Effects of DU-1777, a novel ACE inhibitor, on blood pressure and tissue ACE activity in vivo and ex vivo

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Effects of la,24(R)-dihydroxyvitamin D 3 on the differentiation and the proliferation of epidermal keratinocytes and on the serum calcium level in rats M a t s u n a g a , T., Kiyoki, M., N a r u c h i , T. a n d K u r o k i *, T. Pharmacological Research Department, 2nd Bio-Medical Research Laboratories, Teijin Limited and * Department of Cancer Cell Research~ University of Tokyo, Tokyo, Japan

la,25-dihydroxyvitamin D3(1,25-(OH)2D3) is known to be a hormonally active form of vitamin D 3 in the regulation of intracellular and extracellular calcium levels and of differentiation of myeloid cells. The biological effects of 1,25-(OH)2D 3 are mediated by its binding to a specific receptor for itself, which has been found in almost all tissues examined, including kidney, small intestine and skin. Recently, 1,25-(OH)2D 3 was reported to induce differentiation and inhibit proliferation of epidermal keratinocytes (Hosomi et al., 1950). la,24(R)-dihydroxyvitamin D3(1,24(R)-(OH)2D3) was synthesized as a new analog of 1,25-(OH)2D 3 in our laboratories. In order to evaluate the therapea~ic value of 1,24-(OH)2D 3 m the treatment of psoriatic lesions, we examined the effects of this derivative on the proliferation and the differentiation of primary cultured mouse epidermal keratinocytes and on the serum calcium level in rats. Furthermore, the pharmacokinetic study was performed in rats after the administration of 3H-1,24(R)-(OH)2D3. 1,24(R)-(OH)D 3 binds to the receptor for 1,25-(OH)2D 3 in the mouse epidermis with the same affinity as 1,25-(OH)2D 3. Treatment of mouse epidermal keratinocytes with ],2d~R)-(OH)2D 3 causes the increase of the number of cornified envelopes in these cells in a time- and dose-dependent manner. The activity of epidermal transglutaminase is increased to 150% of ~.ontrol by incubation with 12 nM of 1,24(R)-(OH)2D3. The inhibitory effect of 1,24(R)(OH)2D 3 on 3H-thymidine incorporation to the DNA of epidermal keratinocytes is greater than that of 1,25-(OH)2D3. The doses for 50% inhibition are 0.66 nM of 1,24(R)-(OH)2D 3 and 5.1 nM of 1,25-(OH)2D 3 respectively. These results indicate that 1,24(R)-(OH)2D 3 induces terminal differentiation and inhibits proliferation of mouse epidermal keratinocytes by binding to the specific receptor for 1,25-(OH)2D 3. Although these in vitro activities of 1,24(R)-(OH)2D 3 are similar to or rather more than those of 1,25-(OH)2D 3, when given as a single intravenous injection, it induces less hypercalcemia than l~25-(OH)2D 3. When rats are treated intravenously with 3H-1,24(R)-(OH)2D3 or 3H-1,25-(OH)2D 3 at equal doses, the serum levels of unchanged 1,24(R)( O H ) 2 D 3 are found to be lower and to decrease faster than those of 1,25-(OH)2D 3. Thus, the weak induction of hypercalcemia by 1,24(R)-(OH)2D 3 may be explained by its faster turnover. A recent study shows that 1,24(R)-(OH)2D3 is effective in the treatment of psoriatic lesions when administered topically at a dose of 2/tg per g ointment. The decrease effect of 1,24(R)-(OH)2D 3 in inducing hypercalcemia shows to be advantageous for its clinical use. Reference Hosomi, J. et al., 1983, Endocrinology 113, 1950. P.we.463 1

Effects of DU-1777, a novel ACE inhibitor, on blood pressure and tissue ACE activity in vivo and e x vivo T a k e y a m a , K., N a g a t a , S., Ikeno, A., F u k u y a , F., Nishimura, S. a n d Hosoki, K. Department of Pharmacology, Research Laboratories, Dainippon Pharmaceutical. Co., Ltd. Osaka 564, Japan

The renin-an~otensin system plays an important role for the control of blood pressure and water-sodium balance

1557 and in the pathogenesis of hypertension. Many clinical studies have hitherto proven that angiotensin converting enzyme (ACE) inhibitors are quite effective in the treatment of hypertension. DU-1777, (2S, 3aS, 7aS)-l-(N2-Nicotinoyl-L-lysyl-a-D-glutamyl)octahydro-lH-indole-2-carboxylic acid, is a novel and potent ACE inhibitor with an IC50 of 6.4 nM for inhibiting lung ACE in vitro (Sawayama et al., 1989, 1990). In an attempt to clarify the antihypertensive properties, we examined the effects of DU-1777 on blood pressure in various animal models of hypertension and on ACE activity in plasma and tissues in vivo and ex vivo, in comparison with lis.lnopril, a long-acting potent ACE inhibitor. In renal hypertensive rats and dogs, DU-1777 (1-3 mg/kg, p.o.) itdaibited the pressor response to angiotensin I injected i.v., suggesting ACE inhibition in the blood and lung. This inhibition by DU-1777 began to recover at 5 hr after oral administration. On the other hand, the inhibition by lisinopril of the pressor response at 0.3-1.0 mg/kg, p.o. was more pronounced and longer-lasting than that of DU-1777 at 1-3 mg/kg, p.o., where both agents caused almost identical antihypertensive effects. DU-1777 (1-30 mg/kg, p.o.) caused slow-onset and long-lasting (more than 9 hr) antihypertensive effects in renin-angiotensin dependent renal hypertensive rats and dogs (2-kidney, !-clip type) and in spontaneously hypertensive rats (SHR), similarly to lisinopril. In renin-angiotensin independent hypertensive models (1-kidney, l-clip renal and DOCA-salt hypertensive rats), however, there was a distinct difference between DU-1777 and lisinopril in lowering blood pressure. DU-1777 (1-30 mg/kg, p.o.) caused a significant antihypertensive effect, particularly in 1-kidney renal hypertensive rats, but lisinopril was almost without effect. The antihypertensive potenci~ of DU-1777 in these hypertensive animals decreased in the following order; renal ¢2-kidr,ey type) SHR, renal (l-kidney type)> DOCA-salt hypertensive rats. The maximum antihypertensive effect of DU-1777 (15-50 mmHg) was observed from 7 to 9 hr after oral administration in all of the models and the effect continued ove~"24 hr in renal hypertensive rats and dogs. The fall of blood pressure caused by DU-1777 in DOCA-salt hypertensive rats was not affected by i.v. infusion of saralasin (1.0 g/kg/min), an angiotensin II receptor antagonist. Furthermore, the time-t~urse of ACE inhibition examined ex vivo in the plasma, lung, aorta, and kidneys of ,enal hypertensive rats (2-kidney type) given DU-1777 (10 mg/kg, p.o.) was poorly correlated with that of the re,Juction in blood pressure. These results suggest an involvement of an unknown mechanism(s) in add"tion to ACE inhibition for the antihypertensive action of DU-1777. References Sawayama, T., 1989, Chem. Pharma. Bull. 37, 2417. $awayama, T., 1990, Chem. Pharma. Bull. 38, in press.

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Vascular effects of lysopht ,hatidyl inositol: correlation with |ntracellular calcium Shan, J., Lewanczuk, R.Z., Benishin, C.G., Resnick *, L.M. and Pang, P.K.T. Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7 and * Cardiovascular Center, The New York Hospital-Corneil Medical Center, New York, NY, U.S.A.

Inappropriately high levels of 1,25 dihydroxy-vitamin D (calcitriol) have been noted in some forms of hypertension (Resnick et al., 1986). Some evidence suggests that calcitriol may increase intracellular calcium levels in vascular smooth muscle (Bukoski et al., 1987). In hepatoeytes, calcitriol increases intracellular free calcium. This effect has been shown to be mediated by lysophosphatidyl inositol (LPI), a product of phosphofipase A activation, and seems to be due to release of intracellular calcium (Baran and Kelly, 1988). The purpose of this study was to determine whether LPI also increased intracellular free calcium in vascular smooth muscle. Such an effect would be expected to result in increased blood pressure and might, therefore, establish a link between increased levels of calcitriol and some forms of hypertension. Smooth muscle cells isolated from Sprague-Dawley (SD) rat tail arteries were grown in DMEM for 2a hr. Cells