Development of a discriminative biphasic in vitro dissolution test and correlation with in vivo pharmacokinetic studies for differently formulated racecadotril granules.

The purpose of this study was to discriminate the release behavior from three differently formulated racecadotril (BCS II) granules and to establish an in vitro-in vivo correlation. Three granule formulations of the lipophilic drug were prepared with equivalent composition but prepared with different manufacturing processes (dry granulation, wet granulation with or without binder). In vitro release of the three granules was investigated using a biphasic dissolution system (phosphate buffer pH6.8 and octanol) and compared to the conventional single phase USP II dissolution test performed under sink and non-sink conditions. In vivo studies with each granule formulation were performed in rats. Interestingly, the granule formulations exhibited pronouncedly different behavior in the different dissolution systems depending on different wetting and dissolution conditions. Single phase USP II dissolution tests lacked discrimination. In contrast, remarkable discrimination between the granule formulations was observed in the octanol phase of biphasic dissolution system with a rank order of release from granules prepared by wet granulation with binder>wet granulation without binder>dry granulation. This release order correlated well with the wettability of these granules. An excellent correlation was also established between in vitro release in the octanol phase of the biphasic test and in vivo data (R2=0.999). Compared to conventional dissolution methods, the biphasic method provides great potential to discriminate between only minor formulation and process changes within the same dosage form for poorly soluble drugs.

Quantitative analysis of racecadotril metabolite in human plasma using a liquid chromatography/tandem mass spectrometry.

Orally administered racecadotril is rapidly hydrolyzed to the more potent enkephalinase inhibitor thiorphan in vivo. A sensitive and specific liquid chromatography/tandem mass spectrometry method was developed and validated to quantify thiorphan in human plasma using lisinopril as the internal standard. After a simple protein precipitation with methanol, the post-treatment samples were analyzed on a CN column interfaced with a triple-quadruple tandem mass spectrometer using negative electrospray ionization. The method was validated to demonstrate the specificity, lower limit of quantification, accuracy, and precision of measurements. The assay was linear over the concentration range 9.38-600 ng/mL using a 5 microL aliquot of plasma. The correlation coefficients for the calibration curves ranged from 0.9985 to 0.9995. The intra- and inter-day precisions over the entire concentration were not more than 6.33%. Methanol and water (35:65, v/v) is used as the isocratic mobile phase, with 0.1% of formic acid in water. The method was successfully applied for pharmacokinetic study after a single oral administration of 200 mg racecadotril to 20 healthy volunteers.

Racecadotril.

Racecadotril is an oral enkephalinase inhibitor used in the treatment of acute diarrhoea. It prevents the degradation of endogenous opioids (enkephalins), thereby reducing hypersecretion of water and electrolytes into the intestinal lumen. In a randomised double-blind study in 6 adult volunteers with castor oil-induced diarrhoea, racecadotril significantly reduced stool weight and stool number in comparison with placebo. Similar results have been obtained in treating castor oil-induced diarrhoea in rats. Racecadotril was significantly more effective than placebo in randomised double-blind studies in adults or children with diarrhoea (of infectious origin or in adults with HIV infection). In well controlled trials, racecadotril had efficacy similar to that of loperamide and was generally as effective as loperamide-oxide. Racecadotril had a similar tolerability profile to placebo, and was better tolerated than loperamide, in adults and children with diarrhoea. It caused significantly less constipation after resolution of diarrhoea than loperamide.

Inhalation of substance P and thiorphan: acute toxicity and effects on respiration in conscious guinea pigs.

Substance P is a tachykinin and a biologically active neuropeptide. The peptide produces salivation, neuronal excitation, vasodilatation, increased vascular permeability and contraction of smooth muscles in the respiratory tract. The study was designed to evaluate the acute effects in guinea pigs of inhaled aerosolized Substance P (SP). Apart from the acute toxic effect of the peptide, the distribution in different organs was also investigated. The acute inhalation toxicity of SP (LC50, 15 min) when co-administrated with the neutral endopeptidase inhibitor thiorphan was 368 microg m(-3). The peptide caused an increase in respiratory rate proceeding a decrease in tidal volume. As the exposure proceeded, a decrease in both respiratory rate and further decreases in tidal volume were observed until either the animal died or the exposure was terminated. The decreases in respiratory rate and tidal volume were probably due to bronchoconstriction caused by SP. Eighteen per cent of the inhaled amount of radioactive SP was retained in the body, and the highest concentrations of radioactivity were found in the kidney, lung and liver. Substance P in combination with thiorphan administered as an aerosol is extremely toxic and highly potent. Exposure to the substance at extremely low air concentrations may result in incapacitation in humans.

Pathophysiology and therapy of irinotecan-induced delayed-onset diarrhea in patients with advanced colorectal cancer: a prospective assessment.

PURPOSE: Irinotecan (CPT-11), a camptothecin derivative, has shown efficacy against colorectal cancer. Delayed-onset diarrhea is its main limiting toxicity. The aim of this study was to determine the pathophysiology of CPT-11-induced delayed-onset diarrhea and assess the efficacy of combined antidiarrheal medication in a phase II, prospective, successive-cohorts, open study. PATIENTS AND METHODS: Twenty-eight patients with advanced colorectal cancer refractory to fluorouracil (5-FU) therapy received CPT-11 350 mg/m2 every 3 weeks. The first cohort of 14 consecutive patients explored for the mechanism of diarrhea received acetorphan (a new enkephalinase inhibitor) 100 mg three times daily; the second 14-patient cohort received, in addition to acetorphan, loperamide 4 mg three times daily. Before treatment, and if late diarrhea occurred, patients underwent colon mucosal biopsies for CPT-11 and topoisomerase I levels; intestinal transit time; fecalogram; fat and protein excretion; alpha1-antitrypsin clearance; D-xylose test; blood levels for vasoactive intestinal polypeptide, glucagon, gastrin, somatostatin, prostaglandin E2, and carboxylesterase; CPT-11/SN-38 and SN-38 glucuronide pharmacokinetics; and stool cultures. RESULTS: Delayed-onset diarrhea occurred during the first three treatment cycles in 23 patients (82%). Electrolyte fecal measurements showed a negative or small osmotic gap in nine of nine patients and an increased alpha1-antitrypsin clearance in six of six patients. There were no modifications in stool cultures or hormonal dysfunction. Four of 11 patients (36%) with delayed-onset diarrhea in the first cohort responded to acetorphan, whereas nine of 10 patients (90%) responded to the combination of acetorphan and loperamide (P < .02). CONCLUSION: CPT-11-induced delayed-onset diarrhea is caused by a secretory mechanism with an exudative component. Early combined treatment with loperamide and acetorphan seems effective in controlling the diarrheal episodes.

Pharmacologic profile of CGS 24128, a potent, long-acting inhibitor of neutral endopeptidase 24.11.

We compared the pharmacologic profiles of thiorphan, a neutral endopeptidase (NEP) inhibitor which is cleared rapidly from the circulation, and CGS 24128, an inhibitor with a much longer half-life (t1/2). Thiorphan and CGS 24128 inhibited NEP in vitro with IC50 values of 5.0 +/- 0.2 and 4.3 +/- 0.2 nM, respectively. After administration at 10 mg/kg intravenously (i.v.), the concentrations of CGS 24128 in the plasma were > 500 nM for 4 h but plasma thiorphan was detectable for only 60 min. Thiorphan 3 mg/kg administered intraarterially (i.a.) increased plasma atrial natriuretic peptide immunoreactivity (ANPir) levels by 58 +/- 12% in rats administered exogenous ANP(99-126). This response lasted < 60 min, whereas the same dose of CGS 24128 produced an average increase of 191 +/- 19% in ANPir concentrations that persisted for 4 h. ANP-induced (1 microgram/kg i.v.) natriuresis was significantly potentiated in anesthetized rats pretreated (60 min) with a bolus of CGS 24128 10 mg/kg i.v. The change in urinary sodium excretion (UNaV) produced by ANP was 28.8 +/- 4.0 and 15.8 +/- 1.8 muEq/kg/min in CGS 24128- and vehicle-treated rats, respectively. ANP-induced natriuresis was also greater during continuous infusion of thiorphan (5 mg/kg bolus + 0.1 mg/kg/min i.v.; delta UNaV = 29.2 +/- 5.8 and 13.8 +/- 3.2 muEq/kg/min in drug- and vehicle-treated rats, respectively) but not when thiorphan was administered as a bolus (10 mg/kg i.v.) 60 min before the ANP challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Disposition of thiorphan in DOCA-salt and spontaneously hypertensive rats.

Thiorphan was administered intravenously (i.v.) at 10 mg/kg to conscious rats in two different models of hypertension to allow a comparison of pharmacokinetics. The two models were: 1) Deoxycorticosterone acetate (DOCA)-salt uninephrectomized rats; 2) Spontaneously hypertensive rats (SHR), and their respective normotensive controls; 3) Sprague-Dawley (SD) rats; and 4) Wistar-Kyoto rats (WKY). Pharmacokinetic parameters were calculated for total and unbound thiorphan in plasma. In normotensive SD and WKY rats, the volume of distribution, clearance and plasma protein binding of thiorphan were not significantly different. Furthermore, the apparent elimination half-life was not significantly different for total or unbound thiorphan amongst all models. The volume of distribution and plasma clearance for both unbound and total thiorphan, however, were lower in DOCA-salt rats when compared to normotensive control rats by 61-66% and 46-51%, respectively. In contrast, pharmacokinetic parameters for both unbound and total thiorphan were not significantly different between SHR and WKY rats. These results indicate that reduced clearance of thiorphan in DOCA-salt rats may be due to the co-administration of DOCA-salt or altered renal function of the hypertrophic remaining kidney and not solely due to hypertension.

Evaluation of enkephalinase inhibition in the living mouse, using [3H]acetorphan as a probe.

A novel in vivo binding test was developed in order to evaluate the degree of occupancy of enkephalinase (EC 3.4.24.11), a membrane-bound metallopeptidase, in cerebral and peripheral tissues of mice treated with enkephalinase inhibitors. The probe selected for this purpose was the prodrug [3H]acetorphan, a lipophilic diesterified derivative of the potent enkephalinase inhibitor thiorphan readily releasing the latter by tissue hydrolysis. In order to validate the in vivo binding assay, [3H]thiorphan binding to membranes was first studied in vitro. [3H]Thiorphan binding to cerebral and peripheral tissues (lung and kidney) was saturable over a low nonspecific binding, occurring with a KD of 0.6 nM consistent with the Ki of the compound as enkephalinase inhibitor. [3H]Thiorphan binding varied largely among various tissues and was highly correlated with the catalytic activity of enkephalinase, thus indicating a selective labeling of the peptidase. After the i.v. administration of [3H]acetorphan a large fraction of the radioactivity remained bound to membranes isolated by a rapid filtration assay. Bound radioactivity mainly corresponded to [3H] thiorphan as identified by high-performance liquid chromatography analysis of kidney membranes, whereas unchanged [3H]acetorphan was not detectable. In vivo binding generated by [3H]acetorphan was saturable, with maximum binding sites values which were in rather good agreement with corresponding maximum binding sites values of [3H]thiorphan binding in vitro, particularly in brain. Specific in vivo binding was calculated as the difference between total and a generally low, nonspecific binding evaluated in mice receiving a large dose of nonlabeled acetorphan. Specific in vivo binding varied largely among tissues and generally reflected the abundance of enkephalinase molecules in the latter.(ABSTRACT TRUNCATED AT 250 WORDS)