KR0159990B1 - Pharmaceutical compositions for the treatment of hypertension - Google Patents

Abstract

The present invention relates to a novel hypertension therapeutic composition and a method for treating hypertensive mammals except the human body, wherein the hypertension therapeutic composition and the hypertension except the human body are coadministered with a calcium channel blocker, a calcium supplement, and / or vitamin D. A method of treating a mammal.

The inventors have identified a hypertension circulating factor associated with mammalian hypertension with respect to the regulation of intracellular calcium uptake derived from the parathyroid gland, and thus, the calcium channel blocker, calcium supplement and / or vitamin D are commonly administered to prevent To reduce the side effects of, and to provide a novel hypertension therapeutic composition that can obtain a synergistic effect.

By using a therapeutic composition comprising a calcium channel blocker and a calcium supplement, a greater amount of anti-hypertensive effect can be obtained by using a smaller amount of calcium channel blocker, thereby increasing the therapeutic effect and reducing side effects as well as reducing the calcium channel blocker dose. Therefore, the treatment cost can be significantly reduced.

Description

Hypertension Therapeutic Compositions and Methods of Treatment of Hypertension Mammals Excluding Humans

1 is an elution graph for SHR rat plasma according to Example 3.

2 is a graph of reverse phase HPLC separation of SHR rat plasma samples of FIG.

2 is (b) is an HPLC graph of WKY rat plasma.

3 is a schematic of the mechanism by which calcium enters the cell and its mechanism regulation.

4 shows the mean arterial pressure change of SD rats injected with plasma obtained from SHR, SD and WKY rats.

5 shows the mean arterial pressure change of SD rats injected with plasma obtained from SHR, SD and WKY rats.

FIG. 6 shows ⁴⁵ Ca uptake of tail arterial sections cultured in Krebs buffer containing plasma of SD, WKY, SHR rats.

7 shows the mean arterial pressure change of SD rats subjected to an anomalous fraction of SHR rat plasma separated by molecular exclusion chromatography.

FIG. 8 (a) is a reverse phase HPLC graph of culture medium obtained from parathyroid cell culture using thyroid of SHR rat.

8 (b) is a comparative graph of parathyroid cell medium in WKY rats.

Figure 9 (a) is an SDS-PAGE gel scan using the 14KD standard.

Figure 9 (b) is a gel scan of parathyroid extracts in SD rats after 8 hours of incubation.

Figure 9 (c) is a gel scan of parathyroid extracts of SHR rats after 8 hours of incubation.

Figure 10 (a) is a scan of SDS-PAGE gels in Sprague-Dawley thyroid cell (12 hour culture) culture medium.

Figure 10 (b) is a scan of SHR rat thyroid cell culture medium after 4 hours of incubation.

Figure 10 (c) is a scan of SHR rat thyroid cell culture medium after 12 hours of incubation.

Figure 10 (d) is a gel scan using 14KD standard.

11 and 12 are graphs of the data shown in Table 1.

13 illustrates a method of stimulating vascular smooth muscle contraction by a system for increasing calcium absorption.

14 illustrates the beneficial and damaging effects of calcium supplementation alone.

Figure 15 illustrates the beneficial and damaging effects of calcium antagonists.

Figure 16 illustrates the benefits of combination therapy.

Figure 17 is an isoelectric focusing gel of PHF plasma and cell culture medium.

The present invention relates to a novel hypertension therapeutic composition and a method for treating hypertensive mammals except the human body, wherein the hypertension therapeutic composition and the hypertension except the human body are coadministered with a calcium channel blocker, a calcium supplement, and / or vitamin D. A method of treating a mammal.

The inventors have identified a hypertension circulating factor associated with mammalian hypertension with respect to the regulation of intracellular calcium uptake derived from the parathyroid gland, and co-administered a calcium channel blocker and a calcium supplement and / or vitamin D. To reduce the side effects of, and to provide a novel hypertension therapeutic composition that can obtain a synergistic effect.

Hypertension is generally defined as an increase in systolic and / or diastolic arterial pressure above 140/90 mmHg. Diseases associated with hypertension include atherosclerosis, hypertensive renal failure, stroke, congestive heart failure and myocardial infarction. Although many effective therapies for lowering arterial blood pressure are known, the etiology of essential hypertension is unknown. Although the etiology of hypertension is generally thought to be genetic, the fact that these drugs act to induce different pharmacological responses suggests that there are other primary causes of essential hypertension.

Many studies have suggested that one or more circulatory factors may play an important role in the development or persistence of hypertension. [Ref: Wright et al., Hypertensive substances found in the blood of spontaneous hypertensive rats; Life Sci 1984: 34: 1521-1528; Dahl et al., Humoral Transmission of Hypertension: Demonstration from Parabiosis; Circ Res. 1969; 24/25 (Suppl. I) 21-23; Greenberg et al., Demonstration of circulating factors as a cause of venous hypertrophy in spontaneous hypertensive rats; Am. J.

Physiol. 1981; 241; H421-H430 Tobian et al., Circulating fluid booster in Dahl S rats of hypertensive hypertension; Clin. Sci. 1979; 57: 345s-347s; Axogenic factors in the pathology of essential hypertension, such as Zidek; Klin Wochenschr. 1985; 63 (Suppl. II) D; 94-96; Hirat et al., Hypertensive factor in hypertensive lumbar sensitized rat serum; Hypertension 1984; 6: 709-716].

For example, in by-products and cross-circulation experiments, elevated blood pressure is induced in normal animals by exposure of hypertensive animals to the blood of hypertensive animals. In other words, subcutaneous injection of erythrocyte-related factors obtained from spontaneous hypertensive rats (SHR) into the Wistar-Kyoto (WKY) rats of normal blood pressure induced hypertension. Infusion of (Dahl) rat serum into normal blood pressure-induced rats may cause elevated blood pressure.

There are also reports that circulating factors increase intracellular calcium in both hypertensive rats and hypertensive humans. [Reference ; Banos et al., Two factors associated with increased calcium absorption in platelets in patients with essential hypertension; Clin. Exp. Hypertens. 1987; 9: 1515-1530; Zidek et al., Effect of Plasma on Hypertension on Calcium Transport in Permeable Human Neutrophils; clin Sci. 1988; 74; 53-56; Effect of Circulatory Factors on Intracellular Calcium in Normal Platelets of Linder et al .; N. Eng. J. Med. 1987; 316; 509-513; Increase in cytosolic free calcium in platelets of spontaneous hypertensive rats and patients with essential hypertension; Clin. Sci. 1985; 68; 179-184; Wright et al., Stimulation of aortic tissue calcium uptake by spontaneous hypertensive rat erythrocyte extract with hypertension; Can. J. Physiol Pharmacol. 1986; 64: 1515-1520.]

Since vascular tension is influenced by intracellular calcium concentration, it is reasonable to consider that factors that raise blood pressure and factors that increase intracellular calcium correlate with each other, although it has not been experimentally demonstrated yet. It is considered to be. Evidence suggests that calcium-regulating hormones are involved in many forms of hypertension. [Reference ; L. M. L.M. Resnick, Am. j. Med. 82 (Suppl. 1B) 16 (1987). Parathyroid hormone (PTH) is a calcium regulatory hormone. More than 30% of patients with essential hypertension correspond to a subgroup characterized by increased levels of immunoreactive parathyroid hormone (ir-PTH). [Reference ; Laragh et al., Kidney Int. 34, (Suppl. 35), S162 (1988). An increase in PTH concentration has been reported in the case of SHR rats [see; McCarron et al., Hypertension 3 (Suppl. 1), I162 (1981))] Hyperparathyroid patients often have hypertension, which in most cases is reduced by parathyroidectomy. Can be. [Reference ; Hellstrom et al., Brit. J. Urol. 30, 13 (1958). Similar results of parathyroidectomy have also been reported in SHR rats. [Reference ; Schleiffer et al., Jap. Circ. J. 45, 1272 (1981). Although administering PTH externally reduces blood pressure in mammals and other vertebrates, many researchers have published that PTH promotes essential hypertension [see; Pang et al., Gen. Comp. Endocrinol. 41, 135 (1980)). In addition, the vasodilating action of PTH is related to molecules at specific sites separated from sites showing hypercalcemia effects [see; Pang et al., Endocrinology, 112, 284 (1983). PTH is also referred to as L-type calcium channel [see; Wang et al. (Submitted for publication) to inhibit calcium influx into vascular smooth muscle. [Reference ; Pang et al., Life Sci 42. 1395 (1988)). This contradiction is further evident by the fact that serum ionized calcium levels appear to decrease in hypertensive patients with elevated PTH levels [see; Resnick et al., New Engl. J. Med. 309. 888 (1983); Hvarfner et al., Acta Med. Scand. 219. 461 (1986)]. Serum ionized calcium concentration is presumably elevated when PTH is elevated.

Although it is clear that parathyroidism is involved in essential hypertension, the reference literature on the action of PTH on the vasculature is inconsistent with the role that PTH causes in essential hypertension. Until the present invention was completed, PTH was the only active hormone reported to be produced by the parathyroid glands.

Calcium channel blockers are described in Fleckstein et al. Kreislaufforsch. 56. As reported by 716 (1967), it has been identified as a method for controlling hypertension and has been commonly used for control of hypertension. Three types of calcium channel blockers, Verapamil, Nifedipine and Diltiazem, are currently of clinical importance in the United States, all of which have been shown to inhibit calcium ion influx into vascular smooth muscle. Get high blood pressure effect. The ultimate effect is vasodilation. Calcium channel blockers are useful because they limit calcium absorption into vascular smooth muscle, but are known to stimulate some endocrine systems, such as the RAS system. [Kotchen et al. Am. j. Cardiol. 62 41G (1998); Matsumara et al., J. Pharmacol. Exp. Ther. 241. 1000 (1978); Resnick et al. Fed. Proc. 45 2739 (1986). Calcium channel blockers may cause hypervascular expansion, negative inotrophy, hypothermia of sinus nodal rate, atrioventricular node conduction disorder, and interference with non-vascular smooth muscle contraction. Can be. Therefore, a combination therapy that minimizes the amount of calcium channel blocker required to achieve a predetermined antihypertensive effect is desirable.

The previously unreported presence of circulatory factors originating in the parathyroid gland is demonstrated in many people and SHR rats with essential hypertension. The factor has been shown to regulate intracellular calcium uptake and can be inhibited by increasing dietary calcium levels. The circulating factor was isolated and a method of screening the circulating factor using an antibody generated corresponding to the factor was described. The circulating factor has a molecular weight of about 3,000 to 4,000.

Bioassay data revealed that circulating factors in humans and rats were substantially similar. Circulatory factors appear to be present in a large number of people, but not all in people with essential hypertension, especially those associated with low-renin hypertension. Circulation factor identification in hypertensive patients can be used to plan and monitor therapies to eliminate the effects of hypertension.

In addition, the combination of dietary calcium supplements and calcium channel blockers is an effective method for the treatment of hypertension, and it has been found that the combination therapy using these two substances is more effective and certain than when used alone.

The effect is much larger than the sum of the effects of using each of the two substances and shows synergy. In addition, co-administration of effective forms of vitamin D and calcium channel blockers such as 1α, 25-dihydroxycholecalciferol (1α, 25- (OH) ₂ D ₃ ), which increase intestinal calcium absorption, are also effective treatments. .

Circulatory factors of SHR rat blood are reported in the paper Am. J. Hypertens. 2. Its existence was confirmed in 26-31 (1989).

In this paper, we report an increase in blood pressure in WKY and SD rats when plasma from SHR rats is injected into rats of normal blood pressure by infusion or bolus injection. It was also reported that ⁴⁵ Ca uptake by rat microarterial sites in vitro increased dose dependently with increasing SHR plasma concentrations in buffer media.

These results clearly show that both the increase in blood pressure and the increase in calcium uptake in the cell depend on the amount of available SHR plasma present in the system. Strangely, the early onset of the above two phenomena was delayed and progressive, whereas conventional endogenous boosters such as norepinephrine, angiotensin II, and vasopressin were found to elevate blood pressure upon administration. It became. Another result observed in this paper is that blood pressure drops very rapidly to baseline when SHR plasma injection is stopped and replaced with plasma from normal blood pressure rats. This drop precludes simple quantitative effects.

Related experiments have shown that dialysis plasma obtained from normal and hypertensive subjects causes hypertension when injected into SD rats of normal blood pressure. The plasma of these subjects increased calcium uptake in the rat aorta in vitro.

The source of the circulatory factor is unknown, but some reports suggest that PTH is elevated in hypertensive rats, suggesting parathyroid gland as the study's goal.

Parathyroidctomy of SHR rats was found to reduce blood pressure, and plasma of SHR rats with parathyroid gland resection did not increase blood pressure in normal blood pressure rats.

Conversely, when the parathyroid gland of SHR rats were implanted into Sprague-Dawley rats of normal blood pressure, the blood pressure was elevated and the factors were expressed in plasma as shown by the injection of isolated plasma into other normal blood rats. Brought the result. [See: Pang and Lewananceuk, Amer. J. Hypertens. 2. 898 (1989). Based on these studies, we concluded that the parathyroid gland was the source of circulatory factors and named the foreign body Parathyroid Hypertensive Factor or PHF.

Potential multifactors in the experiments described above may be the presence of calcium and other low molecular weight boosters present in high concentrations in transfused plasma. In order to eliminate this possibility, all transfused plasmas were dialyzed for a long time using membranes to remove molecular weights of about 1000 or less. This process removes calcium and is effective in removing most known endogenous boosters, including the reported ouabain-like factors, having a molecular weight range of 400-500. In addition, all known endogenous boosters act rapidly.

SHR rats, WKY rats and SD rats were decapitated and bled to separate and confirm PHF from plasma, and each type of plasma collected was centrifuged by treatment with heparin. The collected plasma was filtered by primary dialysis (using a dialysis membrane to remove molecular weight less than 1000) and by an ultrafiltration system capable of filtering less than about 5000 molecular weights, and the collected filtrates were concentrated by freeze drying. The concentrated dialysate was passed through a molecular sieve column such as Bio-Gel P-6. Fractions were collected, eluting with 0.05 M NH ₄ OA _c at pH 7.0.

Elution was monitored spectroscopically at 280 nm using a Pharmacia U.V detector, where fractions not found in samples from normal blood pressure rats were identified in plasma of SHR rats. (See Figure 1)

Anomalous fractions of SHR rats were collected, concentrated by lyophilization and injected into rats of normal blood pressure. An increase in blood pressure was observed along with the delayed initial expression (30-45 minutes) of SHR rat plasma. Fractions at the same elution site of normal blood pressure rat plasma specimens did not exhibit this action.

The active ingredient of the above SHR fraction was determined to have a molecular weight of about 3,500 Daltons by molecular exclusion and chromatography using liquid chromatography column and HPLC.

To further characterize the active fractions, lyophilization and sorting while monitoring the elution again at 280 nm with Brownlee RP-P (C-8) reversed phase column HPLC using a 0.1% trifluoroacetic acid: acetonitrile solvent gradient It was. The results are shown in FIGS. 2 (a) and 2 (b).

Parathyroid glands were excised from SHR rats and cultured for 7 days with daily medium change in Hank's medium (Gibco).

The calcium in the medium can be reduced to stimulate PHF production. Similar to the treatment of plasma samples, dialysis and filtered pooled media were lyophilized and sorted by HPLC using a reversed phase column. Figure 8 (a) shows the separation, and Figure 8 (b) shows the results when using the parathyroid gland of WKY rats.

Primary cell cultures prepared from SHR and SD rats were cultured as described above. Samples of the medium were separated and lyophilized at 4 hour intervals. After resuspension with a minimum amount of distilled water, the extract is deposited on an SDS-PAGE gel (15-18% acrylamide / bisacrylamide) and developed for about 1 hour, followed by Bio-Rad Mini-Protein II (Bio-Rad Mini-). Protein II). The gel was scanned after staining with Coomassie blue, preferably silver.

The result is shown in FIG. A unique peak appeared in the medium of SHR rat cells, which was not seen in SD rat cell medium or SHR rat cell medium about 12 hours ago. The peak molecular weight was estimated to be about 3,300 Daltons.

Parathyroid cells were cultured as described above in related experiments. After 8 hours the cells were separated from the medium and homogenized in 50 mM acetic acid followed by Ca. Cell debris was removed by centrifugation at 5,000 x g and 10-18% acrylamide / bisacrylamide gel chromatography was performed. The result is shown in FIG.

The isoelectric point of PHF obtained from SHR parathyroid medium or plasma was determined using a Bio-Rad mini-isoelectric focusing system and a cationic electrolyte gradient of pH 3-10, namely IEF and Measurement was made on SDS-PAGE. The results are shown in FIG. 17 and show typical droplets at about pH 6.

RNA can be extracted from cells producing PHF using conventional techniques (Maniatis, T et al., Molecular Cloning, A Laboratory Method, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982)). . Poly (A) RNA can be isolated using chromatography, such as an olido- (dT) cellulose column, converted to cDNA using reverse transcriptase and conventional techniques [eg, Rand (Land) et al., 9 Nucl. Acids Res., 2251 (1981)] can be double stranded. DNA can be inserted into useful transformation vectors and used to modify appropriate cloning hosts such as E. coli K12. Digested plasmids obtained from the host are used to prepare cDNA libraries and screened by synthetic labeling markers by standard methods. Southern, 98 J. Mol Biol. 503 (1975).] Strongly hybridizing fragments can be used to construct expression vectors. Cells modified with the expression vector can be used as a production source to produce PHF in high yield.

Using purified PHF from plasma, or from cultured or modified cells, polyclonal antibodies and monoclonal antibodies can be generated against PHF, and the antibodies can be used to analyze PHF. Can be.

Viamontes et al., J. Immunol. Male Balb / c mice were immunized by implanting partially purified PHF-attached aminophenolthiol ether disks obtained from parathyroid cultures according to the method of Meth, 94 13-17 (1986). These mice were inoculated with Freund's incomplete adjuvant at two week intervals and antibody titers were evaluated by enzyme-linked immunosorbent assay (ELISA) using PHF isolated from plasma as antigen. A detectable amount of the polyclonal antibody was observed within one month and the titer was then elevated.

Monoclonal antibodies can be prepared from the spleen of polyclonal antibody producing mice.

Certain hybridomas and MCAs are known to Methods in Enzymology 121, 1-947 (1986) of Langone and Van Vunakis, or to those of ordinary skill in the art. It can be obtained using a modification.

Detection methods using polyclonal and / or monoclonal antibodies are not particularly limited and include assay systems based on radioimmunoassay (RIA), enzyme immunoassay, enzyme-linked immunosorbent assay and immunoprecipitate formation. Such an analysis system is particularly desirable in the form of a diagnostic kit (Kit) that can be used in a clinic or clinics in the absence of sophisticated analytical instruments. The methods described above were used to detect hormones and other immune reactants in the liquor. One example is a commercially available kit for early diagnosis of pregnancy. Therefore, PHF can be detected quantitatively or qualitatively using the above-mentioned method.

Representative test kits of enzyme-immunoassays include: a) antibodies against parathyroid hypertensive factors bound to a solid phase; b) a second antibody labeled with an enzyme; c) a substrate for labeling said enzyme with said second antibody; And d) a standard solution of parathyroid hypertensive factor.

Representative test kits for RIA include: a) antibodies against parathyroid hypertensive factors; b) a second antibody against parathyroid hypertensive factor; c) standard solution of radiolabeled parathyroid hypertensive factor; d) standard solution of unlabeled parathyroid hypertension factor; And e) precipitation reagents. Representative test kits for immunoprecipitation assays include: a) antibodies to parathyroid hypertensive factors; b) a solid phase to which the antibody is attached; And c) a standard solution of parathyroid hypertensive factor.

The identification of the PHF has led to the description of unusual research reports that dietary calcium often effectively lowers blood pressure in hypertensive patients and that calcium channel blockers also effectively lower blood pressure in the same patient.

Higher serum calcium ions are believed to result in higher intracellular calcium concentrations and blood pressure. While not currently based on any particular theory, PHF acts to open the calcium channel as shown in FIG. 2, but high levels of dietary calcium appear to inhibit the release of PHF. Indeed, the inventors have shown that calcium deficiency diets in SHR rats result in an increase in plasma PHF which is not seen in the plasma of high calcium dieted SHR rats. [Rwantsk and Fang, Abstract, 4th Annual Meeting of the America Society of Hypertension, 1989].

This hypothesis is supported by infusion studies with other vascular active substances.

Infusion of norepinephrine, arginine vasopressin (AVO) or angiotensin II (A-II) with dialysis SHR serum in SD rats further increased mean arterial pressure (MAP) and peaked hypertensive responses to SHR plasma. It was observed to rise to maximum when it reached.

The presence of PHF has been shown to be characterized by low, low-renin-type or salt-sensitive hypertension, but not high-renin-type or salt-free hypertension. The intracellular source of PHF in the parathyroid gland was identified from parathyroid dissection of SHR rats resulting in a decrease in MAP. In addition, unique cell types not observed in SD or WKY rats were observed in the parathyroid gland of SHR rats.

The novel cells can be observed by both light and electron microscopy.

The cells were stained deeper with cytoplasma using aldehyde fuchsin or iron hematoxylin, confirming that the dense and irregular shape had nuclei.

The use of PHF detection allows physicians to easily perform diagnostic tests to identify specific causes of essential hypertension, select appropriate treatments, and monitor them.

It was found that supplemental calcium inhibits the production of not only PHF but also renin, and that elevated calcium levels are effective in reducing hypertension.

The effect of calcium supplementation is unpredictable due to the fact that high concentrations of blood calcium can limit the antihypertensive effect by increasing the bio-availability of calcium to vascular smooth muscle tissue. In addition, extremely high levels of dietary calcium can cause calcium accumulation and kidney stones in painful joints. Any calcium supplement that increases serum calcium concentration may be used, with calcium carbonate derived from mineral or oyster shells being preferred. Examples of the formulations are Os-Ca ^R (manufactured by Marion) and Biocal ^R (manufactured by Miles).

The use of combination therapies including calcium channel blockers and dietary calcium supplements can lower blood pressure using less calcium channel blockers while gaining greater antihypertensive effects. Calcium channel blocker means a pharmaceutical composition that inhibits the inflow of calcium into the cell or inhibits the movement of calcium from the cell. [See Gilman et al., The Pharmacological Basis of Experimental Therapeutics, 7thed., McMillan, New York, 1985. pp. 816-821.] Representative examples of calcium channel blockers include dihydropyridine and verapamil, such as nifedipine. Benzeneacetonitrile such as Verapamil) and benzothiazepine such as diltiazem.

As a result, not only the therapeutic effect is increased and the side effects are reduced, but also the treatment cost can be considerably reduced since the price of calcium supplement is much cheaper than commercially available calcium channel blockers.

The administration of calcium supplements may be replaced by the administration of an effective form of vitamin D, such as 1α, 25- (OH) ₂ D ₃ , if it contains adequate calcium in the diet. Administration of vitamin D is intended to increase calcium absorption in the duodenum mucosa. When calcium and / or 1α, 25- (OH) ₂ D ₃ are administered in combination with a calcium channel blocker, it is desirable to make the active ingredients into one capsule containing each appropriate unit dose. Having the patient take only one medicine facilitates the taking.

Combination therapy involving calcium channel blockers, calcium and / or effective forms of vitamin D, such as 1α, 25- (OH) ₂ D _3, results in a daily dose of calcium channel blocker less than half the daily dose required. In some cases, the daily dose of calcium channel blockers may be reduced to up to one fifth of the dose required to take calcium channel blockers alone. Such combination therapy may be used in combination with agents for the control of hypertension and angina such as angiotensin converting enzyme (ACE) inhibitors, β-adrenergic antagonists, nitrates and diuretics.

A particular advantage of the combination of calcium channel blockers and calcium and / or 1α, 25- (OH) ₂ D ₃ is the predictability of the treatment. Dose-response curves for nifedipine are unpredictable in individual patients and require considerable time to ascertain the appropriate dosage. The effect of calcium supplementation from the outside cannot be predicted due to various factors such as absorption rate, secretion rate, parathyroid hormone concentration, and PHF intensity. Surprisingly, however, the combination of calcium channel blockers and calcium supplements was found to be synergistic and predictive of dose response. Thus, the duration of patient care needed to find the appropriate dose of the drug is shortened and the potential for side effects is reduced because of the higher therapeutic index.

The dosage of the combination pharmaceutical composition used depends on the needs of the individual patient.

A representative body type is a capsule containing calcium channel blocker corresponding to 1/5 to 1/2 of the usual dosage and 500 mg of calcium carbonate and / or 1α, 25- (OH) ₂ D ₃ 10-25USP units (0.05 μg). Form.

For example, the usual dosage of nifedipine is 10 mg to 20 mg 2-3 times a day, but the dosage according to the present invention is 5 to 10 mg. The typical dose of verapamil compared is 80-120 mg and the dose of diltiazem is 30-60 mg.

The presence of PHF and analysis of PHF are applicable not only to the identification of essential hypertension, but also to the study and treatment of other diseases with or without hypertension as the primary symptom. For example, most non-insulin dependent diabetics are hypertensive. In contrast, patients with hypertension often have reduced glucose tolerance. In both cases, an increase in intracellular free calcium was observed.

PHF was detected in the plasma of Ob / Ob mice with obese, hypertensive and non-insulin dependent diabetes mellitus. The PHF of these mice was isolated from the same subfraction of serum as the PHF of SHR rats.

The detection of PHF is useful in the diagnosis of non-insulin dependent diabetes mellitus (NIDDM) and may open new areas of research on the role of PHF in NIDDM.

Some types of cancer are characterized by an increase in free calcium in the cytoplasm. [Reference: Okazaki et al., Canc. Res., 46 (12Pt 1), 6059-6063 (1986); Lipton and Morris, Canc. Chemother. Pharmacol. 18 (1), 17-20 (1986); Chien and Warren, Canc. Res., 46 (11), 5706-5714; Shirakawa et al., Canc. Res., 46 (2), 658-661 (1986); And Meyer, J. Hypertens., 5 (Supple. 4), S3-S4 (1987)]. Parathyroid activation is also associated with some forms of cancer. [Reference: Palmer et al., Am. J. Epidemiol., 127 (5), 1031-1040 (1998); And Feig and Gottesman, Cancer, 60 (3), 429-432 (1987). PHF is a source of parathyroid gland, and PHF may increase calcium levels in the cytoplasm, so PHF may be involved in many types of cancer. Therefore, screening PHF is valuable not only for understanding the cause of such cancer, but also for developing detection and treatment.

Hereinafter, the present invention will be described based on Examples, but the present invention is not limited by Examples. Various modifications can be made by those skilled in the art without departing from the scope of the present invention.

Demonstration of PHF Presence in SHR Rats

Male rats of the SHR, Wistar-Gyoto (WKY) and Sprague-Dawley (SD) strains were collected and collected. Heparin was added to the mixed blood of each strain (100 IU / ml) for 10 minutes at 4 ° C. Centrifuged at 3K × g. The obtained plasma was dialyzed against distilled water using a membrane to remove molecular weight substances of less than 1,000 Daltons.

Na pentobarbital was injected into the SD rats (50 mg / kg, ip) to anesthetize, and catheters were inserted into the jugular vein for the injection of plasma and drugs, and the carotid artery for the measurement of blood pressure (bp). Inserted into each.

Plasma was injected at a rate of 3 ml / kg / hr or bolus several times at a time of 2.5 ml / kg.

4 shows the change in mean arterial pressure over time during plasma injection of SHR, WKY and SD rats into SD rats. After 105 minutes, the injection of SHR plasma was stopped and SD plasma was returned to the baseline blood pressure within 30 minutes.

The effect of bolus administration of SHR, WKY and SD plasma on SD rats is shown in FIG. The changes in blood pressure and related effects over time were similar to those in FIG.

Example 2

Regulation of calcium absorption

In SD rat arteries, calcium [ ⁴⁵ Ca] uptake was determined using the low-affinity lanthanum resistant pool method described in Pang et al., LIfe Sci., 42, 1935 (1988). Measurement was made in vitro. The arterial slices of rats were equilibrated in oxygenated (95% O ₂ , 5% CO ₂ ) Krebs Hanseleit buffer for 2 hours. After equilibration, the pieces were incubated in 30% plasma Krebs buffer in the presence of 0.1 Ci ⁴⁵ Ca. After the scheduled incubation time the tissues were washed with cold Ca ²⁺ − removal, La ³⁺ containing solution and calibrated and then cooked as described in Pang's paper Life Sci., 42. 1935 (1988). Ca uptake was measured in scintillation counter and shown in FIG.

The absorption of ⁴⁵ Ca follows the upward curve of blood pressure and is dose dependent.

Example 3

Separation of PHF by Exclusion Chromatography

Plasma plasma of SHR, WKY and SD rats overnight in distilled water (using dialysis membrane to remove molecular weight less than 1,000) and using Amicon ultra-filtration cell (using molecular weight less than 5,000 filtration membrane) After filtration and eluting with 0.05M ammonium acetate in a column filled with Bio-Tel P-6, chromatograph was performed, and the fraction eluted at 280 nm was measured using a Pharmacia UV photometer. 6.67 mL aliquotos were collected.

Fractions having a molecular weight of about 3,500 Daltons were observed in SHR plasma but not in plasma of WKY or SD rats (FIG. 1). The molecules and markets used to classify the column were ACTH (m.w. 4541.7), insulin B chain (m.w. 3456), insulin A chain (m.w. 2530) and 13 amino acid synthetic peptide (m.w. 1464).

Unique fractions obtained from SHR rats were concentrated by lyophilization and injected into catheter-inserted SD rats as described in Example 1. The synergistic effect of blood pressure comparable to SHR rat plasma was observed in the initial expression time and intensity, and the results are shown in FIG. 7.

Example 4

Purification of PHF by HPLC

The unique fraction obtained in Example 3 was freeze dried and 0.1% trifluoroacetic acid; A solvent gradient of acetonitrile was used to classify on Brownley RP-P (C-8) reversed phase column HPLC and monitored at 280 nm. Biological activity was found in one peak [see also second (a)] and did not appear in serum samples obtained from WKY rats (see second (b)).

Example 5

Isolation from Cell Cultures

Parathyroid glands were excised from SHR and WKY rats and cultured in Hank's medium with daily change of medium. The medley medium was dialyzed against distilled water and filtered using the same method as for treating blood serum.

Fractions with a molecular weight between 1,000 and 5,000 were freeze dried and 0.1% trifluoroacetic acid; A gradient of acetonitrile was used to sort on a round RP-P (C-8) reversed phase column HPLC and monitored at 280 nm. The active fraction of SHR eluted at the same position as in serum but no corresponding fraction was found in the medium of WKY rat cells.

8 (a) is an elution graph of SHR cell lines medium; 8 (b) shows the case of the WKY rat.

Example 6

Separation of Culture Medium Using SDS-PAGE

Example on 15-18% acrylamide / bisacrylamide (Bio-Rad) containing Mini SDS-PAGE slab gels in Tris buffer (pH8.3) Cell culture media obtained from SHR and SD rat parathyroid cultures prepared as described in 5 were lyophilized and resuspended with water (H ₂ O). The gel was run at 200V for about 1 hour (Bio-Rad Mini-Protein II). The results are shown in FIGS. 10 (a)-(d).

Example 7

Isolation of Cell Extracts Using SDS-PAGE

Parathyroid cells of SHR and SD rats incubated for 8 hours in Hank's medium were isolated, homogenized with 50 mM acetic acid, centrifuged at 5,000 x g, loaded onto 10-18% acrylamide / bisacrylamide plate gel at 200V. Developed for hours. The results are shown in Figs. 9 (a)-(c).

Example 8

Isoelectric Focusing

Analytical isoelectric focusing on column fractions was done using Bio-Rad MOdel 111 Mini-IEFcell. This technique involves separating proteins based on isoelectric points and the results indicate the purity of the column fraction separated by Example 3. The method used is isoelectric focusing theory, methodology and application by Righetti, as modified by the Bio-Rad MOdel 111 Mini IEF Cell Instruction Manual (Bio-Rad Labs., Richmond, CA). Methodology and Application) (Elsevier Biomedical Press, Amsterdam (1983)). A thin sample of polyacrylamide (pH3-10) containing positively charged (pH3-10) was placed on a support film and a purified sample obtained from SHR serum and parathyroid cell cultures was applied to the gel with a standard sample, and then the gel was graphite. Directly connected to the electrode.

Protein was concentrated for 60-90 minutes. Protein bands were stained with a colorant containing both Coomassieblue and Crocein scarlet and the results are shown in FIG. Protein bands at pH 6 were found in both serum and culture media.

Example 9

Preparation of Polyclonal Antibodies

Male Balb / c mice were immunized with partially purified PHF (20-30% purity). Peptide samples each containing PHF in the formulation were subjected to diazo binding to the aminophenyl thioether (APT) inducible disk according to the method set forth by Viamontes et al. (J. Immun. Metho 94, 13-17, 1986). Combined. 0.3 ml of a solution in which NANO ₃ was added at 10 mg / ml in double distilled water was added to 10 ml of 1.2 N HCL. 0.3 ml of NANO ₃ / HCI solution was added to the top of each 6 mm APT disk and the disk was placed in a dish and kept on shaker for 4 minutes at 4 ° C. The disc was washed with cold distilled water and 02M NaOAc buffer (pH 4) and dried. The induction margin disks were then immersed in Freund's complete supplement and then implanted ip. After 2 weeks the titer of anti-PHF was measured by ELISA method and re-inoculated in mice as described above except the disc was immersed in Freund's incomplete supplement. After one week titers were measured by ELISA method, mice were re-inoculated at two week intervals and samples were evaluated by the following ELISA method.

Plates were coated with 100 μl / well of PHF at 500 ng / ml concentration in Tris buffer (pH9.0) and stored overnight, then washed with phosphate buffered saline (PBS) -Tween 20 Washed three times with PBS. Plates were then coated with 0.2% gelatin / PBS and aged at 37 ° C. for 30 minutes and washed three times with PBS-Tween20. Add the immunoserum obtained from the mice described above (100 μl / well of 1% gelatin PBS-Tween 20 in a dilution diluting the conflict between results and process), and the folate was aged at room temperature for 1 hour and then PBS- Wash three times with Twee20. Horse radish peroxidase (1: 2,000) added to 1% gelatin PBS-Twee20 was added in an amount of 100 µl / well, aged at room temperature for 1 hour, and the plates were washed with PBS-Tween 20. . Add 10 µl of freshly prepared citric acid, 0.05 µl, pH 4, 2 µl of 2,2'-azinobis (3-ethylbenzthiazoline sulfonic acid), 40 µl of 20 mg / ml hydrogen peroxide solution in the amount of 100 µl / well And aged at room temperature for 1 hour before reading at 405 nm (Titer-Tek MUlti Scan). The results are shown in Table 1 and shown in FIG. 11 and FIG.

Example 10

Feeding combination drugs

12-week-old male SHR rats obtained from Harlan Sprague-Dawley were divided into 12 or 24 groups and consumed prepared diets containing 0.2%, 0.4% and 0.8% basic calcium, respectively. Also included in the diet were 0, 50, 150 and 300 mg / kg of food containing nifedipine. Distilled water was supplied unlimitedly. Ingestion was continued for 8 weeks, and then average blood pressure of each animal was measured. The data is shown in Table 2.

From Table 2, it can be seen that the SHR rats who consumed the normal diet (0.2% calcium) without taking nifedipine showed an average blood pressure of 176 mmHg. The highest intake of nifedipine (300 mg / kg) results in a drop in blood pressure of nearly 100 mmHg.

Intake of 4 times the usual dietary calcium shows a slight drop in blood pressure. When co-administered the maximum amount of dietary calcium and nifedipine, the blood pressure was reduced to nearly 120 mmHg, which was about 56 mmHg or 32% lower than that of normal blood rats.

The average blood pressure dropped by 20 mmHg when fed a diet containing 50 or 150 mg / kg nifedipine, but the average blood pressure dropped by almost 40-60 mmHg when a diet containing 0.8% calcium was consumed. In combination with nifedipine and dietary supplemental calcium, an elevated effect could be predicted compared with the administration of only one ingredient, and the response to the amount of intake was proportional. The data shows a synergistic relationship between the two components.

Example 11

Composition I

Capsules containing a calcium channel blocker and a calcium supplement can be prepared by conventional methods by the following composition.

Nifedipine 5mg

CaCo₃ 500 mg

Gelatin (Soft) 1,495 mg

Example 12

Composition II

A capsule containing a calcium channel chazan residue and 1α, 25- (OH) ₂ D ₃ can be prepared by a conventional method by the following composition.

Nifedipine 5mg

1α, 25- (OH) ₂ D ₃ 0.05 μg

Gelatin (Soft) 1,745 mg

Reference: 50ng / well

** Normal Mouse Serum

Claims (23)

A method of treating high blood pressure mammals other than the human body, characterized by coadministration of calcium channel blockers and calcium supplements to lower blood pressure. A high blood pressure therapeutic composition, comprising a calcium channel blocker, a calcium supplement, and a pharmaceutically acceptable carrier that lowers blood pressure. The method of claim 1, wherein the calcium channel blocker is dihydropyridine. 4. The method of claim 3, wherein the dihydropyridine is nifedipine. The method of claim 1, wherein the calcium channel blocker is benzeneacetonitrile. 6. The method of claim 5, wherein the benzeneacetonitrile is verapamil. The method of claim 1, wherein the calcium channel blocker is benzothiazepine. 8. The method of treating a hypertensive mammal other than a human according to claim 7, wherein the benzothiazepine is diltiazem. The method of claim 1, wherein the calcium supplement is calcium carbonate. The method of claim 1, wherein the calcium supplement is calcium carbonate. The therapeutic agent composition for hypertension according to claim 2, wherein the calcium channel blocker is dihydropyridine. The hypertension therapeutic composition according to claim 10, wherein the dihydropyridine is nifedipine. 3. The hypertension therapeutic composition according to claim 2, wherein the calcium channel blocker is benzeneacetonitrile. The high blood pressure therapeutic composition according to claim 12, wherein the benzeneacetonitrile is verapamil. 3. The high blood pressure therapeutic composition according to claim 2, wherein the calcium channel blocker is benzothiazepine. The therapeutic composition for hypertension according to claim 14, wherein the benzothiazepine is diltiazem. The method of claim 1, further comprising administering vitamin D. The antihypertensive therapeutic composition according to claim 2, further comprising vitamin D. A method of treating high blood pressure mammals other than the human body, characterized by coadministration of a calcium channel blocker that lowers blood pressure and an effective amount of vitamin D. A high blood pressure therapeutic composition, comprising a calcium channel blocker that lowers blood pressure, an effective amount of vitamin D, and a pharmaceutically acceptable carrier. 19. The method of claim 18, wherein the vitamin D is 1α, 25-dihydroxycholecalciferol. 20. The therapeutic composition for hypertension according to claim 19, wherein vitamin D is 1α, 25-dihydroxycholecalciferol. A method for the treatment of high blood pressure mammals except for the human body, characterized by co-administration of 10-50% calcium channel blockers and calcium supplements. An antihypertensive therapeutic composition comprising a calcium channel blocker, a calcium supplement, and a pharmaceutically acceptable carrier in an effective amount of blood pressure lowering.