Synthesis of phenoxyacetic acid derivatives as highly potent antagonists of gastrin/cholecystokinin-B receptors. III.

In order to improve the biological characteristics of DA-3934 (5), a novel gastrin/cholecystokinin (CCK)-B receptor antagonist, phenoxyacetic acid derivatives replacing the N-methyl-N-phenylcarbamoylmethyl moiety of 5 with various alkyl chains have been synthesized and their biological activity evaluated. The relationship between the structure of these compounds and their human gastrin receptor binding affinity showed that there should be the optimal size among the various N-alkyl chains. Also a significant increase in the receptor binding affinity was achieved by several compounds. Among those compounds, 2-[3-[3- [N-cyclohexylmethyl-N-[2-(N-methyl- N-phenylcarbamoylmethoxy)phenyl]carbamoylmethyl]ureido]pheny l]acetic acid (22c) and (+/-)-2-[3-[3-[N-[2-(N-methyl-N- phenylcarbamoylmethoxy)phenyl]-N-(3-methylpentyl)carbamoy lmethyl]ureido] phenyl]acetic acid (22h) exhibited high affinity for human gastrin receptors and were also more potent inhibitors in a pentagastrin-induced gastric acid secretion model than the parent compound, 5. The ED50 values of these compounds when administered intraduodenally to rats were 0.12 and 0.63 mg/kg, respectively.

Comparative evaluation of DQ-2511, a novel gastroprokinetic agent, with cisapride in ameliorative action on experimentally induced delayed gastric emptying.

We compared the main pharmacological effect of DQ-2511 (3-[[[2-(3,4-dimethoxyphenyl)- ethyl]carbamoyl]methyl]amino-N-methylbenzamide), a novel gastroprokinetic agent, with that of cisapride. Single oral administration of DQ-2511 (3-10 mg kg-1) caused similar significant improvements to delays in gastric emptying of semi-solid meals evoked by cholecystokinin-octapeptide (CCK8: 5 micrograms kg-1, i.v.) in monkeys, to that with cisapride (3 mg kg-1). A 2-week oral treatment of unilaterally vagotomized rats with DQ-2511 (1-10 mg kg-1) lessened delays in gastric emptying, whereas cisapride (0.3-10 mg kg-1) had no effect under the same experimental protocols. In anesthetized rats, bolus intravenous injection of either compound (60 micrograms kg-1) enhanced gastric motility determined by means of strain gauge force transducers. Electrophysiological investigations revealed that bolus injection of DQ-2511 (6-60 micrograms kg-1) depressed the afferent discharge rate of the ventral gastric branch of the vagus nerve, while cisapride showed no effect. These results suggest that the mechanism of ameliorative action of DQ-2511 on delayed gastric emptying may differ from that of cisapride.

Calculation of maximal removal rate of indocyanine green to measure hepatic functional mass in dogs by use of the nine-hour method.

OBJECTIVE: To establish a versatile and reliable procedure for the determination of indocyanine green maximal removal rate (ICG Rmax) to measure hepatic functional mass in dogs within 9 hours (9-hour method). DESIGN: Relation between 9-hour and standard 3-day methods was examined. ANIMALS: 101 healthy dogs. PROCEDURE: On investigation of the optimal technical conditions allowing completion of all procedures in a day, the appropriate IV administered doses of ICG were 0.125, 0.5, and 2.0 mg/kg of body weight, and the best blood sample collection times for obtaining plasma half-life at these 3 doses were before and 3, 6, and 9 minutes after ICG administration. RESULTS: Comparison of the 9-hour with 3-day method yielded a correlation coefficient (r) of 0.84, indicating close (P < 0.01) correlation. In the 9-hour method, mean +/- SD of ICG Rmax in healthy dogs was 0.24 +/- 0.09 mg/kg/min in male (n = 62) and was 0.23 +/- 0.06 mg/kg/min in female (n = 21) Beagles, and was 0.21 +/- 0.10 mg/kg/min in male (n = 11) and 0.20 +/- 0.07 mg/kg/min in female (n = 7) mixed-breed dogs. In Beagles treated orally with carbon tetrachloride (0.1 ml/kg in gelatine capsules) thrice weekly during a 10-week period, plasma alanine transaminase activity plateaued at a high value (> 2,000 IU/L) on day 5, and remained at this value until the end of the study. The ICG Rmax changed accordingly: day 5, 0.17; day 10, 0.11; day 40, 0.05; and day 60, 0.06 mg/kg/min. CONCLUSION: The 9-hour method for determination of ICG Rmax correlates favorably with the 3-day method. CLINICAL RELEVANCE: This procedure may be practically applied in veterinary clinics in terms of prediction of hepatic functional mass, and for diagnosis of hepatotoxicosis induced by certain compounds.

Pharmacological characteristics of DQ-2511 as a prokinetic agent.

The pharmacological characteristics of DQ-2511, a substituted benzamide (3-[[[2-(3,4-dimethoxyphenyl)ethyl]carbamoyl]methyl] amino-N-methylbenzamide), as a prokinetic agent were studied. Cholecystokinin-octapeptide, dopamine, and alpha-calcitonin gene-related peptide, all suppressed gastric emptying in mice. Reversal of the depressed emptying occurred when DQ-2511 was administered by the oral or intraperitoneal route. When the action of eight proposed metabolites of DQ-2511 on the mouse cholecystokinin-octapeptide model was investigated, the main metabolite in plasma, MA-2, showed no effect, although two minor metabolites ameliorated or aggravated the delayed gastric emptying. This finding implies that DQ-2511, as the parent compound itself, exerts the ameliorative action. In dogs treated with cisplatin or copper sulfate, DQ-2511 had no antiemetic activity, as assessed by the number of vomiting episodes. The concern that the mechanism of action of DQ-2511 was blockade of receptors for cholecystokinin-octapeptide, dopamine, serotonin, alpha-calcitonin gene-related peptide, nicotine or muscarine, was resolved by results of radioligand binding studies showing the absence of a DQ-2511 binding to any of these receptor types. Evidence is accumulating that the mechanism of the prokinetic action of DQ-2511 involves the intrinsic and extrinsic autonomic innervation.

DQ-2511, having an antiulcer action, elicits vasodilation through an increase in cyclic GMP contents in rat arteries.

It has been reported that 3-(([2-(3,4-dimethoxyphenyl)ethyl]carbamoyl)methyl)amino-N-methylbenz amide (DQ-2511: ecabapide) effectively increases gastric mucosal blood flow in rats, suggesting that this property may contribute to the antiulcer activity. To clarify the mechanisms underlying the increase in gastric mucosal blood flow, we investigated the dilator response of rat isolated thoracic aorta, mesenteric artery and celiac artery smooth muscle preparations to DQ-2511. This compound prevented noradrenaline-induced contraction in both the presence and absence of endothelium in the arterial specimen, and it (0.01-1 mM) inhibited these contractions induced by noradrenaline in all tissues in a concentration-dependent manner. This inhibitory effect of DQ-2511 was most evident in the celiac artery. The dilator response to DQ-2511 (0.1 mM) was abolished after pretreatment with methylene blue (3 microM), a guanylate cyclase inhibitor. Under the same conditions, methylene blue inhibited the dilator response to acetylcholine (1 microM), but not that to papaverine (10 microM). Furthermore, when DQ-2511 (0.01-1 mM) was incubated with the arterial preparations, this compound increased cyclic GMP content in segments in a concentration-dependent manner. These findings demonstrate that the vasodilation induced by DQ-2511 is independent of the endothelium and is related to the augmentation of intracellular cyclic GMP content, which may consequently contribute to the increased gastric mucosal blood flow.

Enhancing effect of DQ-2511 on gastric emptying of spontaneously hypertensive rats.

The present study was designed to clarify the potential of DQ-2511 (3-[[[2-(3,4-dimethoxyphenyl)ethyl]carbamoyl]carbamoyl]methyl] amino-N-methylbenzamide: ecabapide) as a gastroprokinetic agent in spontaneously hypertensive rats (SHR). The gastric emptying of SHR was clearly retarded relative to that of weight-matched normotensive Wistar-Kyoto (WKY) rats when evaluated by the acetaminophen (APAP) method with the semi-solid test meal. There was, however, no significant difference between both strains in gastric mucosal blood flow (GMBF) determined by means of a laser doppler flowmetry. A 2-week treatment of SHR with DQ-2511 (1 mg/kg, oral) stimulated gastric emptying without affecting body weight gain or indirect systolic blood pressure (SBP), whereas cisapride (3 and 10 mg/kg, oral) had no effect under the same conditions. The pharmacological characteristics of DQ-2511 as a gastroprokinetic agent are discussed on the basis of these results.

The measurement of gastric emptying in conscious rats by monitoring serial changes in serum acetaminophen level.

A method for evaluation of gastric emptying via monitoring serial serum levels of acetaminophen (APAP) as an indicator, without involving animal sacrifice, has been developed in conscious rats. We administered orally suspended test meals of 0.5, 1, 2, or 4 mL/rat each, containing 20 mg APAP. The animals were repeatedly bleed at 15, 30, 45, 60, or 90 min after APAP treatment. Serum APAP level was determined by a high-performance liquid chromatography. As a result, a 2-mL meal was found optimal to attain continuous levels of APAP in serum. The APAP method showed a close correlation (r = 0.77) with the phenol red method, a standard procedure for quantitative detection of gastric emptying entailing rat sacrifice. The blood loss (approximately 2 mL) resulting from the APAP method did not significantly modify the rate of gastric emptying. In addition, data was presented to substantiate the validity of the APAP method in demonstrating enhancement and inhibition of gastric emptying using cisapride and cholecystokinin-octapeptide (CCK), respectively. These results suggest that the APAP method is easy, versatile, and reliable, and requires only a minimal number of animals for the evaluation of sequential changes in gastric emptying.

Inhibitory effect of DS-4574, a peptidoleukotriene antagonist with mast cell stabilizing action, on compound 48/80-induced gastric mucosal lesions in rats.

We evaluated the inhibitory effect of DS-4574, a peptidoleukotriene antagonist with mast cell stabilizing action, on rat gastric mucosal lesions induced by compound 48/80 (C48/80: a mast cell degranulator), in comparison with those of disodium cromoglycate (DSCG: a mast cell stabilizer), LY171883 (a peptidoleukotriene antagonist) and cimetidine (a histamine H2 receptor antagonist). Subcutaneous administration of C48/80 (1 mg/kg) once daily for four consecutive days produced extensive gastric lesions in the fundic mucosa. DS-4574 (20, 50 and 100 mg/kg/day, oral) and DSCG (200 mg/kg/day, intraperitoneal) treatment markedly inhibited formation of these mucosal lesions, but LY171883 (100 and 200 mg/kg/day, oral) and cimetidine (400 mg/kg/day, oral) treatment did not. Moreover, DS-4574 and DSCG significantly suppressed both hyperhistaminemia and histamine release from rat peritoneal mast cells induced by C48/80. These results indicate that the inhibitory effect of DS-4574 on gastric lesions induced by C48/80 may be related to its mast cell stabilizing action, but to neither its antisecretory nor its peptidoleukotriene antagonistic activity.

Protective effect of DS-4574 on gastric mucosal injury induced by acidified ethanol in rats.

We compared the protective effect of DS-4574, a peptidoleukotriene receptor antagonist with mast cell stabilizing action, on rat gastric mucosal injury induced by acidified ethanol to that of LY171883, a selective peptidoleukotriene receptor antagonist. Oral treatment with DS-4574 (1-10 mg/kg) or LY171883 (100 and 300 mg/kg) markedly suppressed this mucosal necrosis. Moreover, DS-4574 (10 mg/kg) significantly inhibited both mucosal edema and degranulation of mucosal mast cells. LY171883 (300 mg/kg) protected only from mucosal edema, but not degranulation of mucosal mast cells. These results suggest that DS-4574 possesses protective actions against gastric injury including the reduction of degranulation of mucosal mast cells, which are different from those of LY171883.

Leukemic blasts detected by the Technicon H-1 blood cell counter.

The authors studied the usefulness of the flagging system of the Technicon H-1 Cell Counter using samples from 45 patients with acute leukemia, 24 with acute lymphoblastic leukemia (ALL), and 21 with acute nonlymphoblastic leukemia (ANLL). Results by the automated method were compared with those by the eye-count method. Four out of 24 specimens were falsely negative; no false positive results were found. For ANLL specimens, there were no false positives or negatives. When 1% or more of the cells were blasts by the eye-count method, the automated system almost always flagged the sample. The results suggested that the flagging system programmed in the H-1 is useful for the diagnosis and follow-up of leukemia.

Effects of phalloidin on platelet release.

The effect on platelet of phalloidin, a reagent forming phalloidin-actin polymer, was studied. Phalloidin suppressed ATP release but did not affect aggregation after the stimulation with ADP or A23187, although the amount of F-actin was increased by stimulation. The suppression was correlated to the preincubation time and to the concentration of phalloidin.

[Combination chemotherapy with cyclophosphamide, adriamycin, vincristine and prednisolone (VEPA) for non-Hodgkin's lymphoma].

Combination Chemotherapy with Adriamycin (VEPA) was applied to 16 patients with non-Hodgkin's lymphoma. The effects of VEPA therapy were compared with those of combination chemotherapy without Adriamycin (non-VEPA). Complete remission rate achieved with VEPA therapy was 37.5% while that with non-VEPA therapy was 27.6%. Histologically, the complete remission rate in cases of large-cell type treated with VEPA therapy was 40%, while that with non-VEPA therapy was 14.3%. No cases of stage IV showed complete remission, whereas the complete remission rate for cases of stage III was 37.5% for VEPA therapy, but 27.6% for non-VEPA therapy. From these results we concluded that VEPA therapy is more effective for non-Hodgkin's lymphoma, especially large-cell type, than non-VEPA therapy.

Inhibition of platelet secretion of ATP by phalloidin.

The involvement of actin in the secretion of ATP by platelets was studied using two stimulants, ADP and A23187, and two actin-mediating reagents, cytochalasin B and phalloidin. The degree of actin polymerization was determined using DNase I. Preincubation of platelets with cytochalasin B suppressed the polymerization of actin and ATP secretion induced by stimulants. In the absence of the stimulant, phalloidin-treated platelets exhibited time-dependent actin polymerization and the maximum level was reached at 5 min. No secretion of ATP was observed. The polymerization was enhanced by phalloidin when the platelets were preincubated for 3 to 5 min with the stimulants, but little ATP was secreted. After a 30-min preincubation, the amount of polymerized actin was lower than that after a 5-min incubation, and no ATP was secreted.

[High dose of methotrexate-leucovorin treatment in advanced non-Hodgkin's lymphoma].

High doses of methotrexate (MTX) and leucovorin (LCV) as a rescue treatment were applied to five patients with non-Hodgkin's lymphoma, refractory to standard combination chemotherapy. Two of 5 patients showed a complete remission, and 2 patients showed a partial remission. One patient with massive pleural effusion showed an improvement by the treatment without any severe side effect under monitoring and controlling the dose of MTX. However, the remission duration of the treatment was short; therefore, additive therapy was required of later. From these results we assumed that the high dose of MTX and LCV could be effective for malignant lymphoma being resistant to other combination therapies.