Gerald M. Walsh

A new class of orally active glycol renin inhibitors containing phenyllactic acid at P3.

We prepared a new series of renin inhibitors based on dipeptide glycols, replacing the P4-P3 subsites with an O-(N-morpholinocarbonyl)-3-L-phenyllactic acid residue. This modification proved bioisosteric with Boc-L-phenylalanine, giving rise to highly potent human renin inhibitors (1-5 nM), e.g., SC-46944 (IC50 = 5 nM). Moreover, this change produced compounds that are orally efficacious in reducing plasma renin activity in salt-depleted marmosets.

Dipeptide glycols: A new class of renin inhibitors

The discovery of a new class of novel renin inhibitors consisting of protected dipeptide amides derived from aminoglycols (Formula I) prompted a study of structure-activity in vitro and efficacy in vivo. Thus, Boc-L-Phe-N-[(1S,2R)-1-benzyl-(2,3-dihydroxy)propyl]-L-leucinamide (1) and the corresponding histidinamide (2) inhibit human renin in vitro (IC50: 8.7 X 10-6 M and 2.6 X 10-6 M, respectively). Compound 1 has a slight inhibitory effect on pepsin and compound 2 does not inhibit pepsin at all (at 10-4M); these compounds are inactive against rat renin. Compound 1 is efficacious in lowering plasma renin activity in the Rhesus monkey (i.v.). Results indicate that this new class of low molecular weight inhibitors is specific for human renin and thus constitutes a new source of drug candidates.

Increased Systemic Vascular Responsiveness to Catecholamines in Spontaneously Hypertensive Rats

Systemic vascular responsiveness to i.v. bolus injections of norepinephrine and tyramine was evaluated in adult male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Changes in total peripheral resistance (TPR) were used as an index of vascular response. Dose-response curves were plotted with 1n-dose on the x-axis and percent of maximum change in TPR on the y-axis, and the following indices of responsiveness were used: slope, 1nED50, 1nx-intercept, and maximum response. Measurements were made before and after ganglionic blockade with pentolinium (5 mg/kg, i.v.). Cardiac output for TPR calculations was obtained from an implanted flow probe on the ascending aorta. The slopes and maximum responses to norepinephrine and tyramine were greater in SHR v. WKY, P less than .05, before and after pentolinium treatment. There were no significant differences in 1nED50 or 1nx-intercept between WKY and SHR for tyramine and norepinephrine prior to pentolinium. After pentolinium 1nED50 and 1nx-intercept were similar for SHR and WKY for norepinephrine, but were greater in SHR for tyramine. The results demonstrate an increased systemic vascular responsiveness to catecholamines in adult SHR, with no evidence of increased systemic vascular sensitivity. These findings are consistent with the concept of increased systemic vascular responsiveness to catecholamines in adult SHR secondary to structural changes in blood vessels.

Cardiovascular Effects of Chronic High-dose Atriopeptin-iii Infusion in Normotensive Rats

Seventy-eight Sprague-Dawley rats received continuous intravenous infusions of either atriopeptin III (APIII), 60 micrograms/kg/hr, or distilled water vehicle for a period of 7 days by means of osmotic minipumps. On Day 7 approximately one-half of the animals (20 vehicle-treated rats and 21 APIII-treated rats) were instrumented for evaluation of cardiac function and terminated for measurement of heart weight. The minipumps remained in place during the evaluation of cardiac function. Also on Day 7, the osmotic pumps were removed from the remaining animals and an additional 7 days were allowed to elapse before heart weight and cardiac function were evaluated. Mean arterial blood pressure (MAP) of rats receiving APIII for 7 days was significantly lower (-9%, p less than 0.05) than that of rats receiving vehicle for 7 days. In addition, reductions (p less than 0.05) of total ventricular weightdry (-7%), left ventricular weightdry (-8%), and right ventricular weightdry (-9%) were observed in the APIII-treated rats (all ventricular weights are normalized for body weight). Hematocrit (HCT) was significantly higher (13%, p less than 0.05) in the APIII-treated group. Chronic APIII infusion did not influence ventricular performance nor did it affect regional vascular resistances. Seven days after termination of the APIII infusion the differences in MAP and HCT between vehicle-treated and APIII-treated animals were no longer evident. Partial recovery of the effect on heart weights was apparent, with total ventricular weightdry and left ventricular weightdry remaining slightly reduced (-4 and -5%, respectively; p less than 0.05). No differences were found between the two recovery groups for any index of cardiac function. In separate experiments, it was demonstrated that APIII, 60 micrograms/kg/hr iv, caused a significant increase in urine volume (p less than 0.05 relative to vehicle) during the initial 24 hr of infusion. The results indicate that chronic infusion of a large diuretic dose of APIII exerts relatively little influence on overall cardiovascular function in normotensive rats.

Enhanced potency dipeptide glycol renin inhibitors: Studies in vitro and in the conscious rhesus

We prepared a series of novel dipeptide amides of the formula Boc-Phe-Leu-X, where X is a 3-amino-3-alkyl-1,2-propanediol with lower alkyl substitutions at C-1, in order to probe accessory binding sites in the enzyme renin. This approach was successful in generating potent inhibitors of human and hog renin in vitro. Moreover, these inhibitors were able to effect in vivo reduction of plasma renin activity (PRA) in the conscious salt-depleted rhesus monkey (i.v. route); this effect was related to the size of the C-1 alkyl group.

Role of intestinal transport and first pass liver extraction on oral delivery of renin inhibitor compounds

Abstract The absolute bioavailabilities of three renin inhibitor compounds, one uncharged (compound I) and two positively charged (compounds II and III), were found to be comparable (1—3%). To determine the role of intestinal transport and first pass liver extraction (FPLE) in the oral delivery of these compounds intravenous, intraportal, intraduodenal and intraperitoneal studies were performed in the rat. In the intraduodenal studies, drug solutions were injected into the duodenum of anesthetized rats and portal and systemic blood was collected. In the intraportal studies, the drug solutions were injected into the portal vein and systemic blood was collected. From the ratio of the area under the drug concentration-time curves (tAUC) for the oral and intraportal studies, the extent of intestinal transport of compounds I–III was estimated as 9.7, 2.2 and 2.2%, respectively. In the intraduodenal studies the maximum portal plasma concentrations of compounds I–III were 2.8, 0.5 and 0.2 μg/ml, respectively. The tAUC of compound I in portal plasma was 8–26-times higher than those for compounds II and III. From comparison of the intraportal and intravenous tAUC values, the FPLE of compounds I–III was estimated as 76 ± 4, 61 ± 3 and 8 ± 23% (mean ± SE), respectively. Overall, the results indicated that the intestinal transport and FPLE of compound I was the highest among the three analogs. Compound II showed low intestinal transport and high FPLE and compound III showed low intestinal transport and low but variable FPLE.

Vascular pressure-flow analysis in normal and hypoxemic spontaneously hypertensive rats.

The pressure-flow relationship of the autoperfused subclavian vascular bed was compared in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) after spinal cord transection. Studies were performed under normoxemic and hypoxemic conditions. In adult SHR, vascular resistance was greater relative to WKY under both conditions. In young (8 week old) SHR vascular resistance was consistently greater over a wide range of perfusion pressures compared to young WKY rats when blood oxygen content was normal. Vascular resistance was not different between young SHR and WKY when the animals were hypoxemic. The results demonstrated that elevated vascular resistance in adult SHR was independent of oxygen availability and supraspinal nerve function; however, in young SHR elevated vascular resistance was dependent upon oxygen availability, although independent of supraspinal nerve function.

Abstract 3428: Effect of a non cardiotoxic Doxorubicin analog, 13-deoxy, 5-imino doxorubicin on decatenation of DNA by Topoisomerase II.

Anthracyclines such as doxorubicin are widely used to treat solid and hematological cancers. However, within the course of treatment they have the potential to cause life threatening chronic cardiotoxicity which limits their potential use. The cause of cardiotoxicity is not thoroughly understood, with previous research indicating the generation of reactive oxygen species and free iron accumulation as possible causes for cardiomyocyte death. Recent publications have shown the interaction between doxorubicin and Topoisomerase IIβ plays a key role in anthracycline induced cardiotoxicity. Topoisomerase enzymes are utilized during the coiling and uncoiling of DNA within the cell and are active targets of anthracyclines not only in tumoricidal action (Topoisomerase IIα) but also in the pathogenesis of cardiotoxicity (Topoisomerase IIβ). In this research we seek to further evaluate the interaction between doxorubicin, doxorubicin analogues and Topoisomerase IIβ. The analogues were selected due to previous chronic in vivo experiments in rabbits where classical manifestations of cardiotoxicity (including reduced left ventricular shortening fraction and pathological changes in cardiac tissue) were not observed. Furthermore, one analogue (13-deoxy, 5-imino doxorubicin) has shown promising clinical data suggesting a strong antineoplastic effect without the cardiotoxic effects. Doxorubicin and this analogue were placed in the presence of catenated DNA for 6 hours at 37°C prior to exposure to Topoisomerase IIα and β enzymes. The reaction was allowed to proceed for 30 minutes at 37°C and analyzed for decatenation of the DNA by the enzyme. This experiment was then repeated in cardiac H9c2 cells to assay possible differential inhibition at the cellular level. Citation Format: Nicole Frank, Richard Olson, Gerald M. Walsh, Todd Talley, Barry Cusack. Effect of a non cardiotoxic Doxorubicin analog, 13-deoxy, 5-imino doxorubicin on decatenation of DNA by Topoisomerase II. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3428. doi:10.1158/1538-7445.AM2013-3428