JP3504697B2 - Polypeptide having PTHrP antagonist activity and therapeutic agent for calcium metabolism containing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a polypeptide having PTHrP antagonistic activity and a therapeutic agent for calcium metabolism containing the same. The antagonist of the present invention is used for treating hypercalcemia and the like.

[0002]

BACKGROUND ART Parathyroid hormone (parathyroid hormone) is a typical example of a calcium metabolism regulator in blood.
One, PTH), calcitonin, vitamin D, etc., but it has been pointed out that none of the above is a causative agent of hypercalcemia caused by cancer. In 1987, a protein exhibiting the same activity as PTH was isolated from human squamous cell carcinoma showing hypercalcemia by Moseley et al.
rathyroid hormone related protein (PTHrP). It was also found that the causative agent of hypercalcemia in cancer patients was this PTHrP.

Furthermore, Suva et al. Have determined the amino acid sequence and cDNA base sequence of PTHrP.
THrP was found to consist of 141 amino acids. Suva et al. Expressed PTHrP in a mammalian cell line from the obtained cDNA and converted it into PTH activity of 48 μg /
The expression of 1 is confirmed.

Rodan et al. Have already chemically synthesized a peptide fragment from the N-terminal side of human PTHrP (hereinafter referred to as "human PTHrP (1-34)") to a cell line ROS17 derived from rat osteosarcoma. PTH on increasing the activity of adenylate cyclase at 2.8 / 2.8
It has been confirmed that it has the same activity as (1-34).

Hypercalcemia in cancer patients is said to occur in about 10% of all cancer patients, and causes various disorders such as pain due to an increase in calcium concentration in blood.

Currently, there is a calcitonin-related peptide as a therapeutic drug for this hypercalcemia, but its effect is transient and its efficacy rate is low. This is because calcitonin binds to the receptors of osteoclasts and suppresses bone resorption, but PTHrP, which is a causative agent of hypercalcemia.
This is because it does not suppress the activity of A. and its effect is therefore limited.

The present applicant has previously found that the amino acid sequences 1 to 6 from the N-terminal of PTHrP are essential for the expression of PTHrP activity and that they can bind to the PTHrP receptor by the amino acid sequences 7 to 34. Therefore, the 7th to 34th amino acid sequence from the N-terminal side of PTHrP (hereinafter, "human PTHrP (7-34)" is PT
The inventors have found that it is useful as an antagonist of HrP and applied for a patent (Japanese Patent Laid-Open No. 207099/1990).

[0008]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention
The PTHrP antagonist described in JP-A-07099 does not have PTHrP activity, but binds to PTHrP receptor competitively with PTHrP and thereby PTHrP.
Antagonize activity. If a PTHrP antagonist having an antagonistic activity higher than that of this known PTHrP antagonist is obtained, it is more effective in treating hypercalcemia.

Therefore, an object of the present invention is to provide the above-mentioned known PT.
It is to provide a human PTHrP antagonist having a higher antagonist activity than that of the HrP antagonist.

[0010]

As a result of earnest research, the present inventors have found that the substitution of several amino acids in human PTHrP (7-34) significantly enhances its antagonistic activity. Completed the invention.

[0011]

The present invention has the activity of antagonizing the physiological action of human PTHrP or a polypeptide having human PTHrP activity without having human PTHrP activity,
A polypeptide comprising an amino acid sequence represented by the following formula [II] is provided. Leu His Asn Leu D-Phe (4-F) Lys Ser Ile
Gln Asp Leu Arg Arg Arg Phe PheLeu His His Leu Il
e Ala Glu Ile His Thr Ala [II]

[0013]

Further, the present invention provides the polypeptide represented by the above formula [II] .

The present invention further provides human PTHrP containing any of the above-mentioned polypeptides of the present invention as an active ingredient.
To provide a therapeutic drug for calcium metabolism against.

The present invention will be described in detail below.

The above formula [II] is described from the N-terminal side. D-Phe (4-F) in the above formula [II] means that the 4-position of the phenyl group in the D-form of phenylalanine is substituted with a fluorine atom. In addition, in the above formula, D-Trp means the D-form of tryptophan. In addition, amino acids not labeled with D and L are L-forms. For the purpose of chemically synthesizing the polypeptide, leucine at the N-terminal may be deaminized to desaminoleucine, and NH2 may be bonded to the carboxyl group of alanine at the C-terminal. As will be clear in
The polypeptide represented by the above formulas has HrP antagonistic activity, and its N-terminal is deaminated,
Also included are C-terminally aminated polypeptides.

Each of the polypeptides represented by the above formula [II] is obtained by substituting several amino acids in the polypeptide consisting of the 8th to 34th amino acid sequences from the N-terminal side of human PTHrP. Surprisingly, this substitution significantly increased the adenylate cyclase inhibitory activity, that is, the human PTHrP antagonistic activity, as will become apparent in the Examples below.

The polypeptide of the present invention has the formula [I
It may be composed of only the amino acid represented by [ I] , or may be one to which another amino acid sequence is added as long as it does not exhibit human PTHrP activity. For example, the amino acid after the 35th position of human PTHrP may be linked to the C-terminal.

The polypeptide of the present invention can be produced by chemical synthesis or genetic engineering techniques. When the number of amino acids is 40 or less, the method of producing by chemical synthesis is convenient, but when the number of amino acids exceeds 40, chemical synthesis becomes difficult, and thus it is preferable to synthesize by a genetic engineering method.

The polypeptide represented by each of the above formulas has 26 or 27 amino acids, and thus can be easily prepared using a commercially available peptide synthesizer. For example, it can be performed by the F _moc method using a Model 430 peptide synthesizer manufactured by Applied Biosystems.

The synthesis of the polypeptide of the present invention by a genetic engineering method includes a region encoding the polypeptide,
It can be carried out by transforming Escherichia coli with an expression vector capable of expressing the polypeptide in Escherichia coli, culturing the transformed Escherichia coli, and recovering the polypeptide from the culture.

In the above expression vector, the region encoding the polypeptide of the present invention may have any base sequence as long as it encodes the polypeptide of the present invention. It is advisable to use frequently used codons to avoid palindromes and homologous sequences.

A promoter which enhances the transcription efficiency in E. coli is present upstream of the polypeptide coding region of the present invention. The promoter is preferably derived from E. coli,
A tryptophan promoter is particularly preferable. The polypeptide coding region of the present invention may be located immediately downstream of the promoter, or the above region may be located downstream of a region encoding an Escherichia coli-derived protein such as E. coli trpE protein. In the latter case, the polypeptide of the invention will be obtained in the form of a fusion protein.

The vector used has an appropriate selectable marker such as antibiotic resistance and an origin of replication for replication in E. coli. Furthermore, a transcription termination codon exists downstream of the polypeptide coding region of the present invention. For example, pUC9, pBR322 and other commercially available E. coli vectors can be used as they are.

The expression vector is prepared by synthesizing the above-mentioned polypeptide coding region of the present invention by a known method such as the phosphoamidide method, and cloning it into a commercially available vector for E. coli or a known vector expressed in E. coli. Can be manufactured by. Transformation using the obtained expression vector and subsequent cultivation of Escherichia coli can be performed by a well-known method. The polypeptide of the present invention produced by transformed Escherichia coli is obtained by collecting bacterial cells by centrifugation or the like, destroying the bacterial cells by well-known lysozyme treatment and / or ultrasonic treatment, etc. It can be separated and purified by subjecting it to gel filtration chromatography or the like.

The therapeutic drug for calcium metabolism of the present invention comprises the above-mentioned polypeptide of the present invention as an active ingredient. The human dose of the therapeutic agent for calcium metabolism of the present invention is usually about 3 × 10 ⁻⁶ mol to 3 × 10 ⁻⁷ mol of the polypeptide of the present invention, and the route of administration is preferably intravenous injection or intramuscular injection. In addition, as a specific formulation example as a calcium metabolism therapeutic agent, the polypeptide of the present invention is added to a physiological saline or a citrate buffer at 1 × 10 ⁻⁵ M to 1
Examples thereof include those containing x10 ^-6 M.

The calcium metabolism therapeutic agent of the present invention can be used for the treatment of various diseases having abnormal calcium metabolism, for example, bone diseases such as hypercalcemia and osteoporosis, and hypercalcemia due to chronic renal failure. .

[0029]

EXAMPLES The present invention will be described more specifically below based on examples.

Example 1 Synthesis of Polypeptide Polypeptides represented by the formulas [I] to [III] were synthesized by a model 430 peptide synthesizer manufactured by Applied Biosystems. However, the N-terminal leucine was deaminized desaminoleucine, and an amino group bound to the carboxyl group of the N-terminal alanine (threonine in formula [III]) was obtained. The results of amino acid analysis of each of the synthesized polypeptides are shown in Table 1 below together with their theoretical values. In addition, HLP of each polypeptide
The retention time in C and the result of FAB-MS are shown in Table 2. The formula [I] and the formula [III] are as follows. Leu His Asn Leu D-Trp Lys Ser Ile Gln Asp Leu Arg
Arg Arg Phe Phe LeuHis His LeuIle Ala Glu Ile His
Thr Ala [I] Leu His Asn Leu D-Trp Lys Ser Ile Gln Asp Leu Arg
Arg Arg Phe Phe LeuHis His LeuIle Ala Glu Ile His
Thr [III]

[0031]

[Table 1]

[0032]

[Table 2]

Example 2 Polypeptide Antagonist Activity (In Vitro Test) The antagonist activity of each polypeptide synthesized in Example 1 was tested in vitro using rat myeloma cell line ROS17 / 2.8.
Tested in. Specifically, the test method was as follows. Sufficient cAM up to near maximum stimulation for ROS cells
5 × 10 ⁻⁹ M PTHrP (1-3 for which P productivity was obtained)
The inhibitory effect against the stimulation of 4) was compared by serially diluting various polypeptides. ROS17 / 2.8 was cultured in Ham's F12 medium for 2 days. After washing the cells with F12 medium, the synthetic polypeptides and PTHrP (1-34) with varying concentrations
Was added and incubated at 37 ° C. for 10 minutes. On ice,
After adding 1N HCl, the cells were collected, and the concentration of the supernatant was measured with a commercially available (Yamasa) cAMP RIA kit. When the polypeptide (I-III) alone is added,
No increase in cAMP (agonist activity) was observed.

The results are shown in FIG. As is clear from this figure, the polypeptide of the present invention is a known human PTHrP.
It was revealed that the inhibitory rate of adenylate cyclase activity was significantly higher than that of (7-34), and that it was superior as a human PTHrP antagonist.

Example 3 Polypeptide Antagonistic Activity (In Vivo Test) 250 pmol of human P per day in 4 mice per group
THrP (1-34) was administered, and at the same time, 500 nmol of the polypeptide of the formula [I] synthesized in Example 1 was administered per day. These routes of administration were subcutaneous continuous administration (Osmotech minipump), and physiological saline containing 2% cysteine-HCl was used as a carrier. The concentration of the active ingredient in the carrier is human PTHrP (1-
34) is 10.4 μM and the polypeptide of formula [I] is 2
It was 0.8 μM and was administered at 24 μl per day. In addition, as a control, the one containing only human PTHrP (1-34) in the above carrier was administered. The calcium concentration in serum was measured before the start of administration and 3 days after the start of administration. The results are shown in Fig. 3.

As is clear from FIG. 2, in the group to which the polypeptide of the present invention was administered, the calcium concentration in the serum was significantly lower than that in the control group, and the in vivo PTHrP antagonist activity of the polypeptide of the present invention was high. confirmed.

[0037]

INDUSTRIAL APPLICABILITY The present invention provides a novel polypeptide having a human PTHrP antagonistic activity superior to the known ones and a therapeutic agent for calcium metabolism containing the same as an active ingredient. Therefore, according to the present invention, various diseases having abnormal calcium metabolism, for example, bone diseases such as hypercalcemia, osteoporosis, and hypercalcemia due to chronic renal failure can be more effectively treated.

[Brief description of drawings]

FIG. 1 shows the adenylate cyclase inhibitory activity of the polypeptides of Examples of the present invention, which was determined by the analysis of human PTHrP (7-34).
It is a figure shown in comparison with.

FIG. 2 is a graph showing the serum calcium concentration-lowering effect of the polypeptide of the present invention in human PTHrP (1-34) -induced hypercalcemic mice.

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Chiharu Oue 1-3-1, Nishitsurugaoka, Oi-cho, Iruma-gun, Saitama Prefecture Tonen Corporation Research Institute (72) Inventor Junichi Matsuzaki Nishitsurugaoka, Oi-cho, Iruma-gun, Saitama Prefecture 1- 3-1 Tonen Co., Ltd. Research Institute (56) Reference JP-A-2-22292 (JP, A) JP-A-4-217997 (JP, A) Pept. Chem. , Sept. 27, 1993, Vol. 1992, p. 547-549 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C07K 14/635 CA (STN) REGISTRY (STN) BIOSIS / WPI (DIALOG)

Claims (3)

(57) [Claims] 1. A human P having no human PTHrP activity.
A polypeptide having an activity of antagonizing the physiological action of a polypeptide having THrP or human PTHrP activity and comprising the amino acid sequence represented by the following formula [II]. Leu His Asn
Leu D-Phe (4-F) Lys Ser Ile Gln Asp Leu Arg Arg Arg
Phe Phe Leu His His Leu Ile Ala Glu Ile His Thr A
la [II] 2. A polypeptide represented by the above formula [II] . 3. A therapeutic agent for calcium metabolism against human PTHrP, which comprises the polypeptide according to claim 1 or 2 as an active ingredient.