Effects of indomethacin on changes in renal blood flow induced by adenosine and its analogues in conscious dogs

Summary The effects of indomethacin on changes in renal blood flow induced by adenosine, NECA (adenosine-5-N-ethyl-carboxamide) and 2,3-dinitro-NECA were investigated in 6 chronically instrumented conscious dogs. Adenosine (187.5, 375 and 750 nmol/kg, i.v.) induced a dose-dependent initial decrease, followed by a reactive increase in renal blood flow. NECA (1.5 nmol/kg, i.v.) also induced an initial decrease, which was, however, followed by a prolonged reactive increase in renal blood fow. 2,3-dinitro-NECA (50 nmol/kg, orally) induced only an increase in renal blood flow. Indomethacin (27.9 mol/kg, i.v.) caused no relevant change of the initial decrease and a significant attenuation of the reactive increase in renal blood flow induced by adenosine. NECA-induced changes in blood flow were affected by indomethacin in the same direction but to a greater extent than were adenosine-induced changes in blood flow. Indomethacin reversed the increase to a decrease in renal blood flow induced by 2,3-dinitro-NECA. Thus, prostaglandins seem to be involved in mediating the response of renal blood flow to adenosine, NECA and 2,3-dinitro-NECA.Part of this study was presented at the fall meeting of the German Pharmacological Society, September 1982 in Vienna, Austria     

A simple method for sampling murine pulmonary and cardiac tissues for analysis of cyclic nucleotide levels

Summary A simple method for the rapid removal and freezing of mouse cardiac and pulmonary tissues is described. Samples thus obtained were judged to be suitable for valid estimation of in vivo levels of cyclic AMP and cyclic GMP based on the following findings: (a) the samples could be obtained and frozen in the very short time period of a few seconds; (b) no indication of adverse effects of the collection procedure was found upon examination of chemical indicators of energy metabolism; (c) the apparent rates of change of cyclic AMP and cyclic GMP levels during the first seconds after tissue isolation could produce small, but acceptable errors; and (d) dose-dependent elevations of pulmonary cAMP levels consistent with known effects in vitro were found after in vivo administration of isoproterenol.Abbreviations cAMP adenosine-3,5-cyclic monophosphate - cGMP guanosine-3,5-cyclic monophosphate     

Hemmung von Adenyl-Cyclase-Präparationen aus der Rattenniere durch Calciumionen und verschiedene Diuretica

Summary Antidiuretic hormone (ADH) increases the permeability to water of certain epithelial membranes. This effect, found in the urinary bladder of the toad and in the distal tubules and the collecting ducts of kidney, is mediated intracellularly by adenosine 35-monophosphate (Ado-35-P). Calcium ions and the diuretic ethacrynic acid are known to inhibit the ADH-induced increase in water permeability of the toad bladder. In adenyl cyclase preparations from rat renal cortex and medulla, the influence of these substances as well as of other diuretics added in vitro has been studied. Adenyl cyclase activity has been determined, excepted as noted, by measuring Ado-35-P formed from 1 mM ¹⁴C-ATP in the presence of 10 mM Mg⁺⁺, an ATP regenerating system, and 5 mM unlabeled Ado-35-P to reduce the enzymatic degradation of the labeled Ado-35-P.Calcium ions reduced the rate of Ado-35-P formation by particles from renal cortex and medulla when the activity was measured in the presence of either Mg⁺⁺ or Mn⁺⁺. With 10 mM Mg⁺⁺, 1 mM Ca⁺⁺ decreased adenyl cylase activity by about 50%. Activities of cortical adenyl cyclase stimulated by parathyroid hormone, thyrocalcitonin or ADH and of medullary adenyl cyclase stimulated by ADH were also reduced by about 50% in the presence of 1 mM Ca⁺⁺. The inhibition was independent of the ATP concentration, but was influenced by the Mg⁺⁺ content of the incubation medium.Adenyl cyclase activities of cortical and medullary membrane preparations were reduced by about 50% by 0.2 mM ethacrynic acid. The extent of this inhibition was essentially the same whether the enzymatic activity was determined in the absence or presence of stimulating hormones. The inhibitory action of ethacrynic acid was partially prevented by simultaneous addition of dithioerythritol (DTE). A derivative of ethacrynic acid, L 589420-0-2, also inhibited renal adenyl cyclase, but its action was not influenced by the addition of DTE. Adenyl cyclase from both parts of the kidney was inhibited by about 90% by 0.2 mM mersalyl. This action was almost completely prevented by the addition of 1 mM DTE. The pharmacological significance of adenyl cyclase inhibition by these diuretics is still uncertain since the role of Ado-35-P in the regulation of sodium transport is as yet unclear.Other diuretics, hydrochlorothiazide, furosemide, mefruside, amiloride, and the non-diuretic benzothiadiazine, diazoxide, had essentially no effect on cortical and medullary adenyl cyclase preparations when they were added in 0.1–0.5 mM concentration.The methylxanthines, theophylline and caffeine, which are known to inhibit nucleoside 35-monophosphate phosphodiesterase, reduced the rate of Ado-35-P formation. The unstimulated and the hormone-stimulated adenyl cyclases were inhibited to the same extent by theophylline. When adenyl cyclases was stimulated by fluoride, however, we found only a very small inhibition by theophylline. Inhibition of the medullary adenyl cyclase was greater than that of the enzyme prepared from renal cortex. At a concentration of 1 mM these methylxanthines significantly inhibited the medullary enzyme, but the inhibition became asymptotic at about 50% when concentrations up to 20 mM were used. Therefore, it is likely that inhibition by these substances varies in different cell types and tissues.Instead of phosphodiesterase inhibitors, unlabeled Ado-35-P can be used in the assay of adenyl cyclase activity to reduce the degradation of enzymatically formed labeled Ado-35-P. This addition, though, can also influence adenyl cyclase activity. In a medullary enzyme preparation 0.2 mM Ado-35-P reduced the adenyl cyclase activity by 13%, 5 mM Ado-35-P by 35%.

---

Abkürzungen Ado-35-P Adenosin-35-monophosphat - Guo-35-P Guanosin-35-monophosphat - ADH antidiuretisches Hormon, Vasopressin - PTH Parathormon - TCT Thyreocalcitonin - DTE Dithioerythrit - EDTA Äthylendiamintetraessigsäure Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.Über einen Teil der Ergebnisse wurde auf der 11. Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft berichtet (Jakobs et al., 1970). Einige der vorliegenden Ergebnisse sind der Inauguraldissertation von K. H. J. (Medizinische Fakultät der Universität Heidelberg, 1971) entnommen.     

Comparative studies on distribution and covalent tissue binding of 2,4,2′,4′- and 3,4,3′,4′-tetrachlorobiphenyl isomers in the rat

The ¹⁴C-labeled tetrachlorobiphenyl (TCB) isomers 2,4,2,4-tetrachlorobiphenyl (2,4,2,4-TCB) and 3,4,3,4-tetrachlorobiphenyl (3,4,34-TCB) were administered orally to rats, and distribution and covalent binding were measured in several organs. Marked differences in distribution and covalent binding of the two TCBs were observed. The accumulation and retention of 2,4,2,4-TCB in adipose tissue were much higher than those of 3,4,3,4-TCB, although the level of radioactivity in the blood was consistently higher in 3,4,3,4-TCB treated rats. The radioactivity bound in covalent linkages with cellular macromolecules in several tissues was also measured. The data obtained indicated that covalent binding was higher in 3,4,3,4-TCB treated rats than in those treated with 2,4,2,4-TCB, particularly in liver and blood components. These results suggest that the two TCB isomers have different pharmacokinetic properties in rats, and the association of covalent binding with 3,4,3,4-TCB-induced toxicities might be important. In addition, we found that repeated oral dosing with the two TCB isomers caused an increase in in vitro liver microsomal generation of reactive metabolites of TCBs, indicating that the microsomal enzyme system is likely to play an important role in the in vivo covalent binding of TCB.     

Evidence for two separate vasoconstriction-mediating nucleotide receptors,both distinct from the P2X-receptor,in rabbit basilar artery: a receptor for pyrimidine nucleotides and a receptor for purine nucleotides

Summary Uridine 5-triphosphate- (UTP-) and adenosine 5-triphosphate-(ATP) induced vasoconstriction was studied in the rabbit basilar artery. The arteries were incubated and perfused at a constant rate of flow. Vasoconstriction was measured as an increase in perfusion pressure.Serotonin, histamine and noradrenaline caused concentration-dependent vasoconstriction, with potency decreasing in that order. Of the nucleotides tested, UTP, UDP, UMP, CTP, ATP, ADP, adenosine 5-O-(3-thio)triphosphate (ATPS), and ,-imido adenosine 5-triphosphate (AMP-PNP) elicited concentration-dependent vasoconstriction, whereas AMP, 2-methylthio-ATP, , -methylene-ATP and ,-methylene-ATP up to 10^–3 mol/l caused no or only a very small increase in perfusion pressure. The order of potency of the pyrimidine nucleotides was: UTP = UDP UMP = CTP; that of the purine nucleotides was: ATPS > AMP-PNP > ATP > ADP > 2-methylthio-ATP = , -methylene-ATP = ,-methylene-ATP. The vasoconstrictor effects of UTP and ATP were not or only to a minor degree influenced by: phentolamine; a mixture of atropine, diphenhydramine and methysergide; indometacin; nordihydroguaiaretic acid; denervation by 6-hydroxydopamine; or mechanical removal of endothelium. Prolonged exposure to ,-methylene-ATP elicited only a very small vasoconstriction and did not change the constrictor effects of UTP or ATP. Prolonged exposure to ATPS elicited marked vasoconstriction; subsequently, responses to ATP were reduced whereas those to UTP were, if anything, slightly enhanced. Reactive blue 2 reduced neither the UTP- nor the ATP-induced vasoconstriction. ATP 10^–3 mol/l elicited marked additional vasoconstriction after precontraction with UTP 10^–3 mol/l, whereas UTP elicited only a very small additional vasoconstriction when its concentration was doubled from 10^–3 to 2 × 10^–3 mol/l.It is concluded that, in the rabbit basilar artery, the vasoconstrictor response to UTP is mediated by a pyrimidine nucleotide receptor which is distinct from the P₂-purinoceptor, and that the vasoconstrictor response to ATP is mediated by a P₂-receptor which is distinct from the known P₂-subtypes.Send offprint requests to I. v. Kügelgen at the above address     

2,2′,3′,4,4′,5,5′-Heptachlorobiphenyl (PCB 180) — on its toxicokinetics,biotransformation and porphyrinogenic action in female rats

The toxicokinetics and biotransformation of 2,2,3,4,4,5,5-heptachlorobiphenyl, as well as its influence on the activity of microsomal and cytosolic enzymes and on the porphyrin pathway in the liver were studied in female rats following oral treatment with 7 mg/kg every other day for 3 months. One day after cessation of treatment the concentration of the compound in liver, spleen, CNS and blood was 100–500 times and in the trachea it was only 5 times less than in the adipose tissue. The daily excretion with the feces and urine amounted to 35 and 1.5 g, respectively. In both excreta, heptachlorobiphenylol was identified as a metabolite. The biotransformation rate was estimated to be about 5%. Investigations of the liver revealed increases in the relative liver weight, total cytochrome P-450 content, O-deethylation of 7-ethoxycoumarin and in the activity of glutathione S-transferases. Disturbances of the hepatic porphyrin pathway were not detected. Only at the end of a post-dosing period of 12 months did the hepatic uroporphyrinogen decarboxylase show diminished activity. Only one of these animals with diminished enzyme activity showed drastically elevated porphyrins. In these animals, the fecal and urinary porphyrins did not differ from controls. At no time did heptachlorobiphenyl influence the urinary excretion of delta-aminolevulinic acid and porphobilinogen. The results indicate 1) that this congener shows expected toxicokinetics with the exception of being accumulated in the trachea and 2) that this congener induces disturbances of the hepatic porphyrin pathway several months after cessation of treatment.     

Evaluation of cardiotoxicity of a new anthracycline derivative: 4′ -deoxy-4′ -iodo-doxorubicin

Summary The present study in rats was performed to evaluate the cardiotoxic activity of 4 -deoxy-4 -iodo-doxorubicin (4-deoxy-4-I-DXR), a new anthracycline derivative with interesting antineoplastic properties and possible lower cardiotoxicity than doxorubicin (DXR). DXR produced ECG alterations consisting of a progressive and irreversible prolongation of SaT and QaT. In contrast, in 4-deoxy-4-I-DXR-treated rats the increase in SaT and QaT duration was significantly lower than that observed in DXR-treated rats and slightly increased over controls.DXR produced significant histologic changes in myocardium consisting of myocyte vacuolization and myofibrillar loss. No significant modifications were observed in mitochondria. In contrast, no significant cardiac lesions were observed in 4-deoxy-4-I-DXR-treated rats. These results suggest that this new anthracycline derivative has a significantly lower degree of cardiotoxic activity than DXR.     

Effect of six virustatic nucleoside analogues on the development of fetal rat thymus in organ culture

The effects of the virustatic agents zidovudine (azidothymidine, AZT) 23-dideoxycytidine (ddC), 23-dideoxyinosine (ddI), acyclovir (ACV), ganciclovir (GCV), and vidarabine phosphate (VP) on the in vitro development of thymic lobes of 17-day-old rat fetuses were tested in an organ culture system. The virustatics were added to the medium for a culture period of 7 days. All nucleoside analogues inhibited the proliferation and differentiation of lymphatic cells. However, differences were observable with respect to the potency of the six drugs to interfere with thymic development. Compared to untreated controls, reduction in the number of thymocytes was significant at concentrations of 30 M AZT and ddI. In the case of ACV, GCV, VP, and ddC concentrations as low as 10 M were sufficient to cause a significant reduction, ddC being the most potent derivate. Increasing concentrations of the nucleoside analogues led to a dose-dependent further inhibition of cell proliferation. At a concentration of 30 M flow cytometry revealed a decrease in the relative number of double positive CD4⁺ CD8⁺ and single positive CD4⁺ CD8^– cells but an increase in the relative number of CD4-CD8⁺ cells. At the same concentration the expression of the CD5 antigen was reduced by the antimetabolites, indicating that maturation of the thymocytes was inhibited. Distribution of the forward light scatter, a cell size-related parameter, showed that the formation of small thymocytes was reduced by the nucleoside analogues. Light and electron microscopic investigations indicated cytotoxic effects of the drugs on the thymocytes, whereas the epithelium was only slightly affected.     

Interaction of adenine nucleotides,UTP and suramin in mouse vas deferens: suramin-sensitive and suramin-insensitive components in the contractile effect of ATP

Summary Effects of various nucleotides, nucleosides and noradrenaline on smooth muscle tension were studied in the isolated mouse vas deferens. ,-Methylene-ATP, ATPS, noradrenaline, ATP and UTP elicited contraction, with potency decreasing in that order; there was no contractile response to adenosine or uridine (up to 100 mol/l). Prolonged incubation with ,-methylene-ATP (concentration increased stepwise from 0 to 15 mol/l) selectively reduced contractions induced by ATP and UTP but not those induced by noradrenaline, and there was cross-tachyphylaxis between ATP and UTP. Suramin (10–300 mol/l) did not alter the response to noradrenaline but shifted the concentration-response curves for ,-methylene-ATP, ATPS, UTP and lower concentrations of ATP (0.1–1 ol/l) to the right. The pA₂-values of suramin were 5.2 against ,-methylene-ATP, 4.8 against ATPyS, 5.1 against UTP and 5.4 against lower concentrations of ATP. The effects of higher concentrations of ATP were largely resistant to suramin. The results indicate that the mouse vas deferens possesses contraction-mediating smooth muscle P_2X-receptors. UTP also acts at this receptor, and there is no evidence for a separate UTP receptor. The selective inhibition of nucleotide- but not noradrenaline-induced contractions by suramin confirms the view that suramin is a selective P₂-antagonist. The resistance against suramin of part of the effect of ATP suggests that ATP activates a suramin-insensitive site in addition to the P_2X-receptor.Send offprint requests to I. von Kügelgen at the above address     

Interaction of 5′-deoxy-5-fluorouridine and methotrexate

Methotrexate (MTX) toxicity is reduced significantly by a non-toxic dose of 5-deoxy-5-fluorouridine (5dFUr). Changes in the hematopoietic system (platelets, erythrocytes, leukocytes, and hematocrit), ileal tissue, and body weight were used as parameters to assess toxicity. MTX treatment alone resulted in: (a) a reduction of body weight; (b) significantly morphological changes in ileal tissue; and (c) a marked decrease in the hematopoietic parameters. Sequential treatment with MTX followed by 5dFUr resulted in reversal of MTX depression of animal body weight and ileal tissue necrosis, and partial reversal in MTX toxicity to the hematopoietic system. Also, for all parameters studied, there were no significant differences between scheduling of MTX after a priming dose of 5dFUr, 5dFUr alone, and control. Hence, this study suggests that 5dFUr is a pharmacological antidote for MTX toxicity, and, therefore, 5dFUr in combination with MTX may provide a basis whereby more intense and effective MTX therapy may be given.This research was supported by MBRS Grant 2S06RR0816-14     

Cardiostimulatory effects of cyclic 3′,5′-adenosine monophosphate and its acylated derivatives

Summary The effects of cyclic 3,5-AMP and of two acylated derivatives, dibutyryl (DBA) and dihexanoyl-3,5-AMP (DHA) were investigated in isolated perfused hearts of guinea pigs, rats and rabbits.In guinea pig hearts, DBA (Ca- and Na-salt) and DHA-Na in high doses (10 moles) produced strong and long lasting increases in the rate and amplitude of contractions, coronary flow, and moderate increases in phosphorylase activity in the majority of experiments. The positive ino- and chronotropic effects occured 3–5 min after injection of the drug, mostly in a fluctuating manner with several maxima. Theophylline augmented the effects of DBA-Na and revealed positive inotropic actions of non substituted 3,5-AMP.In rat hearts, similar, but more pronounced and dose-dependent effects were observed after 1, 5 and 10 moles DBA-Na. Propranolol (50 g) did not block the action of 10 moles DBA-Na. Non substituted 3,5-AMP, 5-AMP and ATP in doses of 10 moles had no significant positive inotropic effects.In rabbit hearts, DBA-Na (50 moles) produced moderate, non fluctuating rises in the amplitude of contraction.The results provide evidence that under certain conditions cyclic 3, 5-AMP itself, like its acylated derivatives DBA and DHA, may produce strong and direct positive inotropic and chronotropic effects in the heart. These findings support the view that cyclic 3,5-AMP is the cellular mediator of the cardiostimulant actions of substances that increase its rate of production in the myocardial cell.The excellent technical help of Mrs. Vera Bauer is gratefully acknowledged by the authors.     

Inhibition by papaverine and eupaverin of 3′,5′-cyclic AMP phosphodiesterase from rabbit skeletal muscle

Summary Papaverine and eupaverin are potent inhibitors of 3,5-cyclic AMP phosphodiesterase from rabbit white skeletal muscle. These drugs inhibit more the activity associated with the particulate fractions than that associated with the soluble fraction.     

Pharmacokinetics of 2′,3′-dideoxyadenosine in dogs

The pharmacokinetics of 2,3-dideoxyadenosine (ddAdo) and 2-3-dideoxyinosine (ddIno) were determined after intravenous bolus administration and long-term intravenous infusion of ddAdo in dogs. ddAdo was rapidly deaminated to ddIno and ddAdo plasma concentrations were only a fraction of ddIno concentrations. The total body clearance of ddAdo exceeded the literature value for the cardiac output of the dog, indicating an extremely rapid metabolism, and the existence of extrahepatic metabolism. Urinary excretion of unchanged ddAdo was a minor route of elimination ( 1%). The pharmacokinetics of ddIno was determined assuming complete conversions of ddAdo to ddIno. ddIno elimination was dose-dependent with total body clearance ranging from 4 to 55 ml/min/kg in individual animals. The plasma half-life was approximately 30 min after most routes of administration, but increased to approximately 60 min in two animals receiving a large intravenous dose of 500 mg/kg. ddIno penetrated into the cerebrospinal fluid to a limited extent, reaching concentrations of 3–11% of those in plasma. Urinary excretion of unchanged ddIno accounted for approximately 20% of the administered dose of ddAdo, while uric acid and hypoxanthine were minor urinary metabolites.Concentrations exceeding the in vitro minimal viral inhibitory concentration (2.4 g/mL) could be safely maintained in plasma for a 10-day period. Infusions which gave cerebrospinal fluid concentrations of 12 to 17 Lg/mL resulted in dose limiting myelosuppression and intestinal toxicity, after less than 10 days of infusion. Orally administered ddAdo was absorbed as ddIno, with bioavailabilities ranging from 28 to 93% in experiments where no emesis occurred. These studies indicate the rapid in vivo conversion of ddAdo to ddIno, and support the selection of ddIno over ddAdo for further drug development.     

Der Einfluß von Hydrochlorothiazid und anderen sulfonamidierten Diuretica auf die 3′,5′-AMP-Phosphodiesterase-Aktivität in der Rattenniere

Summary Vasopressin has been reported to accelerate the conversion of adenosine triphosphate to cyclic 3,5-AMP by stimulating the activity of the adenyl cyclase. According to the view of Orloff and Handler cyclic 3,5-AMP is responsible for the augmentation of osmotic water flow. The cyclic nucleotide ist degraded by the enzyme 3,5-AMP phosphodiesterase (PDE) to 5-AMP. Inhibition of this enzyme by theophylline results in an increase in the concentration of 3,5-AMP and a concomittant increase in osmotic water flow, as shown in the urinary bladder of the toad (Bufo marinus).The experiments presented in this paper derived from a previous observation that furosemide and hydrochlorothiazide inhibit PDE. Both diuretics have been shown to reduce renal PDE activity when injected i.v. to rats in a dose of 25 mg/kg. Following injection of furosemide PDE activity has been found reduced only in the cortex, the effect of hydrochlorothiazide has been shown to be restricted to the inner medulla.Studies on the subcellular distribution of renal PDE revealed two fractions, one third of total activity bound to large particles, probably cell membranes, two third soluble in the hyaloplasm. The two fractions of the enzyme differ in their k _m-value for 3,5-AMP. Subcellular distribution and k _m-values of PDE in the liver have been found to be identical with those in the kidney.Hydrochlorothiazide has been shown to affect both fractions of renal PDE. Because of the restriction of the action of furosemide to the renal cortex no attempt was made to differentiate the effect of the compound with respect to its subcellular localization. Accumulation of 3,5-AMP caused by an impaired degradation of the nucleotide in this region could lead to an increase in the permeability of the distal convoluted tubules to water. As the difference in the osmotic pressure between distal tubular fluid and the surrounding interstitial fluid is relatively small, the increase in water permeability can only result in a small increase in tubular water reabsorption. In view of hydrochlorothiazide reducing PDE activity in the inner medulla and the high difference in the osmotic pressure between the fluid in the collecting tubules and the interstitial fluid it is suggested that a hydrochlorothiazide induced increase in water permeability results in a high increase in water rea-absorption, especially in diabetes insipidus where there is a low osmolarity of the tubular fluid in the collecting duct with an unimpaired cortico-papillary osmotic gradient. This corresponds to the paradoxical antidiuretic effect of diuretics in the treatment of diabetes insipidus centralis and renalis, especially after diuretic induced sodium depletion and reduction of the osmolarity of tubular fluid resulting in an increased osmotic difference between fluids within collecting ducts and interstitium.

Am 31. Oktober 1967 verstorben.     

Localization of Purine Metabolizing Enzymes in Bovine Brain Microvessel Endothelial Cells: An Enzymatic Blood-Brain Barrier for Dideoxynucleosides?

Purpose. The specific activities of the purine and pyrimidine metabolizing enzymes, purine nucleoside phosphorylase (PNP), adenosine deaminase (ADA) and cytidine deaminase (CDA) were determined in bovine brain microvessel endothelial cells (BBMECs), whole cerebral tissue and erythrocytes. In addition, the substrate specificities (K_m and V_max) of purified calf spleen PNP for inosine and 2,3-dideoxyinosine (ddl) and of purified calf intestinal ADA for 2,3-dideoxyadenosine (ddA), 6-chloro-2,3-dideoxypurine (6-Cl-ddP), and 2--fluoro-2,3-dideoxyadenosine (F-ddA) have been explored. Methods. BBMECs were isolated from bovine cerebral cortex by a two step enzymatic dispersion treatment followed by centrifugation over 50% Percoll density gradients. Activities of alkaline phosphatase, -glutamyl transpeptidase, ADA, PNP and CDA were determined in various tissue homogenates (cerebral cortex, BBMECs and erythrocytes). Enzyme kinetic studies were also conducted using commercially available enzymes and several nucleoside analogs of interest. Results. The activities of ADA and PNP were 42-fold and 247-fold higher in the cerebral microvessels than in the cerebral cortex, respectively, while there was no detectable CDA activity in the microvessel fraction and very little overall activity in the cortex. Conclusions. ADA and PNP may serve as an enzymatic blood-brain barrier for some of the anti-HIV dideoxynucleosides. Simulations of brain availability for ddl, ddA, 6-Cl-ddP, and F-ddA demonstrated that the quantitative significance of enzyme localization may vary dramatically, however, depending on the membrane permeability of the drug and its bioconversion rate constant within the endothelial cell.     

The pharmacokinetics of doxifluridine and 5-fluorouracil after single intravenous infusions of doxifluridine to patients with colorectal cancer

Summary The disposition kinetics of a new 5-fluorouracil prodrug, 5-deoxy-5-fluorouridine (5dFUR, doxifluridine), were investigated in six patients with colorectal carcinoma. Each patient randomly received two single intravenous doses of 5dFUR (2 and 4 g · m^–2) on separate days.Plasma concentrations of 5dFUR fell rapidly with terminal half-lives ranging from 16.1 to 27.7 min. A disproportionate increase in the area under the curve with increasing dose was seen in most patients. Doubling the dose resulted in a 40% decrease in nonrenal clearance (0.60 to 0.37 l · min^–1) but no apparent change in renal clearance (0.32 to 0.29 l · min^–1) or steady-state apparent volume of distribution (19.8 to 20.4 l).The mechanism for dose-dependence of 5dFUR appears to be primarily due to nonlinear elimination associated with nonrenal processes rather than nonlinear plasma protein or tissue binding.     

In vivo and in vitro interactions of TCDD and other ligands of theAh-receptor: effect on embryonic and fetal tissues

Interactions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and other ligands of theAh-receptor, had been studied in vivo, in pregnant NMRI mice, and in vitro, in the fetal thymus organ culture system. Benzo(a)pyrene (BP) increased TCDD-induced fetolethality, whereas it did not affect the rate of cleft palate formation. This may indicate that the mechanism of TCDD-induced fetal death is different from that of TCDD-induced cleft palate. 2,3,7,8-Tetrachlorodibenzofuran (TCDBF) and 3,3,4,4-tetrachloroazoxybenzene (TCAOB) were added together to the thymus organ culture, each at a concentration that caused about 25–50% lymphoid development inhibition. Such treatment resulted in an additive effect of about 75%. Similarly, when the slightly toxic -naphthoflavone (BN) was added together with TCDD to the same culture system, it caused a significant increase in the lymphoid inhibitory effect of the latter compound. These may all suggest a common mechanism of action for TCDD and other ligands, which may involve a direct interaction with the receptors present in the thymus.Abbreviations BN -naphthoflavone - BP Benzo(a)pyrene - PAH Polycyclic aromatic hydrocarbons - TCAOB 3,3,4,4-tetrachloroazoxybenzene - TCDBF 2,3,7,8-tetrachlorodibenzofuran - TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin     

Potency of N6-modified N-alkyl adenosine-5′-uronamides at presynaptic adenosine receptors in guinea-pig ileum

Summary The ability of a series of N⁶-modified N-alkyl-5-uronamides to cause presynaptic inhibition of transmitter release was examined in isolated guinea-pig ileum stimulated at 0.2 Hz. These analogs inhibited the twitch responses to nerve stimulation, the majority being full agonists with their inhibitory effects being antagonised by theophylline. These analogs had no significant effects on responses of ileum to carbachol. N-ethyl 5-uronamide substitution resulted in an up to four-fold reduction in activity of N⁶-substituted adenosine analogs, while stereoselectivity of the N⁶-substituted analogs continued to be present. 5-Uronamide substitutions to N⁶-(3-pentyl)-adenosine resulted in a marked loss of activity when there were large alkyl groups at the amide or with amides of secondary amines. It was concluded that adenosine analogs interact with both the N6 and C-5 regions of the adenosine receptor in this tissue, with the interaction being less than additive.Abbreviations CHA N⁶-cyclohexyladenosine - HNBMPR 6[(2-hydroxy-5–nitro)benzylthiol-inosine - NECA N-ethyladenosine-5-uronamide - NCPCA N-cyclopropyl-adenosine-5-uronamide - R-PIA N⁶-R-(1-phenyl-2-propyl)adenosine - S-PIA N⁶-S-(1-phenyl-2-propyl)adenosine Supported by grants from the Medical Research Council of New Zealand and the Auckland Medical Research Foundation (to D. M. Paton) and the Suncoast Chapter, Florida AHA Affiliate, Nelson Research and Development Inc. and the NIH (HL-26611) (to RAO) Send offprint requests to D. M. Paton at the above address     

Bis(carbamoyloxymethyl) Esters of 2′,3′-Dideoxyuridine 5′-monophosphate (ddUMP) as Potential ddUMP Prodrugs

No HeadingPurpose. We previously reported the synthesis of bis(pivaloyloxymethyl) 2,3-dideoxyuridine 5-monophosphate (POM₂-ddUMP) (1a) as a membrane-transport prodrug formulation of the free parent nucleotide, ddUMP. Although successful at delivering ddUMP into cells in culture, POM₂-ddUMP was rapidly degraded by plasma carboxylate esterases after intravenous administration to experimental animals, and therefore has limited therapeutic potential as a systemically administered prodrug. We now report the synthesis of bis(N,N-dimethylcarbamoyloxymethyl)- and bis(N-piperidinocarbamoyloxymethyl) 2,3-dideoxyuridine 5-m onophosphate [DM₂-ddUMP (1b) and DP₂-ddUMP (1c), respectively], analogues of POM₂-ddUMP that were designed to be more resistant to degradation by plasma esterases..Methods. After entering cell by passive diffusion, it was anticipated that loss of one of the carbamoyloxymethyl groups of 1b and 1c would occur by spontaneous chemical hydrolysis to give the intermediate phosphodiesters, 2b and 2c. Cleavage of the remaining carbamoyloxymethyl groups by cellular phosphodiesterase I would generate ddUMP. 1b and 1c were prepared by condensation of 2,3-dideoxyuridine (ddU) with the appropriate bis(N-alkylcarbamoyloxymethyl) phosphate in DMA in the presence of triphenylphosphine and diethyl azodicarboxylate (the Mitsunobo reagent).Results. The half-lives of 1b and 1c when incubated at a concentration of 10^–4 M in human plasma at 37°C were 3.5 h and 3.7 h, respectively, similar to the half-lives observed under the same temperature conditions in 0.05 M aqueous phosphate buffer, pH 7.4. By contrast, the half-life of the POM₂ prodrug, 1a, in plasma was only 5 min. The initial products of degradation of 1b and 1c were the phosphodiesters 2b and 2c. The latter compounds gave rise to ddUMP when incubated with snake venom phosphodiesterase I.Conclusions. These findings support the premise inherent in the design of 1b and 1c, namely that the carbamate prodrugs are far more resistant to hydrolysis by plasma carboxylate esterases than their POM counterparts and can revert to the free parent 5-mononucletides by successive chemical and enzymatic hydrolysis. Further studies of 1b and 1c as membrane-permeable prodrugs of ddUMP are in progress.     

Lysergic acid diethylamide antagonizes shaking induced in rats by five chemically different compounds

Thyrotropin-releasing hormone (TRH), sodium valproate, AG-3-5 (1-[2-hydroxyphenyl]-4-[3-nitrophenyl]-1,2,3,6-tetrahydropyrimidine-2-one), RX336-M (7,8-dihydro-5, 6-dimethylcyclohex-5-eno-1,2,8,14 codeinone), and Sgd 8473 (-[(4-chlorobenzylideneamino)-oxy]-isobutyric acid) each induced repetitive shaking of the body of rats after intraperitoneal injection. This action of the five diverse chemicals appears to be subserved by a common pharmacological component, because pretreatment with d-lysergic acid diethylamide (0.03–1.0 mg kg^-1, s.c.) attenuated the shaking behavior in a dose-related manner, and cross tolerance was found between RX 336-M and TRH, sodium valproate, and AG-3-5.