Discovery of a peptide-based renin inhibitor with oral bioavailability and efficacy.

Peptidic renin inhibitors have been poorly absorbed across the intestine or rapidly eliminated by the liver and have been reported to have oral bioavailabilities of less than 2%. A peptide-based renin inhibitor, A-72517 (molecular mass of 706 daltons), was devised that has oral bioavailabilities of 8, 24, 32, and 53% in the monkey, rat, ferret, and dog, respectively. Dose-related reductions in blood pressure, plasma renin activity, and plasma angiotensin II in parallel with increased plasma drug concentrations were observed after oral administration of A-72517 to conscious, salt-depleted dogs. Thus, peptide-based molecules of sizable molecular mass can be absorbed intact into the systemic circulation of animals. These findings support the potential of peptide-based drugs for oral administration.

Nitric oxide-mediated inhibition of androgen receptor activity: possible implications for prostate cancer progression.

Chronic inflammation increases the risk of cancer and many cancers, including prostate cancer, arise at sites of chronic inflammation. Inducible nitric oxide synthase (iNOS) is an enzyme dominantly expressed during inflammatory reactions. Although synthesis of high amounts of nitric oxide (NO) by iNOS has been demonstrated in pathophysiological processes, such as acute or chronic inflammation, autoimmune diseases or tumorigenesis, the role of iNOS activity in most of these diseases is poorly understood. Analysing prostate cancer biopsies by immunohistochemistry we found iNOS protein expression in tumor cells strongly paralleled by nitrotyrosine suggesting that iNOS is fully active. In vitro, NO inhibits androgen receptor-dependent promoter activity and prostate specific antigen production as well as DNA-binding activity of the androgen receptor (AR) in a concentration-dependent manner. Inhibition of the activity of androgen receptor-dependent reporter constructs is neither owing to diminished AR protein levels nor owing to an inhibition of its nuclear import. In addition, NO inhibits the proliferation of androgen receptor-positive prostate cancer cells significantly more efficiently than proliferation of androgen receptor-negative prostate cancer cells. In summary, our findings suggest that intratumoral iNOS activity favors development of prostate cancer cells that are able to proliferate androgen receptor-independently, thereby promoting prostate tumor progression.

Evidence for a novel keratinocyte fatty acid uptake mechanism with preference for linoleic acid: comparison of oleic and linoleic acid uptake by cultured human keratinocytes, fibroblasts and a human hepatoma cell line.

Keratinocytes require the essential fatty acid (FA), linoleic acid (LA), for the synthesis of stratum corneum membrane lipids. A plasma membrane-FA binding protein (PM-FABP), is postulated to mediate cellular FA-uptake in hepatocytes and several other tissues, but the mechanism whereby exogenous FA are taken up by keratinocytes has not been investigated. This study examines the uptake of LA and oleic acid (non-essential) in cultured human keratinocytes, in comparison to dermal fibroblasts and the human hepatoma cell line, HepG2. As previously reported for hepatocytes, FA-uptake in keratinocytes was curvilinear, with an initial (30 s) rapid cellular influx. The initial uptake component was temperature dependent, exhibited saturable kinetics and was significantly inhibited by pretreatment with trypsin. In contrast, fibroblast FA-uptake lacked an initial rapid uptake component, was relatively temperature insensitive, and was not inhibited by trypsin. Keratinocytes differed from both hepatocytes and fibroblasts by more rapid uptake of LA in comparison to oleic acid during the initial influx phase. Moreover, FA-uptake in keratinocytes was not inhibited by preincubation with a anti-rat liver PM-FABP antibody. These data provide evidence for a PM-FA transporter in keratinocytes that is distinct from the hepatic PM-FABP. The apparent preference of the putative keratinocyte FA transporter for LA may function to ensure epidermal capture of sufficient LA for barrier lipid synthesis.

The RNA binding protein TIAR is involved in the regulation of human iNOS expression.

Human inducible NO synthase (iNOS) expression is regulated by post-transcriptional mechanisms. The 3'-untranslated region (3'-UTR) of the human iNOS mRNA contains AU-rich elements (ARE), which are known to be important for the regulation of mRNA stability. The 3'-UTR of the human iNOS mRNA has been shown to regulate human iNOS mRNA expression post-transcriptionally. One RNA-binding protein known to interact with AREs and to regulate mRNA stability is the T cell intracellular antigen-1-related protein (TIAR). In RNA binding studies TIAR displayed high affinity binding to the human iNOS 3'-UTR sequence. In RNase protection experiments, the cytokine incubation needed for iNOS expression did not change TIAR expression in DLD-1 cells. However, overexpression of TIAR in human DLD-1 colon carcinoma cells resulted in enhanced cytokine-induced iNOS expression. In conclusion, TIAR seems to be involved in the post-transcriptional regulation of human iNOS expression.

End-to-end distribution function of stiff polymers for all persistence lengths.

We set up recursion relations for calculating all even moments of the end-to-end distance of Porod-Kratky wormlike chains in D dimensions. From these moments we derive a simple analytic expression for the end-to-end distribution in three dimensions valid for all peristence lengths. It is in excellent agreement with Monte Carlo data for stiff chains and approaches the Gaussian random-walk distributions for low stiffness.

Self-similar variational perturbation theory for critical exponents.

We extend field theoretic variational perturbation theory by self-similar approximation theory, which greatly accelerates convergence. This is illustrated by recalculating the critical exponents of O (N) -symmetric phi(4) theory. From only three-loop perturbation expansions in 4-epsilon dimensions, we obtain analytic results for the exponents, which are close to those derived recently from ordinary field-theoretic variational perturbational theory to seventh order. In particular, the specific-heat exponent is found to be in good agreement with best-measured exponent alpha approximately -0.0127 of the specific-heat peak in superfluid helium, found in a satellite experiment. In addition, our analytic expressions reproduce also the exactly known large- N behavior of the exponents.

Ultraviolet A1 radiation induces nitric oxide synthase-2 expression in human skin endothelial cells in the absence of proinflammatory cytokines.

Skin exposure to ultraviolet radiation from sunlight causes erythema and edema formation as well as inflammatory responses. As some of these ultraviolet-induced effects are potentially mediated by nitric oxide synthases, we examined the role of cytokines and ultraviolet A1 radiation (340-400 nm) on the expression of the nitric oxide synthase-2 in endothelia of normal human skin biopsies during short-term organ culture as well as expression and activity of the nitric oxide synthase-2 in in vitro cell cultures of human dermal endothelial cells. Both, cytokine challenge (interleukin-1beta + tumor necrosis factor-alpha + interferon-gamma) but also ultraviolet A1 exposure (50 J per cm2) in the absence of cytokines led to the expression of nitric oxide synthase-2 in human skin organ cultures as shown by immunohistochemistry. Moreover, exposing human dermal endothelial cell cultures to proinflammatory cytokines but also to ultraviolet A1 radiation (6-24 J per cm2) in the absence of cytokines resulted in significant nitric oxide synthase-2 mRNA and protein expression as well as enzyme activity. Ultraviolet A1 irradiation of cytokine activated cells led to further increases in nitric oxide synthase-2 mRNA, protein expression, and enzyme activity. Moreover, a reporter gene assay using a human nitric oxide synthase-2 promoter construct provide evidence that ultraviolet A1, in the absence of cytokines, induces nitric oxide synthase-2 expression and activity, as previously shown for cytokines. Thus, the results presented here demonstrate for the first time that in dermal endothelia of human skin ultraviolet A1 radiation alone represents a proinflammatory stimulus sufficient to initiate nitric oxide synthase-2 expression as well as activity comparable with the respective response seen in the presence of proinflammatory cytokines.

Atrial natriuretic factor inhibits angiotensin-, norepinephrine-, and potassium-induced vascular contractility.

We have previously shown that the natriuretic effect of rat atrial extract (AE) may be due, perhaps entirely, to its powerful renal hemodynamic actions. The present study was undertaken to test the hypothesis that mammalian atria contain a substance that behaves as a functional antagonist of endogenous vasoconstrictors, by examining the direct effects of AE and extensively purified atrial "natriuretic" factor on the contractile response of rabbit aortic rings to angiotensin II (AII), norepinephrine (NE), and K+-induced depolarization. Dose-response curves to AII and NE (i.e., change in tension vs log hormone concentration) were determined in the absence or presence of boiled AE or ventricular extracts (VE). Increasing concentrations of boiled AE caused a progressive right-ward shift of the AII and NE dose-response curves, whereas VE was without effect. A similar inhibitory effect was produced after extensive purification of atrial natriuretic factor by gel filtration and reversed-phase high performance liquid chromatography (HPLC). It appeared that this factor antagonized AII-induced contractility to a greater degree than that of NE. Moreover, the partially purified factor also inhibited the contraction induced by depolarization with 15 mM KCl in a concentration-dependent manner. These studies show that a substance present in the atria, but not ventricles, blocks both hormone- (receptor) and depolarization- (nonreceptor) induced vasoconstriction in aortic rings. Moreover, this antagonism is retained following extensive purification of an atrial factor that induces natriuresis in the intact rat and isolated rat kidney, suggesting that both the vasoactive and natriuretic properties of AE may reside in a single substance.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions.

Three isozymes of nitric oxide (NO) synthase (EC 1.14.13.39) have been identified and the cDNAs for these enzymes isolated. In humans, isozymes I (in neuronal and epithelial cells), II (in cytokine-induced cells), and III (in endothelial cells) are encoded for by three different genes located on chromosomes 12, 17, and 7, respectively. The deduced amino acid sequences of the human isozymes show less than 59% identity. Across species, amino acid sequences for each isoform are well conserved (> 90% for isoforms I and III, > 80% for isoform II). All isoforms use L-arginine and molecular oxygen as substrates and require the cofactors NADPH, 6(R)-5,6,7,8-tetrahydrobiopterin, flavin adenine dinucleotide, and flavin mononucleotide. They all bind calmodulin and contain heme. Isoform I is constitutively present in central and peripheral neuronal cells and certain epithelial cells. Its activity is regulated by Ca2+ and calmodulin. Its functions include long-term regulation of synaptic transmission in the central nervous system, central regulation of blood pressure, smooth muscle relaxation, and vasodilation via peripheral nitrergic nerves. It has also been implicated in neuronal death in cerebrovascular stroke. Expression of isoform II of NO synthase can be induced with lipopolysaccharide and cytokines in a multitude of different cells. Based on sequencing data there is no evidence for more than one inducible isozyme at this time. NO synthase II is not regulated by Ca2+; it produces large amounts of NO that has cytostatic effects on parasitic target cells by inhibiting iron-containing enzymes and causing DNA fragmentation. Induced NO synthase II is involved in the pathophysiology of autoimmune diseases and septic shock. Isoform III of NO synthase has been found mostly in endothelial cells. It is constitutively expressed, but expression can be enhanced, eg, by shear stress. Its activity is regulated by Ca2+ and calmodulin. NO from endothelial cells keeps blood vessels dilated, prevents the adhesion of platelets and white cells, and probably inhibits vascular smooth muscle proliferation.

Estrogens increase transcription of the human endothelial NO synthase gene: analysis of the transcription factors involved.

Estrogens have been found to reduce the incidence of cardiovascular disease that has been ascribed in part to an increased expression and/or activity of the vasoprotective endothelial NO synthase (NOS III). Some reports have shown that the level of expression of this constitutive enzyme can be upregulated by estrogens. The current study investigates the molecular mechanism of the NOS III upregulation in human endothelial EA.hy 926 cells. Incubation of EA.hy 926 cells with 17beta-estradiol or the more stable 17alpha-ethinyl estradiol enhanced NOS III mRNA and protein expression up to 1.8-fold, without changing the stability of the NOS III mRNA. There was no enhancement of NOS III mRNA after incubation of EA.hy 926 cells with testosterone, progesterone, or dihydrocortisol or when 17alpha-ethinyl estradiol was added together with the estrogen antagonist RU58668, indicating a specific estrogenic response. Nuclear run-on assays indicated that the increase in NOS III mRNA is the result of an estrogen-induced enhancement of NOS III gene transcription. In transient transfection experiments using a 1.6 kb human NOS III promoter fragment (which contains no bona fide estrogen-responsive element, ERE), basal promoter activity was enhanced 1.7-fold by 17alpha-ethinyl estradiol. In electrophoretic mobility shift assays, nuclear extracts from estrogen-incubated EA.hy 926 cells showed no enhanced binding activity either for the ERE-like motif in the human NOS III promoter or for transcription factor GATA. However, binding of transcription factor Sp1 (which is essential for the activity of the human NOS III promoter) was significantly enhanced by estrogens. These data suggest that the estrogen stimulation of the NOS III promoter could be mediated in part by an increased activity of transcription factor Sp1.

Effects of the renin inhibitor A-64662 in monkeys and rats with varying baseline plasma renin activity.

The efficacy of the potent, primate selective renin inhibitor A-64662 was studied in monkeys and rats with varying baseline plasma renin activity (PRA) to elucidate the relationship between PRA and the hypotensive response induced by this compound. The effect of a single bolus of vehicle or A-64662 at 0.001, 0.01, 0.1, 1.0, and 10.0 mg/kg i.v. was compared in 30 normal and 30 salt-depleted, anesthetized monkeys (n = 5/dose). Baseline mean arterial pressure (MAP) was similar among all groups, but baseline PRA was elevated in salt-depleted monkeys. A-64662 induced a comparable dose-related fall in MAP, affecting the magnitude and duration of action, accompanied by inhibition of PRA, the duration of which was dose-related in both the normal and salt-depleted groups. However, the minimum effective doses required to reduce MAP by approximately 10% were 0.01 mg/kg for the salt-depleted monkeys and 0.1 mg/kg for the normal monkeys. In a second study, three consecutive boluses of vehicle or A-64662 at 0.1, 1.0, and 10.0 mg/kg were administered to anephric monkeys, human renin-infused anephric monkeys, and normal monkeys (n = 4/group). A dose of 0.1 mg/kg was ineffective, but the 1.0 mg/kg dose lowered MAP by 11 +/- 3% (mean +/- SE) in the anephric monkeys. The infusion of renin into anephric monkeys restored the efficacy of A-64662 at the 0.1 and 1.0 mg/kg doses to responses comparable to those of the normal monkeys. A-64662 at 10.0 mg/kg caused a similar fall in MAP of 50 to 60% in anephric, renin-infused anephric, and normal monkeys in the absence of detectable PRA.(ABSTRACT TRUNCATED AT 250 WORDS)

What is the value of home blood pressure measurement in patients with mild hypertension?

To investigate the value of home blood pressure (BP) measurements, the BP was recorded daily by the patient at home and compared with recordings in the physician's office and with a 24-hour BP recording taken with a noninvasive ambulatory BP recorder in a group of 93 patients with mild untreated hypertension. Office BPs (mean 148/94 mm Hg) were higher than either home (138/89 mm Hg) or average 24-hour BPs (131/89 mm Hg). For systolic BP, home and office measurements gave similar correlations with 24-hour BP (0.67 and 0.55). For diastolic BP, however, home readings were lower and more accurate (0.76 vs 0.36). Thus, our findings indicate that home readings reflect the overall level of BP more reliably than office readings, and if due consideration is given to the fact that they are usually lower than office readings, they may be used as an alternative and cost-effective means of evaluating patients with mild hypertension.

Prolonged duration of blood pressure response to enalkiren, the novel dipeptide renin inhibitor, in essential hypertension.

The effects of sustained renin inhibition by repeated administration of enalkiren (A-64662), the novel dipeptide renin inhibitor, were evaluated in a randomized, double-blind, placebo-controlled, parallel-group study of 32 inpatients (eight per group) with essential hypertension who were maintained on a diet containing 60 meq/day sodium. Three different dosage regimens of enalkiren were studied: 1) 1.2 mg/kg quotid., 2) 0.3 mg/kg q.i.d., and 3) 0.1 mg/kg q.i.d. Each patient received an intravenous infusion every 6 hours for 1 week. Placebo infusions were used to mimic the 4 times/day dosing schedule. Blood pressure was measured periodically via 24-hour automated monitoring equipment. Mean plasma renin activity in the patient groups ranged from 1.58 to 2.68 ng angiotensin I/ml/hr. Plasma renin activity was promptly suppressed in all groups receiving enalkiren. Prolonged duration of plasma renin activity suppression (greater than or equal to 24 hours) was demonstrated after the administration of 1.2 mg/kg enalkiren. The 0.3 mg/kg q.i.d. and 1.2 mg/kg quotid. regimens produced statistically significant reductions (p less than or equal to 0.05) in systolic and diastolic blood pressures with clear evidence of persistent antihypertensive activity for 12 hours or more when compared with the placebo group. Despite relatively large reductions in mean systolic and diastolic blood pressure, mean pulse rates were essentially unchanged. The prolonged reduction in blood pressure with enalkiren without evidence of tachyphylaxis after 1 week of treatment suggests that renin inhibitors may emerge as useful therapeutic agents for the treatment of hypertension.

Effect of atrial natriuretic factor on blood pressure, renin, and aldosterone in Goldblatt hypertension.

We previously provided evidence that atrial natriuretic factor (ANF) antagonizes angiotensin II-induced vascular contractility and angiotensin II-stimulated aldosterone production by isolated adrenal cells. To examine the importance of these effects in vivo, synthetic ANF (auriculin A) was administered intravenously (2 micrograms/kg bolus followed by 0.3 microgram/kg/min constant infusion) to conscious, unrestrained two-kidney, one-clip and one-kidney, one-clip rats on normal sodium intake and their sham-operated controls. The one-kidney, one-clip rats also were studied on a sodium-deficient diet. Mean blood pressure, plasma renin activity, and plasma aldosterone levels were measured before and after 60-minute infusion. In saralasin-responsive two-kidney, one-clip rats (n = 10), ANF administration reduced blood pressure (from 187 +/- 11 [SE] to 153 +/- 11 mm Hg; p less than 0.001) and plasma aldosterone levels (from 182 +/- 61 to 125 +/- 60 ng/dl; p less than 0.05), while plasma renin activity increased (from 59 +/- 16 to 82 +/- 20 ng/ml/hr; p less than 0.05). Lesser changes in blood pressure occurred in saralasin-nonresponsive two-kidney, one-clip rats (149 +/- 10 to 143 +/- 8 mm Hg; n = 5), sodium-replete one-kidney, one-clip rats (183 +/- 9 to 170 +/- 11 mm Hg; n = 9), two-kidney sham-operated rats (122 +/- 3 to 115 +/- 4 mm Hg; n = 8), and one-kidney sham-operated rats (117 +/- 3 to 112 +/- 3 mm Hg; n = 7). Control plasma renin and aldosterone levels were not elevated in these latter groups and did not change significantly with ANF administration. In sodium-depleted one-kidney, one-clip rats, which became saralasin responsive, ANF administration significantly reduced blood pressure (from 184 +/- 11 to 156 +/- 12 mm Hg; n = 8), plasma aldosterone levels (from 286 +/- 41 to 179 +/- 36 ng/dl), and plasma renin activity (from 69 +/- 19 to 44 +/- 13 ng/ml/hr).(ABSTRACT TRUNCATED AT 250 WORDS)

Discovery of a well-absorbed, efficacious renin inhibitor, A-74273.

The development of orally active renin inhibitors has been plagued by limited bioavailability in animals and humans. A-74273 is a novel, potent nonpeptide inhibitor of human renin (IC50 = 3.1 nM). This compound was absorbed into the portal and systemic circulations of anesthetized rats, ferrets, monkeys, and dogs after intraduodenal dosing. This favorable pattern also was observed after oral dosing in conscious animals, except in monkeys. Hepatic extraction of A-74273 was more efficient in rats and monkeys than in dogs or ferrets. A-74273 modestly inhibits dog renin, and when given orally as the base (0, 0.3, 1, 3, 10, and 30 mg/kg; n = 8 per dose) to conscious, salt-depleted dogs it induced dose-related reductions in mean arterial pressure and plasma renin activity. Peak falls in mean arterial pressure from normotensive baselines were -14 +/- 1, -26 +/- 3, and -44 +/- 3 mm Hg for the 3, 10, and 30 mg/kg groups, respectively (p < 0.05). Baseline plasma renin activity values (10.9 +/- 1.1-12.7 +/- 1.1 ng angiotensin I/ml/hr) were maximally inhibited, ranging from 43 +/- 8% at 0.3 mg/kg to 98 +/- 1% at 30 mg/kg. Bioavailability in this model was estimated to be 54 +/- 13% when plasma drug levels were determined by a renin inhibitory activity assay, but bioavailability was lower when compared with high-performance liquid chromatographic analysis of A-74273. This discrepancy was accounted for by the identification of structurally similar metabolites that are as active as the parent drug against human renin but much less potent against dog renin.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiovascular effects of atrial natriuretic factor in anesthetized and conscious dogs.

Atrial natriuretic factor lowers blood pressure in normotensive and hypertensive animal models. The present study examined the mechanism of the blood pressure-lowering effect in 10 normotensive dogs. Four awake dogs previously instrumented with electromagnetic flow probes for measurement of cardiac output and catheters for systemic hemodynamic and cardiac dynamic measurements were studied. After a 30-minute control period, a 3 micrograms/kg bolus followed by 0.3 micrograms/min/kg of a 24-residue synthetic atrial natriuretic factor was infused for 30 minutes, followed by a 1-hour recovery period. Mean arterial pressure fell significantly during infusion (control, 125 +/- 4; infusion, 108 +/- 5; recovery, 125 +/- 9 mm Hg; p less than 0.05) and was accompanied by a slight but significant bradycardia (control, 144 +/- 7; infusion, 134 +/- 5; recovery, 145 +/- 7 beats/min; p less than 0.05). Significant reductions in cardiac output (control, 2.66 +/- 0.60; infusion, 2.18 +/- 0.60; recovery, 2.74 +/- 0.60 L/min; p less than 0.05), stroke volume (control, 18.4 +/- 3.9; infusion, 16.0 +/- 4.2; recovery, 19.0 +/- 3.7 ml/beat; p less than 0.05), and maximum increase in rate of change of left ventricular systolic pressure (control, 2475 +/- 200; infusion, 2088 +/- 216; recovery, 2487 +/- 243 mm Hg/sec; p less than 0.05) were also observed during infusion. No significant changes in total peripheral resistance or central venous pressure were noted, although the latter tended to fall during infusion. A similar pattern was observed in six pentobarbital-anesthetized dogs, except that infusion of atrial natriuretic factor did not induce bradycardia.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of chronic infusion of renin inhibitor A-64662 in sodium-depleted monkeys.

The potent and primate-selective renin inhibitor A-64662 (n = 8) or vehicle (n = 6) was administered intravenously for 7 days to sodium-depleted cynomolgus monkeys to investigate the chronic effects on arterial pressure, sodium excretion, and the renin-angiotensin-aldosterone system. A 0.1-mg/kg i.v. bolus followed by a continuous 0.01-mg/kg/min infusion of A-64662 lowered mean arterial pressure from 89 +/- 3 (average of 4 control days) to 75 +/- 4 mm Hg (p less than 0.05) after 1 day of administration. This decrement was associated with marked inhibition of plasma renin activity (PRA) from 57.7 +/- 11.1 to 1.3 +/- 0.6 ng angiotensin I (Ang I)/ml/hr (p less than 0.05). Similar hypotensive levels (range 73 +/- 4 to 77 +/- 4 mm Hg) were observed on days 2-7 of A-64662 infusion and PRA remained suppressed, ranging from 0.6 +/- 0.4 to 1.9 +/- 1.0 ng Ang I/ml/hr. Plasma angiotensin II (Ang II) levels were reduced (p less than 0.05) from the control value of 66.7 +/- 20.2 to 12.4 +/- 3.3 and 26.4 +/- 6.5 pg/ml on the second and seventh days, respectively, of A-64662 infusion. In contrast, infusion of vehicle alone had no discernible effect on mean arterial pressure, PRA, or plasma Ang II concentrations. Plasma aldosterone decreased (p less than 0.05) from control on the second and third days of A-64662 infusion, although differences between the treatment groups were not detected throughout the study. Urinary sodium excretion remained at control levels throughout the infusion of A-64662. Cessation of A-64662 administration resulted in a recovery of mean arterial pressure to preinfusion levels within 1 day. This study indicates that continuous infusion of A-64662 results in a sustained hypotension in sodium-depleted monkeys. This effect appears to be related, at least partially, to inhibition of PRA and lower plasma Ang II levels.

Hemodynamic and humoral effects of the new renin inhibitor enalkiren in normal humans.

The effect of the renin inhibitor enalkiren (Abbott-64662) was evaluated in eight normal volunteer subjects on a standardized sodium diet (100 mmol/day) by measurement of various components of the renin-angiotensin system and drug levels in plasma. On day 1, vehicle and doses of 0.001, 0.003, and 0.01 mg/kg i.v. were administered within 2 minutes at 90-minute intervals. On day 2, vehicle and doses of 0.01, 0.03, and 0.1 mg/kg i.v. were given. With the higher doses, blood pressure tended to decrease slightly with no change in heart rate. Plasma renin activity and plasma angiotensin-(1-8)octapeptide (angiotensin II) fell markedly in a dose-dependent manner. Inhibition of plasma renin activity was maximal 5 minutes after administration of the drug and persisted 90 minutes after the doses of 0.03 and 0.1 mg/kg. Not surprisingly, there was a close correlation between plasma renin activity and plasma angiotensin II levels (r = 0.81, n = 28, p less than 0.001). In contrast, active and total renin measured directly by monoclonal antibodies rose in dose-related fashion in response to renin inhibition. Pharmacokinetic parameters were calculated using the plasma drug concentrations obtained up to 6 hours after the 0.1 mg/kg dose. By means of a two-compartment model, plasma mean half-life of the drug was estimated at 1.60 +/- 0.43 hours.

Sequence and factor requirements for faithful in vitro transcription of human 7SL DNA.

We have analysed the transcription of a functional human 7SL gene by RNA polymerase III (RNAPIII) in S100 extracts in vitro. Accurate and efficient synthesis of 7S L RNA depends on the presence of (i) an upstream sequence and (ii) an internal promoter element located within the first 22 bp of the gene. These findings were substantiated by DNase I footprinting. Mutations of the internal promoter identified the doublet CG [nucleotide (nt) +15/+16] outside the A-box homologue (nt +5 to +14) as being essential for both proper promoter function in the in vitro transcription assay and competition in the template-exclusion assay. Fractionation of S100 extracts identified two fractions required in addition to RNAPIII for faithful transcription of the gene. Each of these two fractions gave rise to one of two footprints observed in DNase I protection experiments, indicating that at least two DNA-binding factors are involved.

Renin inhibitors. Improvements in the stability and biological activity of small peptides containing novel Leu-Val replacements.

We have designed a novel class of potent (0.3-7 nM) renin inhibitors which contain a dihydroxyethylene replacement for what is formally the Leu10-Val11 amide bond. Good potency (0.6 nM), water solubility (greater than 10 mg/ml at 37 degrees C), stability toward degradation by chymotrypsin (t1/2 = 820 min), and in vivo activity in a primate model (15% drop in mean arterial pressure in association with complete inhibition of plasma renin activity) are properties which have been incorporated into compound 10, an interesting new agent to be used in the study of hypertension.