Antimalarial Agents Chloroquine Research Articles

In vitro assessment of the pharmacodynamic properties and the partitioning of OZ277/RBx-11160 in cultures of Plasmodium falciparum

Using synchronous cultures of Plasmodium falciparum malaria, the stage sensitivity of the parasite to OZ277 (RBx-11160), the first fully synthetic antimalarial peroxide that has entered Phase II clinical trials, was investigated in vitro over a concentration range of 1 x to 100 x the IC50. Secondly, partitioning of OZ277 into P. falciparum-infected red blood cells (RBCs) and uninfected RBCs was studied in vitro by measuring its distribution between RBCs and plasma (R/P). The effects of timed in vitro exposure (1, 6, 12 or 24 h) to OZ277 were monitored by incorporation of [3H]hypoxanthine into parasite nucleic acids and by light-microscopic analysis of parasite morphology. Partitioning studies were performed with radiolabelled [14C]OZ277. After 1 h of exposure to OZ277 at the highest concentration (100 x the IC50) followed by removal of the compound, the hypoxanthine assay showed that growth of mature stages of P. falciparum was reduced to below 20%. Young ring forms were slightly less sensitive (43% growth). Similar stage-specific profiles were found for the antimalarial reference compounds artemether and chloroquine. Strong inhibition (< or = 6% growth) of all parasite stages was observed when the parasites were exposed to each of the three compounds for 6 h or longer. After removal of the compounds, the parasites did not recover, indicating that the observed growth inhibitions were cytotoxic rather than cytostatic. Pyrimethamine was confirmed to be active exclusively against young schizonts. Light-microscopic analysis also demonstrated the specificity of pyrimethamine against the schizont forms and showed that OZ277, artemether and chloroquine attenuated parasite growth more rapidly than did pyrimethamine. The R/P for OZ277 was 1.5 for uninfected RBCs and up to 270 for infected RBCs. The present study indicates similar stage-specific profiles for OZ277 and for the more well-established antimalarial agents artemether and chloroquine. Secondly, the study describes a significant accumulation of radiolabelled OZ277 in P. falciparum-infected RBCs.

A novel synthesis of substituted quinolines using ring-closing metathesis (RCM): its application to the synthesis of key intermediates for anti-malarial agents

A method for synthesizing substituted quinolines using ruthenium-catalyzed ring-closing metathesis as a key step has been developed. Substituted 1,2-dihydroquinolines, 4-silyloxy-1,2-dihydroquinoline and 4-methoxy-1,2-dihydroquinoline, were successfully synthesized in excellent yields via ene–ene metathesis and silyl or alkyl enol ether–ene metathesis, respectively. The synthetic intermediates of the antimalarial agents quinine, chloroquine, and PPMP–quinine hybrid were efficiently synthesized by this methodology.

HPLC and HPTLC assays for the antimalarial agents Chloroquine, Primaquine and Bulaquine

A combination Kit for antirelapse treatment of P. vivax malaria, consisting of Chloroquine phosphate tablets and Bulaquine capsules has been recently developed, and marketed under the trade name Aablaquine. Bulaquine is prepared from Primaquine. Several methods of analysis are reported for each drug separately as well two drugs in combination but no method for simultaneous estimation of these three drugs is known. Therefore, the present study was undertaken to develop a sensitive and reproducible high performance liquid chromatographic as well as high performance thin layer chromatographic assay method for the simultaneous estimation of Chloroquine, Primaquine and Bulaquine.

PH-Dependent regulation of camptothecin-induced cytotoxicity and cleavable complex formation by the antimalarial agent chloroquine

Two classes of drugs interact with DNA topoisomerase (topo) I, namely topoi poisons such as the camptothecins, which create DNA single-strand breaks and the catalytic inhibitors, which do not. Here, we demonstrate that the antimalarial agent chloroquine is a catalytic inhibitor of eukaryote topoi, as the drug inhibited topol-mediated DNA relaxation. Chloroquine is known to be a topoll catalytic inhibitor and as such is able to inhibit the activity of a topoll poison, i.e. etoposide. We now show that chloroquine also inhibits the topoi poison camptothecin as camptothecin-stimulated nicking of plasmid DNA was inhibited by chloroquine. These observations also apply to endogenous topoi in whole cells. Accordingly, camptothecin-induced single-strand breaks as well as Cytotoxicity were antagonised by chloroquine. Further, in a band depletion assay in whole cells, chloroquine prevented camptothecin-mediated topoi trapping, indicating that chloroquine inhibits topoi by interfering with the DNA binding step of the enzyme. In contrast to camptothecin, chloroquine is a weak base and therefore does not enter the cell if the extracellular fluid is acidic, as is the case in most solid tumors. This leads to the possibility of directing Cytotoxicity to solid tumors with low extracellular pH by combining a neutral anticancer agent, i.e. camptothecin with a weak base antagonist, i.e. chloroquine. To test the feasibility of this principle, we investigated the drug combination at varying extracellular pH. We found that the antagonising effect of chloroquine on camptothecin-mediated trapping of topol and DNA single-strand break formation was abolished at acidic extracellular pH. In a clonogenic assay, camptothecin in combination with chloroquine selectively killed cells at low pH (6.2), while camptothecin Cytotoxicity was antagonised by chloroquine at normal pH (7.2). In conclusion, we show that the topoi catalytic inhibitor chloroquine inhibits camptothecin and that chloroquine can target the cytotoxic effect of camptothecin to tumor cells in acidic environments.

The antimalarials quinacrine and chloroquine induce weak lysosomal storage of sulphated glycosaminoglycans in cell culture and in vivo

The antimalarial agents quinacrine and chloroquine are well known as potent inducers of lysosomal storage of polar lipids (lipidosis) in cell culture and in vivo. In previous experiments on cultured fibroblasts, chloroquine was shown to additionally cause weak lysosomal storage of sulphated glycosaminoglycans (GAGs) thus inducing mucopolysaccharidosis (MPS). In the present study, quinacrine was investigated for this ability, because we wished to know whether or not the acridine ring system in quinacrine would enhance the M PS-inducing potency as compared to chloroquine carrying an isoquinoline ring system. Tilorone (2,7-bis[2-(diethylamino)ethoxy]fluoren-9-one) known as a potent inducer of MPS served as reference compound. The compounds were compared at a concentration (3 μM) which did not enhance the secretion of the lysosomal enzyme β-hexosaminidase (E.C. 3.2.1.52), since this would be an indication of unspecific drug effects upon the endosomal/lysosomal compartments of the cell. Additionally the liver of quinacrine- and chloroquine-treated rats was examined with the question whether the lysosomal GAG storage induced by either drug in cell culture had an equivalent in intact organisms. Both, in cell culture and in vivo, quinacrine was found to be a more potent inducer of lysosomal GAG storage than was chloroquine. The results suggest that the acridine ring system favours this drug side effect as compared with the bicyclic isoquinoline ring system. On the other hand, quinacrine was significantly less potent than tilorone and the symmetrically substituted acridine derivative 3,6-bis[2-(diethylamino)ethoxy]acridine investigated previously. This suggests that the asymmetric structure of the quinacrine molecule reduces the potency as compared to the symmetrically substituted bisbasic compounds with planary tricyclic ring systems such as tilorone and congeners.

Susceptibility of melanized and nonmelanized Cryptococcus neoformans to the melanin-binding compounds trifluoperazine and chloroquine.

Cryptococcus neoformans is an opportunistic fungal pathogen which becomes heavily melanized in the presence of phenolic substrates such as L-dopa. Various drugs are known to bind to melanin with high affinity, including the antipsychotic agent trifluoperazine and the antimalarial agent chloroquine. We hypothesized that drugs which bind melanin may have different toxicities for melanized and nonmelanized C. neoformans cells. The effects of trifluoperazine and chloroquine or C. neoformans were determined by measuring cell viability after exposure to these drugs. Cell viability was measured by CFU determination and flow cytometry with propidium iodide staining. Melanized cells were more susceptible than nonmelanized cells to the fungicidal effects of trifluoperazine. Chloroquine had no fungicidal effect on either melanized or nonmelanized C. neoformans under the conditions studied. Flow cytometry of trifluoperazine-treated C. neoformans cells stained with the mitochondrial stain dihydrorhodamine 123 revealed fluorescence changes consistent with mitochondrial damage. Our results indicate that melanized and nonmelanized C. neoformans cells can differ in susceptibility to certain drugs and suggest that strategies which target melanin may be productive for antifungal-drug discovery.

Mechanism of action of hydroxychloroquine as an antirheumatic drug

The antimalarial agents chloroquine and hydroxychloroquine have been used widely for the treatment of rheumatoid arthritis and systemic lupus erythematosus. These compounds lead to improvement of clinical and laboratory parameters, but their slow onset of action distinguishes them from glucocorticoids and nonsteroidal antiinflammatory agents. Chloroquine and hydroxychloroquine increase pH within intracellular vacuoles and alter processes such as protein degradation by acidic hydrolases in the lysosome, assembly of macromolecules in the endosomes, and posttranslation modification of proteins in the Golgi apparatus. It is proposed that the antirheumatic properties of these compounds results from their interference with "antigen processing" in macrophages and other antigen-presenting cells. Acidic cytoplasmic compartments are required for the antigenic protein to be digested and for the peptides to assemble with the alpha and beta chains of MHC class II proteins. As a result, antimalarials diminish the formation of peptide-MHC protein complexes required to stimulate CD4+ T cells and result in down-regulation of the immune response against autoantigenic peptides. Because this mechanism differs from other antirheumatic drugs, antimalarials are well suited to complement these other compounds in combination drug therapy.

Antimelanoma activity of chloroquine, an antimalarial agent with high affinity for melanin.

The antimalarial agent chloroquine is known for high affinity for melanin. This 4-aminoquinoline derivative was examined for anti-melanoma activity and uptake into melanoma cells. Chloroquine inhibited growth of cultured melanoma cells; the effect was much greater to a moderately pigmented cell line HMV-II than to a nonpigmented HMV-I. Treatment with chloroquine at a dose of 62 mg/kg i.p. for 12 days prolonged by 71% the life span of mice bearing B16 melanoma, while 24-day treatment at 31 mg/kg resulted in a 81% increase in life span. HMV-II cells showed a two-fold increase in uptake of chloroquine as compared with HMV-I cells. Chloroquine, 24 hr after administration to mice implanted s.c. with B16 melanoma, was selectively accumulated in the pigmented tissues, melanoma and eyes. Other nonpigmented tissues such as the liver, lung, and kidney showed rapid uptake (within 1 hr) and release. These results suggest that chloroquine is toxic to pigmented melanoma cells, the process being partly mediated by binding to melanin.

Enantioselective chromatography of the antimalarial agents chloroquine, mefloquine, and enpiroline on a α1 ‐acid glycoprotein chiral stationary phase: Evidence for a multiple‐site chiral recognition mechanism

AbstractThe effect of mobile phase pH and dimethyloctylamine (DMOA) on the retention (k') and stereoselectivity (α) of antimalarial agents mefloquine, enpiroline, and chloroquine on the α1‐acid glycoprotein chiral stationary phase (AGP‐CSP) was investigated. An increase of k' with increasing pH was observed while the effect on α was a function of the solute. The magnitude and direction of changes induced by DMOA depended on pH and the structure of the solute.The results of this study are consistent with a change of the conformation of the AGP between pH 5 and 7. At pH 7, the effect of DMOA on mefloquine was relatively well described by a competitive displacement from one enantioselective site. The effect on chloroquine and enpiroline suggests a multiple‐site mechanism in which both competitive and allosteric interactions are involved.

Chloroquine as a hyperthermia potentiator

The antimalarial agent chloroquine (CQ) inhibits DNA and RNA polymerase and interferes with lysosomal function. We sought to determine if these properties make chloroquine effective as a hyperthermia sensitizer. B16F10 melanoma cells were treated for 180 min at 37 or 41°C with 0.005 m M CQ, 0.01 m M CQ, 0.05 m M CQ, or 0.1 m M CQ and colony formation evaluated at 7 days. CQ was cytotoxic at 37 or 41°C in a dose-dependent fashion. A significant increase in cytotoxicity was seen with 0.5 and 0.1 m M CQ at 41°C compared to 37°C ( P < 0.01). The influence of treatment time on CQ cytotoxicity was examined by treating cells with 0.05 m M CQ at 37 or 41°C for 30-min intervals from 30 to 180 min. Increasing length of exposure to CQ increased cytotoxicity at both 37 and 41°C. For each interval studied treatment at 41°C significantly decreased colony formation compared to treatment at 37°C ( P < 0.01). Complete cell kill was achieved after 180 min of 41°C treatment compared to 80% cell kill at 37°C. We conclude that in this model CQ is an effective potentiator of hyperthermia.

Antimalarial activity of new water-soluble dihydroartemisinin derivatives.

The usefulness of sodium artesunate (3), a water-soluble derivative of artemisinin (1), is impaired by its poor stability in aqueous solution. To overcome the ease of hydrolysis of the ester group in 3, a new series of derivatives of dihydroartemisinin (2) was prepared in which the solubilizing moiety, which contains a carboxylate group, is joined to dihydroartemisinin by an ether rather than an ester linkage. The new derivatives were prepared in good yield by treatment of dihydroartemisinin with an appropriate alcohol under boron trifluoride etherate catalysis at room temperature. All major condensation products are the beta isomer. Hydrolysis of the esters with 2.5% KOH/MeOH gave the corresponding potassium salts, which were converted to free acids (8b-d) by acidification. The derivatives were tested in vitro against two clones of human malaria, Plasmodium falciparum D-6 (Sierra Leone clone) and W-2 (Indochina clone). No cross-resistance to the antimalarial agents mefloquine, chloroquine, pyrimethamine, sulfadoxine, and quinine was observed. In general, the new compounds are more effective against the W-2 than the D-6 strain. Esters (5a-d) possess activity comparable to that of the parent compounds 1 and 2; however, conversion of the esters to their corresponding carboxylates (7a-d) or acids (8b-d), with the exception of artelinic acid (8d), drastically decreases the antimalarial activities in both cell lines. Artelinic acid, which is both soluble and stable in 2.5% K2CO3 solution, possesses superior in vivo activity against Plasmodium berghei than artemisinin or artesunic acid.

The anticholinesterase activity of mefloquine.

The characteristics of the inhibition of electric eel acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) by the antimalarial agents mefloquine, chloroquine, amodiaquine and amopyroquine were determined. The antimalarials were found to be non-competitive inhibitors of both AChE and BChE. In both enzyme systems, inhibitory potencies were in the order amodiaquine greater than amopyroquine greater than chloroquine greater than mefloquine. The low inhibitory potency of mefloquine may account in part for the appearance of gastrointestinal and central nervous system disturbances only at high doses of the drug.

Mutagenicity evaluation of the two antimalarial agents chloroquine and mefloquine, using a bacterial fluctuation test

The two antimalarial agents chloroquine and mefloquine have been tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1537 and TA1538. Chloroquine was found to revert strain TA1537 at concentrations of 100 and 250 μg/ml, most likely due to intercalation. No mutagenicity was found with mefloquine at concentrations up to 2.5 μg/ml, neither without nor with metabolic activation by Ca 2+-precipitated rat liver microsomes. Higher concentrations of mefloquine and chloroquine inactivated the bacteria.

Behavioral toxicity and equivocal suicide associated with chloroquine and its derivatives

Although the antimalarial agents chloroquine, hydroxychloroquine, and amodiaquine are widely used to treat a variety of medical conditions, their behavioral toxicity and lethality are not generally recognized. Therapeutic doses sometimes cause psychosis, delirium, personality change, and depression. Since moderately low overdoses of chloroquine can result in rapid death, such behavioral effects could lead to accidental or state-dependent overdosage and death.