Biomedical subjects
D Farquhar
Publications and source records attributed to D Farquhar.
Synthesis and antitumor evaluation of bis[(pivaloyloxy)methyl] 2'-deoxy-5-fluorouridine 5'-monophosphate (FdUMP): a strategy to introduce nucleotides into cells.
The bis[(pivaloyloxy)methyl] [PIV2] derivative of 2'-deoxy-5- fluorouridine 5'-monophosphate (FdUMP) was synthesized as a potential membrane-permeable prodrug of FdUMP. The compound was designed to enter cells by passive diffusion and to revert to FdUMP after removal of the PIV groups by hydrolytic enzymes. The most convenient preparation of PIV2FdUMP was by condensation of 2'-deoxy-5-fluorouridine (FUdR) with PIV2 phosphate in the presence of triphenylphosphine and diethyl azodicarboxylate (the Mitsunobo reagent). PIV2FdUMP was stable in the pH range 1.0-4.0 (t1/2 > 100 h). It was also fairly stable at pH 7.4 (t1/2 = 40.2 h). In 0.05 M NaOH solution, however, it was rapidly degraded (t1/2 < 2 min). In the presence of hog liver carboxylate esterases, PIV2FdUMP was converted quantitatively to the mono-[(pivaloyloxy)methyl] [PIV1] analogue PIV1FdUMP. After a 24 h incubation, only trace amounts of FdUMP (1-3%) were observed, indicating that PIV1FdUMP is a poor substrate for carboxylate esterases. In mouse plasma, PIV2FdUMP was rapidly metabolized, first to PIV1FdUMP and then to FdUMP. With continued incubation, FUdR was formed, presumably due to further catabolism of FdUMP by plasma phosphatases or 5'-nucleotidases. Since PIV1FdUMP is a poor substrate for carboxylate esterase, the cleavage of the second PIV group is most likely mediated by plasma phosphodiesterases. The rate of degradation of PIV2FdUMP in the presence of acid and alkaline phosphatase, 5'-nucleotidase, or spleen phosphodiesterase was the same as that in buffer controls, indicating that the compound is not a substrate for these nucleotide catabolizing enzymes. The concentration of PIV2FdUMP and its 3'-O-acetyl ester (PIV2 3'-O-Ac-FdUMP) required to inhibit the growth of Chinese hamster ovary (CHO) cells in vitro to less than 50 cells per colony was 5 x 10(-6) M, the same as that required for 5-fluorouracil (FU). Both nucleotide prodrugs showed the same growth-inhibitory potency against a mutant CHO cell line that was 20-fold resistant to FU (CHO/FU). Administered intraperitoneally at optimal dosage for 5 consecutive days, PIV2FdUMP and PIV2 3'-O-Ac-FdUMP were as effective as FU at prolonging the life spans of mice bearing intraperitoneally implanted P388 leukemia. Both prodrugs retained full therapeutic activity against a P388 subline resistant to FU. Collectively, these data indicate that PIV2FdUMP and PIV2 3'-O-Ac-FdUMP are effective membrane-permeable prodrugs of FdUMP.
The dual use of opioids and temazepam by drug injectors in Glasgow (Scotland).
In recent years much attention has been drawn to the use of buprenorphine (Temgesic) by heroin injectors in Glasgow and elsewhere. Glasgow has also witnessed a parallel increase in use of the benzodiazapine temazepam, often used as a 'cocktail' with buprenorphine. This paper presents new evidence that, although buprenorphine use among Glasgow drug injectors may now be declining, the use of temazepam-opioid cocktails has continued.
N-(5,5-diacetoxypent-1-yl)doxorubicin: a new intensely potent doxorubicin analogue.
N-(5,5-Diacetoxypent-1-yl)doxorubicin (DAPDOX) (3), a new, water-soluble analogue of doxorubicin, has been synthesized by coupling doxorubicin with 5-oxopentane-1,1-diacetate in the presence of NaBH3CN. This analogue was designed to be converted to the corresponding aldehyde, N-(5-oxopent-1-yl)doxorubicin, in the presence of carboxylate hydrolases, enzymes that are ubiquitous in tissue. DAPDOX had a half-life of several days in 0.05 M phosphate or 0.05 M acetate buffer solution at pH 4.0. However, in 0.05 M phosphate buffer at pH 7.4 in the presence of 20 unit equiv of porcine liver carboxylate esterase, the half-life of DAPDOX was less than 1 min. N-(5-acetoxypent-1-yl)doxorubicin (4), which should give rise to N-(5-hydroxypent-1-yl)doxorubicin on esterase-mediated hydrolysis, and N-(pent-1-yl)doxorubicin (5), were also prepared for comparative biological studies. DAPDOX was 150 times more potent than doxorubicin at inhibiting the growth of Chinese hamster ovary (CHO) cells in culture. The compound retained the same degree of potency against a CHO subline 100-fold resistant to doxorubicin (CHO/DOX) that expressed elevated levels of P-glycoprotein. Compounds 4 and 5, on the other hand, were no more effective than doxorubicin at inhibiting the growth of CHO cells and were 4-7-fold less potent against the CHO/DOX subline. DAPDOX is representative of a new structural class of doxorubicin analogues with unique chemical and biological properties.
Selective action of 2',3'-didehydro-2',3'-dideoxythymidine triphosphate on human immunodeficiency virus reverse transcriptase and human DNA polymerases.
This study used DNA primer extension and sequencing gel analyses to evaluate the molecular action of 2',3'-didehydro-2',3'-dideoxythymidine triphosphate (D4TTP), in comparison with 3'-azido-2',3'-dideoxythymidine triphosphate (AZTTP), on DNA strand elongation by human immunodeficiency virus reverse transcriptases (HIV-RT) and human DNA polymerases alpha (pol alpha) and epsilon (pol epsilon) purified from T-lymphoblastoid CEM cells. D4TTP was preferentially incorporated into the T sites of the elongating DNA strand by HIV-RT and terminated DNA synthesis at the incorporation sites. The DNA chain termination activity of D4TTP was equipotent to that of AZTTP. In contrast, D4TTP was a poor substrate for pol alpha and pol epsilon. The analogue was incorporated into DNA by the human enzymes about 10,000- to 20,000-fold less efficiently than by HIV-RT, whereas the incorporation of AZTTP by pol alpha and pol epsilon was not detectable by the DNA primer extension assay. Pol epsilon, an enzyme with 3'----5'-exonuclease activity, was unable to remove the incorporated 2',3'-didehydro-2',3'-dideoxythymidine monophosphate (D4TMP) from the 3'-end of the DNA strand, whereas 3'-azido-2',3'-dideoxythymidine monophosphate was excised from DNA by pol epsilon at about 20% of the rate for normal deoxynucleotide excision. The preferential incorporation of D4TTP by HIV-RT appears to be a molecular basis for the selective anti-HIV activity of D4T, whereas the inability of pol epsilon to remove D4TMP from DNA may be related to the cytotoxicity of this compound.
Membrane-permeable dideoxyuridine 5'-monophosphate analogue inhibits human immunodeficiency virus infection.
2',3'-Dideoxyuridine (ddU) is ineffective at controlling human immunodeficiency virus type 1 (HIV-1) infection in human T cells, because it is not biotransformed to the active 5'-triphosphate. The metabolic block resides in the poor substrate affinity of ddU for cellular nucleoside kinases. This problem cannot be overcome by supplying the preformed nucleotides, because such compounds are unable to penetrate cells. To circumvent the requirement of ddU for enzymic phosphorylation, we have prepared bis(pivaloyloxymethyl) 2',3'-dideoxyuridine 5'-monophosphate (piv2 ddUMP), as a potential membrane-permeable prodrug of ddUMP, and investigated its metabolism and anti-HIV activity in two human T cell lines, one with wild-type thymidine kinase activity (MT-4) and the other deficient in thymidine kinase activity (CEM-tk-). The 5'-mono-, di-, and triphosphates of ddU were formed in both cell lines after exposure to piv2-ddUMP. In contrast, phosphorylated metabolites were not observed in cells treated with ddU or ddUMP alone. piv2-ddUMP also reduced the cytopathic effects of HIV-1 in MT-4 cells (ED50, 4.75 microM) and inhibited virus production in culture fluid (ED50, 20 microM). In addition, piv2-ddUMP protected CEM-tk- cells from HIV-1 infection, as demonstrated by inhibition of intracellular p24 antigen levels (ED50, 3 microM) and reverse transcriptase activity in culture medium (Ed50, 2.5 microM). Based on these findings, we propose that the "masked nucleotide" strategy may make available for development nucleoside analogues hitherto considered inactive because of failure to undergo biotransformation to the corresponding 5'-monophosphates. Moreover, by circumventing metabolic dependency on nucleoside kinases, the strategy may overcome acquired resistance to nucleoside analogues caused by the loss or depletion of nucleoside kinases.
N-(5,5-diacetoxypentyl)doxorubicin: a novel anthracycline producing DNA interstrand cross-linking and rapid endonucleolytic cleavage in human leukemia cells.
The cytotoxic and DNA-damaging effects of a novel alkylating anthracycline, N-(5,5-diacetoxypentyl)doxorubicin, were quantified in HL-60 human leukemia cells and in an intercalator-resistant daughter line, HL-60/AMSA. The new drug was cytotoxic to both lines at doses as low as 50 nM for 1 h. N-(5,5-Diacetoxypentyl)doxorubicin produced DNA interstrand cross-linking in both lines. The cross-linking appeared to increase in both lines following drug treatment, but the increase was greater in the resistant line. This appeared to be due to an underestimation of cross-linking, particularly in sensitive HL-60, secondary to time-dependent DNA fragmentation that followed drug removal. This time-dependent DNA fragmentation was probably endonucleolytic cleavage (a feature of apoptosis) as characteristic nucleosomal ladders were produced by N-(5,5-diacetoxypentyl)doxorubicin treatment in a cotemporal time-dependent fashion. This novel anthracycline is the first of a family of alkylating anthracyclines designed to be water soluble, easy to formulate, and capable of producing DNA interstrand cross-linking. Because this last characteristic has previously been associated with doxorubicin analogues of great potency and low toxicity, these newer, more readily formulated drugs may have great clinical utility.
Aldophosphamide acetal diacetate and structural analogues: synthesis and cytotoxicity studies.
The synthesis of aldophosphamide acetal diacetate and a number of structural analogues is described. These compounds are designed to undergo biotransformation to the corresponding aldehydes in the presence of carboxylate esterases, enzymes that are ubiquitous in mammalian tissue. Several of these aldehydes can theoretically exist in pseudoequilibrium with the 4-hydroxyoxazaphosphorine tautomers; others lack this capability. The half-lives of the acetals in 0.05 M phosphate buffer, pH 7.4, at 37 degrees C ranged from 1 to 2 days. In the presence of 2 unit equiv of porcine liver carboxylate esterase, all of the compounds were hydrolyzed with half-lives of less than 1 min. Although closely structurally related, the compounds exhibited a wide range of cytotoxicities to L1210 murine leukemia cells in vitro.
Doxorubicin analogues incorporating chemically reactive substituents.
Doxorubicin (1) analogues 2-5, incorporating the following alkylating or latent alkylating substituents, R, on the 3'-position of the daunosamine sugar have been synthesized as potential antitumor agents: 2, R = NHCOC6H4(p)SO2F; 3, R = NHCOCH2Br; 4, R = NHCOCH2Cl; 5, R = NHCON(NO)CH2CH2Cl. These compounds were designed on the premise that alkylating anthracyclines might bind covalently to critical intracellular target macromolecules and overcome resistance to the parent agent attributable to reduced cellular drug accumulation. Growth inhibitory studies of the analogues were conducted in vitro against mouse leukemia cells (L1210 and P388) and human uterine sarcoma cells that are sensitive (MES-SA) and resistant (MES-SA/DOX) to doxorubicin. The analogues were 5-100-fold less potent than doxorubicin against the sensitive cell lines. However, they were only marginally cross-resistant with doxorubicin against MES-SA/DOX. Compounds 3 and 5 were also evaluated against a human myelocytic cell line (KBM-3) and a subline (KBM-3/DOX) resistant to doxorubicin. They were equally potent against both cell lines, indicating a complete lack of cross-resistance with doxorubicin. Alkylating anthracyclines may have potential for the treatment of tumors resistant to the parent agents.
Selective action of 3'-azido-3'-deoxythymidine 5'-triphosphate on viral reverse transcriptases and human DNA polymerases.
The action of 3'-azido-3'-deoxythymidine 5'-triphosphate (N3dTTP) on DNA strand elongation catalyzed by human immunodeficiency virus type 1 reverse transcriptase was evaluated in comparison with human DNA polymerase alpha and proliferating cell nuclear antigen-independent DNA polymerase delta. Sequencing gel analysis demonstrated that the human immunodeficiency virus 1 reverse transcriptase preferentially incorporated N3dTTP into the T sites of the growing DNA strands and caused chain termination in a dose-dependent manner. This effect was observed even when the N3dTTP concentration was 0.3 microM, 100-fold less than dTTP. Studies with reverse transcriptases from avian myeloblastosis virus and Moloney murine leukemia virus showed that N3dTTP was also efficiently incorporated into DNA by these enzymes and terminated DNA strand elongation. In contrast, human DNA polymerases alpha and delta did not incorporate detectable amounts of N3dTTP into the DNA and were not inhibited by 300 microM N3dTTP. The selective incorporation of the chain-terminating nucleotide by the viral reverse transcriptases appears to be a molecular basis for the positive therapeutic index of 3'-azido-3'-deoxythymidine.
Cyproterone acetate/ethinyl estradiol in the treatment of acne. A comparative dose-response study of the estrogen component.
The investigators compared 2 mg cyproterone acetate (CPA) in combination with either 0.035 mg or 0.050 mg ethinyl estradiol (EE2) (Diane -35 versus Diane -50) in the treatment of acne. Both formulations of Diane were highly effective in improving acne, even in women who had been refractory to other types of medication. Cycle control with both formulations was excellent and adverse effects were generally mild and confined to the first two cycles of treatment. Mean plasma lipid levels increased with both treatments, yet most individual values remained within normal limits after one year of therapy while the LDL-cholesterol/HDL-cholesterol ratio was stable throughout the study period. Plasma testosterone and DHEA-S levels paralleled the decline in the clinical severity of the acne. There was no loss of clinical effectiveness with Diane -35 and it provided the advantage of a 30% decrease in the amount of estrogen.
Potent DNA chain termination activity and selective inhibition of human immunodeficiency virus reverse transcriptase by 2',3'-dideoxyuridine-5'-triphosphate.
2',3'-Dideoxyuridine (ddUrd) exhibits poor if any anti-human immunodeficiency virus (HIV) activity in ATH8 and MT-4 cells. This is in agreement with the failure of ddUrd to be efficiently anabolized intracellularly to its 5'-triphosphate metabolite. However, 2',3'-dideoxyuridine-5'-triphosphate (ddUTP) proved to be a potent and selective inhibitor of the reverse transcriptase of HIV (Ki, 0.05 microM) and avian myeloblastosis virus (Ki, 1.0 microM). Bacterial DNA polymerase I, mammalian DNA polymerase alpha, terminal deoxyribonucleotidyl transferase, and Moloney murine leukemia virus reverse transcriptase were resistant to ddUTP. ddUTP is incorporated into the growing DNA chain principally at dTTP sites and inhibits further elongation. The potential of ddUTP as an anti-HIV therapeutic agent merits further investigation. However, to achieve this goal, it will be necessary to resort to techniques capable of delivering preformed phosphorylated ddUrd to the susceptible cells.
Suitability of a new stable acetal analogue of aldoifosphamide for purging leukemic cells from human bone marrow.
The in vitro cytotoxic properties of acetaldoifosphamide, a new chemically stable bis-acetate analogue of aldoifosphamide that requires enzymatic activation by cellular carboxylate esterases, has been compared with that of 4-hydroperoxycyclophosphamide (4-HC). On a molar basis, acetaldoifosphamide was 8-10 times more potent than 4-HC against two different human leukemic myeloid cell lines, but only twice as potent as 4-HC against normal bone marrow granulocyte-macrophage colony-forming cells (GM-CFC). Acetaldoifosphamide retained its activity against leukemic cell lines that were highly resistant to the antileukemic drugs doxorubicin and m-AMSA. GM-CFC doubling times after exposure of bone marrow to high concentrations of acetaldoifosphamide in suspension cultures were 6-12 hours. Similar doubling times were obtained after incubation of marrow with 4-HC. Acetaldoifosphamide has a sparing effect on hematopoietic stem cells that is similar to that found for 4-HC; however, it is considerably more potent than 4-HC. Acetaldoifosphamide is different from 4-HC in its chemical stability and its unique requirement for carboxylate esterase activation. We conclude that acetaldoifosphamide may have advantages over 4-HC for in vitro purging of leukemic cells from human bone marrow.
Evidence for acyloxymethyl esters of pyrimidine 5'-deoxyribonucleotides as extracellular sources of active 5'-deoxyribonucleotides in cultured cells.
Cells commonly resist growth inhibition by purine and pyrimidine bases and nucleosides by restricting intracellular formation of the corresponding 5'-mononucleotides. Nucleotide derivatives that can act as effective membrane-transport precursors of the poorly membrane-permeable nucleotides have not been identified so far. We studied the bis(pivaloyloxymethyl)ester (I) of FdUMP (5-fluoro-dUMP) and a cyclic phosphodiester (II) of FdUMP derived from 1,3-dihydroxyl-1-C-(pivaloyloxy-methyl)propane which are active in vivo against a 5-fluoro-2'-deoxyuridine (FUdR)-resistant mouse leukemia and are attacked by carboxylic esterases under physiological conditions to produce FdUMP by elimination of formaldehyde and acrolein respectively. The assay for intracellular FdUMP was the inhibition of DNA synthesis due to inhibition of TMP synthetase in cultured mouse LM(TK-) fibroblasts genetically devoid of thymidine kinase (TK) and thus unable to convert FUdR directly to FdUMP. At 10(-6)M, I, II, or FUdR inhibited DNA synthesis in 2 hr by 99, 80, and 35% respectively; at 10(-5)M. maximal inhibition was attained after less than 15, 30 and 90 min respectively. Inhibition of DNA synthesis in TK+ cells by 10(-5) M I, II, or FUdR was reversed completely by 10(-5)M thymidine (TdR) but unaffected by 10(-5)M UdR, confirming TMP synthetase as the locus of inhibition. At 10(-5)M, bis(pivaloyloxymethyl) esters of phenyl phosphate or a p-substituted benzylphosphonic acid did not inhibit significantly DNA synthesis in TK+ cells. From this finding, and from effects produced by V (see below), we conclude that pivalic acid and CH2O arising from I contribute little to its above inhibitory effects. In TK- cells in which DNA synthesis is prevented by blockade of TMP synthetase with aminopterin, the bis(pivaloyloxymethyl) ester (V) of TMP, at 0.9 x 10(-4) M, induced a 4-fold faster rate of DNA synthesis than did 10(-3)M TMP, whereas 10(-3) M TdR did not affect the rate. After 3 hr the rate with V was 80% that in the absence of aminopterin. In the above systems the nucleotide diesters I, II and V appear to be acting as effective extracellular sources of active intracellular FdUMP and TMP, in processes that involve loss of the two esterifying groups.
The effects of acetaldophosphamide, a novel stable aldophosphamide analogue, on normal human and leukemic progenitor cells in vitro: implications for use in bone marrow purging.
Acetaldophosphamide (A-ALD), a novel in vitro active and stable derivative of aldophosphamide, kills human bone marrow-derived granulocyte-macrophage colony-forming cells (GM-CFC) independent of the cell cycle. The surviving fraction of GM-CFC is an exponential function of the drug concentration and time of exposure. Variation of marrow light-density cell concentration between 2 x 10(6) and 10 x 10(6)/ml does not significantly influence its GM-CFC toxicity. Marrow depleted of GM-CFC by A-ALD subsequently generates GM-CFC when grown in suspension cultures. During the early period after treatment with A-ALD the number of surviving GM-CFC (size of surviving GM-CFC compartment) does influence the speed of the GM-CFC repopulation in suspension cultures. The importance of the number of surviving GM-CFCs for the growth and maintenance of GM-CFC population in such suspension cultures diminishes with time. No significant differences are observed after 2 wk, indicating that the ancestor stem cell population and its regenerative potential responsible for in vitro hematopoiesis have not been significantly affected by the drug treatment. A-ALD-treated progenitor cells retain their ability to integrate with the previously established marrow stromal cell layer and generate GM-CFC within this layer to an extent comparable to that of untreated marrow cells. The effect of A-ALD on human hematopoiesis is comparable to that of 4-hydroperoxycyclophosphamide. Its advantage over 4-hydroperoxycyclophosphamide is a greater stability in vitro. It has sparing effect on GM-CFC ancestor cells. Its toxicity to myeloid leukemia cell line (KBM-3)-derived clonogeneic cells is higher than to the GM-CFC. It is similar in doxorubicin-sensitive (KBM-3) and -resistant (KBM-3/DOX) leukemic cells. Thus, A-ALD appears to be a promising drug for in vitro purging of bone marrow cells.
The effect of the experimental antitumor agent caracemide on brain choline acetyltransferase.
Caracemide was found to inhibit choline acetyltransferase (CAT) from rat brain. A concentration of 0.5 mM caracemide inhibited the enzyme by 93%, whereas a degradation product from caracemide, N-(methylcarbamoyloxy)acetamide, produced only a 50% inhibition. Two other degradation products, N-(methyl-carbamoyloxy)-N'-methylurea and N-hydroxy-N'-methylurea, lacked any inhibitory activity. With bovine brain CAT, caracemide showed noncompetitive inhibition with the substrate choline, Km 337 microM, Ki240 microM, Vmax 2.83 nmol acetylcholine formed/min/mg protein and mixed inhibition with the substrate acetyl-CoA, Km 21 microM, Ki 146 microM, Vmax 3.85 nmol acetylcholine formed/min/mg protein.
Release of methyl isocyanate from the antitumor agent caracemide (NSC-253272).
In recent phase 1 clinical trials, caracemide [N-acetyl-N-(methylcarbamoyloxy)-N-methylurea; NSC-253272] has demonstrated a marked potential to produce severe central nervous system (CNS) toxicity. Recent in vitro studies of this antitumor agent have presented indirect evidence indicating that methyl isocyanate is a likely metabolite which results from incubation of caracemide with either phosphate buffer or human plasma. This report presents evidence that methyl isocyanate is formed from caracemide in a concentration- and time-dependent manner. These data implicate the caracemide-mediated formation of methyl isocyanate as at least a plausible explanation for the CNS toxicity exhibited by this drug.
Biochemical pharmacology of N-acetyl-N-(methylcarbamoyloxy)-N'-methylurea (caracemide; NSC-253272).
Preclinical pharmacologic studies of caracemide [N-acetyl-N-(methylcarbamoyloxy)-N'-methylurea; CAR] have demonstrated a marked instability of this compound in the presence of either phosphate buffer (pH 7.4) or human plasma. Using [1-14C-acetyl]CAR and [3H-methylcarbamoyloxy]CAR, three CAR degradation products were identified: product A, N-(methylcarbamoyloxy)acetamide; product B: N-(methylcarbamoyloxy)-N'-methylurea; and product C: N-hydroxy-N'-methylurea. CAR degradation in human plasma was demonstrated by high-performance liquid chromatography (HPLC) to occur in a time- and temperature-dependent manner. A 30-min incubation (37 degrees) of CAR (10(-4) M) with human plasma resulted in degradation of more than 55% of parent compound; at 1 hr, more than 75% of original CAR was degraded. Incubation of [1-14C-acetyl]CAR with rat brain homogenate resulted in the formation of 14CO2. This reaction was partially inhibited by coincubation with physostigmine (10(-3) M). CAR inhibited acetylcholinesterase activity in neuroblastoma cells with an IC50 of 14 microM. In mechanism of action studies, CAR was found to inhibit ribonucleotide reductase activity but only at nine times the IC50 of hydroxyurea. In contrast to hydroxyurea, CAR was found to be non-cell-cycle phase-specific and non-cross-resistant with two CHO cell lines resistant to hydroxyurea. These data demonstrate the instability of CAR; moreover, they suggest that its mechanism of cytotoxicity is distinctly different from that of hydroxyurea and that the neurotoxicity associated with CAR administration may be caused in part by inhibition of acetylcholinesterase activity.