Pharmaceutical component based on human parathyroid hormone and a pharmaceutical composition for intranasal administration comprising the component Number:7,087,248 from the United States Patent and Trademark Office (PTO) owispatent

Title: Pharmaceutical component based on human parathyroid hormone and a pharmaceutical composition for intranasal administration comprising the component

Abstract: A medical component comprising a human parathyroid hormone peptide or its derivative, and acetic acid contained at a concentration less than its chemical equivalent with respect to the human parathyroid hormone peptide or to its derivative. Since in the medical component acetic acid, which is present as a salt of or attached to the peptide or its derivative, has been reduced to an amount less than chemical equivalent with respect to the human parathyroid hormone peptide or its derivative, a medical component, which is highly stable and will ensure an excellent use feeling when introduced into a pharmaceutical composition, is obtained.

Patent Number: 7,087,248 Issued on 08/08/2006 to Minamitake,   et al.

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Inventors:	Minamitake; Yoshiharu (Gunma, JP), Ono; Tetsu (Tatebayashi, JP), Kawanishi; Koji (Tatebayashi, JP), Suzuki; Yuji (Ashikaga, JP)
Assignee:	Daiichi Asubio Pharma Co., Ltd. (Tokyo, JP)
Appl. No.:	10/312,726
Filed:	June 29, 2001
PCT Filed:	June 29, 2001
PCT No.:	PCT/JP01/05674
371(c)(1),(2),(4) Date:	December 30, 2002
PCT Pub. No.:	WO02/02136
PCT Pub. Date:	January 10, 2002

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Foreign Application Priority Data

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Jun 30, 2000 [JP]			2000-237717
Jun 30, 2000 [JP]			2000-237718

Current U.S. Class:	424/562 ; 514/12; 514/21; 530/324
Current International Class:	A61K 35/55 (20060101); A61K 38/00 (20060101); C07K 14/00 (20060101)
Field of Search:	424/562 514/2,12 530/324

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References Cited [Referenced By]

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U.S. Patent Documents

	4086196		April 1978		Tregear
	5856138		January 1999		Fukuda
	5977070		November 1999		Piazza et al.

Foreign Patent Documents

	515228		Nov., 1992		EP
	672682		Sep., 1995		EP
	64-16799		Jan., 1989		JP
	97/14429		Apr., 1997		WO

Other References

W M. Law, Jr., et al. "Preparation of Synthetic Bovine Parathyroid Hormone Fragment 1-34 for Pareneral use in Human Studies;" Journal of Clinical Endocrinology and Metabolism, vol. 56, No. 6, pp. 1335-1337 (1983). cited by other . Muawia M. Ibrahim, et al. "Maintenance of Normocalcemia by Continuous Infusion of the Synthetic Bovine Parathyroid Hormone (1-34) in Parathyroidectomized Rats;" Calcified Tissue International, vol. 34, No. 6, pp. 553-557 (1982). cited by other.

Primary Examiner: Carlson; Karen Cochrane
Assistant Examiner: Tsay; Marsha
Attorney, Agent or Firm: Armstrong, Kratz, Quintos, Hanson & Brooks, LLP

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Claims

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The invention claimed is:

1. A pharmaceutical component comprising a human parathyroid hormone peptide or its derivative, and acetic acid contained in an amount less than its chemical equivalent amount with respect to the human parathyroid hormone peptide or to its derivative, as calculated from the number of excess basic amino acid residues in the molecule of the human parathyroid hormone peptide or its derivative.

2. A pharmaceutical component as described in claim 1 comprising a salt of the human parathyroid hormone peptide or of its derivative with acetic acid wherein acetic acid is contained in an amount less than its chemical equivalent amount with respect to the human parathyroid hormone peptide or to its derivative.

3. A pharmaceutical component as described in claim 1, wherein the human parathyroid hormone peptide or its derivative is a peptide comprising the amino acid sequence of SEQ ID NO: 1.

4. A pharmaceutical component as described in claim 3, wherein the pharmaceutical component comprises 3 weight % or less acetic acid with respect to the weight of the peptide.

5. A pharmaceutical component as described in claim 1, wherein the pharmaceutical component is a lyophilized composition.

6. A pharmaceutical component as described in claim 1, wherein the pharmaceutical component is for intranasal administration.

7. A prior-to-use dissolvable pharmaceutical product comprising a lyophilized portion and a dissolving solution portion attached thereto wherein the pharmaceutical component as described in claim 1 is contained in the lyophilized portion.

8. A pharmaceutical component as described in claim 1, wherein the human parathyroid hormone peptide or its derivative is a peptide comprising the amino acid sequence of SEQ ID NO: 2.

9. A pharmaceutical component as described in claim 8, wherein the pharmaceutical component comprises 4 weight % or less acetic acid with respect to the weight of the peptide.

10. A pharmaceutical component as described in claim 8, wherein the pharmaceutical component comprises 6 weight % or less acetic acid with respect to the weight of the peptide.

11. A pharmaceutical composition for intranasal administration comprising a human parathyroid hormone peptide or its derivative and acetic acid contained in an amount less than its chemical equivalent amount with respect to the human parathyroid hormone peptide or to its derivative, as calculated from the number of excess basic amino acid residues in the molecule of the human parathyroid hormone peptide or its derivative.

12. A pharmaceutical component comprising a human parathyroid hormone peptide, and acetic acid contained in an amount less than its chemical equivalent amount with respect to the human parathyroid hormone peptide or to its derivative, as calculated from the number of excess basic amino acid residues in the molecule of the human parathyroid hormone peptide; wherein the human parathyroid hormone peptide is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.

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Description

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FIELD OF THE INVENTION

The present invention relates to a pharmaceutical component based on human parathyroid hormone which has an excellent stability, and ensures a good use feeling when used as a component of a pharmaceutical composition. In a further aspect, this invention relates to a pharmaceutical composition based on human parathyroid hormone for intranasal administration which is suitable for a long term use.

BACKGROUND ART

The peptides of human parathyroid hormone (to be referred to as "hPTH" hereinafter) are biologically active peptides which are responsible for bone metabolism, and have an strong activity to develop bones (Aurbach et al., Recent Progr. Horm. Res., 1972, vol. 28, p. 35). The hPTH is a peptide typically composed of 84 amino acid residues (hPTH(1-84)). A derivative, hPTH(1-34), from hPTH(1-84) composed of 34 amino acid residues designated as amino acid Nos. 1-34 of hPTH(1-84) has been also known to have the same pharmacological activity as does hPTH(1-84) (Tregear et al., Hoppe-Seyler's Z. Physiol. Chem., 1974, vol. 355, p. 415). The amino acid sequences of hPTH(1-84) and hPTH(1-34) are given as Sequence Nos. 1 and 2 in the Sequence Listing, respectively.

Calcitonin and bisphosphonate or therapeutic agents used for the treatment of osteoporosis exhibit their therapeutic effects by inhibiting bone resorption, whereas hPTH(1-84) and hPTH(1-34) stimulate bone formation, or bone metabolism involved in the bone formation. Thus, those peptides have been expected to serve as a new therapeutic agent of osteoporosis (Lane et al., J. Clin. Invest., 1998, vol. 102, pp. 1627 1633).

It has been reported, when hPTH(1-34) is subcutaneously applied to humans at a certain dose once weekly, it will increase the bone mineral content, while, when the same agent is similarly applied at a dose one fifth the previous one but once daily for five days successively, it will also increase the bone of mineral density (BMD) significantly (Sone et al., Miner Electrolyte Metab., 1995, vol. 21, pp. 232 235). In animal experiments it has been shown that hPTH, when given subcutaneously at a certain dose once weekly, will cause less BMP to bones than is observed when it is given divisional (Tawaragi et al., Osteoporosis International, vol. 6, suppl. 1, 1996, p. 245). This suggests a maximum therapeutic effect from hPTH will be obtained when hPTH is given daily at a small dose over a long period than when it is given at a large dose for a short period with a long interval between successive doses. If a small dose of hPTH given continuously over a long period will ensure an therapeutic effect equal to or more excellent than does a large dose given intermittently for a short period, this prescription will be also desirable from the view-point of small doses of hPTH being probably free from the adverse effects on the digestive and cardiovascular organs which are known for high dose administrations of hPTH.

Also, injections would be unsuitable to be used for the treatment of the patient with osteoporosis who will usually require a long term treatment, because the patient should then receive the treatment under the management of a physician; feel a more or less pain during treatment; and visit the physician's office regularly for treatment which would be a burden to the patient.

In view of this, there is a need for a nasal drug which would allow the patient to easily take it at home daily over a long period with no undue pain and burden imposed upon the patient.

However, for a nasal drug to be safely used continuously over a long period it is absolutely necessary that the drug should be smoothly absorbed through the nasal mucosa; have no irritating action against the nasal mucosa; and give an excellent use feeling, because the nasal mucosa is very sensitive to the irritating action of a medicine or an additive thereof. Particularly, for such a drug as the one prepared from hPTH which is expected to give a therapeutic effect when used over a long period, an excellent use feeling is very important when it is intended to be used for intranasal administration. In order to prepare a nasal drug which is acceptable even when used continuously over a long period, it is important to select the active agent in the form of a medicament or of its salt, and proper additives, to determine their effective concentrations, and to optimize their combination. The factors responsible for the use feeling of a nasal drug involve the odor and irritating activity of the drug. Thus, the kinds of medicaments or additives used for a nasal drug and their concentrations are very limited.

There is a commercially available product prepared from hPTH, that is, an injection containing a 5-acetate of hPTH(1-34) which is used as a diagnostic agent for assaying the functional activity of parathyroid (whose generic name is teriparatide acetate and which is provided by Asahi Kasei Kogyo Corp.). However, no intranasal drug based on hPTH is available that will give a satisfactory use feeling in terms of odor and irritability.

Japanese Patent Laid-Open No. 64-16799 describes that, when hPTH(1-34) is purified, it is mixed with acetic acid which is used for the purification process, and the acetic acid content in individual products vary widely from lot to lot, making it difficult to obtain products containing a uniform amount of acetic acid, and that introduction of acetic acid to the product will lead to a reduced activity of the product.

The same document further discloses a method suitable for improving the stability of hPTH(1-34) wherein a lyophilized composition of hPTH(1-34) based on the use of tartaric acid is utilized. However, tartaric acid is so highly acidic that an agent containing it will not be suitable for intranasal administration.

Japanese Patent Laid-Open No. 2-111 discloses a powdery composition for intranasal administration based on a water soluble organic acid which has been developed to improve the nasal absorption of hPTH(1-34) a biologically active peptide such as hPTH (1-34). However, the composition would not be suitable for a long term use, because it will directly irritate the nasal mucosa, depending on the kind and amount of coexistent organic acid.

As discussed above, no intranasal drug based on hPTH that is acceptable even when used over a long period, and ensures an excellent use feeling as well as good stability and absorption has been developed.

DISCLOSURE OF INVENTION

This invention aims at providing a pharmaceutical component based on hPTH which is highly stable, when used as a component of a pharmaceutical composition, and gives an excellent use feeling. In a still other aspect, this invention aims at providing a pharmaceutical composition for intranasal administration based on hPTH which will allow a long term use.

To meet the object of providing a pharmaceutical composition for intranasal administration as described above, the present inventors had studied hard, and reached a finding that an hPTH preparation conventionally prepared is unsatisfactory because it will give an acidic odor and irritation when administered to the nasal mucosa, and that this inconvenience is ascribed to acetic acid which is used in the purification process and exists in very small amount as the constituent of a salt of hPTH or an adherent. Based on this finding, they prepared a pharmaceutical component whose acetic acid content is reduced as compared with the previous one, assessed it and surprisingly found that the component in question is highly stable, gives an excellent use feeling when incorporated in a pharamceutical composition for, and presents with a property of being safely combined with the appropriate amounts of functional components which will be added for the improvement of absorption and stability, as well as with a carrier and excipient which are usually used during preparing medicines. Thus, they achieved this invention.

To put it more specifically, the present invention relates to a pharmaceutical component comprising hPTH and acetic acid whose content is kept less than a certain chemical equivalent with respect to the weight of hPTH. In a still other aspect, this invention relates to a pharamceutical composition for intranasal administration which contains hPTH as its active ingredient, and which also contains acetic acid whose content is kept less than a certain chemical equivalent with respect to the weight of hPTH.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a reverse phase HPLC chromatogram obtained from an hPTH(1-34) preparation as acetic acid content being 9.5% after it has been stored at 40.degree. C. for six months.

FIG. 2 shows a reverse phase HPLC chromatogram obtained from an hPTH(1-34) preparation as acetic acid content being 2.9% after it has been stored at 40.degree. C. for six months.

FIG. 3 shows the amounts of decomposition products (by-products) B, C and D after hPTH(1-34) preparations contained various acetic contents acid have been stored at 80.degree. C. for 15 hours.

FIG. 4 shows the purity levels of hPTH(1-34) of the hPTH(1-34) preparations contained various acetic acid content after the preparations have been stored at 80.degree. C. for 15 hours.

FIG. 5 shows a reverse phase HPLC chromatogram of an hPTH(1-84) preparation as acetic acid content being 12.3% after it has been stored at 80.degree. C. for 15 hours.

FIG. 6 shows the amounts of decomposition products (by-products) after hPTH(1-84) preparations contained various acetic acid contents have been stored at 80.degree. C. for 15 hours.

FIG. 7 shows the purity levels of hPTH(1-84) of the hPTH(1-84) preparations containing various acetic acid content after the preparations have been stored at 80.degree. C. for 15 hours.

DESCRIPTION OF THE EMBODIMENTS

According to this invention, hPTH includes peptides which are involved in bone metabolism, have a strong stimulating effect on the formation of bones, and have an activity to increase the concentration of calcium in serum, that is, a natural type hPTH(1-84) comprising 84 amino acid residues, and its derivatives.

For example, the hPTH may include hPTH(1-84) (Biochemistry 17, 5723(1978); Kimura et al., Biochem. Biophys. Res. Commun., vol. 114, p. 493, 1983), hPTH(1-38) (Japanese Patent Laid-Open No. 57-81448), hPTH(1-34) (Japanese Patent Laid-Open No. 9-29600; Takai et al., Peptide Chemistry, p. 187, 1979), hPTH(1-34)NH.sub.2 (Japanese Patent Laid-Open No. 58-96052), [Nle.sup.8,18]hPTH(1-34) and [Nle.sup.8,18, Tyr.sup.34]hPTH(1-34) (Japanese Patent Laid-Open No. 55-113753), [Nle.sup.8.18]hPTH(1-34)NH.sub.2 (Japanese Patent Laid-Open No. 61-24598), [Nle.sup.8,18, Tyr.sup.34]hPTH(1-34)NH.sub.2 (Japanese Patent Laid-Open No. 60-34996), hPTH(1-37) (Japanese Patent Presentation [Kohyo] No. 5-505594), hPTH(2-84), hPTH(3-84), hPTH(4-84), hPTH(5-84), hPTH(6-84), hPTH(7-84), and hPTH(8-84) (Japanese Patent Presentation [Kohyo] No. 4-505259), etc. These hPTHs can be obtained by the methods based on genetic engineering techniques or chemical synthesis techniques as described in the above documents, or by the methods equivalent with the former.

Generally speaking, when a physiologically active peptide is purified based on the genetic engineering technique or chemical synthesis technique, column chromatography is used. However, because hPHT is a basic peptide, it would be adsorbed to resin constituting a column if it were unscrupulously applied to the column. To prevent the adsorption of hPTH and raise its solubility, an acid is used as an eluent. For a peptide to serve as a material of a pharmaceutical composition, the peptide must be incorporated in a lyophilized composition which will serve as a starting material for preparation of the pharmaceutical composition. To meet this requirement, the acid must be volatile, which limits the range of usable acids.

For illustration, let's assume, for example, hydrochloric acid is used for the present purpose. It is highly acidic even at a low concentration, readily causes accessory reactions such as hydrolysis, and is highly corrosive. Thus, hydrochloric acid is not suitable for the present purpose. If an organic acid is employed for the present purpose, it may be selected from trifluoroacetic acid, formic acid and acetic acid that have a low boiling point. However, it is undesirable to incorporate trifluoriacetic acid in a pharmaceutical composition in terms of safety. Formic acid is also limited in its use because of its reducing activity, and is not compatible with a peptide such as hPTH.

By contrast, acetic acid is the most appropriate to serve as a material for a pharmaceutical composition, on account of its safety and chemical properties, and has been used as an acid indispensable for the final stage purification of a basic peptide. For example, the purification process based on reverse phase high-performance liquid column chromatography (reverse phase HPLC) or on size exclusion column chromatography has been performed using an eluate containing acetic acid with a favorable result. During the process, acetic acid is added at a concentration sufficient to prevent hPTH from being adsorbed to the column, and thus a sample eluated from an aqueous solution of acetic acid, a target peptide fraction, and a lyophilized composition prepared therefrom contain acetic acid at a concentration above the level at which acetic acid exists only as the constituent of a salt of the basic amino acid residues, that is, acetic acid exists not only as the constituent of hPTH salt, but as an adherent attached.

Namely, acetic acid existing in a pharmaceutical component based on hPTH takes two forms: it exists as the constituent of a salt of hPTH, and an adherent attached. Since acetic acid is a volatile substance, it is difficult to keep the content of acetic acid in a lyophilized composition at a constant level, because the content of acetic acid in a lyophilized composition varies depending on the lyophilization condition, the concentration of acetic acid in the original solution prior to lyophilization, with respect to the concentration of hPTH coexistent in the original solution.

The pharmaceutical component of this invention comprises hPTH and acetic acid, in which the content of acetic acid existing as the constituent of a salt of hPTH and an adherent is reduced. This component, because of having a reduced content of acetic acid, improves the stability of hPTH, and ensures a good use feeling when incorporated in a pharmaceutical composition for intranasal administration, and used as such.

In the pharmaceutical component of this invention, acetic acid with a reduced content is defined as acetic acid whose content is reduced below a certain specified chemical equivalent.

Because hPTH(1-34) contains nine basic amino acid residues (including tryptophan residue), one molecule of it can bind with nine molecules of monovalent acid (acetic acid and others) at maximum to form a salt. However, it also contains four acidic amino acid residues which may bind with the basic residues to form a salt within the same molecule. Therefore, with regard to hPTH(1-34) of this invention, the remaining five basic amino acid residues are considered as available for binding with acetic acid, from which the expected weight of acetic acid to bind with one molecule of hPTH(1-34) or a chemical equivalent of acetic acid to hPTH(1-34) is derived. The acetic acid content can be obtained by using the equation I: the weight of acetic acid.times.100(%)/the weight of human parathyroid hormone peptide, on the weight of acetic acid and human parathyroid hormone peptide. The chemical equivalent of acetic acid to hPTH(1-34) is about 7.3% (weight % unless otherwise stated) as the acetic acid content.

Similarly, since hPTH(1-84) contains 19 basic amino acid residues (including tryptophan) and 12 acidic amino acid residues, in preparing a hPTH(1-84) preparation according to this invention, it is assumed that the seven excess basic amino acid residues are available for binding with acetic acid in one molecule of hPTH(1-84), which will give the expected weight of acetic acid to bind with one molecule of hPTH or a chemical equivalent of acetic acid to hPTH(1-84). The acetic acid content can be obtained by using the equation I, the chemical equivalent of acetic acid to hPTH(1-84) is about 4.5% as the acetic acid content.

Namely, according to this invention, acetic acid contained in an amount less than its chemical equivalent means that of an amount of acetic acid omitted as an adherent, but only as the constituent of a salt of hPTH whose percent weight is below the chemical equivalent.

The present invention provides a stable pharmaceutical component based on hPTH by controlling the content of acetic acid thereof, and a pharmaceutical composition for intranasal administration containing the pharmaceutical component.

Further, the present invention provides a pharmaceutical component based on hPTH in which the content of acetic acid is controlled such that it is kept at a specified level, and a medicinal composition for intranasal administration containing the pharmaceutical component.

Since a peptide is generally unstable in a solution, its lyophilized product is used as a material for a pharmaceutical component. If a peptide such as hPTH which exists as a salt containing acetic acid or a volatile substance as the constituent of the salt, is dissolved in water or in diluted acetic acid, and the lyophilized product thereof is used as a material for preparing a pharmaceutical component, the content of acetic acid in the component will not stay at a constant level, which poses a problem. This invention enables preparation of an aqueous solution of hPTH with a reduced content of acetic acid, thereby enabling the production of an hPTH-based pharmaceutical component consistently containing a specific amount of acetic acid in stable. Thus, this invention is advantageous also from the aspect of manufacturing stability.

For a pharmaceutical component according to this invention, the acetic acid content with respect to the weight of hPTH is kept below the chemical equivalent. For example, for the hPTH(1-34)-based component acetic acid content is kept below about 7.3% with respect to the weight of hPTH(1-34), more preferably about 6.0% or less particularly about 4.0% or less from the view-point of stability and utility, or more preferably about 4.0% or less, particularly about 3.0% or less from the view-point of manufacturing stability. It is not preferable that the content of acetic acid were kept at a too low level from the view-point of manufacturing stability, since the component, although it would give an excellent stability and use feeling, would readily become insoluble at a high pH: hPTH(1-34) has an isoelectric point at 8.2 (pI=8.2). The content of acetic acid is preferably kept at about 0.5% or higher, particularly about 1.0% or higher. On the other hand, for hPTH(1-84)-based component, the acetic acid content is kept below about 4.5%, preferably about 3.0% or less from the view-point of stability and utility, more preferably about 0.1% or higher from the view-point of manufacturing stability.

The pharmaceutical component of this invention can be produced by any publicly known method or by any method equivalent with the former. Namely, reduction of the content of acetic acid existing as the constituent of a salt of hPTH or as an adherent below a specified level can be achieved by appropriately introducing a known method such as dialysis, electrodialysis, ion exchange chromatography, size exclusion column chromatography, reverse phase HPLC, etc., into the purification process of hPTH which has been obtained by a genetic engineering-based technique or a chemical synthesis-based technique.

When the excess content of acetic acid is reduced by a method such as dialysis, electrodialysis, ion exchange chromatography, etc., adjustment of the content of acetic acid to any desired level may be achieved by directly monitoring the pH of the hPTH solution, or the concentration of acetic acid in the solution, so that an hPTH solution containing acetic acid at a desired concentration may be obtained.

For example, adjustment of the acetic acid content in an aqueous solution of hPTH may be achieved based on the relation of the acetic acid content in the solution to the pH of the solution.

Dialysis may occur as follows: an aqueous solution of hPTH which has been prepared by a genetic engineering-based or chemical synthesis-based technique, or the same aqueous solution whose pH has been adjusted to pH5 9, with the addition of an alkaline solution such as an aqueous solution of sodium hydroxide or ammonia is placed in a dialysis membrane in the form of a cylinder which will pass low molecular weight components; the solutes in the solution is subject to dialysis based on simple diffusion; and the acetic acid content is removed by this process. For example, a solution of hPTH(1-34) (acetic acid content being 2%) is obtained by subjecting a starting solution to dialysis until the solution outside the dialysis membrane comes to have a pH of about 6.5.

Electrodialysis may occur as follows: an aqueous solution of hPTH which has been prepared by a genetic engineering-based or chemical synthesis-based technique, or the same aqueous solution whose pH has been adjusted to pH5 9, with the addition of an alkaline solution such as an aqueous solution of sodium hydroxide or ammonia is allowed to circulate between two dialysis membranes exposed to an electric field which will pass components having a molecular weight of 300 or less; and acetic acid ions will migrate to the cathode to accumulate there while free hPTH basic ions will migrate to the anode to accumulate there; and acetic acid ions with a low molecular weight are allowed to pass through the membranes to the outside, while free hPTH basic ions with a large molecular weight are allowed to circulate within the dialysis system. It will be possible to produce a solution of hPTH containing a desired constant amount of acetic acid, by monitoring the pH or ionic strength of dialysis solution, thereby checking the reduced acetic acid content. For example, an hPTH(1-34) solution (acetic acid content being about 2%) will be obtained by applying electrodialysis to a starting solution until the pH of dialysis solution comes to have a pH of about 6.5.

In ion exchange chromatography, acetic acid is adsorbed by binding to a basic ion exchange resin to be removed. For example, an aqueous solution of hPTH which has been prepared by a genetic engineering-based or chemical synthesis-based technique, is applied to a basic ion exchange resin column made of a quaternary or secondary ammonium resin; acetic acid is allowed to be bound to the resin through ion-to-ion binding; and a non-adsorbed fraction simply passing through the column is recovered to give an hPTH solution with a reduced acetic acid content. It is possible to obtain an hPTH solution with a specified acetic acid content by altering the amount of ion exchange resin with respect to the weight of hPTH in the solution. For example, in order to obtain an hPTH(1-34) solution (acetic acid content being about 2%), a resin may be used that has a weight sufficiently large to alter the pH of eluate to about pH6.5.

Size exclusion column chromatography may occur as follows: an aqueous solution of hPTH which has been prepared by a genetic engineering-based or chemical synthesis-based technique, or the same aqueous solution whose pH has been adjusted to pH5 9, with the addition of an alkaline solution such as an aqueous solution of sodium hydroxide or ammonia is applied to a column; an aqueous solution containing an organic solvent such as acetonitrile is used for eluation; and acetic acid is thereby removed. It is possible to obtain an hPTH solution with a specified acetic acid content by altering the pH of the aqueous solution of hPTH to be applied to a column. For example, to obtain an hPTH(1-34) solution (acetic acid content being about 2%), an aqueous solution of hPTH having been so adjusted as to give a pH of about 6.5, is applied to a column, and a fraction consisting of hPTH(1-34) eluate is recovered.

In reverse phase HPLC, an aqueous solution of hPTH which has been prepared by a genetic engineering-based or chemical synthesis-based technique, or the same aqueous solution whose pH has been adjusted to pH5 9, with the addition of an alkaline solution such as an aqueous solution of sodium hydroxide or ammonia is served. The solution is applied to a C18 or C4 column initialized with water; and for example water is used as an eluate to elute inorganic salts. Then, an aqueous solution containing an organic solvent such as acetonitrile is allowed to flow to elute hPTH adsorbed to the column; and an hPTH solution with a reduced acetic acid content is thereby obtained.

It is possible to obtain an hPTH solution with a specified acetic acid content by adjusting the pH of an aqueous solution of hPTH to be applied to a column. It is also possible to obtain an hPTH solution with a specified acetic acid content by preparing an hPTH solution with a too small acetic acid content, for example as low a level as permitted to the method, and then adding a necessary amount of acetic acid to give an hPTH solution with a specified acetic acid content. For example, an hPTH(1-34) solution from which acetic acid has been excessively removed, is diluted with water to 10 mg/mL; acetic acid is added to the solution until the pH of the solution becomes pH6.5; and an hPTH(1-34) solution (acetic acid content being about 2%) is thereby obtained.

An aqueous solution of hPTH obtained by the method as described above is lyophilized by a conventional method to produce a pharmaceutical component of this invention.

The medicinal component of this invention may include water-soluble organic acids, or preferably at least one selected from citric acid, adipic acid and glycolic acid, so as to improve the mucosal absorption of the component. A pharmaceutical component further including such an organic acid will ensure a high stability, and will also ensure an excellent use feeling, when administered through a route other than parenteral routes, or particularly when administered nasally.

Accordingly, the pharmaceutical component of this invention may be used as a component of a pharmaceutical composition for intranasal administration suitable for a long term use.

Moreover, the pharmaceutical composition of this invention for intranasal administration has a property of being compatible with widely varied functional components as well as with a carrier, excipient, viscosity-increasing agent, preserver, stabilizer, anti-oxidant, binder, disintegrant, humectant, lubricant, colorant, flavoring agent, corrigent, suspendmolding agent, emulsifying agent, solubilizer, buffering agent, tonicity agent, detergent, soothing agent, Sulfur-containing reducing agent etc. Thus, the pharmaceutical composition of this invention well tolerates the addition of various functional components which may be introduced to improve absorption, solid stability, etc., as appropriate.

The carrier or excipient may include substances well or sparingly soluble to water such as sugars, polysaccharides, dextrins, celluloses, synthesized or semi-synthesized polymers, amino acids, polyamino acids, proteins, and phospholipids.

The sugars (monosaccharides, oligosaccharides) may include, for example, D-mannitol, glucose, lactose, fructose, inositol, sucrose, maltose, etc., while the polysaccharides may include dextran, pullulan, alginic acid, hyaluronic acid, pectic acid, phytic acid, phytin, etc. The dextrins may include .alpha.-cyclodextrin, .beta.-cyclodextrin, .gamma.-cyclodextrin, dextrin, hydroxypropylstarch, hydroxyethylstarch, etc.

The celluloses may include methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.

The synthesized or semi-synthesized polymers may include polyvinyl alcohol, carboxyvinyl polymer, polyethylene glycol, polyvinyl pyrrolidone (PVP), sodium polyacrylate, polyactic acid, etc.

The amino acids may include glycine, taurine, while the polyamino acids may include polyglutamic acid, polyaspartic acid, polyglycine, polyleucine, etc.

The proteins may include gelatin and others. In addition, chitin and chitosan may be included.

Of these carriers or excipients, particularly preferred are sucrose, maltose, .alpha.-cyclodextrin, .beta.-cyclodestrin, dextrin, D-mannitol, inositol, lactose, dextran, methylcellulose, hydroxypropylcellulose, polyvinyl alcohol, pullulan, etc.

Besides them, sorbic acid; benzalconium chloride; cetylpyridinium chloride; benzethonium chloride; parabens such as methyl paraoxybenzoate, ethyl paraoxybezoate, propyl paraoxybenzoate, butyl paraoxybenzoate, and others; gum acacia; sorbitol; magnesium stearate; talc; silica; microcrystalline cellulose; starch; calcium phosphate; vegetable oil; carboxymethylcellulose; sodium lauryl sulfate; water; ethanol; glycerin; and syrup.

Typical examples of surfactants are listed below. Among these, single or combination of more than two of these surfactants can be added to the formulation in the invention.

Nonionic surfactants may include sorbitan esters of fatty acids, for example, sorbitan monocaprilate, sorbitan monolaurate, sorbitan monopalmitate, etc, and glycerol esters of fatty acids, for example, glyceryl monocaprilate, glyceryl monomyristate, glyceryl monostearate, etc, and polyglycerol esters of fatty acids, for example, decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate, etc, and polyoxyethylene sorbitan esters of fatty acids, for example, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate, etc, and polyoxyethylene sobitol esters of fatty acids, for example, polyoxyethylene sobitol tetrastearate, polyoxyethylene sobitol tetralaurate, etc, and polyoxyethylene glycerol esters of fatty acids such as polyoxyethylene glyceryl monostearate, and polyethylene glycerol esters of fatty acids such as polyethylene glyceryl distearate, and polyoxyethylene alkyl ether such as polyoxyethylene lauyl ether, and polyoxyethylene polyoxypropylene alkyl ether, for example, polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propylether, polyoxyethylene polyoxypropylene cetyl ether, etc, and polyoxyethylene alkylphenyl ether such as polyoxyethylene nonylphenyl ether, and polyoxyethylene caster oils, for example, polyoxyethylene caster oil, polyoxyethylene hydrogenated caster oil, and polyoxyethylene yellow beeswax derivatives such as polyoxyethylene sorbitol yellow beeswax, and polyoxyethylene lanolin derivatives such as polyoxyethylene lanolin, and polyoxyethylene amide of fatty acids with HLB 6 to 18 such as polyoxyethylene stearylamide.

Anionic surfactants may include alkyl sulfate (C.sub.10 to C.sub.18) salts, for example, sodium cetyl sulfate, sodium lauryl sulfate, sodium oleylsulfate, etc, and polyoxyethylene alkylether sulfate salts whose average moles of added ethyleneoxide is 2 to 4 and carbons of alkyl groups is 10 to 18, such as sodium polyoxyethylene lauryl ether sulfate, and alkyl sulfo succinate ester salts whose length of alkyl groups is 8 to 18 such as sodium lauryl sulfo succinic acid ester.

Naturally occurring sulfactants may include lecithin, and glycerol lipid phosphate, and sphingolipids such as sphingomyelin, and sucrose esters of fatty acids (C.sub.12 to C.sub.18).

Sulfur-containing reducing agents may include N-acety cysteine, N-acety homocysteine, thioctic acid, thioethanol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, thioalkanic acids (C.sub.1 to C.sub.7) having sulfhydryl group.

Anti-oxidants may include erysorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, alpha-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbyl palmitate, L-ascorbyl stearate, sodium bisulfite, sodium sulfite, triamyl gallate, propyl gallate, and chelating agents, for example, calcium disodium edetate (EDTA), sodium pyrophospate, sodium metaphosphate, etc.

For a pharmaceuticl composition of this invention, hPTH may exist at about 0.01 20%, preferably at about 0.05 10%, and an organic acid may be added as appropriate. The content of the latter prior to use is about 0.05 99.5%, preferably about 0.1 99.0%. A carrier or excipient which is usually added during preparation of a medicinal product may be added as appropriate, or may exist, for example, at about 0.01 99.5% prior to use. Other various functional components may be added as appropriate, or may exist, for example, at about 0.05 99.5% prior to use.

Preparation of the pharmaceutical composition for intranasal administration of this invention may be achieved by any known method.

For example, an hPTH-based pharamceutical component in which the acetic acid content has been reduced may be served as a pharmaceutical composition. Alternatively, to an hPTH-based pharmaceutical component in which the acetic acid content has been reduced, may be added as appropriate a carrier or excipient which is usually added during preparation of a pharmaceutical product, and an organic acid and other various functional components, and the resulting compound may be used as a pharmaceutical component. Addition of an organic acid may take place to replace acetic acid, or simply for addition. For example, an hPTH pharmaceutical component to which are added as appropriate a carrier or excipient which is usually added during preparation of a pharmaceutical preparation, an organic acid, and various functional components; a resulting mixture is dissolved for one time in distilled water; the solution is lyophilized; and a uniform composition is thereby obtained.

Alternatively, an hPTH pharmaceutical component, and a carrier or excipient which is usually used during preparation of a pharmaceutical preparation are dissolved for one time in distilled water; an organic acid and various functional components are then added to the solution; the resultant solution is lyophilized; and a uniform composition is thereby obtained. As a further variant, an hPTH pharmaceutical component, an organic acid, and various functional components are dissolved for one time in distilled water; the solution is lyophilized; a desired amount of the lyophilized compound is dissolved as needed in combination with a carrier or excipient which is usually used during preparation of a pharmaceutical preparation; and a uniform composition is thereby obtained.

The pharmaceutical component of this invention may take various dosage forms depending on its expected administration routes: it may take a form appropriate for being applied to the mucosa of the rectum, nasal cavity, oral cavity, etc. The pharmaceutical composition for intranasal administration of this invention is preferably applied in a form appropriate for intranasal use.

A preferred example of the pharmaceutical composition for intranasal administration of this invention may occur as an on-demand dissolvable form of which a lyophilized portion contains a pharmaceutical composition of this invention provided lyophilized and a dissolving solution portion is attached to the former.

An organic acid such as citric acid, adipic acid or glycolic acid which is added for promoting absorption may exist as the constituent of a salt of hPTH, an adherent, or an additive. Alternatively, the organic acid may be dissolved in the dissolving solution portion.

Administration of a pharmaceutical composition for intranasal administration of this invention may be achieved by any known method. For example, spraying a pharmaceutical composition for intranasal administration of this invention is applicable: the composition may be placed in a container; a nebulizer is attached to the container; the tip of nozzle is inserted into the nasal cavity; and the pharmaceutical composition is sprayed.

The dose of a pharmaceutical composition of this invention may vary depending on the kind of disease, the age and weight of the patient, the severity of disease, and the route through which the composition is administered. If, for example, an hPTH-based composition is applied nasally, it may be applied once daily or several times daily with each dose reduced in proportion, successively for a period. A single dose of hPTH(1-34)-based composition preferably occurs in the range of 10 5,000 .mu.g. During treatment, a so-called wash-out may be inserted, and treatment may then be resumed.

EXAMPLE

The present invention will be detailed below by means of Examples, but this invention should not be limited to those examples.

The testing methods and apparatuses used in Examples are based on what is described below, unless otherwise stated.

1. Analysis of hPTH by HPLC

Determination of the content of a studied peptide in a composition, and checking whether any decomposition products (by-products) are present in the composition was achieved by reverse phase HPLC using the apparatuses and conditions specified below. Apparatus: LC-9A system from Shimadzu Ltd. Column: YMC Protein-RP (4.6 mmo.times.150 mm) Column temperature: 40.degree. C. Eluate: the concentration of acetonitrile in 0.1% trifluoroacetic acid is linearly varied from 25% to 40% in 30 minutes. Flow rate: 1 mL/min Detection: UV(210 nm) Injection amount: 50 .mu.L 2. Analysis of Acetic Acid

The contents of acetic acid of dialysis solutions and of lyophilized compositions were determined by ion exchange chromatography under the conditions as specified below. Apparatus: LC-91 system from Shimadzu Ltd. Column: IC-A1 from Shimadzu (4.6 mmo.times.100 mm) Column temperature: 40.degree. C. Eluate: 1:1 mixture of 0.84% aqueous solution of phthalic acid and 0.58% aqueous solution of tris hydroxymethyl aminomethane Flow rate: 1.5 mL/min Detection: electric conductivity detector Injection amount: 10 .mu.L 3. Mass Analysis

Determination of the masses of hPTH, decomposition products (by-products) of hPTH, and their enzymatic digests was achieved with the apparatuses under conditions as specified below. Apparatus: MAT TSQMS from Finnigan Ion source: ESI Detection mode: positive Spray voltage: 4.5 kV Capillary temperature: 250.degree. C. Mobile phase: (1:1) mixture of 0.2% acetic acid and methanol Flow rate: 0.2 mL/min Scan range: m/z 550 850 4. Sequencing Amino Acids

Determination of the amino acid sequences of decomposition products (by-products) of hPTH and their enzymatic digests was achieved with the following apparatuses. Apparatus: type 477A sequencer from PerkinElmer 5. Determination of Amino Acid Composition

Determination of the amino acid compositions of hPTH, decomposition products (by-products) of hPTH, and their enzymatic digests was achieved with the following apparatuses. Apparatus: type L-8500 amino acid analyzer from Hitachi 6. Storage of Specimens (Stability Test)

The test specimens were stored in a depository kept under the conditions as specified below. Apparatus: LH-30-14 from Nagano Science Co. Ltd. Temperatures set: 1) 40.+-.1.degree. C., 2) 60+1.degree. C., 3) 80.+-.2.degree. C. 7. Lyophilization Apparatus: RL-903BS from Kyowa Vacuum Engineering, Ltd. Vial: 15 mL glass vial

Reference Example 1

Production of hPTH(1-34) (1)

The expression plasmid pG117S4HPPH34 (Japanese Patent Laid-Open No. 9-29660) containing a gene coding for a chimera protein of hPTH(1-34) obtained by connecting a DNA fragment coding for a derivative of .beta.-galactosidase derived from E. coli with a DNA fragment coding for hPTH(1-34) through the intervention of a DNA fragment coding for a linker containing a cleavage motive (Lys-Arg) of Kex2 protease or a processing enzyme, was introduced into the cells of M25 strain E. coli (w3110/ompT: Sugimura et al., Biochem. Biophys. Res. Commun., vol. 153, 1988, p. 753 759). The transformed cells of M25 strain E. coli were cultivated on a medium containing 2% yeast extract in a 20 L culture tank.

The cultivation was continued until the density of cells became OD.sub.660=12. The recovered cells were broken to pieces with a high pressure homogenizer (Manton-Gaullin) in 10 mM Tris-HCl buffer (pH8.2) supplemented with 1 mM EDTA, centrifuged, and washed, to produce about 625 mL of suspension containing about 100 g of inclusion bodies filled with the chimera protein. To 250 mL of suspension containing 40 g of inclusion bodies, were added 100 mL of 1M Tris-HCl buffer (pH8.2), 50 mL of 5M NaCl, 500 mL of deionized water, and 900 g of urea, and the mixture was agitated at 30.degree. C. to allow the inclusion bodies to dissolve.

The solution was diluted with deionized water to 5 L, to which was added 50 mL of 250 mM CaCl.sub.2. Then, to the solution was added Kex2-660 comprising amino acid residues designated an amino acid Nos. 1 660 (Japanese Patent Laid-Open No. 10-229884) which is a derivative from Kex2 protease, until it existed at 20 kU/mL or higher. The mixture was gently stirred for two hours, and hPTH(1-34) was cleaved from the chimera protein. The reaction solution was adjusted to pH6.4 with addition of acetic acid; it was then diluted two fold with deionized water, thereby allowing the chimera protein and .beta.-galactosidase derivative remaining unreacted to precipitate; and the yield was centrifuged to give a supernatant containing 6.7 g of hPTH(1-34). The supernatant was adjusted to pH5.0 with addition of acetic acid; the solution was applied to a cation exchange resin (SP Toyopearl from Tosoh Corporation) previously equilibrated with 10 mM sodium acetate to allow hPTH(1-34) to be adsorbed to the resin; the resin was washed with 10 mM sodium acetate buffer; and 0.4M NaCl was used to give a fraction containing 6.0 g of hPTH(1-34).

To this fraction was added acetic acid to 3 v/v %; the solution was applied to a column for low pressure reverse phase ODS (Soken ODS-W from Soken Chemicals Co.) previously equilibrated with 3 v/v % acetic acid; and 30 v/v % acetonitrile containing 3 v/v % acetic acid was used to eluate hPTH(1-34). The eluate containing hPTH(1-34) was enriched under a reduced pressure; the yield was applied to a column for reverse phase HPLC (TSKge10DS120T with a size of 55 mm.times.600 mm from Tosoh Corp.); and solution of acetonitrile in 5 v/v % acetic acid was allowed to flow at 40 mL/min for 60 minutes with the concentration of acetonitrile being linearly varied from 16% to 32% in the mean time, to eluate hPTH(1-34). Thus, a purified fraction containing 4 g of hPTH(1-34) was obtained.

A 60 g of the remaining inclusion bodies was similarly treated, and another purified fraction containing 5 g of hPTH(1-34) obtained therefrom was combined with the former; the mixture was removed of acetonitrile under a reduced pressure; and the yield was diluted with 5 v/v % acetic acid such that the concentration of hPTH(1-34) fell to 10 mg/mL. A 15 ml of the solution was placed in each glass vial; and all the vials containing the solution were lyophilized to give 9 g of hPTH(1-34) in total (150 mg.times.60 vials).

ESI-MS: 4117.7 (theoretical value being 4117.8). Amino acid composition after being hydrolyzed with 6N hydrochloric acid: Asx-4.0(4); Ser-2.6(3); Glx-4.9(5); Gly-1.0(1); Val-3.0(3), Met-2.0(2); Ile-1.0(1); Leu-5; Phe-1.1(1); Lys-30(3); His-3.0(3); Arg-2.0(2); and Trp-not detected (1).

Reference Example 2

Production of hPTH(1-34) (2)

Similar live microbes to those used in Reference Example 1 were cultivated in a 200 L culture tank. The cultivation was continued until the density of cells became OD.sub.660=160. The recovered cells were broken to pieces with a high pressure homogenizer in 10 mM Tris-HCl buffer (pH8.2) supplemented with 1 mM EDTA, centrifuged, and washed, to produce about 10 L of suspension containing about 5 kg of inclusion bodies filled with the chimera protein.

To 4.0 L of suspension containing 2 kg of inclusion bodies, were added 1.6 L of 1M Tris-HCl buffer (pH8.2), 0.8 L of 5M NaCl, 15 L of deionized water, and 13 kg of urea, and the mixture was agitated at 30.degree. C. to allow the inclusion bodies to dissolve.

The solution was diluted with deionized water to 80 L, to which was added 0.8 mL of 250 mM CaCl.sub.2. Then, to the solution was added Kex2-660 (Japanese Patent Laid-Open No. 10-229884), until it existed at 10 kU/mL or higher. The mixture was gently agitated for one hour, and hPTH(1-34) was separated by cleavage from the chimera protein. The reaction solution was adjusted to pH6.3 with addition of acetic acid; it was then diluted two fold with deionized water, thereby allowing the chimera protein and .beta.-galactosidase derivative remaining unreacted to precipitate; and the yield was subjected to pressurized filtration to give a supernatant containing hPTH(1-34).

The supernatant was adjusted to pH5.0 with addition of acetic acid; the solution was applied to a cation exchange resin column (5 L)(Poros 50HS from PerSeptive Biosystems, USA) previously equilibrated with 10 mM sodium acetate buffer to allow hPTH(1-34) to be adsorbed to the column; the column was washed with 10 mM sodium acetate buffer; and 0.4M NaCl with a concentration gradient was used to give a fraction containing hPTH(1-34). To this fraction was added acetic acid to 3 v/v %; the solution was applied to a column (5 L) for low pressure reverse phase ODS (Soken ODS-W from Soken Chemicals Co.) previously equilibrated with 3 v/v % acetic acid; and 30 v/v % acetonitrile containing 3 v/v % acetic acid was used to eluate hPTH(1-34).

The eluate containing hPTH(1-34) was enriched under a reduced pressure; the yield was filtrated through a 0.22 .mu.m filter; the filtrate was applied to a column for reverse phase HPLC (TSK ODS 80Ts 20 .mu.m, 105 mmID.times.550 mm from Tosoh Corp.); and acetonitirile with a concentration gradient was used in the presence of 3 v/v % acetic acid to eluate hPTH(1-34). Several eluates thus obtained were combined; and the solution was removed of acetonitrile through distillation under a reduced pressure, to give 10.4 L of conc. hPTH(1-34) solution containing 70 g of hPTH(1-34) at 99.6%. Out of 3 kg of the remaining inclusion bodies, 2 kg was subjected to the same purification process, to give 11.6 L of conc. hPTH(1-34) solution containing hPTH(1-34) at 99.6%.

Reference Example 3

Production of hPTH(1-84)

The expression plasmid pGP#19 (Japanese Patent Laid-Open No. 9-29660) containing a gene coding for a chimera protein of hPTH(1-84) obtained by connecting a DNA fragment coding for a derivative of .beta.-galactosidase derived from E. coli with a DNA fragment coding for hPTH(1-84) through the intervention of a DNA fragment coding for a linker containing a cleavage motive (Lys-Arg) of Kex2 protease or a processing enzyme, was introduced into the cells of M25 strain E. coli. The transformed cells of M25 strain E. coli were cultivated at 37.degree. C. in a 3 L culture tank. The cultivation was continued until the turbidity (OD.sub.660) of culture solution became OD.sub.660=1. Then, isopropyl beta-thiogalactoside (IPTG) was added to 1.0 mM.

The cultivation was further continued for four hours. The cells were recovered by centrifugation; and the cells were then suspended in TE (10 mM Tris and 1 mM EDTA at pH8.0). The cells were broken to pieces with a French press, subjected to a repetition of centrifugation and resuspension, and washed, to produce inclusion bodies. A suspension of the inclusion bodies was added for dissolution to a solution (pH8.0) containing 8.0M urea and 10 mM Tris, and centrifuged; the supernatant was loaded onto a Toyopearl column (Toso Corp.); and NaCl with a concentration gradient of 0 0.4M was used to eluate enriched hPTH(1-84). The inclusion body dissolving enriched solution was further concentrated by ultrafiltration with a removal limit of 10,000 MW. The yield was diluted to give a solution in which the constituents became 50 mM for BisTris at pH6.8, 1.0 mM for CaCl.sub.2, and 5 mg/mL for the chimera protein. Kex2-660 was added to 2 kU/mL, and reaction was allowed to proceed at 30.degree. C. for one hour to separate hPTH(1-84) by cleavage.

The reaction solution was adjusted to pH5.0 with addition of acetic acid; it was then diluted two fold with deionized water, thereby allowing the chimera protein and .beta.-galactosidase derivative remaining unreacted to precipitate; and the yield was centrifuged to give a supernatant containing hPTH(1-84). To the supernatant was added acetic acid to 3 v/v %; and the solution was applied to TSK gel ODS 80Ts (21 mmID.times.250 mm, Tosoh Corporation) previously equilibrated with 3 v/v % acetic acid for purification. Fractions containing 98% or higher hPTH(1-84) were combined; the resulting solution was removed of solvent; and the residue was lyophilized to give 500 mg of hPTH(1-84). The molecular weight and amino acid composition of this substance are as indicated below. Based on those data, the substance was identified as hPTH(1-84).

ESI-MS: 9424.7 (theoretical value being 9424.7). Amino acid composition after being hydrolyzed with 6N hydrochloric acid: Asx-10.1(10); Thr-1.1(1); Ser-6.3(7); Glx-11.0(11); Pro-2.9(3); Gly-4.1(4); Ala-7.0(7); Val-8.0(8); Met-1.9(2); Ile-1.0(1); Leu-10; Phe-1.0(1); Lys-8.9(9); His-3.9(4); Arg-5.0(5); and Trp-not detected (1).

Example 1

Removal of Acetic Acid from hPTH(1-34) (1)

(1) An amount of preparation corresponding to 150 mg as hPTH(1-34) obtained in Reference Example 1 was dissolved in distilled water (30 mL), to give an aqueous solution (pH4.7) of hPTH(1-34) at 5 mg/mL. This solution was dialyzed against distilled water (100 mL) with a GI micro acilyzer (Asahi Kasei Corp.) incorporating an electrodialysis membrane AC-130-10 (Asahi Kasei Corp.). The dialysis was continued for removal of acetic acid until the pH of the dialysis solution became pH5.0. The enriched solution containing 6.8% acetic acid was lyophilized, and dried mass corresponding to about 150 mg as hPTH(1-34) was obtained as acetic acid content being 4.8%.

(2) An amount of preparation corresponding to 150 mg as hPTH(1-34) obtained in Reference Example 1 was dissolved in distilled water (30 mL), to give an aqueous solution (pH4.7) of hPTH(1-34) at 5 mg/mL. This solution was subjected to electrodialysis as in Example (1): the dialysis was continued for removal of acetic acid until the pH of the dialysis solution became pH5.5. The enriched solution containing 4.6% acetic acid was lyophilized, and dried mass corresponding to about 150 mg as hPTH(1-34) was obtained as acetic acid content being 3.8%.

(3) An amount of preparation corresponding to 150 mg as hPTH(1-34) obtained in Reference Example 1 was dissolved in distilled water (30 mL), to give an aqueous solution (pH4.7) of hPTH(1-34) at 5 mg/mL. This solution was subjected to electrodialysis as in Example (1): the dialysis was continued for removal of acetic acid until the pH of the dialysis solution became pH5.9. The enriched solution containing 3.1% acetic acid was lyophilized, and dried mass corresponding to about 150 mg as hPTH(1-34) was obtained as acetic acid content being 2.9%.

(4) An amount of preparation corresponding to 150 mg as hPTH(1-34) obtained in Reference Example 1 was dissolved in distilled water (30 mL), to give an aqueous solution (pH4.7) of hPTH(1-34) at 5 mg/mL. This solution was subjected to electrodialysis as in Example (1): the dialysis was continued for removal of acetic acid until the pH of the dialysis solution became pH7.0. The enriched solution containing 1.6% acetic acid was lyophilized, and dried mass corresponding to about 150 mg as hPTH(1-34) was obtained as acetic acid content being 1.6%.

From above results it was demonstrated that with the reduction of acetic acid content, the decrement of acetic acid content by lyophilization becomes smaller, and that with the reduction of acetic acid content, the fractional decrement of acetic acid content becomes smaller. Accordingly, reduction of the content of acetic acid in an hPTH component is useful for preparing a pharmaceutical hPTH component containing a specified amount of acetic acid, which has been difficult to obtain by conventional techniques.

Example 2

Removal of Acetic Acid from hPTH(1-34) (2)

To a solution containing preparation corresponding to about 150 mg as hPTH(1-34) obtained in Reference Example 2 was added 5N NaOH to pH5.5. One fourth of the solution was loaded onto a column for low pressure reverse phase ODS (Soken ODS-W (800 mL) from Soken Chemicals Co.) previously equilibrated with 5 v/v % aqueous solution of acetonitrile, to allow hPTH(1-34) to be adsorbed to the column. A 4 L of 5 v/v % aqueous solution of acetonitrile was flowed to eluate sodium acetate; and 50 v/v % aqueous solution of acetonitrile was then flowed to eluate hPTH(1-34). This process was repeated for each of the four parts; the hPTH(1-34) rich fractions of the four parts were combined to give 8.2 L of nearly acetic acid free fraction containing 122.5 g of PTH(1-34) was obtained (1.13% acetic acid content to hPTH(1-34)).

To this fraction was added distilled water to give an hPTH(1-34) solution at 10 mg/mL; to this solution was added 2.3 g of acetic acid such that the resulting solution contains 122.5 g of PTH(1-34) with 3% acetic acid content at 3%; and the mixture was well stirred. The solution was distributed to vials such that each vial contains 150 mg of hPTH(1-34); and the hPTH(1-34) solution in vial was lyophilized, which gave hPTH(1-34) as acetic acid content being 2.1%.

Example 3

Removal of Acetic Acid from hPTH(1-34) (3)

(1) An amount of preparation corresponding to 300 mg as hPTH(1-34) obtained in Reference Example 1 was dissolved in distilled water (30 mL), to give an aqueous solution of hPTH(1-34) at 10 mg/mL. This solution (pH4.7) containing 9.5% acetic acid was placed in three glass vials, 2 mL for each vial; and they were placed in an FZ-6 lyophilizer (Laboconco C