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Biomedical subjects

J M Collins

Publications and source records attributed to J M Collins.

At least 109 records · Page 6Linked to original sources
PubMed1986

Preferential binding of DNA primase to the nuclear matrix in HeLa cells.

Studies of the spatial organization of DNA replication have provided increasing evidence of the importance of the nuclear matrix. We have previously reported a relationship between rates of DNA synthesis and the differential binding of DNA polymerase alpha to the nuclear matrix over the S-phase. We now report the detection of DNA primase bound to the HeLa nuclear matrix. Matrix-bound primase was measured both indirectly, by the incorporation of [32P]dAMP into an unprimed single-stranded template, poly(dT), and directly, by the incorporation of [3H]AMP into matrix DNA. Characteristics of this system include a requirement for ATP, inhibition by adenosine 5'-O-(thiotriphosphate), a primase inhibitor, and insensitivity to aphidicolin and alpha-amanitine, inhibitors of polymerase alpha and RNA polymerase, respectively. Subcellular quantification of primase and polymerase alpha activity revealed that while most (approximately 72%) primase activity is bound to the matrix, only a minority (approximately 32%) of polymerase alpha activity is matrix-bound. Treatment of the nuclear matrix with beta-D-octylglucoside allowed the solubilization of approximately 54% of primase activity and approximately 39% of the polymerase alpha activity. This data provides further evidence of a structural and functional role for the nuclear matrix in DNA replication. The ability to solubilize matrix-bound replicative enzymes may prove to be an important tool in the elucidation of the spatial organization of DNA replication.

Adenosine Triphosphate↗
PubMed1986

Clinical pharmacokinetics of suramin in patients with HTLV-III/LAV infection.

Suramin has been reported to inhibit the reverse transcriptase activity of a number of retroviruses and to reduce the in vitro infectivity and cytopathic effect of HTLV-III/LAV, the etiologic agent of acquired immune deficiency syndrome (AIDS). The clinical pharmacokinetics of suramin were investigated as part of a pilot study to evaluate the safety and efficacy of this drug for the treatment of patients with diseases caused by HTLV-III/LAV. A dose of suramin 6.2 g was given intravenously over a five-week period to four patients. After the last dose, the plasma half-life of suramin was 44 to 54 days. This is among the longest half-lives reported for any therapeutic substance given to humans. Total plasma levels of suramin were greater than 100 micrograms/mL for several weeks. In vitro activity of suramin was found at concentrations as low as 50 micrograms/mL. Metabolites were not found in plasma, and urinary excretion accounts for elimination of most of the drug. Suramin is approximately 99.7% bound to plasma proteins. The results from these initial clinical pharmacokinetic studies might assist the design of further therapeutic trials of suramin, especially the selection of frequency of dosing and adjustments for renal impairment.

Acquired Immunodeficiency Syndrome↗
PubMed1986

Ca2+ induced phase separations in phospholipid mixtures.

We have probed the character of the observed phase separation in mixtures of phosphatidylcholines (PC) and/or phosphatidylethanolamines (PE) in the presence of CaCl2 solutions. Egg yolk phosphatidylethanolamine (EYPE) and a 1:1 molar ratio of dioleoylphosphatidylcholine/dioleoylphosphatidylethanolamine (DOPC/DOPE) were observed to undergo phase separation in CaCl2 solutions, as was previously observed for egg yolk phosphatidylcholine (EYPC) (L.J. Lis et al. Biochemistry, 20 (1981) 1771-1777). However, the mixed chain lipid, palmitoyloleoyl-PC, yielded only a single phase in water or CaCl2 solution. We hypothesize that two lipid species are necessary for the observed phase separation to occur, but that the separation itself is not a function of the individual lipid species, but of the mixture.

Calcium↗
PubMed1986

Oligodendroglia development in cell culture as monitored with a monoclonal antibody.

A new marker for young oligodendrocytes has been identified by a monoclonal antibody (mOg-1, IgM isotype) prepared from cerebellar plasma membrane stimulated mouse lymphocytes. mOg-1 reactive cells in the mouse cerebellum first appear at day 19 of gestation. Future white matter layers of fixed sections of neonatal rat cerebellum were labeled with mOg-1. Although EM analysis has shown cell-surface binding by presumptive oligodendroglia in neonatal cerebellum, the antibody does not bind to compact myelin. In cell cultures prepared from 6-d-old mice, 1.1% of the cells bound mOg-1 after 3 d in culture, but up to 5% of the cells bound mOg-1 after 2 weeks in culture. Of these same Og-1-positive cells, 69% bound anti-galactocerebroside and 65% bound anti-myelin basic protein. After a week in culture Og-1-positive cells often produced lamellar sheets extending a millimeter over the polylysine substratum in the absence of normal myelin formation. mOg-1 recognizes a cell-surface determinant distinct from well-characterized oligodendroglial molecules (galactocerebroside, sulfatide and myelin basic protein) that is expressed early in oligodendrocyte development. The antibody has been used to follow the maturation of oligodendrocytes in cultures of both normal and jimpy mouse cerebellum.

Animals↗
PubMed1986

Pharmacokinetics of intraventricular and intravenous N,N',N''-triethylenethiophosphoramide (thiotepa) in rhesus monkeys and humans.

The cerebrospinal fluid (CSF) and plasma pharmacokinetics of N,N',N"-triethylenethiophosphoramide (thiotepa), an alkylating agent used for treatment of carcinomatous meningitis, were determined in rhesus monkeys in order to assess the relative advantage of intraventricular versus systemic administration of the drug. Following an i.v. thiotepa dose of 0.9 mg/kg (11 mg/sq m), peak plasma levels of parent drug reached approximately 1 microgram/ml. Thiotepa was rapidly equilibrated with lumbar and ventricular CSF. Systemic, lumbar, and ventricular exposure to the drug, measured as area under the curve (AUC), were similar in all cases. After a 1-mg intraventricular dose of thiotepa, peak ventricular levels were greater than 100 micrograms/ml. However, peak levels in the lumbar CSF at 1 h after intraventricular administration were less than 10 micrograms/ml. The AUC for ventricular CSF was nearly 100-fold greater for the intraventricular route than for the i.v. route; however, the AUC for lumbar CSF following intraventricular delivery was only 5% of the AUC for ventricular CSF. N,N',N''-Triethylenephosphoramide, an active metabolite of thiotepa observed in all fluids, appeared to have a much slower total body clearance than thiotepa. Comparison of the data obtained from monkey experiments with data from a patient with meningeal disease supports the use of the monkey as a model for intraventricular pharmacokinetics. The data presented indicate that there is no relative advantage to intraventricular administration of thiotepa at the doses currently used in clinical trials.

Animals↗
PubMed1986

Potential roles for preclinical pharmacology in phase I clinical trials.

Concepts elucidated from preclinical pharmacology studies have made a substantial impact on the clinical use of anticancer drugs. However, the majority of animal pharmacology results have not been available until after drugs have entered clinical trials. Since clinical pharmacokinetic measurements are already part of many phase I trials, human data could be directly compared with mouse data if mouse pharmacology studies were completed before clinical trials were initiated. Once the starting dose in a phase I clinical trial has been evaluated, subsequent doses are escalated until the maximum tolerated dose is reached. The rate of escalation is empirically defined by a modified Fibonacci series. This universal escalation scheme is applied to all drugs, with no modifications based upon pharmacology or other factors. If the starting dose is far removed from the maximum tolerated dose, a large number of dose escalations are required. Consequently, most patients receive subtherapeutic doses, and the amount of resources allocated to each drug increases. We are exploring potential strategies for controlling the rate of dose escalation based upon pharmacokinetic determinations in mouse and man. Retrospective analyses indicate that 20%-50% savings in the total number of dose escalations are possible.

Aminoacridines↗
PubMed1986

Incorporation of iododeoxyuridine into DNA of granulocytes in patients.

Iododeoxyuridine (IdUrd) competes with thymidine for incorporation into DNA. In order to measure the incorporation of the drug in vivo, granulocytes were isolated from peripheral blood of patients at various times during and after 9- to 14-day IdUrd i.v. continuous infusions. DNA was extracted and enzymatically hydrolyzed. Both thymidine and IdUrd were separated and measured by high-performance liquid chromatography. Thymidine substitution by IdUrd was less than 1% prior to the fifth day of infusion and then increased rapidly to achieve a maximal value between 7 and 17% at the end of the infusion. In our protocol, thrombocytopenia was the most frequent dose-limiting systemic toxicity. For our group of patients, there was a clear overall relationship between the extent of substitution by IdUrd and the hematological toxicity. To our knowledge, these data provide the first direct quantitative determination of substitution by a drug into DNA in vivo without the use of radiolabeled compounds. The ability to directly monitor drug incorporation into DNA may provide the basis for monitoring and improving the selectivity of therapy.

DNA↗
PubMed1986

Alteration of the secondary structure of newly synthesized DNA from murine bone marrow cells by 5-fluorouracil.

The present study evaluates the effects of 5-fluorouracil (FUra) on the structure of newly synthesized DNA purified from bone marrow cells. DNA synthesis was decreased by 30 and 45% of control in the presence of 19 and 100 microM FUra, respectively. Furthermore at these concentrations of FUra, the DNA strand sizes were smaller as determined by alkaline sucrose gradients. Enzymatic digestion of the DNA demonstrated that most of the FUra (greater than 90%) was localized in the internucleotide linkage and not at the chain terminus. As the concentration of FUra was varied, the percentage of FUra at the chain terminus was unchanged, suggesting that the decrease in chain size as well as inhibition of DNA synthesis was not due to chain termination. DNA that had been synthesized in the presence of FUra was shown to fragment after increasing time as demonstrated by alkaline sucrose gradient analysis. This time-dependent fragmentation was associated with an increased number of strand breaks as determined by neutral and alkaline sucrose gradient analysis. A parallel study demonstrated a time-dependent excision of FUra from DNA over this same time period. In summary, these studies demonstrate an association between the excision of FUra from DNA and the changes in secondary structure of newly synthesized DNA.

Animals↗
PubMed1985

Effects of suramin on HTLV-III/LAV infection presenting as Kaposi's sarcoma or AIDS-related complex: clinical pharmacology and suppression of virus replication in vivo.

Suramin was given to ten outpatients with acquired immunodeficiency syndrome (AIDS) presenting as Kaposi's sarcoma (KS) or as an AIDS-related complex (ARC). Side-effects associated with the administration of 6.2 g of suramin over 5 weeks included fevers, rashes, urinary abnormalities, and transient rises in hepatic aminotransferases. Peak serum levels of over 100 micrograms/ml were attained. There was evidence of HTLV-III infectivity and replication in lymphocytes from four patients before therapy. The detectable virus level fell in each case by the time of the last dose, and in three cases it became undetectable at the end of therapy. In each case, viral replication was again detected in the weeks or months following the administration of suramin. Despite this in-vivo virustatic effect, no significant clinical or immunological improvement was observed using this short-term regimen. However, the results provide a rationale for investigating longer-term regimens.

Acquired Immunodeficiency Syndrome↗
PubMed1985

The interrelation between DNA synthesis rates and DNA polymerases bound to the nuclear matrix in synchronized HeLa cells.

Quantitative rates of DNA synthesis can be determined by DNA:propidium fluorescence measurements of synchronized cells progressing through S-phase. We have previously reported that HeLa cells have discontinuous rates with values of about 2.9, 1.6, and 4.4 pg of DNA/h for early, middle, and late S-phase, respectively. In attempts to understand why two peaks of DNA synthesis rates are observed, we have examined the nuclear DNA polymerases alpha and beta over the S-phase. Nuclear matrices isolated from HeLa cells contained 2% of the alpha polymerase and 12% of the beta polymerase that was present in cell lysates, and about 2% of the original DNA. The amounts of endogenous DNA synthesis in isolated nuclear matrices were comparable to the amounts observed when exogenous DNA was added. DNase treatment abolished the endogenous DNA synthesis but not the exogenous DNA synthesis, suggesting that polymerase alpha binding does not depend on matrix-bound DNA. As synchronized cells progressed through the S-phase, there appeared two peaks of enzymatic activity of alpha polymerase bound to the nuclear matrix which correlated with in vitro DNA synthesis in these nuclear matrices and with the two peaks of quantitative DNA synthesis rates. Two peaks of alpha polymerase activity were also observed with isolated nuclei, but not with cell lysates or cytosol. Our results suggest that, over the S-phase, the differential binding of polymerase alpha to the nuclear matrix determines the differential rates of DNA synthesis.

Cell Nucleus↗
PubMed1985

Reduction of DNA synthesis in aging but still proliferating cells.

It is well known that cell proliferation (and hence, DNA synthesis) declines in human diploid fibroblast-like cells with increasing passage number. It is not clear whether DNA synthesis declines in the remaining cells that are still actively proliferating. Estimations of cell kinetic parameters permitted extrapolations to be made that reflected the declining numbers of cells still capable of DNA replication. DNA synthesis declined with culture age in intact cells, permeabilized cells, and in the isolated nuclear matrix even when corrected for declining numbers of proliferating cells. With age, DNA polymerase alpha and beta activity in cell lysates declined, but when corrected for the remaining proliferating cells, only polymerase alpha activity declined; DNA polymerase alpha and beta activity bound to the nuclear matrix declined, but when corrected for declining proliferation, no decline was apparent for either enzyme. There was an increase in the number of S1-nuclease sensitive sites and breaks in the parental DNA of the dividing cells in older cultures. It is suggested that in aging cultures, not only does overall DNA synthesis decline owing to decreasing cell proliferation, but also that DNA synthesis declines in the remaining proliferating cells, that this decline is not due to decreasing amounts of DNA polymerase bound to the nuclear matrix, and that alterations in DNA structure occur.

Cell Cycle↗
PubMed1985

Phase I and pharmacokinetic study of tiazofurin (TCAR, NSC 286193) administered by continuous infusion.

Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide, TCAR) is a synthetic C-nucleoside that demonstrated significant in vivo activity against a variety of animal tumors as well as in vitro activity against human tumor-derived cell lines. Thirteen patients were treated with TCAR administered as a 5-day continuous infusion in this Phase I trial. Seventeen complete cycles were administered in three dose levels ranging from 550 to 1450 mg/M2. Dose-limiting toxicities were myelosuppression and neurotoxicity including severe lethargy. Other toxicities including superficial skin peeling, myalgias, and tearing were seen at all doses. One patient had chest pain on day 4 resulting in stopping the drug, however, there was no evidence of cardiac or pericardial disease. Uric acid levels rose within one day in the absence of allopurinol treatment. There were no treatment related deaths. HPLC measurement of drug levels demonstrated steady-state plasma levels during the infusion, and a half-life following the infusion of 7.7 +/- 0.6 hours. Minor abnormalities in renal function were associated with dramatic changes in pharmacokinetics and toxicity. No clinical responses were observed in this trial.

Adult↗
PubMed1985

Pharmacokinetics of 5-fluorouracil infusions in the rat: comparison with man and other species.

Saturable elimination of 5-FU is exhibited in rats during constant infusions. Over the range of 3-480 mg/m2/h, total-body clearance of 5-FU decreases from 600 ml/min/m2 to less than 90 ml/min/m2. Previously published values for catabolism of 5-FU by rat hepatocytes can be used to simulate the plasma concentrations of 5-FU that were measured over this range of infusions. The qualitative pattern of nonlinear pharmacokinetics for 5-FU is similar in rats, dogs, monkeys, and humans. However, there are major quantitative differences in total-body clearance values, expressed in terms of surface area (ml/min/m2) or as a fraction of cardiac output. Rat and monkey have substantially lower 5-FU clearance values than dog and man. Although 5-FU clearance values were similar in dogs and humans, total body clearance values for thymidine are 18-fold higher in humans than in dogs. The selection of an appropriate animal model to pursue the clinical observations of high total body clearance of pyrimidines remains uncertain.

Animals↗
PubMed1985

Clinical pharmacology of high-dose cisplatin.

Although nephrotoxicity has frequently limited conventional treatment with cisplatin to doses of 100-120 mg/m2 per cycle, vigorous chloruresis can permit the administration of high-dose cisplatin (200 mg/m2 per cycle) with minimal nephrotoxicity. Systemic toxicities are worsened, but therapeutic response seems to be enhanced. The pharmacokinetics of cisplatin in plasma and urine were examined to assess the causes of these effects. Plasma disappearance of ultrafiltrable platinum was well-described by a single exponential for each patient. The mean t1/2 was 50% longer for patients receiving high-dose cisplatin than for patients receiving conventional doses. The total systemic exposure was three times greater in the high-dose group, which tends to explain the systemic toxicity and improved tumor efficacy, but not the lack of nephrotoxicity. It is suggested that the kidneys of patients in the high-dose group were relatively protected by dilution of active Pt species in the urine in the tubule lumen as well as by high chloride ion concentrations in the urine.

Adult↗
PubMed1985

Proliferation-associated changes in in vitro mRNA translation in the HL60 cell line.

In order to characterize patterns of gene expression during the proliferation cycle of HL60 cells, we have analysed changes in the population of mRNA available for translation in vitro. HL60 cells were separated into cell cycle phases by centrifugal elutriation, monitoring the separation with flow cytometry. RNA was extracted from cell fractions highly enriched in G1, S or G2+M phases and translated in vitro. Translation products were analysed by two-dimensional electrophoresis followed by autoradiography. Autoradiograms were analysed by a computer-assisted method utilizing a drum-scanning microdensitometer. Spots were identified by their relative positions on the films and their relative intensity was estimated. Of the 159 peptides studied for cell cycle-associated changes in synthesis, nine showed phase-associated changes. The most significant changes were the accumulation of four peptides that showed maximal synthesis only in G2+M phases. An additional four peptides were synthesized maximally in both S and G2+M phases. One peptide showed maximal synthesis in S phase. These changes in gene expression suggest that these relatively abundant transcripts are regulated primarily at a quantitative level during proliferation and may be related to the doubling of structural proteins prior to mitosis.

Cell Line↗
PubMed1985

A phase I study of intravenous iododeoxyuridine as a clinical radiosensitizer.

Twenty-four patients with locally advanced (19 patients) or metastatic (5 patients) tumors were treated in a Phase I study combining constant intravenous infusions of iododeoxyuridine (IUdR) and hyperfractionated radiation therapy. IUdR was given as a constant infusion for 12 hours/day for two separate 14-day infusion periods in most patients. The dose of IUdR was escalated from 250 to 1200 mg/m2/12-hour infusion in this study. The initial tumor volume was treated to 45 Gy/1.5 Gy BID/3 weeks followed by a cone-down boost to 20-25 Gy/1.25 Gy BID/2 weeks after a planned 2-week break. THe IUdR infusion preceded the initial and cone-down irradiation by 1 week. Local acute toxicity (within the radiation volume) was uncommon and few patients required an alteration of the planned treatment schedule. Two patients developed late local toxicity with one patient showing clinical signs of radiation hepatitis and another patient developing a large bowel obstruction that required surgical bypass. Dose-limiting systemic toxicity was confined to the bone marrow with moderate to severe thrombocytopenia developing on Day 10-14 of infusions at 1200 mg/m2/12 hours. Mild stomatitis and partial alopecia occurred in some patients at this dose level. No systemic skin toxicity was seen. Pharmacology studies revealed steady-state arterial plasma levels of IUdR of 1 to 8 X 10(-6) M over the dose range used. In vivo IUdR incorporation into tumors was studied in three patients with high-grade sarcomas using an anti-IUdR monoclonal antibody and immunohistochemistry and demonstrated incorporation in up to 50-70% of tumor cells. The preliminary treatment results, particularly in patients with unresectable sarcomas, are encouraging. In comparison to our previous experience with intravenous bromodeoxyuridine, this Phase I study of IUdR shows less systemic toxicity (especially to skin), higher (2-3X) steady-state arterial levels, and comparable in vivo tumor cell incorporation.

Biopsy↗
PubMed1985

Clinical pharmacology of 5-iodo-2'-deoxyuridine and 5-iodouracil and endogenous pyrimidine modulation.

We describe the clinical pharmacology and metabolism of 5-iodo-2'-deoxyuridine (IdUrd) during and after a 12-hour infusion. The kinetics of IdUrd were linear between 250 and 1200 mg/m2. The plasma IdUrd concentration reached steady state in less than 1 hour. Total body clearance of IdUrd was 750 ml/min/m2 and the disappearance t1/2 at the end of the infusion was less than 5 minutes. The primary metabolite, 5-iodouracil (IUra), did not reach steady state during the infusion. At the end of the 1200 mg/m2 infusion, the maximum plasma IUra concentration was 100 mumol/L, or about 10 times the simultaneous IdUrd plasma concentration. During the infusion there was at least a fifty- to 100-fold increase in uracil and thymine plasma concentrations. After the infusion, IUra disappearance from plasma was nonlinear, with an apparent Michaelis constant of 30 mumol/L. Plasma uracil and thymine levels slowly decreased after the IdUrd infusion until IUra fell to less than 30 mumol/L. There was subsequently a parallel and more rapid decrease in the plasma concentrations of uracil and thymine. Uridine, 2'-deoxyuridine, and thymidine plasma levels did not change significantly as a result of IdUrd therapy. These changes in endogenous pyrimidine pools are consistent with competitive inhibition of dihydrouracil dehydrogenase by IUra. An in vitro human bone marrow assay was used to determine the relative toxicity of IdUrd and IUra. Although exposure to IUra was tenfold higher than that to IdUrd, IdUrd was at least 100 times more cytotoxic to marrow cells.

Chromatography, High Pressure Liquid↗