Design and synthesis of novel, potent and selective hypoxanthine analogs as adenosine A1 receptor antagonists and their biological evaluation.

Multipronged approach was used to synthesize a library of diverse C-8 cyclopentyl hypoxanthine analogs from a common intermediate III. Several potent and selective compounds were identified and evaluated for pharmacokinetic (PK) properties in Wistar rats. One of the compounds 14 with acceptable PK parameters was selected for testing in in vivo primary acute diuresis model. The compound demonstrated significant diuretic activity in this model.

High-performance liquid chromatographic assay for N2-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-arginine (ST 789) in plasma by cyclization with benzoin and fluorimetric detection.

This paper describes a new highly sensitive assay for N2-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-arginine, an immunomodulatory agent, required for clinical pharmacokinetic investigation. A pre-column derivatization by cyclization with benzoin in aqueous medium produces the fluorescent 2-substituted amino-4,5-diphenylimidazole fluorescing at 450 nm (excitation wavelength 310 nm). L-Arginine-acetyl-L-carnitinamide chloride (ST 857, II), another arginine derivative, was used as an internal standard. A C18 DB column (5 microns, 250 mm x 4.6 mm I.D.) and a 45:55 (v/v) mixture of 0.05 M ammonium phosphate at pH 7.2 and methanol as mobile phase were used. Linearity was ascertained in the range 5-100 ng. Extraction recovery from plasma proved to be higher than 90% in the range 5-50 ng/ml. Intra-day precision, expressed as coefficient of variation, was in the range 4.7-6.0%. The limit of quantification proved to be 5 ng/ml and the limit of detection 2 ng/ml at a signal-to-noise ratio of 5. The method is specific.

Effect of lactate infusion on renal transport of purine bases and oxypurinol.

To investigate whether or not lactic acid inhibits the renal transport of oxypurines and oxypurinol, we administered physiological saline containing 0.2 mol sodium lactate to 5 normal subjects intravenously. Lactate infusion decreased the fractional clearance of uric acid, but the fractional clearances of hypoxanthine, xanthine and oxypurinol were not affected. These results suggest that uric acid and lactic acid share the renal transport system of organic acids but hypoxanthine, xanthine and oxypurinol do not. It is further suggested that allopurinol treatment is reasonable in subjects with hyperuricemia accompanied by hyperlactatemia since only the urinary excretion of uric acid and not oxypurines (hypoxanthine and xanthine) was inhibited by lactate infusion.

Pharmacokinetic investigation of ST 789 in rats and mice.

Pharmacokinetics of ST 789 were investigated in rats and mice after oral, intravenous, subcutaneous and intramuscular routes. A HPLC method validated for pharmacokinetic studies allowed the Authors to assay ST 789 concentration in plasma, urine and tissues. ST 789 interacted poorly with albumin and plasma proteins. Blood-to-plasma concentration ratio proved to range on average from 1.3 to 2.0 in both in vivo and in vitro studies. Plasma concentration-time behaviour after i.v. injection fitted according to the open three-compartment model; after subcutaneous and intramuscular routes two phases were observed and after oral route the absorption and one elimination phases were detected. Pharmacokinetics of ST 789 proved to vary linearly with the dose administered. Cumulative urinary excretion after parenteral administration ranged on average 60-80% and cumulative biliary excretion was 9.47% of the dose given. Oral administration allowed only 2.5% of the drug given to be excreted in urine, this leading to conclude that this drug is poorly absorbed through the intestine wall. After oral administration ST 789 produced relatively high concentration in lungs and lymphatic tissues, this leading to hypothesize a lymphatic component in its enteral absorption.

Substrate specificity of urate transporter in rat renal brush border membranes.

To further demonstrate the substrate specificity of urate-anion exchanger in rat renal brush border membrane vesicles, the hydroxyl ion gradient-dependent [2-14C] urate uptake was studied by a rapid filtration technique. The [2-14C] urate uptake was more sensitive to unlabeled urate than to unlabeled xanthine and hypoxanthine. In addition, urate derivatives which are methylated at the positions 3 and 9 hardly inhibited the urate uptake. Because of the substrate specificity, the urate-anion exchanger in brush border membranes appears to selectively use urate as the endogenous substrate.

[Clearance of oxypurines in normal subjects and in gout patients subjected to a purine-free diet. Effects of allopurinol]. / Clearance delle ossipurine nei soggetti normali e nei soggetti gottosi sottoposti a dieta apurinica. Effetti dell'allopurinolo.

The clearance of uric acid, hypoxanthine and xanthine has been examined in gout patients and in normal subjects compared to creatinine, after a purine-free diet. The treatment decreased the clearance in normal subjects, but showed an opposite effect in gout patients. The clearances both of uric acid, hypoxanthine and xanthine were enhanced by allopurinol. The interpretation of the observed variations is discussed.

[Clearance of oxypurines and uric acid in patients with gout]. / Clearance delle ossipurine e dell'acido urico in soggetti gottosi.

The clearance of uric acid, hypoxanthine and xanthine has been examined in gouty patients and in normal subjects comparatively to the creatinine clearance. The clearance of the three purine compounds was lower in the gouty patients, while the creatinine clearance showed a normal behavior. These results indicate that either the tubular excretion or the carriers of the considered metabolites probably undergo specific alterations in the gout.

High-performance liquid chromatographic evaluation of PCF 39, a new immunomodulator agent.

An high-performance liquid chromatographic analysis of PCF 39, N2-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-arginine, with ultraviolet detection, has been devised and validated. The main pharmacokinetic results encountered for rats treated intravenously with PCF 39 at a dose of 100 mg/kg are described.

[Purine transport through the blood-brain barrier in hypoxanthine phosphoribosyltransferase deficiency]. / Transporte de purinas a través de la barrera hematoencefálica en la deficiencia de hipoxantina fosforribosiltransferasa.

The transfer of purines through the hematoencephalic barrier is poorly understood. Allopurinol inhibits the enzyme xanthine oxidase and increases xanthine and hypoxanthine plasma levels, but it should not increase the cerebrospinal fluid (CSF) levels of these purines owing to the absence of xanthine oxidase in the central nervous system (CNS). In the present study we evaluated the plasma and CSF concentrations of uric acid, hypoxanthine, xanthine and inosine in the baseline state and after 7 days of allopurinol administration (5-10 mg/kg/24 h) in 4 patients with hypoxanthine phosphoribosyltransferase (HPRT) deficiency. The CSF uric acid level was positively correlated with its plasma level (r = 0.93, p less than 0.01). The CSF hypoxanthine and xanthine concentrations were, as a mean, 5 and 2 times higher, respectively, in patients with HPRT deficiency than in 4 control individuals. As hypoxanthine basically comes from adenine nucleotides, while xanthine comes from guanine nucleotides, this finding suggests that in the CNS of patients with HPRT deficiency there is a higher degradation level of adenine nucleotides than of guanine nucleotides. Allopurinol increased plasma concentration of hypoxanthine, xanthine and inosine 4, 10 and 3 times, respectively, in relation to baseline values. In CSF, the mean increase of hypoxanthine and xanthine concentration was 17.5 mumol and 7.7 mumol, respectively, whereas inosine level was unchanged. These results suggest that in HPRT deficiency hypoxanthine and xanthine may be transferred to the brain.

6-ethylmercaptopurine-mediated growth inhibition of HL-60 cells in vitro irrespective of purine salvage.

A variety of purine analogs inhibit the growth and induce the differentiation of human promyelocytic leukemia (HL-60) cells that lack the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Mechanisms by which purine analogs induce differentiation offer unique potential for cancer chemotherapy. The guanine analogs, 6-thioguanine and 8-azaguanine, induce granulocytic differentiation of HGPRT-deficient HL-60 promyelocytes. Although these compounds are useful as model purine analogs that induce differentiation in HGPRT-deficient HL-60 cells, they suffer the disadvantage that they are highly cytotoxic to wild-type cells. We studied the effect of the hypoxanthine analog 6-ethylmercaptopurine on wild-type and HGPRT-deficient HL-60 cells. 6-Ethylmercaptopurine inhibits growth and produces a specific terminal end-cell in both types of HL-60 cells. The mechanism appears to be independent of the normal modes of cytotoxic activation through HGPRT or adenine phosphoribosyltransferase (APRT), since no new peaks were seen in HPLC chromatograms of the nucleotide pools. Furthermore, hypoxanthine and adenine failed to prevent growth inhibition by 6-ethylmercaptopurine, and inhibition of IMP dehydrogenase and the consequential alteration of the guanine nucleotide pools does not appear to be involved. The mechanism differs from that of guanine analog-induced differentiation in HGPRT-deficient HL-60 cells.

Expression of a novel high affinity purine base transport system in mutant mouse S49 cells does not require a functional nucleoside transporter.

A novel type of somatic mutation that causes the expression of a high affinity purine base permease has been inserted into murine S49 lymphoma cells that are deficient in nucleoside transport. Two classes of mutants expressing this nucleobase permease were generated. The first, as exemplified by the AE1HADPAB2 cell line, possesses an augmented capacity to transport low concentrations of the three purine bases, hypoxanthine, guanine, and adenine. The second class of mutants, as typified by the AE1HADPAB5 clone, possesses an augmented capability to translocate low levels of hypoxanthine and guanine, but not adenine. Neither the AE1HADPAB2 nor the AE1HADPAB5 cells can transport nucleosides suggesting that the expression of the high affinity base transporter did not revert the mutation in the nucleoside transport system. Fusion of the AE1HADPAB2 and AE1HADPAB5 cell lines with wild type cells indicated that the expression of the high affinity base transporter behaved in a dominant fashion, while the nucleoside transport deficiency was a recessive trait. These data suggest that the high affinity purine base transporter of mutant cells and the nucleoside transport function of wild type cells are products of different genes and that expression of the former probably requires the unmasking or alteration of a specific genetic locus that is silent or different in wild type cells.

Erythrocyte adenine PRPP availability in two types of APRT deficiency using silicon oil method.

Erythrocyte phosphoribosylpyrophosphate availability for adenine was measured by silicon oil method previously described. The homozygotes of Japanese type APRT deficiency (n = 6, from 4 families) showed 4.3 +/- 2.7% (mean +/- standard deviation) of adenine PRPP availability and the heterozygotes (n = 5) showed 86.0 +/- 6.0% of adenine PRPP availability. All homozygotes of Japanese type APRT deficiency from 4 unrelated families show the equally decreased adenine PRPP availability and it supports the presumption of the presence of the similar defect of APRT in all families. In a Japanese family of complete APRT deficiency, adenine PRPP availability of the homozygote was undetectable and that of the heterozygote was normal low (54.3% of normal mean activity). The adenine PRPP availability of the heterozygote of complete APRT deficiency was diagnostically different from that of the homozygotes of Japanese type APRT deficiency, despite, these two conditions showed almost the same erythrocyte APRT activity. These results prove that the silicon oil method previously written is the rapid and useful method for differential diagnosis between two types of APRT deficiency.

Effect of nutritional state and allopurinol on purine metabolism in the rat small intestine.

The effect of fasting and refeeding on the uptake and retention of purines by the small intestine of the rat was studied in vivo. Short-term uptake and incorporation into nucleotides of the purine bases adenine, guanine and hypoxanthine and the nucleoside inosine were evaluated in the proximal jejunum. After 5 min, more label was recovered in the intestinal contents in fasted rats, indicating that total absorption was reduced. However, intestinal retention of purines (50 nmol dose) was elevated with fasting (27.2 vs. 16.6 nmol/g for adenine, 5.7 vs. 3.0 nmol/g for guanine and 16.1 vs. 7.4 nmol/g for hypoxanthine, for fed vs. fasted, respectively). After 1 day of refeeding, retention remained elevated for adenine (27.4 nmol/g) and guanine (5.5 nmol/g). After 3 days of refeeding intestinal weight and retention of labeled purines returned to the unfasted levels. Nucleotide formation from all purine bases was greater in the intestinal tissue of fasted as compared to fed rats (25.4 vs. 11.4 nmol/g for adenine, 1.32 vs. 0.24 nmol/g for guanine, and 2.84 vs. 0.82 nmol/g for hypoxanthine). At a higher dose (3000 nmol) hypoxanthine and inosine were retained to a greater extent in the fasted than in the fed state. Pretreatment with allopurinol (a xanthine oxidase inhibitor) reduced the absorption of hypoxanthine, increased the retention of label in the tissue 4-fold or more, and elevated nucleotide formation 10-fold or more. Fasting and allopurinol treatment, both known affectors of xanthine oxidase activity, enhanced both the retention of dietary purine and nucleotide formation.