .mu.m, preferably about 4 to about 7 .mu.m.

In the case where the complex is dispersed into an organic solvent containing hereinafter-mentioned biodegradable polymer, particles having a small diameter are very useful for obtaining an enhanced entrapment ratio of GH and an improved release. For example, an entrapment ratio of GH in the sustained-release preparation is preferably more than about 90% and with regard to the sustained-release of GH, an initial burst ratio of GH is preferable less than about 50%.

The content of the complex of GH and zinc in the sustained-release preparation of the present invention is normally about 0.1% (W/W) to about 40% (W/W), preferably about 1% (W/W) to about 20% (W/W).

The biodegradable polymer is exemplified by high-molecular polymers being slightly soluble or insoluble in water, such as aliphatic polyesters (e.g., homopolymers, copolymers or mixtures thereof synthesized from one or more .alpha.-hydroxycarboxylic acids such as glycolic acid, lactic acid, hydroxybutyric acid etc.), hydroxydicarboxylic acids such as malic acid etc., hydroxytricarboxylic acids such as citric acid etc. and others, poly-.alpha.-cyanoacrylic acid esters, polyamino acids such as poly-.gamma.-benzyl-L-glutamic acid and so on. These may be used in mixture at appropriate ratios. The type of polymerization may be random, block or graft.

The biodegradable polymer is preferably an aliphatic polyester (e.g., a homopolymer, copolymer or mixture thereof synthesized from one or more .alpha.-hydroxycarboxylic acids such as glycolic acid, lactic acid, hydroxybutyric acid etc., hydroxydicarboxylic acids such as malic acid etc., hydroxytricarboxylic acids such as citric acid etc. and others).

Among the above-mentioned aliphatic polyesters, homopolymers or copolymers synthesized from one or more .alpha.-hydroxycarboxylic acids (e.g., glycolic acid, lactic acid, hydroxybutyric acid etc.) are preferred from the viewpoint of reliable biodegradability and biocompatibility. More preferably, the aliphatic polyester is a copolymer synthesized from one or more .alpha.-hydroxycarboxylic acids (e.g., glycolic acid, lactic acid, hydroxybutyric acid etc.). Also, these copolymers may be used in mixture.

Although the above-described .alpha.-hydroxycarboxylic acid may be of the D-, L- or D,L-configuration, it is preferable that the ratio of the D-/L-configuration (mole %) falls within the range from about 75/25 to about 25/75. The ratio of the D-/L-configuration (mole %) is more preferably about 60/40 to about 30/70.

Examples of copolymers of the above-described .alpha.-hydroxy-carboxylic acid include copolymers of glycolic acid with another .alpha.-hydroxy acid, which is preferably lactic acid, 2-hydroxybutyric acid etc.

The .alpha.-hydroxycarboxylic acid copolymer is preferably a lactic acid-glycolic acid copolymer, a 2-hydroxybutyric acid-glycolic acid copolymer etc., more preferably, the .alpha.-hydroxycarboxylic acid copolymer is a lactic acid-glycolic acid copolymer etc.

With respect to the lactic acid-glycolic acid copolymer (hereinafter generally called lactic acid-glycolic acid polymer or PLGA), it is preferable that the content ratio (lactic acid/glycolic acid ratio, hereinafter called L/G) (mole/mole %) be about 100/0 to about 40/60. The content ratio is more preferably about 90/10 to about 45/55, and more preferably about 80/20 to about 45/55. The weight-average molecular weight of the lactic acid-glycolic acid copolymer is about 3,000 to about 20,000, preferably about 3,000 to about 16,000 more preferably about 3,000 to about 14,000.

Also, the degree of dispersion of the lactic acid-glycolic acid copolymer (weight-average molecular weight/number-average molecular weight) is preferably about 1.2 to about 4.0, more preferably about 1.5 to about 3.5.

The lactic acid-glycolic acid copolymer can be synthesized by a known process, such as the method described in Japanese Patent Unexamined Publication No. 28521/1986. It is preferable that the copolymer be synthesized by catalyst-free dehydration polymerization condensation.

With respect to the 2-hydroxybutyric acid-glycolic acid copolymer, it is preferable that glycolic acid accounts for about 10 to about 75 mole % and 2-hydroxybutyric acid for the remaining portion. More preferably, glycolic acid accounts for about 20 to about 75 mole %, and still more preferably about 30 to about 70 mole %. The weight-average molecular weight of the 2-hydroxybutyric acid-glycolic acid copolymer is preferably about 2,000 to about 20,000. The degree of dispersion of the 2-hydroxybutyric acid-glycolic acid copolymer (weight-average molecular weight/number-average molecular weight) is preferably about 1.2 to about 4.0, more preferably about 1.5 to about 3.5. A 2-hydroxybutyric acid-glycolic acid copolymer can be synthesized by a known process, such as that described in Japanese Patent Unexamined Publication Nos. 28521/1986 and 112465/1993. It is preferable that the copolymer be synthesized by catalyst-free dehydration polymerization condensation.

Preferable example homopolymers of the above-described .alpha.-hydroxycarboxylic acid include homopolymer of lactic acid. The weight-average molecular weight of the homopolymer of lactic acid is about 3,000 to about 20,000, preferably about 3,000 to about 14,000.

A homopolymer of lactic acid can be synthesized by a known process, such as that described in Japanese Patent Unexamined Publication No. 28521/1986. It is preferable that the homopolymer be synthesized by catalyst-free dehydration polymerization condensation.

The above-described 2-hydroxybutyric acid-glycolic acid copolymer may be used in a mixture with polylactic acid. Although the polylactic acid may be of the D- or L-configuration or a mixture thereof, it is preferable that the ratio of the D-/L-configuration (mole %) fall within the range from about 75/25 to about 20/80. The ratio of the D-/L-configuration (mole %) is more preferably about 60/40 to about 25/75, and still more preferably about 55/45 to about 25/75. The weight-average molecular weight of polylactic acid is preferably about 1,500 to about 20,000, more preferably about 1,500 to about 10,000. Also, the degree of dispersion of the polylactic acid is preferably about 1.2 to about 4.0, more preferably about 1.5 to about 3.5.

For producing polylactic acid, two methods are known: ring-opening polymerization of lactide, a dimer of lactic acid, and dehydration polymerization condensation of lactic acid. For obtaining a polylactic acid of relatively low molecular weight for the present invention, direct dehydration polymerization condensation of lactic acid is preferred. This method is, for example, described in Japanese Patent Unexamined Publication No. 28521/1986.

When a 2-hydroxybutyric acid-glycolic acid copolymer and polylactic acid are used in mixture, their mixing ratio is about 10/90 to about 90/10 (% by weight). The mixing ratio is preferably about 20/80 to about 80/20, and more preferably about 30/70 to about 70/30.

In the present specification, weight-average molecular weight is defined as the molecular weight obtained by gel permeation chromatography (GPC) with 9 polystyrenes as reference substances with respective weight-average molecular weights of 120,000, 52,000, 22,000, 9,200, 5,050, 2,950, 1,050, 580 and 162. Number-average molecular weight based on GPC measurement is also calculated. The degree of dispersion is calculated from the weight-average molecular weight and the number-average molecular weight. Measurements are taken using a GPC column KF804L.times.2 (produced by Showa Denko) and an RI monitor L-3300 (produced by Hitachi, Ltd.) with chloroform as the mobile phase.

The above-described copolymer synthesized by catalyst-free dehydration polymerization condensation, usually has a terminal carboxyl group.

In the present invention, the biodegradable polymer preferably has a terminal carboxyl group.

A biodegradable polymer having a terminal carboxyl group is a polymer in which the number-average molecular weight by GPC determination and that by terminal group determination almost agree.

By terminal group quantitation, number-average molecular weight is calculated as follows:

About 1 to 3 g of the biodegradable polymer is dissolved in a mixed solvent of acetone (25 ml) and methanol (5 ml), and the solution is quickly titrated with a 0.05 N alcoholic solution of potassium hydroxide while being stirred at room temperature with phenolphthalein as an indicator to determine the terminal carboxyl group content; the number-average molecular weight based on terminal group quantitation is calculated using the following equation: Number-average molecular weight based on terminal group quantitation=20,000 A/B

A: Weight mass (g) of the biodegradable polymer

B: Amount (ml) of the 0.05 N alcoholic solution of potassium hydroxide added until the titration end point is reached.

For example, in the case of a polymer having a terminal carboxyl group synthesized from one or more .alpha.-hydroxy acids by catalyst-free dehydration polymerization condensation, the number-average molecular weight based on GPC measurement and the number-average molecular weight based on terminal group quantitation almost agree. On the other hand, in the case of a polymer having essentially no terminal carboxyl group synthesized from a cyclic dimer by ring-opening polymerization using a catalyst, the number-average molecular weight based on terminal group quantitation is significantly higher than the number-average molecular weight based on GPC determination. This difference makes it possible to clearly differentiate a polymer having a terminal carboxyl group from a polymer having no terminal carboxyl group.

While the number-average molecular weight based on terminal group quantitation is an absolute value, the number-average molecular weight based on GPC determination is a relative value that varies depending on various analyti-cal conditions (e.g., kind of mobile phase, kind of column, reference substance, slice width chosen, baseline chosen etc.); it is therefore difficult to have an absolute numerical representation of the latter. However, the fact that the number-average molecular weight based on GPC determination almost agrees with the number-average molecular weight based on terminal group quantitation means that the number-average molecular weight based on terminal group quantitation falls within the range from about 0.5 to about 2 times, preferably from about 0.8 to about 1.5 times as high as the number-average molecular weight based on GPC determination. Also, the fact that the number-average molecular weight based on terminal group quantitation is significantly higher than the number-average molecular weight based on GPC determination means that the number-average molecular weight based on terminal group quantitation is about 2 times or more as high as the number-average molecular weight based on GPC determination.

As the biodegradable polymer of the present invention, a metal salt (also referred to as a complex) of the above-described biodegradable polymer is preferably used. For instance, a polyvalent metal salt of each kind of biodegradable polymer disclosed in WO 97/01331, preferably a divalent metal salt, especially a zinc salt of lactic acid-glycolic acid copolymer, is preferably used. Biodegradable polymers can be produced by a process described in WO 97/01331 and modifications thereof.

When the polyvalent metal salt of the biodegradable polymer is zinc, the polymer may be produced by reacting the biodegradable polymer with zinc oxide in an organic solvent.

In said process, the biodegradable polymer and zinc oxide are first allowed to exist together in an organic solvent to prepare a solution of a complex of a biodegradable polymer and zinc oxide in the organic solvent. Although the concentration of the biodegradable polymer in the solution varies depending on the molecular weight and the type of the organic solvent, it is, for instance, about 0.1 to about 80% (W/W), preferably about 1 to about 70% (W/W) and more preferably about 2 to about 60% (W/W). Although the amount of zinc oxide added varies depending on the type of organic solvent, it is, for instance, about 0.001 to about 2% (W/W), preferably about 0.01 to about 1.5% (W/W) and more preferably about 0.1 to about 1% (W/W), based on the amount of the biodegradable polymer.

Regarding the order of addition of the biodegradable polymer and zinc oxide to the organic solvent, zinc oxide both in a powder state or in a dispersed state in the organic solvent can be added to a solution of the biodegradable polymer in the organic solvent, conversely, a solution of the biodegradable polymer in the organic solvent can be added to a dispersion of zinc oxide in the organic solvent. Furthermore, the organic solvent can be added after the biodegradable polymer and zinc oxide both in a powder state have been admixed.

The conditions required to produce a solution of a complex of a biodegradable polymer and zinc oxide, such as complex of PLGA and zinc oxide, from a biodegradable polymer and zinc oxide can be changed according to the type of biodegradable polymer used, the particle diameter of zinc oxide, the type of organic solvent, and the composition ratio. When PLGA is, for example, employed as the polymer, a complex of PLGA and zinc oxide can be obtained by the above reaction usually at about 0 to about 30^oC., preferably about 2 to about 25^oC., for about 1 to about 168 hours, preferably about 12 to about 96 hours, more preferably about 24 to about 72 hours. The production of a complex of PLGA and zinc oxide in the present invention can be confirmed visibly since zinc oxide which is in a dispersed state at the time of addition dissolves in the organic solvent to give a clear solution. The reaction time is not limited to the above ranges and can be determined using turbidity as an index.

Although this reaction proceeds simply by the co-presence of PLGA and zinc oxide in the organic solvent, the reaction carried out under stirring or shaking advantageously reduces of the reaction time. Furthermore, the reaction carried out under ultrasonication is equally preferred. As the reaction temperature becomes higher, the reaction time becomes shorter.

The thus obtained complex of biodegradable polymer and zinc oxide is applied to the next process, preferably as a solution in an organic solvent, or if necessary as a solid after removal of the organic solvent.

The sustained-release preparation of the present invention is produced by removing the organic solvent from dispersion, preferably a solid-in-oil dispersion in which a GH and zinc containing complex at molar ratio of about 1:1.6 to about 1:2.4, preferably as a powder, is dispersed into a solution of a biodegradable polymer (hereafter also means "biodegradable polymer" including a metal salt of the biodegradable polymer) in an organic solvent (oil phase). Methods of producing a sustained-release preparation include the in-water drying method, phase separation method, spray drying method, and modifications thereof.

Claim 1 of 10 Claims

What is claimed is:

1. A sustained-release preparation, comprising a complex of human growth hormone and zinc containing the human growth hormone and the zinc at a molar ratio, of about 1:1.6 to about 1:2.4and a biodegradable polymer.

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