David J. Holbrook

The inhibition in vitro of bacterial DNA polymerases and RNA polymerase by antimalarial 8-aminoquinolines and by chloroquine

Abstract The effects of various 8-aminoquinolines, two of their hydroxylated potential metabolites, and chloroquine on the in vitro activities of DNA polymerase of Micrococcus luteus (Micrococcus lysodeikticus) and of Escherichia coli and RNA polymerase of E. coli were determined. The antimalarial 8-aminoquinolines (including primaquine, and two hydroxylated potential metabolites) inhibited the activity of each bacterial DNA polymerase, and the levels of inhibition were not decreased appreciably by increasing the DNA concentration. Although the inhibition by chloroquine of the DNA polymerases was markedly decreased as the Mg 2+ concentration was increased, the inhibition by the 8-aminoquinolines was insensitive or only moderately sensitive to variations in the Mg 2+ concentration. At low Mg 2+ concentrations (0.3–0.6 mM), the inhibition of the DNA polymerases by chloroquine exceeded the inhibition by any of the 8-aminoquinolines but at higher Mg 2+ concentrations (1.4–7.0 mM) the inhibition by several of the 8-aminoquinolines approached or exceeded the inhibition by chloroquine. The inhibition of M. luteus DNA polymerase by the hydroxylated 8-aminoquinolines and chloroquine was markedly decreased by increases in the polymerase concentration. For M. luteus DNA polymerase, it was concluded that the inhibition was predominantly due to the interaction of aminoquinolines with the polymerase although a lesser contribution from the interaction of the aminoquinolines with the template (or primer) DNA was not excluded. No differences were observed in the inhibition of E. coli DNA polymerase by samples of dextrorotatory, levorotatory, and racemic chloroquine. With the exception of chloroquine and pentaquine, the aminoquinolines had little effect on the activity of E. coli RNA polymerase, and the inhibition of RNA polymerase was much less than the inhibition of the DNA polymerases.

In vitro inhibition of mouse hepatic mixed-function oxidase enzymes by helenalin and alantolactone

The sesquiterpene lactones (STL) helenalin and alantolactone were effective in vitro inhibitors of the mouse hepatic microsomal mixed-function oxidase (MFO) enzymes, aminopyrine demethylase (APD), aniline hydroxylase (ANH) and 7-ethoxyresorufin deethylase (ERD). Helenalin and alantolactone concentrations of 0.5 mM produced a 50-60% inhibition of APD and ERD, and a 20-30% inhibition of ANH. An increase in substrate (aminopyrine) concentration from 0.5 to 25 mM decreased STL inhibition of APD by 12-32%. APD was also inhibited at low aminopyrine concentrations (0.5 mM) by the helenalin derivative 2,3,11,13-tetrahydrohelenalin (tetrahydrohelenalin). The STL produced type I binding spectra with oxidized microsomes; Ks values for helenalin and alantolactone were 161 and 9 microM respectively. These results suggest that STL inhibition of the MFO system results, in part, from STL binding to the substrate-binding site of cytochrome P-450. It has been reported that the irreversible alkylation of protein cysteinyl residues is responsible for STL inhibition of several different enzymes, and second-order rate constants for the reaction of helenalin and alantolactone with glutathione were 25.1 and 1.80 mM-1.hr-1 respectively. Tetrahydrohelenalin did not react with glutathione. However, the subsequent addition of 3.0 mM thiols, i.e. L-cysteine, N-acetylcysteine or glutathione, to STL-treated (0.5 mM) microsomes reversed helenalin and alantolactone inhibition of APD and ERD by 50-80%. The ability of thiols to reverse STL inhibition of APD was decreased 20-43% by the coincubation of STL and microsomes with an NADPH-generating system. In addition, established effects of sulfhydryl-reactive compounds on the MFO system, i.e. inhibition of NADPH-cytochrome c reductase and conversion of cytochrome P-450 to cytochrome P-420, were not observed after addition of helenalin (1.0 mM) or alantolactone (0.5 mM) to mouse hepatic microsomes. These results suggest that STL inhibition of MFO enzymes may not be dependent upon the reactivity of the STL towards sulfhydryl groups. Instead, we suggest that STL binding to the substrate-binding site of cytochrome P-450 and subsequent metabolism of the STL may contribute to inhibition of the MFO system.

Comparisons of tetrachloro(d,l-trans)1,2-diaminocyclohexaneplatinum(IV) biotransformations in the plasma of Fischer 344 rats at therapeutic and toxic doses

SummaryPlasma biotransformations of tetrachloro(d,l-trans) 1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) were determined in vivo at both therapeutic (3 mg/kg) and toxic (12 mg/kg) doses in Fischer 344 rats. Tetraplatin was rapidly converted to dichloro(d,l-trans)1,2-diaminocyclohexaneplatinum(II) [PtCl2(dach)]. This conversion was complete at the earliest time measured (7.5 min) at the therapeutic dose, but some unreacted tetraplatin was detectable in the circulation at the toxic dose. Three other major biotransformation products were observed in plasma: (d,l-trans)1,2-diaminocyclohexaneaquachloroplatinum(II) [Pt(H2O)(Cl)(dach)]+, the Pt-methionine complex, and another biotransformation product tentatively identified as either the Pt-cysteine or Pr-ornithine complex. Several other minor plasma biotransformation products were detected. Two of these were most likely formed intracellulary from tetraplatin. Two or more other platinum complexes appeared to lack the diaminocyclohexane carrier ligand and were most likely formed intracellulary by trans-labilization of the carrier ligand. Tetraplatin, PtCl2(dach), and [Pt(H2O)(Cl)(dach)]+ all rapidly disappeared from the circulation. The other biotransformation products were persistent through at least 3h and could be responsible for the delayed toxicity of tetraplatin. Although some minor differences were observed between tetraplatin biotransformations at the toxic vs therapeutic doses, most biotransformation products were simply present at much greater concentrations at the toxic dose than at the therapeutic dose. Thus, our data suggest that dose-dependent differences in tetraplatin toxicity are probably attributable to the amount, rather than the type, of biotransformation products present in the plasma.

Incorporation of glycine into protein fractions of nuclei of liver and hepatoma.

Summary A procedure is described for the separation of the proteins of cell nuclei into several fractions which differ in solubilities and in glycine content. Twenty hours after intraperitoneal injection of glycine-l-C14 into adult rats bearing subcutaneously transplanted hepatomas the specific activities of the protein fractions of the nuclei of liver and tumor were in the descending order: residual lipoprotein > globulins > histone fraction III > histone fraction II. Each of the protein fractions of the nuclei of liver cells had higher specific activities than corresponding protein fractions of the hepatoma with the notable exception of the histones which were in the reverse relationship.

Inhibition of aminoacylation and polypeptide synthesis by chloroquine and primaquine in rat liver in vitro

Abstract The effects of chloroquine and primaquine on aminoacylation and polypeptide synthesis in subcellular rat liver systems were investigated. Both drugs exhibited concentration-dependent inhibition of phenylalanine incorporation into aminoacyltRNA. Under conditions where the phenylalanyl-14C-tRNA concentration remained at a “steady state” level, even in the presence of chloroquine or primaquine, the poly U-dependent polyphenylalanine synthesis exhibited concentration-related inhibition by the drugs. When choroquine or primaquine was added several minutes after addition of poly U, the inhibition was not abolished. The drugs did not bring about premature chain termination. Both chloroquine and primaquine inhibited peptidyl transferase, as measured by transfer of polypeptide to puromycin-3H.

INHIBITION OF PRECURSOR INCORPORATION INTO NUCLEIC ACIDS OF MAMMALIAN TISSUES BY ANTIMALARIAL AMINOQUINOLINES

1 Chloroquine, primaquine and ethidium inhibited thymidine incorporation into deoxyribonucleic acid of rat tissues when administered concurrently with the labelled precursor. 2 Chloroquine and primaquine inhibited the incorporation of uridine and adenine, but not orotate, into various ribonucleic acid fractions of liver of rats and mice. These drugs had no effect on leucine incorporation into hepatic protein in rats or mice. 3 Although chloroquine and primaquine are active against different stages in the life cycle of the malarial parasites, the two aminoquinolines exert similar effects in rodent tissues.

Inhibition of inosine monophosphate dehydrogenase by sesquiterpene lactones

Inosine monophosphate (IMP) dehydrogenase had previously been determined to be a likely target enzyme for the sesquiterpene lactones, a class of potential anti-neoplastic drugs. IMP dehydrogenase was purified approx. 770-fold from the P-388 lymphocytic leukemia tumor cell line. The Km values for the substrates, IMP and NAD, were determined to be 12 microM and 25 microM, respectively. Xanthine monophosphate (XMP) was shown to be a competitive inhibitor with a Ki of 67 microM. Mycophenolic acid gave mixed-type inhibition with a Ki of 8 nM for the noncompetitive component and a Ki of 2 nM for the competitive component. Dissociation constants (Kd) and rate constants for inhibition of IMP dehydrogenase by nine different sesquiterpene lactones were determined. The highest Kd was seen with 2,3-dihydrohelenalin while the lowest Kd was observed with bis-helenalinyl malonate. Binding of the drugs by IMP dehydrogenase increased as the size of the drug increased. Also, changes in structure at position 6 had a relatively large effect on the Kd. There was no correlation with hydrophobicity, as determined by octanol/water partition. The first-order rate constants for the reaction of the sesquiterpene lactones with IMP dehydrogenase (k1) and the second-order rate constants for the reaction of the sesquiterpene lactones with glutathione (k2) were also determined. The rate constants for most of the sesquiterpene lactones with the alpha-methylene-gamma-lactone moiety were similar and were approximately twice as great as the rate constants for those sesquiterpene lactones with only the alpha, beta-unsaturated cyclopentenone ring. Microlenin had approximately 5-times the reactivity of the other sesquiterpene lactones towards IMP dehydrogenase, but had approximately the same reactivity towards glutathione, suggesting that it was bound to the enzyme in a way which facilitated its reaction with one or more essential sulfhydryls. The same procedure was used for a series of N-substituted maleimide compounds with the N-substituent ranging in size from a methyl group to a benzyl group. The binding of the maleimide compounds was generally tighter than for the sesquiterpene lactones and there was an increase in binding with size.

Interaction of Antimalarial Aminoquinolines (Primaquine, Pentaquine, and Chloroquine) with Nucleic Acids, and Effects on Various Enzymatic Reactions in Vitro

Two classes of aminoquinolines are effective as antimalarial drugs. The 4-aminoquinolines, including the prototype chloroquine, are active against the erythrocytic stage of Plasmodia. In contrast, the 8-aminoquinolines, including as representatives primaquine and pentaquine, are active against the tissue stages of the malaria parasite (Powell, 1966). In addition, chloroquine is also used for several other parasitic diseases and for rheumatoid arthritis.

Regular articleAcute toxicity of helenalin in BDF1 mice

The acute toxicity of helenalin, a sesquiterpene lactone isolated from Helenium microcephalum, was examined in male BDF1 mice. The 14-day LD50 for a single ip dose of helenalin in male mice was 43 mg/kg. A single ip injection of 25 mg helenalin/kg increased serum alanine aminotransferase (ALT), lactate dehydrogenase (LDH), urea nitrogen (BUN), and sorbitol dehydrogenase within 6 hr of treatment. Multiple helenalin exposures, ip injection of 25 mg helenalin/kg for 3 days, increased differential polymorphonuclear leukocyte counts and decreased lymphocyte counts. Serum ALT, BUN, and cholesterol levels were also increased by multiple helenalin exposures at 25 mg helenalin/kg/day. Helenalin significantly reduced liver, thymus, and spleen relative weights and histologic evaluation revealed substantial effects of multiple helenalin exposures on lymphocytes of the thymus, spleen, and mesenteric lymph nodes. No helenalin-induced histologic changes were observed in the liver or kidney. Multiple helenalin exposures (25 mg/kg/day) significantly inhibited hepatic microsomal enzyme activities (aminopyrine demethylase and aniline hydroxylase) and decreased microsomal cytochromes P-450 and b5 contents. Three concurrent days of diethyl maleate (DEM) pretreatment (3.7 mmol DEM/kg, 0.5 hr before helenalin treatment) significantly increased the toxicity of helenalin exposure. The present studies indicate that the hepatic microsomal drug metabolizing system and lymphoid organs are particularly vulnerable to the effects of helenalin. In addition, helenalin toxicity is increased by DEM pretreatments which have been shown to decrease glutathione concentrations.

Reduction of Tetrachloro(dl-trans)1,2-diaminocyclohexaneplatinum(IV) (Tetraplatin) Toxicity by the Administration of Diethyldithiocarbamate (DDTC), S-2(3-Aminopropylamino)ethylphosphorothioic Acid (WR-2721), or Sodium Selenite in the Fischer 344 Rat,

Diethyldithiocarbamate (DDTC), S-2(3-aminopropylamino)ethylphosphorothioic acid (WR-2721), and sodium selenite have all been shown to effectively reduce cisplatin toxicity. As a result, we have investigated the efficacy of these compounds to reduce the toxicity associated with tetrachloro(dl-trans)1,2-diaminocyclohexaneplatinum(IV) (tetraplatin), a second-generation platinum compound recently approved for phase I/II clinical trials. The dose-limiting toxicities associated with tetraplatin (16.5 mg/kg) in the Fischer 344 male rat were nephrotoxicity, myelosuppression, and gastrointestinal toxicity. The nephrotoxicity in Fischer 344 rats was effectively reduced by treatment with either DDTC (750 mg/kg ip) 0.5 hr after tetraplatin or WR-2721 (200 mg/kg ip) 0.5 hr before tetraplatin as determined by blood urea nitrogen and creatinine values. Diarrhea was evident in 95% of the rats treated with tetraplatin alone while it was not evident in any of the DDTC- or WR-2721-protected rats. Only DDTC was moderately effective in preventing tetraplatin-induced decreases in platelet and lymphocyte counts. Histopathology confirmed DDTC protection of renal, intestinal, and lymphoid tissues and WR-2721 protection of renal and intestinal tissues. Sodium selenite was ineffective in reducing tetraplatin-induced damage when administered 4 hr before tetraplatin at doses of 0.5, 1.0, or 2.0 mg/kg. The results suggest that DDTC may allow for increased dosages of tetraplatin by ameliorating the toxic side effects of the drug. WR-2721 may also have some usefulness in tetraplatin therapy, but it does not reduce as wide a variety of toxic side effects as DDTC.