Increased thrombopoietin levels in idiopathic thrombocytopenic purpura patients with a poor response to steroid therapy.

The serum thrombopoietin (TPO) levels in 61 idiopathic thrombocytopenic purpura (ITP) patients were found to be slightly increased compared with those of 29 normal subjects. The TPO levels of the 15 ITP patients who had a poor response to steroid therapy (i.e. an unchanged platelet count) were higher than those of the 22 ITP patients who had a good response to steroid therapy (i.e. an increased platelet count) and the normal subjects. The TPO levels in the 15 ITP patients whose platelet count was higher than 10 x 10(4)/microl after the discontinuation of steroid therapy significantly higher than those of the normal subjects. The platelet-associated immunoglobulin G (PAIgG) levels in the ITP patients who had a poor response to steroid therapy were slightly increased compared with the normal subjects and the ITP patients who had a good response to the steroid therapy and the nine ITP patients who did not undergo the steroid therapy. The serum TPO level was negatively correlated only with the megakaryocyte count in the ITP patients, and the megakaryocyte count in the ITP patients who had good responses to the steroid therapy was higher than that in those who had poor responses. These data suggest that serum TPO levels might be important for the prediction of the outcome of ITP patients who receive steroid therapy.

[An elderly case whose residual malignant lymphoma was completely remitted by oral etoposide treatment using unusual dosing schedule].

In a 72-year-old man with malignant lymphoma (Hodgkin's disease, nodular sclerosis type, Stage III S), complete remission was induced by intravenous drip of adriamycin and oral administration of etoposide. After that, a maintenance dose of etoposide (25 mg) was administered every 3 days without observable side effects. From these results, we considered that the dose level and interval of administration are both important for chemotherapy in elderly patients.

New alpha-thiol dipeptide dual inhibitors of angiotensin-I converting enzyme and neutral endopeptidase EC 3.4.24.11.

Dual inhibitors of the two zinc metallopeptidases, neutral endopeptidase (NEP, EC 3.4.24.11) and angiotensin-I converting enzyme, have been the focus of much clinical interest for the treatment of hypertension and congestive heart failure. A novel series of alpha-thio dipeptides containing central cyclic non-natural amino acids were prepared and were evaluated for their ability to inhibit these two metallopeptidases in vitro and in vivo. Most of these compounds were found to be excellent dual inhibitors of ACE and NEP in vitro and several were also found to inhibit angiotensin-I (AI) pressor response in conscious rats when given by intravenous administration. Compound 6n, one of our most potent dual inhibitors in vitro, was found to be more efficacious than captopril in the AI pressor experiment when administered orally to conscious rats. This compound was also found to inhibit plasma NEP activity following oral administration to conscious rats and was more efficacious than acetorphan. The structure-activity relationships and biological activity of these dual inhibitors will be discussed.

Dicarboxylic acid dipeptide neutral endopeptidase inhibitors.

The synthesis of three series of dicarboxylic acid dipeptide neutral endopeptidase 24.11 (NEP) inhibitors is described. In particular, the amino butyramide 21a exhibited potent NEP inhibitory activity (IC50 = 5.0 nM) in vitro and in vivo. Blood levels of 21a were determined using an ex vivo method by measuring plasma inhibitory activity in conscious rats, mongrel dogs, and cynomolgus monkeys. Free drug concentrations were 10-1500 times greater than the inhibitory constant for NEP over the course of a 6 h experiment. A good correlation of free drug concentrations was obtained when comparing values determined by the ex vivo analysis to those calculated from direct HPLC measurements. Plasma atrial natriuretic factor (exogenous) levels were elevated in rats and dogs after oral administration of 19a. Urinary volume and urinary sodium excretion were also potentiated in anesthetized dogs treated with 21a.

A rapid indirect method to determine the plasma concentrations of neutral endopeptidase inhibitors.

The selection of therapeutic agents for clinical development is principally based upon pharmacodynamic and pharmacokinetic criteria. Although many compounds are routinely tested in pharmacologic assays, direct pharmacokinetic assessment is difficult and not applicable to a large number of agents. Therefore, we have developed a rapid indirect method based on enzyme inhibition for determining the unbound concentration of NEP 24.11 inhibitors in rat plasma. In the present study, drug levels of compounds from three different classes of NEP 24.11 inhibitors: mercaptoalkyl, carboxyalkyl and aminophosphonates were compared. Studies were carried out in conscious, unrestrained rats. Arterial blood samples were obtained 10, 30, 60, 120, and 240 min after drug administration at 10 mg/kg i.v. or 30 mg/kg p.o. The blood was collected in EDTA and plasma prepared immediately. Protein bound NEP inhibitor was separated from plasma by centrifugation through an ultrafiltration membrane. Following acidification and serial dilution, the concentration of unbound inhibitor was determined in the plasma ultrafiltrate using the in vitro assay for NEP 24.11 inhibition. The results of this study indicated that the mercaptoalkyl inhibitor thiorphan was cleared rapidly from plasma, whereas, the plasma concentrations of the carboxyalkyl inhibitor CGS 23880A (UK-69,578), and the plasma concentrations of the aminophosphonate, CGS 24128, were maintained at high levels for at least 4 hours. Furthermore, the ratio of the NEP inhibitor concentration/IC50 value correlated well with the pharmacologic activity of these compounds.

Differential structure-activity relationships of phosphoramidon analogues for inhibition of three metalloproteases: endothelin-converting enzyme, neutral endopeptidase, and angiotensin-converting enzyme.

The structure-activity relationships of phosphoramidon analogues for inhibition of endothelin-converting enzyme (ECE), neutral endopeptidase 24.11 (NEP), and angiotensin-converting enzyme (ACE) were compared. Phosphoramidon inhibited ECE, NEP, and ACE activities with IC50 values of 3.5, 0.034, and 78 microM, respectively. Removal of the rhamnose moiety of phosphoramidon (dipeptide 3) reduced the potency for ECE (IC50 = 70 microM), whereas the potencies for NEP (0.003 microM) and ACE (0.20 microM) were increased. Addition of 2-(2-naphthyl)ethyl to dipeptide 3 improved the potency for ECE (0.55 microM) but weakened the potency for NEP (0.02 microM), and had no significant change for ACE. Interchange between Leu and Trp abolished the inhibitory activities for ECE and NEP, but the compound remained active for ACE. These results suggest that a hydrophobic group in the P1 position of phosphoramidon analogues increases the potency for ECE significantly, whereas compounds containing a free phosphonic acid are optimal for inhibition of NEP and ACE. Furthermore, an aromatic group in the P'2 position is essential for the inhibition of ECE and NEP, but not ACE.

N-Phosphonomethyl dipeptides and their phosphonate prodrugs, a new generation of neutral endopeptidase (NEP, EC 3.4.24.11) inhibitors.

Inhibitors of the zinc protease neutral endopeptidase (NEP, EC 3.4.24.11) offer significant therapeutic interest as antihypertensives due to their ability to potentiate the biological action of the circulating natriuretic hormone ANF (atrial natriuretic factor). N-Phosphonomethyl dipeptides bearing a central (4-phenyl)phenylalanine residue have been designed to exert potent and selective NEP inhibition. In particular, (S)-3-[N-[2- [(phosphonomethyl)amino]-3-(4-biphenylyl)propionyl]amino]propionic acid (10a) (CGS 24592) displayed high inhibitory potency in vitro (IC50 = 1.9 +/- 0.1 nM) and a long plasma half-life in rats but lacked oral bioavailability. This drawback was overcome by using esterase-sensitive (acyloxy)alkyl phosphonates. More remarkable, several diaryl phosphonate derivatives of 10a also performed as effective prodrugs. Specifically, the structurally simple diphenyl phosphonate 18 (CGS 25462) induced potent inhibition of NEP ex vivo for at least 8 h after oral administration to rats (30 mg/kg). Its antihypertensive effect was demonstrated in DOCA-salt rats. At 30 mg/kg orally, 18 caused a significant reduction in mean arterial pressure measuring -35 +/- 7 mmHg at 5-h postdosing. The alpha-aminomethyl phosphonate 18 represents a new generation of selective NEP inhibitors that combine high potency, long duration of action, and oral bioavailability. Therefore, it holds promise as a novel therapeutic agent for the treatment of human hypertension and congestive heart failure.

Design and synthesis of an orally active macrocyclic neutral endopeptidase 24.11 inhibitor.

A potent macrocyclic inhibitor of neutral endopeptidase (NEP) 24.11 was designed using a computer model of the active site of thermolysin. This 10-membered ring lactam represents a general mimic for any hydrophobic dipeptide in which the two amino acid side chains bind to an enzyme in a contiguous orientation. The parent 10-membered ring lactam was synthesized and exhibited excellent potency as an NEP 24.11 inhibitor (IC50 = 3 nM). In order to improve oral bioavailability, various functionality was attached to the macrocycle. These modifications lead to CGS 25155, an orally active NEP 24.11 inhibitor that slows down the degradation of the cardiac hormone atrial natriuretic factor, producing a lowering of blood pressure in the DOCA-salt rat model of hypertension.

Heterocyclic lactam derivatives as dual angiotensin converting enzyme and neutral endopeptidase 24.11 inhibitors.

A series of 13- and 14-membered ring lactam derivatives 9a,b, 10, 11, and 12a-c was prepared from L-cysteine. Compounds 9a,b and 12a,b were tested in vitro for inhibition of neutral endopeptidase 24.11 (NEP) and angiotensin converting enzyme (ACE) inhibition. The structure-activity profile of the series is discussed. Compound 9b, a 13-membered ring macrocyclic lactam, had an NEP IC50 of 18 nM and an ACEIC50 of 12 nM in vitro and showed dual plasma inhibition after intravenous or oral administration.

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.

The biological activity of atrial natriuretic factor cleaved by endoprotease 3.4.24.11.

Ring cleavage of atrial natriuretic peptide (ANF) between Cys7 and Phe8 by endoprotease 3.4.24.11 yields X-ANF. Since endoprotease 3.4.24.11 may contribute to ANF metabolism in vivo, the present study determined if X-ANF exhibits reduced biological activity in comparison to the parent molecule.

Characterisation of neutral endopeptidase 3.4.24.11 (NEP) in the kidney: comparison between normotensive, genetically hypertensive and experimentally hypertensive rats.

Neutral endopeptidase 3.4.24.11 (NEP) has been identified as the major atrial natriuretic factor (ANF) degrading enzyme in rat kidney, therefore, suggesting a possible role for this enzyme in blood volume and pressure regulation. Various experimentally induced and genetically hypertensive rat models have been used to test NEP inhibitors. The presence of different isoforms of NEP in the various hypertensive rat models would have relevance when searching for novel NEP inhibitors. Therefore, we compared the properties of NEP in kidney cortex homogenates in order to test for possible differences in the following hypertensive rat models and their appropriate controls: spontaneously hypertensive rats (SHR), Wistar Kyoto strain (WKY), DOCA-salt hypertensive rats, and Sprague Dawley control rats (SD). No relevant differences were found when comparing the following parameters: (1) specific activity (mean: 204 U/mg protein), (2) Michaelis constant (mean: 280 microM), (3) IC50 of thiorphan (mean: 6.5 nM) and phosphoramidon (mean: 54 nM), (4) pH profiles (optimum at pH 8.0), (5) heat inactivation profiles (half-life 20 min at 65 degrees C), (6) immunotitration of kidney cortex homogenates, (7) molecular weight as determined by gel filtration (92,000 Dalton) and (8) affinity chromatography with concanavalin A. Without evidence for the presence of different NEP isoforms, it is unlikely that divergent findings in DOCA-salt rats and SHR using a given NEP inhibitor are due to isoforms of NEP.

Carnitine metabolism and morphometric change of liver mitochondria in valproate-treated rats.

The effect of the administration for 7 or 28 days of 50 mg/kg/day valproate (VPA) on carnitine metabolism and morphological changes of liver mitochondria in immature rats was evaluated. The dose of VPA was almost the same as that we clinically used. Carnitine concentrations in serum, red blood cells (RBC), muscle, liver and urine were measured. The rats treated with VPA for 7 days showed no significant change in carnitine concentration in each tissue examined or by morphology. In the serum, RBC and muscle of rats treated with VPA for 28 days, free carnitine levels decreased, while acylcarnitine levels and the ratio of acylcarnitine to free carnitine (acyl/free ratio) increased. Mitochondrial enlargement was also induced and urinary acyl/free ratio of VPA treated rats was higher than that of control rats after the 14th day of the treatment. These results suggest that carnitine deficiency and morphometric changes in mitochondria occur time dependent even if the dose of VPA is clinically appropriate.

Enkephalinase inhibitors. 1. 2,4-Dibenzylglutaric acid derivatives.

The synthesis of two new series of dicarboxylic acid dipeptides and two sulfhydryl-containing inhibitors are described. The in vitro enkephalinase inhibition data and some in vivo analgesic data are presented for these compounds. For the dibenzylglutaric acid series structure-activity relationships and in vivo analgesic activity are discussed. The reverse amides, i.e., 4-amino-2,4-dibenzylbutyric acid derivatives, are also discussed. Two sulfhydryl-containing inhibitors showed good in vivo potency in the mouse jump-latency hot-plate test after peripheral administration at moderate low doses.

Identification of protease 3.4.24.11 as the major atrial natriuretic factor degrading enzyme in the rat kidney.

We have identified a metalloendoprotease from rat kidney cortex that cleaves the cysteine-phenylalanine bond (Cys7-Phe8) within the 17 amino acid ring structure of atrial natriuretic factor (ANF). Cleavage at this site represents the major ANF degradative activity in rat kidney, and is inhibited by the known metalloendoprotease inhibitors, thiorphan, phosphoramidon and zincov with IC50 values in the nanomolar range. Since these are specific inhibitors of protease 3.4.24.11, both protease 3.4.24.11 and ANF degrading activities were monitored during purification. Both activities copurified at each chromatographic step. Furthermore, purified protease 3.4.24.11 cleaved ANF specifically at the Cys7-Phe8 bond. It is concluded from this work that the major ANF degrading enzyme in rat kidney is protease 3.4.24.11.

Carnitine metabolism in valproate-treated rats: the effect of L-carnitine supplementation.

The effect of the administration for 7 days of valproate (500 mg/kg/day) or valproate (500 mg/kg/day) plus L-carnitine (200 mg/kg/day) on carnitine concentrations in serum, red blood cells, muscle, liver, and urine was evaluated. In the serum and muscle of the valproic acid (VPA) group, free carnitine levels decreased, while acyl-carnitine levels and acyl/free ratio increased, when compared to those of the control. When L-carnitine was given to the VPA group, the free carnitine levels increased in the serum, muscle, and liver, and the acyl/free ratio decreased in all tissues when compared to those of the VPA group. The mean of free carnitine level in urine of the VPA group was not different but acylcarnitine increased when compared to values of controls, and after the supplementation with L-carnitine the acylcarnitine (from day 4 to 7) levels were decreased compared to the VPA group. The serum beta-OH-butyrate level in the VPA group was decreased when compared to those of controls and VPA plus L-carnitine groups. These results indicate that L-carnitine supplementation protects against the alteration in carnitine metabolism induced by the administration of VPA.

Degradation of atrial natriuretic factor by kidney cortex membranes. Isolation and characterization of the primary proteolytic product.

Synthetic rat atrial natriuretic factor (r-ANF, 1-28) was incubated with rat kidney cortex membranes, and a predominant degradation product was identified by reverse-phase high performance liquid chromatography. The degradation product was subjected to amino acid analyses and found to have a composition identical to r-ANF. Amino-terminal sequence analyses identified two distinct amino-terminal residues and suggested that cleavage had occurred between the cysteine-phenylalanine bond (residues 7 and 8) of r-ANF. This degradative process could be inhibited by 1,10-phenanthroline and EDTA, suggesting that the enzyme responsible for proteolysis is a metalloendoprotease. The enzyme exhibits a Michaelis-Menten constant of approximately 10 microM for the metabolism of r-ANF and has a broad pH optimum between 8.5 and 9.5. These findings suggest that ANF may be initially degraded in the kidney at a single cleavage site within the 17-residue ring structure.

Hepatotoxicity in rat following administration of valproic acid: effect of L-carnitine supplementation.

The effect of prolonged administration (7 days) of valproate (VPA, 500 mg/kg/day), or VPA (500 mg/kg/day) with L-carnitine (200 mg/kg/day) on blood carnitine levels and the appearance of liver mitochondria were assessed in the rat. VPA-treated rats showed hypocarnitinemia and enlarged mitochondria when compared with saline-injected control rats. In rats treated with both VPA and L-carnitine, serum and liver carnitine levels were increased by the L-carnitine supplement and the liver mitochondria were not enlarged. L-Carnitine supplement in VPA-medicated patients seems to prevent hepatotoxicity, especially mitochondrial dysfunction.