Genetic toxicology testing of the antimalarial drugs chloroquine and a new analog, AQ-13

AQ-13 ([N1-(7-chloro-quinolin-4yl)-3-(N3,N3-diethylamino)propylamine] dihydrochloride trihydrate) is an aminoquinoline antimalarial drug that is effective against chloroquine-resistant strains of Plasmodium falciparum. It is structurally similar to the widely used chloroquine diphosphate (CQ). We evaluated these drugs in the three assays currently recommended by the International Conference on Harmonization (ICH): bacterial mutagenesis in Salmonella typhimurium and Escherichia coli, mammalian cell mutagenesis in L5178Y mouse lymphoma cells, and micronucleus induction in rat bone marrow. A small but statistically significant increase in revertant colonies was produced by CQ with Salmonella tester strain TA98 without metabolic activation (MA) and by AQ-13 with strain TA1537 both with and without MA. In L5178Y cells, testing of CQ and AQ-13 up to cytotoxic concentrations with and without MA produced no increase in mutant colonies and no increase in the numbers of small colonies. Slight decreases in the ratio of polychromatic erythrocytes (PCE) to red blood cells (RBC) were observed in male and female rats treated with CQ and in females only treated with AQ-13; however, none of these changes was statistically significant. No increases in the frequency of micronucleated PCE were observed at any dose level of CQ or AQ-13. Although both CQ and AQ-13 showed weak bacterial mutagenicity, this mutagenic effect was not confirmed in either the mouse lymphoma mutagenesis assay or the micronucleus assay. These results indicate that CQ and AQ-13 should pose minimal risk of genotoxic damage in human populations being administered these drugs.     

Comparative analysis of in vitro rat liver metabolism of the antimalarial primaquine and a derived imidazoquine

The present study provides proof-of-concept regarding the expectedly high enzymatic stability of primaquinederived imidazolidin-4-ones, imidazoquines, formerly developed as alternatives to the parent antimalarial with potentially improved oral bioavailability [J. Med. Chem., 2009, 52, 7800-7807]. This study provides relevant experimental evidence on the remarkably low propensity of imidazoquines to undergo metabolic conversions mediated by rat liver enzymes. This, together with favourable key ADME parameters previously predicted for these compounds [Bioorg. Med. Chem. Lett. 2009, 19, 6914-6917], and proven lack of acute toxicity in mice, further reinforces the role of imidazoquines as reference leads for the development of novel primaquine surrogates. This is a particularly relevant issue in the present status of malaria chemotherapy worldwide, where primaquine remains the sole drug in clinical use able to block transmission between infected persons and the insect vector and to effectively act on liver-stage parasite forms, including hypnozoites.     

Synthesis and antimalarial activity of new amino analogues of amodiaquine

Amodiaquine remains one of the most prescribed antimalarial 4-aminoquinoline. To assess the importance of the 4'-hydroxyl group and subsequent hydrogen bond in the antimalarial activity of amodiaquine (AQ), a series of new analogues in which this functionality was replaced by various amino groups was synthesized. The incorporation of a 3'-pyrrolidinamino group instead of the 3'-diethylamino function of AQ allowed the development of a parallel series of amopyroquine derivatives. The compounds were screened against both chloroquine (CQ)-sensitive and -resistant strains of Plasmodium falciparum and their cytotoxicity evaluated upon the MRC5 cell line. Antimalarial activity in a low nanomolar range was recorded showing that the 4'-hydroxy function can be successfully replaced by various amino substituents in terms of activity without any influence of the level of CQ-resistance of the strains. Furthermore the ability of the compounds to inhibit beta-hematin formation was measured in order to discuss the mechanism of action of these new compounds. Compounds 7d and 8d exhibit a high selectivity index and may be considered as promising leads for further development.     

Evaluation of the genotoxic effects of pyrimethamine, an antimalarial drug, in the in vivo mouse

Pyrimethamine is an antimalarial drug and a known teratogenic agent. With this drug, positive and negative results have been reported by various investigators in in vivo and in vitro genotoxicity/mutagenicity assays. In this investigation the genotoxic effects of pyrimethamine (PY) were tested in mice in vivo systems, using the bone marrow micronucleus test (MNT) and the transplacental MN test (TMNT). PY at the highest dose (40 mg/kg) induced statistically significant MN in bone marrow cells at 24 and 48 h. In the transplacental MN test, PY did not induce significant MN in fetal liver or in maternal bone marrow. Teratogenesis Carcinog. Mutagen. 20:65-71, 2000.     

In vitro evaluation of the genotoxic and cytotoxic effects of artesunate, an antimalarial drug, in human lymphocytes

Artesunate is one of the main antimalarial drugs used in several countries. It is a semisynthetic compound derived from artemisinin, a substance extracted from the Chinese plant, Artemisia annua L. Despite the widespread use of artesunate as an antimalarial drug, there is a lack of data regarding its genotoxic effects in human lymphocytes. Therefore, in this study, we used the comet assay and micronucleus test to evaluate the possible genotoxic effects of artesunate in cultured human lymphocytes. In addition, cell death by necrosis and apoptosis was also assessed. Cells exposed to different concentrations of artesunate showed a significant concentration-dependent increase (P < 0.05) in DNA damage index and micronuclei frequency. A significant increase in the frequency of apoptotic and necrotic cells was also observed. Our results showed that artesunate is a genotoxic and cytotoxic compound in cultured human lymphocytes.     

Enhanced antimalarial effects of chloroquine by aqueous Vernonia amygdalina leaf extract in mice infected with chloroquine resistant and sensitive Plasmodium berghei strains

Background

The emergence and spread of Plasmodium falciparum with resistance to chloroquine (CQ), the safest and cheapest antimalarial drug coupled with the increasing cost of alternative drugs especially in developing countries have necessitated the need to optimize antimalarial actions of plant extracts and restore chloroquine efficacy.

Objective

The present study determines the ability of Vernonia amygdalina leaf extract to enhance the prophylactic and therapeutic efficacy of chloroquine against Plasmodium berghei malaria in mice.

Methods

Chloroquine sensitive (P. berghei^S) and resistant (P.berghei^R) ANKA clones of Plasmodium berghei maintained by serial passage in mice were used to develop respective experimental rodent malaria models based on intraperitoneal injection of 10⁶ parasitize erythrocyte suspension in PBS (pH 7.2) and subsequent development of parasitaemia. These models were then used to investigate the prophylactic enhancement of chloroquine (CQ) at 5 mg/kg via combination with selected doses (31.25, 62.5, 125mg/kbw) of Vernonia amygdalina leaf extracts using a 4-day suppression test. Effect of these combinations on the therapeutic efficacy of CQ at 30mg/kg over 3 days were evaluated. Treatment outcomes including parasite clearance (PCT) and rescrudescent time (RT) were compared with CQ-chlorpheniramine combination. The acute toxicity of the extract-CQ combinations was also determined enzymatically.

Results

Prophylatically, chloroquine (5mg/kg) in combination with vernonia extracts achieved a dose-dependent (57.2–72.7%) suppression of parasitaemia due to CQ sensitive and resistant P berghei strains in the experimental animals. Therapeutically, chloroquine (30mg/kg for 3 days) combined with vernonia to dose-dependently shorten the parasite clearance times (2.6–4.4 vs. 4.8 days; P < 0.05 for CQ-V62.5/125 combination), prolong the recrudescent times (8.9–18.9 vs. 7.2 days; P < 0.05) and improve day 14 cure rate (66.7–100 vs. 58.3%) in the treated P. berghei^S infected mice compared to CQ monotherapy. Whereas CQ monotherapy failed, resolution of parasitaemia due to the CQ resistant parasite with day 14 cure rates of 25 – 100% were also observed with these combinations. In therapeutic terms, the potencies of CQ-V125 combination were comparable to those of CQ-chlorpheniramine (0.25mg/kg, 12hourly, 7 days) in the infected animals. Toxicity testing indicates that these combinations elicited mild to - moderate increases in the liver enzymes measured when administered orally to mice for 7 days.

Conclusion

This study indicates that Vernonia amygdalina leaf extract dose - dependently restore the efficacy of CQ against CQ resistance P. berghei malaria in mice.     

Formaldehyde-induced cytotoxic, genotoxic and mutagenic response in human lymphocytes and Salmonella typhimurium

The incidences of chromatid-type aberrations and sisterchromatidexchanges were significantly increased in human lymphocytestreated with formaldehyde (FA) in vitro. In the presence ofthe mammalian metabolic activation system, i.e. S9 mix, theyields were reduced, although not to control levels. With S9mix the structural chromosome damage induced by exposure to1.0 mM FA was qualitatively and quantitatively identical tothat induced by 0.05 mM cyclophosphamide (used as positive controlfor metabolic activation). Cell proliferation was clearly reducedwith or without the presence of S9 mix. In a plate assay withSalmonella typhimurium strain TA100 in the absence and presenceof S9 mix, a weak mutagenic response was observed. Using thepre-incubation method, FA induced without S9 mix a 1.6-foldand with S9 mix a 2.7-fold increase of revertant numbers overcontrols.     

Comparative study of the mutagenic and genotoxic activity associated with inhalable particulate matter in Rio de Janeiro air

We have determined the genotoxic and mutagenic activities associated with inhalable particulate matter (IPM) collected in Rio de Janeiro, Brazil, Camden, NJ, and Caldecott Tunnel, CA, and used these results to compare three different bioassays. Samples collected every 12 hr (Rio) or every 24 hr (Camden) were extracted sequentially with cyclohexane (CX), dichloromethane (DCM), and acetone (ACE), for a rough fractionation by polarity, and composites of the extracts were tested for mutagenicity using the Salmonella frame shift (TA98) and base substitution (TA100) tester strains, as well as for genotoxicity using the Rossman Microscreen bioassay based on the induction of lambda-prophage in a lysogenic Escherichia coli strain. All samples were tested without and with S9 metabolic activation. Maximum mutagenic and genotoxic activities were in the nonpolar (CX) and polar (ACE) fractions, respectively, indicating that these two assays detect different classes of compounds with different efficiencies. Oxidative aging of the Rio aerosol is indicated by a shift in activities in both tests from the less polar fractions in the day to the polar (ACE) fraction at night. The Rio TA98 mutagenic (18 rev/m3) and genotoxic (1.4 x 10(5) PFU/m3) activities were higher than those for Camden, an Eastern U.S. city, by factors of 1.4 and 2.8, respectively.     

Comparative efficacy of chloroquine and amodiaquine (25 and 35 mg/kg) in school children infected with P. falciparum (Brazzaville, March 1990)

The efficacy of 4 therapeutic schedules was compared in March and April 1990 in Brazzaville school children, aged between 6 and 8 years, with parasitaemia of at least 1,000 trophozoites of Plasmodium falciparum per mm3. It was possible to interpret 125 simplified in vivo tests. The results showed that the activity of amodiaquine is still relatively satisfactory. The activity of chloroquine was slightly lower with the schedule of 25 mg/kg but was good at 35 mg/kg. Although these results were obtained in children who were mostly asymptomatic, they show that the use of amino-4-quinolines is still justified, at least in the initial treatment of uncomplicated malaria in semi-immune congolese subjects.     

Nucleus may be the key site of chloroquine antimalarial action and resistance development

The first proposed hypothesis about the mechanism of chloroquine (CQ) action on malaria parasites is DNA intercalation hypothesis which indicates that the site of CQ action is within nucleus. Later on the interest of research was shifted from nucleus to lysosome due to the report of CQ accumulation within lysosome. The current opinions about CQ action and resistance are mainly based on the results of more than 30 years' studies on lysosome, which can be used to explain some facts but still remains incomplete and controversial. Based on recently published papers and our related data it is possible that the key CQ target protein may exist in nucleus. Development of CQ resistance is probably mainly due to the alteration in the CQ target protein or certain mechanism which prevents CQ from reaching its target protein in nucleus. In conclusion, the key site of CQ action may be in nucleus though it has not been well explored while CQ action in lysosome which has been well studied may be secondarily important in CQ action and resistance.     

The side effects of antimalarial drugs indicates a polyamine involvement in both schizophrenia and depression

The nature of the side effects produced by the antimalarials and a common moiety present in these drugs (spermidine) links the polyamines to both schizophrenia and depression. The raised incidence of psychosis in systemic lupus erythematosus and during pregnancy both associated with raised blood polyamine levels, makes the involvement of the polyamines in psychosis more likely.     

Inhibition of diamine oxidase by antimalarial drugs

The antimalarial drugs amodiaquine, quinacrine and chloroquine inhibit the catabolism of putrescine by the rat. Amodiaquine, the most potent of the three, does so in a dose-dependent fashion. This is attributed to the action in vivo of the drugs on diamine oxidase, an enzyme that is inhibited by them in vitro.     

In vitro activity of various antimalarials (chloroquine, amodiaquine, quinine and mefloquine) against 32 isolates of Plasmodium falciparum in French Guiana

The in vitro susceptibility of various Plasmodium falciparum isolates obtained from patients infected in French Guiana was tested in a semi-microtest, using four antimalarial drugs. 90.6% of 32 isolates were resistant to chloroquine, 40% to amodiaquine and 17% to quinine. All the 17 isolates were susceptible to mefloquine.     

Inhibiting effects of morphine,chloroquine, nonsteroidal and steroidal anti-inflammatory drugs on electrically-induced contractions of guinea-pig ileum and the reversing effect of prostaglandins

Twelve different non steroidal antiinflammatory drugs (NSAID), five steroidal antiinflammatory drugs, morphine and chloroquine have been added at various concentrations to in vitro electrically-stimulated preparations of guinea-pig ileum. They all inhibit the electrically-induced contractions. Prostaglandins E as well as nicotine reverse this inhibition. These reversing effects are less evident on totally inhibiting drug concentrations than on partially inhibiting drug concentrations. It is suggested that this inhibiting effect could be due mostly to nervous as well as muscular membrane permeability changes induced by these drugs and not to inhibition of prostaglandin synthesis which could be only proposed as partial explanation for NSAID effects. The reversing action of nicotine could be related to a release of acetylcholine while a sensitization of guinea-pig ileum smooth muscle to acetylcholine could explain the reversing properties of prostaglandins.