Molecular basis for hycanthone drug action in schistosome parasites.

Hycanthone (HYC) is a retired drug formerly used to treat schistosomiasis caused by infection from Schistosoma mansoni and S. haematobium. Resistance to HYC was first observed in S. mansoni laboratory strains and in patients in the 1970s and the use of this drug was subsequently discontinued with the substitution of praziquantel (PZQ) as the single antischistosomal drug in the worldwide formulary. In endemic regions, multiple organizations have partnered with the World Health Organization to deliver PZQ for morbidity control and prevention. While the monotherapy reduces the disease burden, additional drugs are needed to use in combination with PZQ to stay ahead of potential drug resistance. HYC will not be reintroduced into the schistosomiasis drug formulary as a combination drug because it was shown to have adverse properties including mutagenic, teratogenic and carcinogenic activities. Oxamniquine (OXA) was used to treat S. mansoni infection in Brazil during the brief period of HYC use, until the 1990s. Its antischistosomal efficacy has been shown to work through the same mechanism as HYC and it does not possess the undesirable properties linked to HYC. OXA demonstrates cross-resistance in Schistosoma strains with HYC resistance and both are prodrugs requiring metabolic activation in the worm to toxic sulfated forms. The target activating enzyme has been identified as a sulfotransferase enzyme and is currently used as the basis for a structure-guided drug design program. Here, we characterize the sulfotransferases from S. mansoni and S. haematobium in complexes with HYC to compare and contrast with OXA-bound sulfotransferase crystal structures. Although HYC is discontinued for antischistosomal treatment, it can serve as a resource for design of derivative compounds without contraindication.

Molecular analysis of the TK locus in L5178Y large and small colony mouse lymphoma cell mutants induced by hycanthone methanesulfonate.

Mouse lymphoma cells of the L5178Y TK + /-3.7.2C line were exposed to varying concentrations of the anti-schistosomal drug hycanthone methanesulfonate. The trifluorothymidine (TFT)-resistant cells fell into two classes based on colony size. Southern blot analyses were performed using NcoI-digested DNA from a number of large and small mutant colonies from each treatment group. Two different restriction fragment banding patterns were identified in these analyses, those colonies that contained the 6.4 kb NcoI restriction fragment and those that did not. A total of 471 mutant colonies were analyzed and 84.5% (398) of these colonies did not exhibit the 6.4 kb fragment. There did not appear to be a hycanthone methanesulfonate dose response effect in the number of colonies that did not contain the 6.4 kb fragment among the treated groups. In addition, 82% (154 out of 188) of spontaneous mutants did not contain the 6.4 kb fragment. The results imply that greater than 80% of all spontaneous mutations found in the mouse lymphoma assay regardless of colony size do not contain the 6.4 kb fragment and each may be considered to be a large scale mutation. In addition, greater than 80% of the hycanthone induced mutations in the mouse lymphoma assay do not contain the 6.4 kb fragment and thus may be considered to be a large scale mutation.

Sex differences in micronucleus induction with hycanthone methanesulfonate in bone marrow cells of mice.

The clastogenic effect of the antischistosomal drug hycanthone methanesulfonate was studied with the micronucleus test in mouse bone marrow cells. Male and female (102/El x C3H/El)F1 mice were treated with single i.p. injections. Bone marrow was sampled 18, 24 and 30 h after treatment with 100 mg/kg. The highest micronucleus yield occurred at 24 h. The dose response for micronucleus induction at 24 h after treatment was non-linear for doses between 5 and 300 mg/kg. The lowest effective dose was 5 mg/kg for females and 10 mg/kg for males. The experiments revealed a significantly higher sensitivity of female mice for the induction of micronuclei in polychromatic erythrocytes by hycanthone methanesulfonate. This result supports the recommendation to use both sexes for quantitative assessment of genotoxicity in the micronucleus test.

Female-specific mutagenic response of mice to hycanthone.

Male and female gametogeneses differ markedly in all mammals. While male germ cells are continuously being produced from stem cells throughout the reproductive life span, the number of female germ cells is fixed during prenatal development and, soon after birth, all of the oocytes are arrested in a modified diplotene, or dictyate, stage. Following puberty, dictyate oocytes are hormonally triggered to mature either singly or in groups, resulting in ovulation and the completion of the first meiotic division. It has been hypothesized that female mice are more susceptible to dominant lethal effects of intercalating agents than male mice because oocyte chromosomes, which are arrested in a diffuse state, are generally more accessable to intercalation than are the more condensed chromosomes present within most male germ cell stages. This hypothesis was further tested using the intercalating agent hycanthone methane-sulfonate. Effects of hycanthone were studied in maturing and primordial oocytes and in male germ cells throughout spermatogenesis. No induction of dominant lethality was observed for treated males while a significant increase in embryonic death, expressed around the time of implantation, was observed in females that mated within 4.5 days after treatment. These effects were the result of dominant lethal mutations induced in maturing oocytes and not of maternal toxicity as indicated by the presence of chromosomal aberrations observed at first-cleavage metaphase of zygotes obtained from treated females. These results add support to the hypothesis that certain intercalating chemicals, which are not mutagenic to male mice, may be mutagenic to females and point to a need for more in-depth studies of female-specific mutagenesis.

Chromosome analysis of small and large L5178Y mouse lymphoma cell colonies: comparison of trifluorothymidine-resistant and unselected cell colonies from mutagen-treated and control cultures.

Mutagenesis assays at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells frequently yield mutant colonies with a bimodal size distribution. The objectives of this study were to determine whether a relationship exists between mutant colony size and chromosomal aberrations and whether the colony-size distributions obtained from this assay can indicate the clastogenic activity of a test chemical. Cells from 8 different types of L5178Y mouse lymphoma cell colonies were examined for chromosomal abnormalities within 10 cell generations after colony isolation. The colonies included small (sigma) and large (lambda) unselected cell (UC) and trifluorothymidine-resistant (TFTr) colonies derived from TK +/- cell cultures treated with the solvent dimethyl sulfoxide (DMSO) or hycanthone methanesulfonate (HYC). Chromosome abnormalities were present in cells from 12% (7/60) of the UC colonies, but there was no apparent relationship between colony diameter and the presence of chromosomal abnormalities. Abnormalities affecting chromosome 11, which is believed to be the site of the TK gene, were not observed in cells from UC colonies. Abnormalities affecting chromosome 11 were observed only in cells from sigma-TFTr colonies irrespective of whether they were spontaneous (5/15 colonies) or induced by HYC (4/15 colonies). Overall, 30% (9/30) of sigma-TFTr colonies had cells with an abnormal chromosome 11 and 10% (3/30) had abnormalities affecting other chromosomes. Abnormalities affecting chromosome 11 were not observed in cells from lambda-TFTr colonies (0/30 colonies). The observation of only 30% of sigma-TFTr colonies with chromosome damage affecting chromosome 11 indicates that other mechanisms, in addition to chromosome damage at the level of resolution used in this study (i.e., 200-300 chromosome bands). contribute to small TFTr colony size.

Binding of tritiated hycanthone and hycanthone N-methylcarbamate to macromolecules of drug-sensitive and drug-resistant schistosomes.

Adult Schistosoma mansoni of the hycanthone-sensitive and of the hycanthone-resistant strain were exposed in vitro to tritium-labeled hycanthone. The drug was taken up in similar amounts by the two strains, a result which is not compatible with hypothetical mechanisms of resistance based on reduced drug entry into the schistosomes. Labeled hycanthone was found to bind irreversibly to macromolecules of sensitive schistosomes, whereas the binding was minimal in resistant worms. In particular, the DNA of sensitive schistosomes showed high levels of tightly bound hycanthone, while the corresponding fraction of resistant schistosomes failed to do so. Female schistosomes and immature worms, which are less sensitive to hycanthone, showed a diminished drug-DNA binding with respect to adult males. Tritiated hycanthone N-methylcarbamate, which is effective against sensitive and resistant schistosomes, bound in similar amounts to the DNA of both strains. These results strongly support a previously proposed mechanism of action of hycanthone, which is based essentially on the alkylation of worm macromolecules by a drug derivative produced in sensitive schistosomes.

Preparation and antischistosomal and antitumor activity of hycanthone and some of its congeners. Evidence for the mode of action of hycanthone.

The synthesis of a series of esters of hycanthone (HC) and 7-hydroxyhycanthone, their antitumor activity, and their antischistosomal effects on HC-sensitive and HC-resistant schistosomes are reported. Binding studies using tritium-labeled HC and hycanthone N-methylcarbamate (HNMC) with calf thymus DNA provided evidence that HNMC but not HC alkylated the DNA. Tritiated HNMC also bound to the DNA of intact HeLa cells exposed to the drug while very little tritiated HC bound to DNA under the same conditions. The mechanism proposed previously to account for the antischistosomal action of HC, namely, drug esterification followed by alkylation of DNA, applies also to the antitumor action of the drug as shown in Scheme I.

Stable dicentric chromosomes induced by chemical mutagens in L5178Y mouse lymphoma cells.

Stable, tandem dicentric chromosomes were discovered in two mutant cell colonies resulting from exposure of L5178Y mouse lymphoma cells to chemical mutagens. These unusual dicentrics were present in all metaphase cells examined from these colonies, even after approximately 65 cell generations in culture. Observation of cells in metaphase and anaphase suggests that the interstitial centromere in these dicentrics is non-functional, and that the terminal centromere is solely responsible for their orderly anaphase segregation.

Antimuscarinic activities of hycanthone analogs: possible relationship with animal toxicity.

The antimuscarinic activity of hycanthone and five antischistosomal analogs was determined in three biological assays of cholinergic systems. A linear relationship was established between the LD50 values of hycanthone analogs in mice and 1) the Ki values obtained from the inhibition of [3H]quinuclidinyl benzilate binding to the muscarinic receptors of N4TG1 neuroblastoma cells; 2) the I50 values obtained from the inhibition of alpha-amylase secretion induced by carbachol in pancreatic acini cells; and 3) the KB values obtained from the inhibition of guinea-pig ileum contraction induced by acetylcholine. The linear relationship established between antimuscarinic potency and toxicity in mice suggests that a possible relationship exists between the toxicity of the hycanthone analogs and their antimuscarinic activities. On the other hand, no correlation was established between antischistosomal efficacy and antimuscarinic potency. The Ki and I50 values ranged from 10(-7) to 10(-5) M for the inhibition of the binding of [3H]quinuclidinyl benzilate to the muscarinic receptors and for the inhibition of alpha-amylase secretion. The KB values determined by the guinea-pig ileum assays were approximately 10(-5) to 10(-6) M. The ranking of antimuscarinic potency of the compounds in the three different assays were in good agreement.

Analogues of hycanthone and lucanthone as antitumor agents.

Hycanthone analogues (5 and 6) containing 7-substituted hydroxyl groups were prepared and evaluated as antitumor agents. These compounds were significantly more active than the corresponding unsubstituted derivatives. The 7-hydroxylated 4-(hydroxymethyl)-9H-xanthen-9-ones, 11 and 12, were also active antitumor agents. However, the 7-hydroxy-9H-xanthen-9-one counterparts of the 7-hydroxylucanthones were totally devoid of antitumor activity. Results obtained thus far are consistent with the hypothesis that 4-hydroxymethyl substituents in the 9H-xanthen-9-one and 9H-thioxanthen-9-one series are required for antitumor activity.

Mutagenicity of hycanthone in Drosophila: additional results and a comparison with some analogs.

The data reported in this paper extend earlier results on the effects of hycanthone in Drosophila. The main findings are the following. (1) A refined brood-pattern analysis of hycanthone-induced sex-linked recessive lethals confirmed the specific sensitivity of mid- and late spermatids. Injection of young males 0--20 h old) did not cause a shift in the brood pattern, but tended to produce higher rates of recessive lethals than injection of 4-day-old males, although the difference was not significant. (2) An autosomal recessive lethal test (chromosome 2) similarly showed a low sensitivity of premeiotic stages. (3) Feeding of hycanthone was much less effective than injection. This difference was not observed for the methyl analog lucanthone. From the observation that hycanthone- and lucanthone-induced mutations exhibited different germ-cell-stage sensitivity patterns, it was concluded that lucanthone does not (at least not exclusively) act via metabolic activation to hycanthone. (4) After injection, the hycanthone analogs IA-3-N-oxide and IA-4-N-oxide were marginally mutagenic. (5) It was shown previously that hycanthone was ineffective in producing breakage events, in Drosophila. In this report, hycanthone is shown to be weakly active in inducing ring-X chromosome loss. This emphasizes the relative sensitivity of the ring-X-loss test, in comparison with the tests that detect translocations or dominant lethals.

Genetic analysis of adenine-3 mutants induced by hycanthone, lucanthone and their indazole analogs in Neurospora crassa.

Ad-3 mutants induced by hycanthone, lucanthone and their indazole analogs, IA-3, IA-4 and IA-5 were studied to characterize the genetic alterations produced by these agents in Neurospora crassa. The results of genetic analysis indicate that, in marked contrast to past experiments with chemical mutagens on heterokaryon 12, all of these antischistosomal agents induce a very high frequency of multilocus deletions.

Action of antischistosomal drugs, hycanthone and its analog 1A-4 N-oxide, on viral interferon induction.

Two antischistosomal drugs, hycanthone and its indazole analog 1A-4 N-soxide, of which the former is a potent mutagen, differed markedly in their ability to depress interferon induction by influenza virus in rhesus monkey kidney (LLC-MK2) cell cultures. At the concentration of 10 micron/ml, hycanthone reduced interferon yields as much as 73%; the same quantity of analog had no adverse effect on interferon induction. Pretreatment of cell cultures with the analog negated the inhibitory activity of hycanthone on viral interferon induction; however, this did not occur when the sequence was reversed. Interferon-mediated antiviral cellular resistance was not affected when cell cultures were pretreated with either hycanthone or 1A-4 N-oxide. A possible association may exist between the mutagenicity of antischistosomal drugs and their ability to affect interferon synthesis.

Experimentally produced resistance of Schistosoma mansoni to hycanthone.

Genetically transferred resistance to the antischistosomal drug hycanthone has been observed in several strains of Schistosoma mansoni: 1) in the progeny of worms to whose hosts hycanthone had been administered 54 to 70 days after exposure to cercariae (Type I); 2) in the progeny of worms to whose hosts hycanthone had been administered when the worms were still in an immature stage (27 to 29 days after percutaneous cercarial exposure) (Type II); and 3) in the progeny of worms from hosts that had been infected with cercariae of one sex followed by infection with the opposite sex 2 to 58 weeks later (Type III). In types I and II, drug resistance was transferred maternally. Hycanthone-resistant schistosomes were cross-resistant to antischistosomal drugs structurally related to hycanthone, such as oxamniquine and two chloro-indazole analogs of hycanthone, but not to niridazole and to another nitroheterocyclic compound.

The activity of a benzothiopyranoindazole (IA-4 N-oxide) against Schistosoma mansoni infection in rhesus monkeys.

The prophylactic and therapeutic efficacies of IA-4 N-oxide against Schistosoma mansoni in rhesus monkeys (Macaca mulatta) have been evaluated. Ten monkeys were divided into 5 groups of 2 monkeys each. All monkeys were exposed to S. mansoni cercariae on day 0 and treatment groups were established as follows: untreated controls, oral prophylactic administration, intramuscular prophylactic administration, oral therapeutic administration, and intramuscular therapeutic administration. Analysis of parasitologic and pathologic criteria indicate that the compound is most effective when administered as a therapeutic regimen. Complete cures were effected in these monkeys. Prophylactic treatments resulted in a delay in onset of patency and reductions in fecal egg excretion, worm burdens, and tissue damage.

Mutagenic activities in vitro and in vivo of five antischistosomal compounds.

Five antischistosomal compounds--hycanthone, two of its chloroindazole analogs (IA-4 and IA-4 N-oxide), oxamniquine, and metrifonate--were tested for mutagenic activity, using Salmonella typhimurium strains TA 98 and TA 100 under in vitro and in vivo (host-mediated) conditions. In all assay systems hycanthone exhibited by far the highest mutagenic potency. Although oxamniquine and metrifonate had low metagenic activity in vitro and although their administration resulted in urine of low metagenic activity, their host-mediated mutagenic activities on strain TA 100 were fairly high. Confirming earlier studies with a less sensitive Salmonella strain, TA 1535, IA-4 N-oxide was found to be less metagenic than IA-4. Orally administered IA-4 and IA-4-oxide were less mutagenic under in vivo conditions than an equal dose administered intramuscularly. By contrast, the antihistosomal activity of a given dose of each compound was the same, regardless of which of these two routes was used, suggesting that mutagenic and antischistosomal effects are produced by different metabolites. The observations reported in this paper provide additional evidence that mutagenic activities can be dissociated from desired chemotherapeutic effects by suitable structural modifications.