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Biomedical subjects

A R Paterson

Publications and source records attributed to A R Paterson.

At least 73 records · Page 4Linked to original sources
PubMed1983

Modification by nitrobenzylthioinosine-5'-monophosphate of pseudoisocytidine pharmacokinetics in mice and rats through inhibition of membrane transport.

In isolated, perfused mouse livers, initial rates of uptake of [2-14C]pseudoisocytidine (PIC), measured during the first 15 secs of perfusion were markedly reduced when the perfusion medium contained 5 X 10(-6) M nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport. A similar inhibition of PIC uptake occurred when mice were treated with NBMPR-P (the 5'-monophosphate of NBMPR) at doses greater than 0.2 mg/kg ip injected 30 mins prior to the liver perfusion assay. However, in vivo studies showed that a late effect of NBMPR-P was enhancement in PIC levels in liver and other tissues in mice and rats, relative to levels in animals that had not received NBMPR-P. Increases in incorporation of PIC into RNA reflected the NBMPR-P-induced increases in tissue levels of PIC. NBMPR-P and other inhibitors of nucleoside transport may have therapeutic applications in manipulation of the pharmacokinetic behavior and toxicity of nucleoside drugs.

Animals↗
PubMed1982

Species differences in nucleoside transport. A study of uridine transport and nitrobenzylthioinosine binding by mammalian erythrocytes.

A kinetic study of the inward transport of uridine in erythrocytes of rabbit, human, mouse, rat and guinea-pig demonstrated that the apparent Km of this process was similar (about 0.2mM) in these cell types, but Vmax. values differed markedly. In this array of cell types, Vmax. values were proportional to the number of transport-inhibitory, high-affinity binding sites present per cell of each type. Transport of uridine or adenosine was not detected in dog erythrocytes, nor was saturable, high-affinity binding of nitrobenzylthioinosine demonstrable. These findings demonstrate that species differences in nucleoside transport capacity are attributable to differences in the cell-surface content of functional nucleoside transport sites, rather than to differences in the kinetic properties of these sites.

Animals↗
PubMed1982

Manipulation of toxicity and tissue distribution of tubercidin in mice by nitrobenzylthioinosine 5'-monophosphate.

The i.v. administration of tubercidin, an analog of adenosine, in a single dose of 45 mg/kg caused death in about 90% of B10D2F1 mice so treated. Serum and urine analysis, as well as histological examination of tissues, related the lethality of tubercidin to hepatic injury, which was markedly reduced when mice were treated with the inhibitor of nucleoside transport, nitrobenzylthioinosine 5'-monophosphate (NBMPR-P), at i.p. doses higher than 10 mg/kg 30 min prior to tubercidin injection. With high NBMPR-P doses (100 mg/kg, i.p.) followed by tubercidin injection (45 mg/kg, i.v.), kidney damage and high mortality occurred. The tissue distribution of 3H following (( G-3H]tubercidin administration paralleled hepatic or renal injury: NBMPR-P treatment decreased the content of tubercidin-derived 3H in liver and increased that in kidney. Furthermore, the half-life of the decline in tubercidin levels in serum during the first minute after[3H]tubercidin administration was longer in NBMPR-P-treated mice (26 sec) than in untreated mice (10 sec), with the result that 3H levels in serum were more than ten times higher in the former than in the latter at an early stage during the distribution of tubercidin. Within 15 min after i.p. administration, the tissue distribution of (( 3H]tubercidin was complete. The i.p. administration of tubercidin caused ascites and the appearance of amylase in the peritoneal fluid evidently because of peritonitis and pancreatic injury. Administration of NBMPR-P by the i.p. route, but not by the i.v. route, prevented these injuries and shifted the LD50 of i.p. injected tubercidin (5 mg/kg) to markedly higher values (a 4-fold increase with NBMPR-P at 100 mg/kg). The protection of mice by NBMPR-P against lethal injuries caused by i.p. injected tubercidin was consistent with the inhibition by NBMPR-P of tubercidin accumulation in mesentery and pancreas. The tissue specificity of the NBMPR-P influence on the tissue distribution of tubercidin may reflect differences in NBMPR-P pharmacokinetics and/or in properties of the nucleoside permeation mechanism among various tissues.

Animals↗
PubMed1982

Uptake of cytidine by isolated, perfused mouse liver.

Mouse livers were perfused at 22 degrees C with an oxygenated salts medium containing [5-3H]cytidine and [carboxyl-14C]inulin. The cellular uptake of cytidine was determined from the 3H content of liver samples less that present in the extracellular (inulin) space of the samples. Time courses of cytidine uptake were biphasic with initial phases which were approximately linear for 15 s and had time zero values that approximated the extracellular space. Rates of cytidine uptake derived from the initial phase of uptake evidently represented rates of membrane transport because (i) initial rates were saturable, and (ii) cytidine uptake was blocked by the nucleoside transport inhibitor, nitrobenzylthioinosine (NBMPR). Treatment of mice with the 5'-monophosphate of NBMPR (greater than 0.2 mg/kg, injected i.p.) 30-40 min prior to the perfusion also blocked cytidine entry into livers. An apparent half-saturation constant of about 10(-3) M and a maximum rate of about 1 mumol . g-1 . min-1 were estimated for the NBMPR-sensitive transport of cytidine into mouse liver cells.

Adenine Nucleotides↗
PubMed1982

Cytosine arabinoside influx and nucleoside transport sites in acute leukemia.

Although cytosine arabinoside (araC) can induce a remission in a majority of patients presenting with acute myeloblastic leukemia (AML), a minority fail to respond and moreover the drug has less effect in acute lymphoblastic leukemia (ALL). The carrier-mediated influx of araC into purified blasts from patients with AML, ALL, and acute undifferentiated leukemia (AUL) has been compared to that of normal lymphocytes and polymorphs. Blasts showed a larger mediated influx of araC than mature cells, since mean influxes for myeloblasts and lymphoblasts were 6- and 2.3-fold greater than polymorphs and lymphocytes, respectively. Also, the mean influx for myeloblasts was fourfold greater than the mean for lymphoblasts. The number of nucleoside transport sites was estimated for each cell type by measuring the equilibrium binding of [(3)H]nitrobenzylthioinosine (NBMPR), which inhibits nucleoside fluxes by binding with high affinity to specific sites on the transport mechanism. The mean binding site numbers for myeloblasts and lymphoblasts were 5- and 2.8-fold greater, respectively, than for the mature cells of the same maturation series. The mean number of NBMPR binding sites for myeloblasts was fourfold greater than for lymphoblasts. Patients with AUL were heterogeneous since blasts from some gave values within the myeloblastic range and others within the lymphoblastic range. The araC influx correlated closely with the number of NBMPR binding sites measured in the same cells on the same day. Transport parameters were measured on blasts from 15 patients with AML or AUL who were then treated with standard induction therapy containing araC. Eight patients entered complete remission, while seven failed therapy, among whom were the three patients with the lowest araC influx (<0.4 pmol/10(7) cells per min) and NBMPR binding (<3,000 sites/cell) for the treated group. In summary, myeloblasts have both higher araC transport rates and more nucleoside transport sites than lymphoblasts and this factor may contribute to the greater sensitivity of AML to this drug. AraC transport varied >10-fold between leukemic blasts and normal leukocytes, but transport capacity related directly to the number of nucleoside transport sites on the cell. Finally, low araC transport rates or few NBMPR binding sites on blasts were observed in a subset of patients with acute leukemia who failed to achieve remission with drug combinations containing araC.

Acute Disease↗
PubMed1982

Initial rate kinetics of the transport of adenosine and 4-amino-7-(beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (tubercidin) in cultured cells.

A procedure is described for determining early time courses of nucleoside uptake by cultured cells in suspension. Replicate samples of cell suspensions were exposed to medium containing 3H-nucleosides for brief intervals (sec) ended by addition of nitrobenzylthioinosine, a potent inhibitor of nucleoside transport that terminated nucleoside uptake virtually instantaneously. Time courses of nucleoside uptake were constructed from the cellular content of nucleoside acquired by the replicate samples during graded intervals of exposure to the labeled permeant. Such time courses were definitive of cellular uptake of nucleosides during the first few sec of exposure to permeant and yielded initial rates of uptake of adenosine and 4-amino-7-(beta-d-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (tubercidin). Defining initial rates of nucleoside uptake as rates of inward transport, relationships between transport rates and extracellular concentrations of these permeants were evaluated in HeLa cells and in two cultured lines of mouse lymphoma L5178Y cells that differ in their abilities to phosphorylate adenosine and tubercidin. Transport rates for these permeants were similar in the two L5178Y cell types and were saturable in the 3 cell lines with Km values between 14 and 38 microM. Adenosine and tubercidin were mutually competitive permeants in L5178Y cells, indicating that they are substrates for the same transport mechanism.

Adenosine↗
PubMed1981

Absence of binding sites for the transport inhibitor nitrobenzylthioinosine on nucleoside transport-deficient mouse lymphoma cells.

Cells of an adenosine-resistant clone (AE1) of S49 mouse lymphoma cells were compared with cells of the parental line with respect to (a) characteristics of nucleoside transport, (b) high affinity binding of the inhibitor of nucleoside transport, nitrobenzylthioinosine (NBMPR), and (c) the antiproliferative effects of the nucleoside antibiotics, tubercidin, arabinosyladenine and showdomycin. Rates of inward transport of uridine, thymidine, adenosine, 2'-deoxyadenosine, tubercidin, showdomycin, and arabinosyladenine in AE1 cells were less than 1% of those in cells of the parental S49 line. The inhibitor of nucleoside transport, NBMPR, reduced rates of inward nucleoside transport in S49 cells to levels comparable to those seen in the transport-defective mutant. S49 cells possessed high affinity sites that bound NBMPR (6.6 X 10(4) sites/cell, Kd = 0.2 nM), whereas site-specific binding of NBMPR to AE1 cells was not demonstrable, indicating that loss of nucleoside transport activity in AE1 cells was accompanied by loss of the high affinity NBMPR binding sites. Relative to S49 cells, AE1 cells were resistant to the antiproliferative effects of tubercidin and showdomycin, but differences between the two cell lines in sensitivity toward arabinosyladenine were minor, suggesting that nucleoside transport activity was required for cytotoxicity of tubercidin and showdomycin, but not for that of arabinosyladenine.

Adenosine↗
PubMed1981

Binding of nitrobenzylthioinosine to high-affinity sites on the nucleoside-transport mechanism of HeLa cells.

Nitrobenzylthioinosine (NBMPR) binds reversibly, but with high affinity (Kd 0.1--1.2 nM), to inhibitory sites on nucleoside-transport elements of the plasma membrane in a variety of animal cells. The present study explored relationships in HeLa cells between NBMPR binding and inhibition of uridine transport. The Km value for inward transport of uridine by HeLa cells in both suspension and monolayer culture was about 0.1 mM. The affinity of the transport-inhibitory sites for uridine (Kd 1.7 mM), inosine (Kd 0.4 mM) and other nucleoside permeants was low relative to that for NBMPR. The pyrimidine homologue of NBMPR, nitrobenzylthiouridine, also exhibited low affinity for the NBMPR-binding sites. Pretreatment of HeLa cells with p-chloromercuribenzene sulphonate (p-CMBS) or N-ethylmaleimide (NEM) decreased binding of NBMPR to its high-affinity sites and inhibited uridine transport, indicating the presence of thiol groups essential to both processes. NEM, a more penetrable reagent than p-CMBS, inhibited binding and transport at much lower concentrations than the latter compound. Pretreatment of cells with concentrations of p-CMBS that alone had no effect on either NBMPR binding or uridine transport increased the sensitivity of transport to NBMPR inhibition and changed the shape of the NBMPR concentration-effect curve, suggesting synergistic inhibiton of uridine-transport activity by these two agents.

4-Chloromercuribenzenesulfonate↗
PubMed1981

Treatment of mouse neoplasms with high doses of tubercidin.

Previous studies from this laboratory demonstrated that a potent inhibitor of nucleoside transport, nitrobenzylthioinosine (NBMPR), protected cultured cells against cytotoxic nucleosides (nebularine, tubercidin, and toyocamycin). NBMPR and its 5'-monophosphate (NBMPR-P) also protected mice against potentially lethal dosage of these agents. This report describes protection of mice from potentially lethal dosages of tubercidin by administration of NBMPR-P and the use of combinations of these agents in treatments of mice bearing transplanted neoplasms. Treatment of mice bearing i.p. implants of the Ehrlich ascites carcinoma, leukemia L1210/TG8, and colon carcinoma 26 with potentially lethal dosages of tubercidin administered together with host-protecting dosages of NBMPR-P resulted in substantial kill of neoplastic cells and long-term survivors. In these experiments, therapeutic effects were achieved at optimal dosages of NBMPR-P, which protected host vital tissues but did not protect neoplastic cells in ascitic fluids (Ehrlich ascites carcinoma cells and leukemia L1210/TG8 cells). However, at supraoptimal dosages of NBMPR-P, the occurrence of therapeutic failures which were neoplastic deaths indicated that NBMPR-P also protected the neoplastic ascites cells against tubercidin cytotoxicity. Thus, the selectivity of tubercidin toxicity toward cells of the Ehrlich ascites carcinoma and leukemia L1210/TG8 was modified by NBMPR-P dosage.

Animals↗
PubMed1981

Therapy of mouse leukemia L1210 with combinations of nebularine and nitrobenzylthioinosine 5'-monophosphate.

Earlier reports from this laboratory showed that: (a) in the presence of nitrobenzylthioinosine (NBMPR), a potent, tightly bound inhibitor of nucleoside transport, cells proliferating in culture were protected against a number of cytotoxic nucleosides; and (b) mice were protected against potentially lethal dosages of nebularine (and other toxic nucleosides) by coadministration of NBMPR. The present study, which used nitrobenzylthioinosine 5'-phosphate (NBMPR-P), a readily soluble "prodrug" form of NBMPR, extended the in vivo protection studies and showed that the half-life of the protection effect was about 4 hr. In chemotherapy experiments, mice bearing transplanted neoplasms were treated with high dosages of nebularine together with protecting doses of NBMPR-P. When mice bearing leukemia L1010 were treated with a potentially lethal regimen of nebularine administered together with NBMPR-P, a substantial kill of leukemic cells resulted (some mice were long-term survivors). The therapeutic effect was optimal at dosage levels of the protecting agent in excess of those required in nonleukemic mice for protection against the lethal nebularine dosages used, suggesting that the therapeutic effect was due to the joint presence in the leukemic cells of a metabolite of NBMPR-P and nebularine; NBMPR-P protection of the leukemic host against nebularine lethality was necessary for the therapeutic effect to be manifested.

Animals↗
PubMed1979

Fluctuations in nucleoside uptake and binding of the inhibitor of nucleoside transport, nitrobenzylthioinosine, during the replication cycle of HeLa cells.

Binding of the potent nucleoside transport inhibitor 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine (NBMPR) and rates of uptake of several nucleosides were examined at 4-hr intervals during the replication cycle of HeLa S3 cells. Monolayer cultures of synchronous cells, obtained by mitotic detachment, were assayed for high-affinity binding of NBMPR and for rates of uptake of thymidine, uridine, cytidine, adenosine, inosine, and guanosine. The number of NBMPR binding sites per cell doubled between 4 and 16 hr after detachment (late G1 and S phase); during this interval, V max 'S for uptake of cytidine and adenosine doubled, and for uridine and thymidine uptake increased about 4- and 8-fold, respectively. Rates of inosine and guanosine uptake at extracellular concentrations below saturation increased 2-fold between G1 and S phase of the cell cycle. Km 'S for cellular uptake of thymidine, uridine, cytidine, and adenosine did not change with progress through the cycle. The results presented suggest that changes in nucleoside uptake during the HeLa cell cycle were due, in part, to changes in the activity of NBMPR-sensitive transport elements in the membrane.

Binding Sites↗
PubMed1979

Drug sequence-dependent toxicity and small bowel mucosal injury in mice treated with low doses of 3-deazauridine and 1-beta-D-arabinofuranosylcytosine.

The toxicity to mice of combinations of 1-beta-D-arabinofuranosylcytosine and 3-deazauridine was investigated. The drugs were administered daily i.p. on Days 1 to 5, each drug at 10 mg/kg body weight; these dosages are small fractions of the dosages at which 10% of the treated animals died when either drug was administered alone on the foregoing schedule. This drug combination was severely toxic when 3-deazauridine was administered 2 to 8 hr prior to 1-beta-D-arabinofuranosylcytosine; most mice treated in this way died within 3 days of the last treatment. Histological examination showed that severe damage to the small bowel mucosa resulted from treatment with the drugs in the above, lethal sequence. In contrast, treatments with this drug combination at the same dosages were tolerated when the two agents were administered simultaneously or when 1-beta-D-arabinofuranosylcytosine preceded 3-deazauridine. Under the latter conditions, small bowel mucosal injury was much less severe. Female mice were more sensitive to the toxic treatment regimen than were male mice and were protected against the latter when either the 3-deazauridine or the 1-beta-D-arabinofuranosylcytosine component was preceded by treatment with nitrobenzylthioinosine (100 mg/kg), a potent inhibitor of nucleoside transport.

3-Deazauridine↗