JP2000327583A - Bone-directional hormone derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound being a conjugate of a specific hormone preparation and specific peptide and exhibiting the osteoporosis therapeutic effect through distributing only in the bone tissue and for a long time. SOLUTION: This compound is a bone-directional hormone derivative, which is a conjugate of a hormone preparation selected from preferably a female hormone preparation such as (17β-)estradiol, preferably a male hormone preparation such as testosterone and preferably a protein assimilatory hormone preparation such as oxymetholone, and a peptide composed of an acidic amino acid, of preferably formula I (A is the moiety of the above-mentioned hormone preparation; X is the crosslinking agent moiety between A and the peptide moiety; N is 4-30; n is 1-3), preferably a compound of formula II. The above-mentioned derivative is obtained, for instance, by dehydrocondensation of a peptide synthesized through a normal peptide synthesis, composed of e.g. aspartic acid and having a protected carboxylic acid residue at the end of an amino acid side chain with a product formed by reaction between e. g. malonic anhydride and a hydroxyl group of the hormone molecule followed by deprotection.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an osteotropic hormone derivative specifically transported to bone tissue and a therapeutic agent for osteoporosis containing the same.

[0002]

BACKGROUND OF THE INVENTION Osteoporosis is defined as a condition in which the bone mass is low and the microstructure of the bone tissue changes, making the bone brittle and prone to fracture. In general, osteoporosis is classified into primary osteoporosis and secondary osteoporosis.

[0003] Various hormonal agents have received attention as therapeutic agents for osteoporosis, including female hormone agents (Christia).
nsen, C. & Christiansen, MS, Lancet, 1: 459, 1981
Etc.), androgenic drugs (Shin Nawada, History of Medicine,
143: 56, 1987; Arata Nawada, Ryoichi Takayanagi, Latest Medicine, 50: 1
433, 1995, etc.), anabolic hormone drugs (Need, AGet)
al., WMWHeft, 14/15: 392, 1993; Hiroyo Morii, The latest medicine, 50: 1438, 1995, etc.).

[0004] However, when a drug such as a hormonal agent is systemically administered, its transferability to bone tissue is poor, and this has been a major problem in performing drug treatment for bone diseases. In particular, it has been known that long-term systemic administration of sex hormones and anabolic hormones for the purpose of treating osteoporosis causes a wide variety of reproductive disorders and cardiac / circulatory disorders.

On the other hand, it has been reported that peptides composed of aspartic acid and glutamic acid have a high binding affinity to hydroxyapatite which is a component of bone (Bern).
ardi, G., Method Enzymol., 27: 471, 1973; Fujisawa,
R. et al., Biochim. Biophys. Acta, 1292: 53, 1996.
Such).

[0006]

It is expected that a peptide having a binding affinity with hydroxyapatite as described above will be used as a carrier for selectively transporting a drug to bone tissue or bone marrow. However, to date, such a conjugate of a peptide and a hormone having a specific translocation property to bone tissue has been prepared, and an example of selective translocation to bone tissue and sustained retention in bone has been confirmed. Not reported.

[0007]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that female hormone agents,
The present invention has been completed by finding that a conjugate of an androgenic agent or an anabolic hormone agent and a peptide comprising an acidic amino acid is distributed only in bone tissue for a long period of time and exhibits an effect of treating osteoporosis.

[0008] That is, the present invention provides an osteotropic hormone derivative which is a conjugate of a hormonal agent selected from the group consisting of a female hormone agent, an androgen agent and an anabolic hormone agent, and a peptide comprising an acidic amino acid. I do.

[0009] The present invention further provides a remedy for osteoporosis containing the above-mentioned osteotrophic hormone derivative.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Osteotrophic hormone derivatives Preferred osteotropic hormone derivatives of the present invention have the following general formula (1):

[0011]

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Wherein A is a hormonal agent portion selected from the group consisting of a female hormonal agent, an androgenic agent and an anabolic hormonal agent, and X is a crosslinking agent portion between the hormonal agent portion A and the peptide portion. Yes, N is 4-30,
n is 1-3).

The hormonal agent represented by A in the above formula used in the osteotropic hormone derivative of the present invention is selected from the group consisting of female hormonal agents, male hormonal agents, and anabolic hormonal agents which are effective in treating osteoporosis. Is done. Steroid hormones having a hydroxyl group for forming a conjugate with a peptide comprising an acidic amino acid are preferred.

A preferred female hormone agent is (17β-)
Estrogens such as estradiol, estrone, estriol, equilin, echirenin, ethinyl estradiol, and stilbenes such as diethylstilbestrol and hexestrol are particularly preferred. (17β-) estradiol and estriol are particularly preferred. Preferred androgens are testosterone,
Androgens such as androstenedione, dehydroepiandrosterone and fluoxymesterone,
Particularly preferred is testosterone. Preferred anabolic hormone agents are oxymetholone, methinolone acetate and stanozolol.

[0015] The peptide moiety used in the osteotropic hormone derivative of the present invention comprises N acidic amino acids. Preferably N is 4 to 30, more preferably 4 to 20, further preferably 6 to 20, and most preferably 6 to 12. Fmoc, which is a fluorescent amino acid protecting group, can be added to various lengths (N
= 2,4,6,8,10), and tested for affinity to hydroxyapatite by binding to a peptide consisting of aspartic acid and glutamic acid. As a result, it was observed that the affinity increased as N increased.

As the acidic amino acids, aspartic acid (n = 1), glutamic acid (n = 2) and 2-aminoadipic acid (n = 3) are preferred. The acidic amino acids used in the peptide portion may be the same or different, for example, a peptide in which aspartic acid and glutamic acid are mixed. The acidic amino acid is L
-Or D-form.

In the above formula, X is a cross-linking agent portion between the hormone agent portion A and the peptide portion, and may be prepared using any cross-linking agent known to those skilled in the art. For _{example, -CH 2 -, - (CH} 2) 2 -, - (CH 2) 3 -, -
_{CH 2 CO -, - (CH} 2) 2 CO- or - (CH _{2) 3} C
O- or the like can be suitably used.

Preferred osteotropic hormone derivatives of the present invention have the following formula (2) or (3):

[0019]

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Where N and n are as defined above. One example of a preferred osteotropic hormone derivative of the invention has the formula:

[0021]

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Method for Producing Osteotrophic Hormone Derivative The following general method can be used to produce the osteotropic hormone derivative of the present invention. Also, various alternatives known to those skilled in the art can be used. 1) Solid-phase method (FMOC method,
BOC method) or aspartic acid, glutamic acid or 2-peptide by a general peptide synthesis method such as a liquid phase method.
A peptide consisting of aminoadipic acid or a peptide of an appropriate length in which these amino acids are mixed is synthesized.

Subsequently, the N-terminal FMOC group or B
The OC group is removed by an appropriate method to obtain a peptide in which the carboxylic acid residue at the amino acid side chain terminal is protected (protected peptide).

Examples of the FMOC raw material include Nα-
FMOC-L (or D) -aspartic acid β-t-butyl ester, Nα-FMOC-L (or D) -glutamic acid γ-t-butyl ester, Nα-FMOC-L (or D) -2-aminoadipic acid δ-t
-Butyl esters. Also, BOC
As an example of the raw material of the method, t-butyl ester, benzyl ester, cyclohexyl ester, etc. of Nα-t-BOC-L (or D) -amino acid can be used. 2) Separately, a malonic anhydride, succinic anhydride or glutaric anhydride is reacted with a hydroxyl group of a hormone molecule in a suitable alkali solvent such as pyridine to form a crosslink. 3) Protected peptide of the product obtained in 1) and the product obtained in 2) using a dehydration condensing agent such as carbodiimide.
-Synthesize the hormone conjugate and finally add the appropriate acid,
The protecting group is eliminated by an alkali or the like. Further, the desired peptide-hormone conjugate is obtained by using liquid chromatography or the like.

As the crosslinking agent, monochloroacetic acid, halogenated propionic acid, halogenated methylpropionic acid and the like can also be used. Therapeutic agent for osteoporosis containing bone-tropic hormone derivative The present invention also provides a therapeutic drug for osteoporosis containing the above-described bone-tropic hormone derivative. Therapeutic agents include a therapeutically effective amount of an osteotropic hormone derivative. The dose of the therapeutic agent for osteoporosis of the present invention is generally 0.1 μg to 10 mg / kg body weight per day, preferably 1.0 μg to 1 mg / kg body weight, more preferably 10 μg to 1 mg / kg body weight. The specific dose is determined by a physician in consideration of the type of the bone tropic hormone derivative to be used, the sex and weight of the patient, the severity of the disease, and the like.

The therapeutic agent for osteoporosis of the present invention can be administered in the form of solutions, solid preparations, emulsions, suspensions, etc., by intravenous, subcutaneous, intramuscular injection, oral administration, nasal administration and the like. Solution preparations for injection may contain solubilizers, surfactants, emulsifiers, pH adjusters and the like known in the field of formulation. Alternatively, it may be administered in a form encapsulated in a liposome or the like.

The present invention will be described in more detail by the following examples. The scope of the present invention is not limited to the invention described in the examples, but includes various changes and modifications that can be made by those skilled in the art based on the disclosure of the present specification.

[0028]

EXAMPLES Example 1 Affinity of Peptides Consisting of Acidic Amino Acids to Hydroxyapatite 1) Synthesis Method of FMOC-Acid Amino Acid Peptide Using an automatic peptide synthesizer, N-FMOC-L-aspartic acid β-t- Butyl ester or N-FMOC-L-glutamic acid β-t
-Butyl ester is used as a raw material to extend the peptide chain sequentially, and the number of each amino acid residue is different (2-10
A) protected peptide resin was constructed. These were treated with trifluoroacetic acid to simultaneously cleave from the resin and remove the t-butyl protecting group of the amino acid side chain, and the resulting crude product was purified by HPLC. 2) Affinity test method for hydroxyapatite 2 mg of hydroxyapatite (HA, Bio-Gel HTP, Bio-Rad) in various concentrations of FMOC-peptide dissolved in TBS solution (50 mM Tris / HCl, 150 mM NaCl) Was suspended for 1 hour and centrifuged at 10,000 rpm for 5 minutes.
And measured at an excitation wavelength of 264 nm and a trend wavelength of 314 nm.
The amount of unbound material was measured. In addition, 100 mM
The amount of HA-binding substance released by adding EDTA was measured in the same manner.

FIG. 1 shows the obtained results. A tendency was observed that the affinity increased as the number of amino acids increased in both the aspartic acid peptide and the glutamic acid peptide.

Example 2 Synthesis of Osteotrophic Hormone Derivative Estradiol- (L-Asp) ₆ conjugate 1) Using an automatic peptide synthesizer, N-FMOC-L-aspartic acid β-t-butyl ester was used as a starting material. The peptide chain was sequentially extended to construct a protected peptide resin in which six residues of aspartic acid were bonded. 2) Subsequently, the FMOC group is removed in the presence of piperidine,
A protected (Asp) ₆ peptide resin was obtained. 3) Separately, β-estradiol 17-hemisuccinate was synthesized by the following method (note that β-estradiol 17-hemisuccinate was synthesized).
Commercial products of hemisuccinate are also available).

[0031]

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4) The protected (Asp) ₆ peptide resin synthesized in 2) and the β-estradiol 17-hemisuccinate (0.13 mmol) obtained in 3) are carbodiimide (1.6 mmol) in DMF.
And at room temperature for 12 hours to effect dehydration condensation.Finally, exclusion from the resin by trifluoroacetic acid treatment and elimination of the t-butyl protecting group of the amino acid side chain were performed simultaneously, and the obtained crude product was subjected to HPLC. Attached and intended estradiol
-(L-Asp) ₆ conjugate (29 μmol, 29 mg) was obtained.

[0033]

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Example 3 Distribution of Osteotrophic Hormone Derivative In Vivo 1) Preparation and Administration of Test Solution Estradiol- (L-Asp) ₆ conjugate synthesized in Example 2 was dissolved in olive oil and 100 μg After administration to ddY mice (12-week-old) subcutaneously on the back of the mice at a dose of / kg, plasma and femurs were collected over time. 2) Measurement of 17β-estradiol An equivalent amount of 1N NaOH was added to plasma, and the plasma was treated at 60 ° C for 1 hour and neutralized with hydrochloric acid. Femurs were placed in 6N HCl for 6 hours.
After treatment at 0 ° C. for 1 hour, the mixture was neutralized with NaOH. 17β-estradiol in these samples was
The measurement was performed using an oassay kit (Diagnostic Products Co.). 3) Test results The results obtained are shown in FIG. The estradiol concentration in the plasma rapidly increased after administration, but after 8 hours from the administration, the plasma concentration dropped to the normal estradiol concentration level. On the other hand, in the femur, the estradiol concentration increases rapidly after the administration, but gradually decreases after 2 hours from the administration. However, the rate of decrease was extremely slow, and the elimination half-life was about 2 weeks.

Example 4 Therapeutic Effect of Osteotrophic Hormone Derivative on Osteoporosis The therapeutic effect of the osteotrophic hormone derivative of the present invention on osteoporosis was tested using ovariectomized mice as an osteoporosis model. 1) Preparation and administration of test solution Estradiol- (L-Asp) ₆ conjugate synthesized in Example 2 was dissolved in olive oil, and 30,100,300 μg /
kg ddY mice (12 weeks old,
Female) was administered subcutaneously to the back once a week for 5 weeks. As a control drug, 17β-estradiol was dissolved in olive oil and subcutaneously administered at 100 μg / kg once every three days for 5 weeks. Separately, a sham operation group (a group of mice subjected to a surgical operation until immediately before the removal of the ovaries) and an ovariectomy group (the mice were anesthetized with pentobarbital, and the abdomen was incised about 5 mm,
After completely removing the ovaries on both sides, a group of mice in which the incised portion was sutured was prepared. 2) Measurement of bone density and organ weight After the experiment was completed, femurs were excised from the mice of each experimental group, and the bone density was measured using a bone density measurement device (AlokaDCS-600R). In addition, each organ (uterus, brain, lung, heart, liver, spleen, digestive tract, skin and muscle at the administration site) was extracted, and its wet weight was measured. 3) Test results Except for the ovarian weight, the weights of other major organs showed no clear difference between the estradiol- (L-Asp) ₆ conjugate-administered group and the estradiol-administered group, and between the sham-operated group and the ovariectomized group. Did not. The total ovarian weight was significantly lower in the ovariectomized group than in the sham-operated group, and the estradiol-administered group completely suppressed this reduction (see FIG. 3). But,
Estradiol- (L-Asp) ₆ conjugate administration group did not increase ovarian weight at any dose.

On the other hand, the bone density of the femur was significantly lower in the ovariectomized group than in the sham-operated group, and
The-(L-Asp) ₆ conjugate administration group suppressed the decrease in bone density in a dose-dependent manner, similarly to the estradiol administration group.

[0037]

The osteotropic hormone derivative of the present invention is specifically distributed in bone tissue and stays for a long period of time, so that once administered once a week, a sufficient therapeutic effect on osteoporosis was exhibited. Moreover, the osteotropic hormone derivative does not affect the ovarian weight, which also indicates that the osteotropic hormone derivative of the present invention acts selectively on bone tissue. Therefore, the bone tropic hormone derivative of the present invention is expected to be a useful therapeutic agent for osteoporosis without side effects on other organs such as the genital organ even when used for a long period of time.

[Brief description of the drawings]

FIG. 1 is a graph showing the affinity of a peptide comprising an acidic amino acid for hydroxyapatite.

FIG. 2 is a graph showing changes in plasma and femoral estradiol concentrations after administration of estradiol- (L-Asp) ₆ conjugate.

FIG. 3. Estradiol- (L-Asp) ₆ was added to ovariectomized mice.
The bone density of the femur after administration of the conjugate or estradiol is a graph showing ovarian weight.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Ryuichi Fujisawa 4-2-15-301 Kita-37-Nishi 4-chome, Kita-ku, Sapporo-shi, Hokkaido F-term (reference) 4C084 AA02 AA07 BA31 BA36 BA37 DB01 DC50 MA01 NA14 ZA962 ZA972 ZC032 ZC102 ZC112 4H045 AA10 AA30 BA14 BA15 BA16 BA55 DA30 EA27 EA30 FA34 FA57 FA61 GA24

Claims (11)

[Claims] 1. An osteotropic hormone derivative which is a conjugate of a hormonal agent selected from the group consisting of a female hormonal agent, an androgenic agent and an anabolic hormonal agent, and a peptide comprising an acidic amino acid. 2. A compound represented by the following general formula (1): (Where A is a hormonal agent portion selected from the group consisting of a female hormonal agent, an androgenic agent, and an anabolic hormonal agent, X is a crosslinking agent portion between the hormonal agent portion A and the peptide portion, and N Is from 4 to 30, and n is from 1 to 3.). 3. The method according to claim 1, wherein the female hormone agent is selected from the group consisting of (17β-) estradiol, estrone, estriol, equilin, equilenin, ethinylestradiol, diethylstilbestrol and hexestrol. Osteotrophic hormone derivative. 4. The osteotropic hormone derivative according to claim 1, wherein the androgen is selected from the group consisting of testosterone, androstenedione, dehydroepiandrosterone and fluoxymesterone. 5. An anabolic hormone agent comprising: oxymetholone;
The osteotropic hormone derivative according to claim 1 or 2, which is selected from the group consisting of methinolone acetate and stanozolol. 6. The osteotropic hormone derivative according to claim 2, wherein N is 6 to 12. 7. X is _{-CH 2 -, - (CH 2} ) 2 -, - (C
_{H 2) 3 -, - CH} 2 CO -, - (CH 2) 2 CO- or -
(CH ₂₎ osteotropic hormone derivative according to claim 2 wherein the ₃ CO-. 8. The following general formula (2) or (3): 3. An osteotropic hormone derivative according to claim 2, having the formula wherein N and n are as defined above. 9. The following formula (4): The osteotropic hormone derivative according to claim 2, which has the following formula: [10] An agent for treating osteoporosis, comprising the bone tropic hormone derivative according to [1]. [11] An agent for treating osteoporosis, comprising the bone tropic hormone derivative according to [2].