Glutathione protects cardiac and skeletal muscle from cyclophosphamide-induced toxicity.

Administration of cyclophosphamide at a dose which is lethal to 10% of control athymic nude mice resulted in sudden death within 3 h in all mice that had been pretreated with the glutathione synthesis inhibitor L-buthionine-SR-sulfoximine. In Fischer 344 rats pretreated with L-buthionine-SR-sulfoximine, the cyclophosphamide dose producing 100% acute toxicity was lowered from 500-150 mg/kg; cardiac monitoring revealed ventricular fibrillation to be the cause of death. These and additional studies reported demonstrate that cytoplasmic glutathione is an important protectant against the cardiac and skeletal muscle toxicity of cyclophosphamide and indicate that such toxicity may be substantially increased by glutathione depletion. Since diet and many drugs (including cyclophosphamide itself) are known to affect glutathione levels, the present studies suggest that cardiac and skeletal muscle glutathione content is likely to be a clinically significant determinant of the frequency and severity of the adverse drug interactions and systemic toxicity sometimes observed during cyclophosphamide therapy.

Sequence of gene choB encoding cholesterol oxidase of Brevibacterium sterolicum: comparison with choA of streptomyces sp. SA-COO.

The nucleotide (nt) sequence of the cholesterol oxidase (Cho)-encoding gene (choB) cloned from Brevibacterium sterolicum ATCC21387 was determined. The sequence contained an open reading frame with a G + C content of 64.9 mol% that would encode a protein of 552 amino acids (aa). Comparison of the nt sequence of choB and deduced aa sequence to those of the Cho-encoding gene (choA) of Streptomyces sp. strain SA-COO showed identities of 64% and 58%, respectively. N-terminal aa sequence analysis of the extracellular enzyme of B. sterolicum confirmed that the mature enzyme consisted of 507 aa with a predicted Mr of 54,902, and was preceded by a 45-aa signal sequence.

New antiarrhythmic agents. N-aryl-8-pyrrolizidinealkanamides.

The synthesis and antiarrhythmic activity of N-aryl-8-pyrrolizidinealkanamides are described. The target compounds were evaluated for their ability to protect against chloroform-induced fibrillation in mice. Many of them were found to have antifibrillatory activity comparable to that of lidocaine; several are more potent than lidocaine. N-(2,6-Dimethylphenyl)-8-pyrrolizidineacetamide which was found to be more potent and less toxic (LD50) than lidocaine, also showed a long duration of action in dogs with ventricular arrhythmias after oral administration.

Synthesis and selective coronary vasodilatory activity of 3,4-dihydro-2,2-bis(methoxymethyl)-2H-1-benzopyran-3-ol derivatives: novel potassium channel openers.

A variety of compounds having a benzopyran such as levcromakalim generally exhibit potent antihypertensive activity. During extensive investigations aimed toward identifying K+ channel openers having selective coronary vasodilation without potent hypotensive and tachycardiac effects, we synthesized a series of 3,4-dihydro-2H-1-benzopyran-3-ol derivatives modified at positions 2, 4, and 6 in the benzopyran ring. Initially, compounds having two methoxymethyl groups at position 2 were found to show a selective effect on coronary blood flow (CoBF) relative to mean arterial pressure (MAP) in anesthetized dogs. To find more potent vasodilators, various benzopyran derivatives modified at position 4 were synthesized and structure-activity relationships were examined by evaluation of the extent and duration of the increase in CoBF in anesthetized dogs. As a result, compounds having a (1,6-dihydro-6-oxopyridazin-3-yl)amino group at position 4, in addition to the two methoxymethyl groups at position 2, were found to be more potent and to have an improved duration of action. Among these compounds, JTV-506, (-)-(3S,4R)-6-cyano-3,4-dihydro-4-[(1,6-dihydro-1-methyl-6-oxopyridaz in-3-yl)amino]-2,2-bis(methoxymethyl)-2H-1-benzopyran-3-ol, exhibited good selectivity for its effect. Administration of this compound (0.03 mg/kg, p.o.) elicited an increase of CoBF without a change of systemic blood pressure and heart rate (HR) in conscious dogs. Further evaluation was performed with respect to (i) the selectivity of its action on the coronary artery versus the aorta and (ii) its effects on MAP, HR, and electrocardiographic ST elevation. As a result, JTV-506 was selected as a potent and selective coronary vasodilator with various pharmacological features favoring clinical development.

Dihydropyrimidines: novel calcium antagonists with potent and long-lasting vasodilative and antihypertensive activity.

The novel calcium antagonists 3-N-substituted-3,4-dihydropyrimidines 1 and 9 and 3-N-substituted-dihydro-pyrimidin-2(1H)-ones 8 were regioselectively synthesized in good yields. Compounds 1 [especially 1s [R1 = (CH2)2N(benzyl)(2-naphthylmethyl), R2 = i-Pr, X = 0-NO2] and 1t [R1 = (CH2)2N(benzyl)(3,4-dichlorobenzyl), R2 = i-Pr, X = 0-NO2]] exhibited not only more potent and longer lasting vasodilative action but also a hypotensive activity with slow onset as compared with dihydropyridines. Moreover, some dihydropyrimidines [1q [R1 = (CH2)2N(benzyl)(3-phenylpropyl), R2 = CH2(cyclopropyl), X = 0-NO2], 1s, and 1t] were weaker in blocking atrioventricular conduction in anesthetized open-chest dogs and less toxic than the dihydropyridines.

Purification and properties of lipase from Rhizopus japonicus.

Lipase [EC 3.1.1.3] was purified from the culture supernatant of Rhizopus japonicus KY 521 by ethanol precipitation, chromatography on Column-lite, affinity chromatography on heparin-Sepharose 4B, and separation into two lipases, I and II, by isoelectric focusing. The purified lipases I and II were found to be homogeneous by disc electrophoresis, and showed isoelectric points at pH 7.4 and pH 7.9, respectively. They both had an apparent molecular weight of about 42,000, hydrolyzed tricaprin very rapidly, and exhibited a pH optimum around pH 7.0-8.5. These lipases were inhibited by the addition of serum to the reaction mixtures. These lipases were enhanced slightly in the absence of serum by high concentrations of NaCl and protamine, but were inhibited strongly by these compounds in the presence of serum.

Substitutions of Ser for Asn-163 and Pro for Leu-264 are important for stabilization of lipase from Pseudomonas aeruginosa.

The lipase gene from Pseudomonas aeruginosa was randomly mutated by error-prone PCR to obtain thermostable mutants, followed by screening for thermostable mutant lipases. Out of about 2,600 transformants, four thermostable clones were obtained. Their nucleotide sequences showed that they had two or three amino acid substitutions. Analysis of the thermal stabilization of these mutant lipases indicated that Asn-163 to Ser and Leu-264 to Pro mutations were essential for the increased stability of the lipase. We expressed a mutant lipase (StLipA-5) having only the Asn-163 to Ser mutation and another (StLipA-6) having only the Leu-264 to Pro mutation in P. aeruginosa PAO1161, purified them, and then confirmed that the temperature which causes a 50% decrease in the activity of the non-treated enzyme on treatment for 30 min was increased by 1.5 and 3 degrees C, respectively, compared to the wild-type enzyme. However, the thermal stability of the mutant lipase (StLipA-7) having both mutations was increased only by 2.5 degrees C. These mutant lipases were stabilized through a decrease in activation entropy. Kinetic studies showed that the Kcat/K(m) values of StLipA-5, StLipA-6, and StLipA-7 were decreased by 14.4, 52.9, and 26.0%, respectively. Interestingly, the pH-stabilities of StLipA-6 and StLipA-7 were also increased, especially at alkaline pH. Based on these results, the tertiary structure and mechanism of stabilization of the lipase were discussed.

Expression and imprinting status of human PEG8/IGF2AS, a paternally expressed antisense transcript from the IGF2 locus, in Wilms' tumors.

A large imprinted gene cluster in human chromosome 11p15.5 has been implicated in Beckwith-Wiedemann syndrome and Wilms' tumor. We have identified a paternally expressed imprinted gene, PEG8/IGF2AS, in this locus. It is transcribed in the opposite direction to the IGF2 transcripts and some genomic regions are shared with the IGF2 gene, as in the case of the mouse imprinted Igf2as gene reported previously by T. Moore et al. As to the relationship between these genomic regions, the human and mouse genes are very similar but there is no homology in their middle parts. Interestingly, PEG8/IGF2AS and IGF2 were found to be overexpressed in Wilms' tumor samples, at levels over ten and a hundred times higher than that in normal kidney tissues neighboring the tumors, respectively. These findings indicate that PEG8/IGF2AS is a good marker of Wilms' tumor and also suggest the possibility of PEG8/IGF2AS being one of the candidate Wilms' tumor genes.

Effects of sitting position on uterine activity during labor.

To determine which components of uterine activity are affected by different positions of labor, 116 intrauterine pressure records in the sitting and supine positions were analyzed in order to measure resting, contraction, and bearing down pressures. The resting pressure in the sitting position showed consistent elevation compared to the supine position, while the contraction pressure did not differ strikingly in the two positions. The bearing down pressure in the sitting position for nulliparas during the second stage and for multiparas at the time of the 8- to 10-cm dilation was significantly higher than that in the supine position. Also, the sitting position led to a significantly shorter duration of the second stage in nulliparas and the 5- to 10-cm dilation period in multiparas. These findings suggest that the maternal position does not affect uterine contractility, that the increased resting pressure in the sitting position is of some importance in supplementing the downward delivery force, and that the increased bearing down pressure in the sitting position could help to significantly shorten the duration of labor.

Production of trehalose phosphorylase by Catellatospora ferruginea.

A range of microorganisms was screened for new and high producer strains of trehalose phosphorylase (EC 2.4.1.64). Trehalose phosphorylase activity was found in cells of actinomyctes of the genera Actinomadura, Amycolata, Catellatospora, Kineosporia, and Nocardia. Among them, Catellatospora ferruginea showed the highest enzyme activity. Trehalose phosphorylase from C. ferruginea was able to catalyse both the phosphorolysis of trehalose into beta-glucose 1-phosphate and D-glucose and the synthesis of trehalose from beta-glucose 1-phosphate and D-glucose.

Effect of JTH-601, a novel alpha(1)-adrenoceptor antagonist, on prostate function in dogs.

We examined the effect of JTH-601 (3-¿N-[2-(4-hydroxy-2-isopropyl-5-methylphenoxy)ethyl]-N-methylaminom ethyl¿-4-methoxy-2,5,6-trimethylphenol hemifumarate), a new alpha(1L)-adrenoceptor antagonist, on prostatic function in isolated canine prostate and in anesthetized dogs. In the contraction study, phenylephrine and noradrenaline produced concentration-dependent contractions in canine prostate and carotid artery, respectively. In these tissues, JTH-601, prazosin (a non-selective alpha(1)-adrenoceptor antagonist), and tamsulosin (an alpha(1A)-adrenoceptor antagonist) competitively antagonized contraction in a concentration-dependent manner. The pA(2) (pK(B)) values with prostate were 8.49+/-0.07 for JTH-601, 7.94+/-0.04 for prazosin and 9.42+/-0.22 for tamsulosin. The ratio of pA(2) (carotid artery/prostate), i.e. prostatic selectivity, was 10.471 for JTH-601, 0.008 for prazosin and 0.371 for tamsulosin, respectively. In anesthetized dogs, JTH-601 (1 mg/kg, i.d.) significantly decreased urethral pressure by 15% without affecting blood pressure or heart rate. Tamsulosin (0.1 mg/kg, i.d.) decreased urethral pressure to the same extent as did JTH-601, but with a significant effect on blood pressure and heart rate. JTH-601 showed higher selectivity for canine prostate both in vitro and in vivo. In prostate, an important role of the alpha(1L)-adrenoceptor is suggested in the smooth muscle contraction mediated by alpha(1)-adrenoceptors. JTH-601 is expected to be an effective alpha(1)-adrenoceptor antagonist for the treatment of urinary outlet obstruction by benign prostatic hypertrophy with a minimum effect on the cardiovascular system.

Antithrombotic effects of a synthetic inhibitor of activated factor X, JTV-803, in animals.

JTV-803, 4-[(2-amidino-1,2,3,4-tetrahydroisoquinolin-7-yloxy)methyl]-1-(4-pyridinyl)piperidine-4-carboxylic acid monomethanesulfonate trihydrate, at > or = 0.1 mg/kg/h inhibited the increase in plasma thrombin-antithrombin III complex in response to continuous infusion of thromboplastin in rats. JTV-803 inhibited thrombus formation in an arteriovenous shunt model by intravenous infusion at > or = 0.3 mg/kg/h and prolonged the occlusion time of photochemically induced arterial thrombus in the middle cerebral artery at >1.5 mg/kg/0.5 h. Activated partial thromboplastin time was prolonged at 10 mg/kg/h. Intravenous administration of JTV-803 prolonged bleeding time at 30 mg/kg/h, a dose 10-100 times higher than the dose that inhibited thrombus formation. Compared with thrombin inhibitor, JTV-803 had less of an effect on the bleeding time. In the arteriovenous shunt model in cynomolgus monkey, JTV-803 prolonged the occlusion time when administered by continuous infusion at 0.3 mg/kg/h or orally at 10 mg/kg. These results suggest that the human factor Xa inhibitor JTV-803 is an orally active anticoagulant that does not affect bleeding time and is useful for the prevention of thrombus.

Inhibitory effect of JTV-803, a new cyclic guanidine derivative, on factor Xa in vitro and in vivo.

JTV-803, 4-[(2-amidino-1,2,3,4-tetrahydroisoquinolin-7-yloxy)methyl]-1-(4-pyridinyl)piperidine-4-carboxylic acid monomethanesulfonate trihydrate showed a competitive inhibitory effect on human factor Xa, with a K(i) value of 0.019 microM. This compound was 100 times more selective in inhibiting human factor Xa as compared to its inhibitory activity against thrombin, plasmin, and trypsin. JTV-803 was also examined for its inhibitory effect on activated factor Xa obtained from plasma of various animal species. JTV-803 exerted a potent inhibitory effect on human factor Xa (IC(50): 0.081 microM). JTV-803 prolonged activated partial thromboplastin time and prothrombin time in a dose-dependent manner. Oral anticoagulant efficacy of JTV-803 was examined ex vivo for its inhibition of human factor Xa in cynomolgus monkeys. JTV-803 produced more than 20% inhibition of human factor Xa for 8 h. Taken together, the results indicate JTV-803 is a long-acting oral anticoagulant which exerts its effect via specific inhibition of human factor Xa.

Effect of JTH-601, a novel alpha1-adrenoceptor antagonist, on the function of lower urinary tract and blood pressure.

In the present study, we investigated the effect of JTH-601 (3-{N-[2-(4-hydroxy-2-isopropyl-5-methylphenoxy)ethyl]-N-methylaminomethyl}-4-methoxy-2,5,6-trimethylphenol hemifumarate), a novel alpha1-adrenoceptor antagonist, in vitro and in vivo. JTH-601 (10(-9)-3 x 10(-8) M) competitively antagonized phenylephrine-induced contraction in lower urinary tract tissues (prostate, urethra and bladder trigon) in a concentration-dependent manner. The mean pA2 values for JTH-601 were 8.59+/-0.14, 8.74+/-0.09 and 8.77+/-0.11 for prostate, urethra and bladder trigon, respectively. In anesthetized rabbits, intraduodenal administration of JTH-601 (0.3-3 mg/kg), prazosin (0.03-0.3 mg/kg) and tamsulosin (0.03-0.3 mg/kg) dose dependently inhibited the phenylephrine-induced increase in urethral pressure for 3 h. Although these drugs also decreased mean blood pressure, JTH-601 was less potent than prazosin or tamsulosin. In conscious rabbits, administered JTH-601 (0.01-1 mg/kg, i.v.) had a tendency to augment orthostatic hypotension, but dose dependency was not evident. Prazosin (0.01-1 mg/kg) and tamsulosin (0.001-1 mg/kg) dose dependently augmented orthostatic hypotension. These results indicate that JTH-601 antagonized alpha1-adrenoceptor-mediated contractile responses more potently than prazosin or tamsulosin in rabbit lower urinary tract both in vitro and in vivo. JTH-601 is therefore expected to be effective in the treatment of urinary outlet obstruction in benign prostatic hypertrophy.

The role of alpha 1L-adrenoceptor in rat urinary bladder: comparison between young adult and aged rats.

We examined the role of the alpha1L-adrenoceptors in the urinary bladder of young adult and aged rats in vitro. In the isolated body of the urinary bladder (corpus vesicae), phenylephrine-induced contractions were significantly facilitated in aged rats. Either prazosin, a non-selective alpha1-adrenoceptor antagonist, or JTH-601, an alpha1L-adrenoceptor antagonist, competitively inhibited the phenylephrine-induced contraction of isolated body of the urinary bladder. The antagonistic effect of JTH-601 was almost equipotent between young adult and aged rats (pA2 values were 9.61+/-0.12 and 9.79+/-0.07, respectively), although a statistically significant difference was noted for that of prazosin (pA2 values were 9.49+/-0.09 and 9.19+/-0.06, respectively). In macroscopic autoradiographic studies, specific binding of [3H]JTH-601 (5nM) was seen widely in the muscle layer of urinary bladder, but no differences were noted between young adult and aged rats. In the present study, there was no evidence to suggest a role of the alpha1L-adrenoceptors in the body of rat urinary bladder. On the other hand, alpha1A-adrenoceptors may play an important role in an age-related increase of alpha1-adrenoceptors response in this tissue. These results suggest that a facilitation of contractile response mediated by alpha1A-adrenoceptors may be a cause of unstable bladder in aged persons.

Vasodilatory and diuretic actions of alpha-human atrial natriuretic polypeptide (alpha-hANP).

Vascular and diuretic actions of synthetic alpha-human atrial natriuretic polypeptide (alpha-hANP) were studied using anesthetized dogs and isolated canine arterial strip preparations. alpha-hANP, when given intra-arterially or intravenously, dilated the renal artery more selectively than the vertebral, femoral, common carotid and coronary arteries. alpha-hANP selectively relaxed the high K+-contracted renal artery strip as compared with the basilar, coronary and femoral arterial strips. Intravenous alpha-hANP also increased urine volume and urinary excretion of electrolytes at doses, at which it increased renal blood flow and lowered systemic blood pressure without changing heart rate. It is concluded that alpha-hANP has a vasodilatory property relatively specific for the renal artery, and that it possesses diuretic, natriuretic, kaliuretic, magnesiuretic, calciuretic and chloruretic activities concomitantly with a definite hypotensive activity.

Distribution of alpha1L-adrenoceptors in canine prostate: characterization by quantitative autoradiography.

Our aim was to determine the distribution of alpha1L-adrenoceptors in canine prostate by an autoradiographic technique using [3H]JTH-601 (an alpha1L-adrenoceptor antagonist) and [3H]JTH-601-G1 (an active metabolite of JTH-601). Prostates were removed from three male beagle dogs. Several slices of the specimens were incubated with 5 nM of [3H]JTH-601, [3H]JTH-601-G1 and [3H]tamsulosin (an alpha1A-adrenoceptor antagonist). For macroscopic autoradiography, visualization was performed using an imaging plate and image-analyser. To examine microscopic localization of binding sites, preparations were exposed, developed and fixed. Specific binding of [3H]JTH-601 and [3H]JTH-601-G1 was observed diffusely throughout the entire interstitium on macroscopic autoradiography. Specific binding of [3H]tamsulosin was also recognized although the binding was weaker than that of [3H]JTH-601. On microscopic autoradiograms, the grains of each ligand were mainly distributed on smooth muscle. These results indicate morphologically that specific binding sites of JTH-601 and JTH-601-G1 exist in canine prostate, suggesting the distribution of alpha1L-adrenoceptors in this tissue, in addition to alpha1A-adrenoceptors.

Determination of 1,5-anhydro-D-glucitol on the basis of its inhibitory effect on trehalase activity.

1,5-Anhydro-D-glucitol (1,5-AG) was found to inhibit trehalase and trehalose phosphorylase activities competitively, because of its structural similarity with D-glucose. Trehalase from Nocardia sp., one of the most 1,5-AG-sensitive enzymes, was used in the determination of 1,5-AG concentration, which is a useful marker for the diagnosis of diabetes. A good linear relationship was observed between 1,5-AG concentration in the range of 0.02 to 1.0 mM and the extent of trehalase inhibition by 1,5-AG. The 1,5-AG concentration range could be determined by estimating enzymatically the amount of the reaction product, D-glucose, produced by the trehalase.

Enzymatic synthesis of novel disaccharides using disaccharide phosphorylases.

A method for the synthesis of novel disaccharides was developed. It involved the following two steps. The first step consisted of two continuous reactions: the conversion of maltose to beta-D-glucose-1-phosphate and D-glucose by the phosphorolytic activity of maltose phosphorylase and the specific consumption of only D-glucose by incubation with glucose-consuming yeast cells. The second step involved the addition of an appropriate carbohydrate and its condensation with the remaining beta-D-glucose-1-phosphate by the synthetic activity of maltose phosphorylase or trehalose phosphorylase. Several factors affecting the yields of disaccharides were optimized. Using this method, five maltose-like derivatives and two trehalose-like derivatives were synthesized from maltose and the corresponding carbohydrates. Among these, 4-O-alpha-D-glucopyranosyl-L-fucopyranose (Glc(alpha1-4)Fuc) and alpha-d-glucopyranosyl alpha-D-fucopyranoside (Glc(alpha1-1alpha)Fuc) were purified, and identified by 1H NMR and 13C NMR.

Effect of high-dose progestogen on hemostatic properties of blood in patients with endometrial cancer.

Fifteen cases of endometrial cancer were administered daily doses of 600 mg of MPA after surgery to prevent the recurrence of cancer. The initiation times of coagulation (time necessary for fibrin network formation) were measured with a highly sensitive damped oscillation rheometer and compared with those of 15 control patients who were not administered MPA. Biochemical studies of blood coagulation and fibrinolysis were also done. The initiation times of coagulation were 19.0+/-1.8 minutes (min mean +/- standard deviation) after 3-6 months and 16.0+/-2.0 min after 9-12 months of MPA administration, both times being significantly shorter compared with the controls (24.0+/-2.5 min). Hematocrit values, platelet counts and fibrinogen levels were similar between the two groups. Activated partial thromboplastin time (APTT) was significantly decreased and antithrombin III activity (AT III), thrombin-antithrombin complex (TAT), plasminogen level, plasmin-alpha(2) plasmin inhibitor complex level (PIC) and the fibrin degradation product level (FDP) were significantly increased in the MPA group compared with the control group. Accelerated coagulation of blood was definitely induced by high-dose MPA but antithrombin and fibrinolytic activities were also induced, and, thus, thromboembolic complications were prevented.