Integrated in vivo pharmacology using an instrumented, unrestrained and conscious rat platform: application to rat models of diabetes.

INTRODUCTION: We define the technical and methodological aspects that led to a practical and reproducible biological in vivo platform allowing the measurement of more than 65 physiobiochemical parameters on a daily basis in freely moving conscious animals. Such a platform presents the ability to unleash incremental information in the hands of modern-day pharmacologists and physiologists. METHODS: To validate this platform, we fully characterized three rat models of Type 1 and Type 2 diabetes and their respective controls. Control, streptozotocin- and alloxan-diabetic Wistar rats in addition to ZDF-Lean and ZDF-Fatty rats were chronically implanted with an arterial catheter and kept in metabolic cages. The catheter was connected to a minipump infusing saline at a constant rate to maintain patency and used to collect blood and measure hemodynamic parameters on a daily basis. RESULTS: Catheter implantation was successful in over 95% of animals and catheter patency was successfully maintained for 30 days in about 75% of animals. The three diabetic rat strains showed elevations in food and water consumption, urinary output, plasma glucose, blood urea nitrogen, triglycerides and cholesterol. The two Type I models also showed a depressed body weight and hemodynamic function. The STZ model differed from the alloxan-model by elevations in liver enzyme activities (AST, ALT, and bilirubin) and a more severe dyslipidemia (triglycerides and total cholesterol). The ZDF-Fatty rats distinguished themselves by higher body weight and elevated white blood cell counts. DISCUSSION: This integrated platform represents a significant improvement in standard in vivo evaluations and could greatly improve the pace of development of potential new drugs.

Triple vasopeptidase inhibition normalizes blood pressure in conscious, unrestrained, and spontaneously hypertensive rats.

BACKGROUND: CGS 35601 is a potent triple vasopeptidase inhibitor (VPI) of angiotensin-converting enzyme (ACE), neutral endopeptidase (NEP), and endothelin-converting enzyme (ECE). The aim of the study was to determine the effects of this VPI on the hemodynamic profile of conscious, instrumented, unrestrained spontaneously hypertensive rats (SHR), in comparison to selective inhibitors of ACE and ACE + NEP, than +ECE combined. Circulating plasma concentrations of vasoactive mediators and reactive oxygen species were measured. METHODS: Old SHR male were instrumented (arterial catheter) and placed in a metabolic cage for daily hemodynamic measurements and blood samplings. Seven days after surgery, SHR received 1) saline vehicle; 2) increasing doses of the triple CGS 35601 (0.01, 0.1, 1, and 5 mg/kg/d, intra-arterially (i.a.) infusion for 5 d/dose) followed by a 5-day washout period; 3) benazepril (ACE inhibitor), ACE inhibitor + CGS 24592 (NEP inhibitor) and ACE inhibitor + NEP inhibitor + CGS 35066 (ECE inhibitor) (1 or 5 mg/kg/d i.a. infusion for 5 d/combination) followed by a 5-day washout period. RESULTS: The lowest dose of CGS 35601 had no effect. Doses at 0.1, 1, and 5 mg/kg/d reduced mean arterial blood pressure by 10%, 22%, and 40%, respectively. Heart rate was unaffected in all groups. CGS 35601 decreased concentrations of angiotensin II (Ang II), endothelin-1 (ET-1), and pro-atrial natriuretic peptide (proANP), and increased those of big ET-1, atrial natriuretic peptide (ANP), bradykinin (BK), and hydrogen peroxide (H2O2) dose dependently. CONCLUSIONS: The blood pressure-lowering effect of this triple VPI was superior to that of the other VPI in this preclinical rat model of hypertension. Further experiments are needed to assess triple VPI to other combinations in other models with regard to efficacy and angioedema. Only then it may constitute a first-in-class approach for the treatment of hypertension and other cardiovascular disorders.

Profile of endothelin isopeptides and markers of oxidative stress alongside the onset of streptozotocin-type I diabetes in rats.

Type I diabetes is associated with vascular endothelial abnormalities. Vasoactive mediators such as endothelins and reactive oxygen species are modulated in diabetic patients. We studied the hemodynamic profile and the release of mediators alongside the onset of streptozotocin-induced type I diabetes in rats. Arterial plasma samples were collected from chronically instrumented, unrestrained and conscious normotensive versus streptozotocin-diabetic rats. Streptozotocin-diabetic rats developed severe hypoinsulinemia and subsequent hyperglycemia within 5 days. Mean arterial blood pressure and heart rate decreased from 107 to 87 mmHg (19%) and from 386 to 282 beats per minute (bpm) (27%), respectively over 21 days. On day 20 post-streptozotocin administration, markers of oxidative stress (reactive oxygen species: hydrogen peroxide, total peroxides) and related vasodilatory nitric oxide metabolites, increased. Plasma concentrations of atrial natriuretic peptide were not affected, while vasocontractile endothelin-1 and big endothelin-1 increased in streptozotocindiabetic rats versus chronically instrumented, unrestrained and conscious normotensive rats. In addition, the ratios of endothelin-1 : big endothelin-1 and nitric oxide : endothelin-1 were increased. The depressed hemodynamic profile may result from an imbalance between vasocontracting and relaxing factors. Their interactions with reactive oxygen species may affect vascular tone and lead to vascular complications prevalent in this pathological condition. Defining the complex regulation and roles of these factors merits further investigations, especially in the later endstages of vascular complications, because the development of these complications is linked to the duration of the diabetic state.

Triple vasopeptidase inhibition of angiotensin-converting enzyme/neutral endopeptidase/endothelin-converting enzyme activities on the hemodynamic profile of chronically instrumented unrestrained conscious spontaneously hypertensive rats.

Inhibition of the renin-angiotensin system with an angiotensin-converting enzyme inhibitor (ACEi) is an effective therapy in hypertension. Vasopeptidase inhibition was initially proposed with compounds inhibiting both angiotensin-converting enzyme and neutral endopeptidase (omapatrilat), but clinical trials revealed that reducing angiotensin II while blocking the degradation of vasodilatory peptides was not without concerns. We have previously investigated the combination of an ACEi with an endothelin-converting enzyme inhibitor (ECEi); now we add a neutral endopeptidase inhibitor (NEPi) toward triple vasopeptidase inhibition. Male spontaneously hypertensive rats were surgically implanted with a vascular catheter and treated with an ACEi (benazepril), a NEPi (CGS 24592) and an ECEi (CGS 35066) (continuous intra-arterial infusion at 1 or 5 mg/kg/day x 5 days each). After 15 days, drugs administration was stopped for 3 days. ACEi (1 mg/kg per day) reduced the mean arterial blood pressure by 8.4%. The addition of a NEPi and an ECEi at the same dose did not shown any added benefit. The mean arterial blood pressure came back to baseline upon cessation of treatment. ACEi (5 mg/kg per day) reduced the mean arterial blood pressure by 28%. The mean arterial blood pressure remained attenuated by 21% and 19% with the addition of the NEPi and the ECEi. Again, the mean arterial blood pressure rose back to 148 +/- 4 mmHg following cessation of treatment. Daily biochemical and hematological analysis of plasma did not reveal any signs of toxicity, except for a rapid elevation in K (40%) after 1 day of ACEi. Thus, angiotensin II inhibition plays a primary role in controlling the blood pressure of spontaneously hypertensive whereas additional NEPi and ECEi did not provide further benefits under the present dose combinations. The normalizing effect of the higher dose of ACEi by itself made it impossible to discriminate the role of neutral endopeptidase and endothelin-converting enzyme-modulated peptides and to further define the paradigm of triple vasopeptidase inhibition toward better control of vascular hemodynamics. Additional studies are underway.

Myocardial blood flow after chronic cardiac decentralization in anesthetized dogs: effects of ACE-inhibition.

Coronary blood flow regulation was studied in dogs with an intact or chronically decentralized intrinsic cardiac nervous system. We also examined the effect of angiotensin-converting enzyme inhibition (ACEI) on coronary autoregulatory pressure-flow relations and distribution of blood flow since the renin-angiotensin system may play a critical role in vasoregulation. Myocardial oxygen demand was reduced in the chronic decentralized dogs compared to the control dogs. The lower pressure limit of the autoregulatory pressure-flow relation was similar for the control and chronic decentralized dogs (47+/-2 and 44+/-7 mm Hg, respectively; p = NS). After ACEI, the lower pressure limit shifted leftward to 40 mm Hg (p=0.001) in both groups. Concomitant blockade of cyclooxygenase, bradykinin catabolism and nitric oxide synthase had no further effect on the lower pressure limit. Total myocardial blood flow was lower (p=0.001) in the chronic decentralized dogs compared to the control dogs, while transmural distribution of blood flow was preserved in both groups. The results show that even though myocardial oxygen requirements are lower in the chronically decentralized heart compared to controls, coronary autoregulation is maintained at levels observed in normally innervated hearts. The present findings indicate that intrinsic cardiac neurons contribute to coronary autoregulatory control and myocardial blood flow distribution even in the absence of cardiac connections to the central nervous system. In addition, in the chronic decentralized dog, ACEI allows the heart to work at lower coronary perfusion pressures while myocardial blood flow distribution is preserved.

Angiotensin inhibition and coronary autoregulation in a canine model of LV hypertrophy.

In humans with hypertension and LV hypertrophy, beneficial effects of angiotensin inhibition may be associated with preserved autoregulatory capacity. We studied the effect of acute angiotensin converting enzyme (ACE) inhibition on coronary autoregulatory pressure-flow relations and transmural distribution of blood flow in sham and LV hypertrophy dogs. Heart/body weight ratio increased (p = 0.001) from 5.5 +/- 0.7 in sham to 6.9 +/- 0.5 in LV hypertrophy dogs. The lower coronary pressure limit (LPL) on the pressure-flow relation was 47 +/- 2 mmHg in sham and 57 +/- 6 mmHg (p = 0.001) in LV hypertrophy dogs; after acute ACE-inhibition the LPL was reduced to 40 +/- 5 mmHg and 49 +/- 6 mmHg (p = 0.001), respectively. Transmural distribution of blood flow was preserved at the LPL in both groups before and after acute ACE-inhibition. Concomitant blockade of prostaglandin and nitric oxide release and bradykinin catabolism had no additional effects on the LPL and distribution of blood flow. After acute ACE-inhibition in LV hypertrophy dogs, distribution of blood flow across the LV wall was preserved and subendocardial vascular reserve was maintained even though the LPL was significantly lower. Preservation of autoregulatory capacity by ACE inhibitors contributes to beneficial outcome in patients with hypertension and LV hypertrophy.

Effects of benazepril, an angiotensin-converting enzyme inhibitor, combined with CGS 35066, a selective endothelin-converting enzyme inhibitor, on arterial blood pressure in normotensive and spontaneously hypertensive rats.

Continuous intra-arterial administration of a selective endothelin-converting enzyme (ECE) inhibitor CGS 35066 at a dose of 30 mg/kg decreased the mean arterial blood pressure (MABP) in conscious unrestrained normotensive rats and spontaneously hypertensive rats (SHRs). At that dose, the magnitude of the antihypertensive effects was greater in SHRs than in normotensive rats. Additional administration of an angiotensin-converting enzyme (ACE) inhibitor benazapril (lotensin) further reduced MABP in normotensive rats and completely blocked hypertension in SHRs. However, when the selective ECE inhibitor was subsequently removed, blood pressure was less inhibited in normotenive rats whereas it remained strongly inhibited in SHRs by the ACE inhibitor alone. These results imply that simultaneous treatment with benazepril and CGS 35066 gave additive antihypertensive effects in normotensive rats but not in SHRs, when both compounds were administered at a dose of 30 mg/kg. Our results suggest that: (i) the endothelin (ET) system together with the renin-angiotensin system contribute to the maintenance of blood pressure in normal healthy rats; (ii) while an ECE inhibitor acts as an antihypertensive agent on its own, the sole efficacy of ACE inhibitor at that dose is sufficient to block MABP without the participation of the ET system in SHR.

Effects of a selective endothelin A receptor antagonist, ABT-627, in healthy normotensive anaesthetized rats developing acute pulmonary air embolism.

Acute pulmonary air embolism (APAE) injures the vascular endothelium in the lung and results in pulmonary hypertension (PH). Endothelins (ETs), a family of potent vasoactive peptides, are known to be associated with PH of various aetiologies. We evaluated the effects of ABT-627, a selective ET(A) receptor (ET(A)-R) antagonist in a rat model of APAE over 3 h. APAE rats developed a higher right ventricular systolic pressure (RVSP), lower mean arterial blood pressure (MABP), and had lower PaO(2). At 3 h, arterial plasma levels of ET-1 were increased. ABT-627-treated controls showed no effects. However, ABT-627 significantly lowered RVSP during APAE, abolished the short recovery phase (within 10-25 min) of MABP without affecting the subsequent lowering of MABP, and improved oxygen saturation in APAE rats. These results show that ET(A)-R subtype is involved in the pathogenesis of APAE since a blockade of this receptor subtype attenuated the cardiopulmonary deterioration and improved blood gas exchanges in rats with this disease.