Non-iron porphyrins inhibit beta-haematin (malaria pigment) polymerisation.

Infrared spectroscopy was used to evaluate the effect of non-iron porphyrins (protoporphyrin IX and haematoporphyrin) on haematin polymerisation to beta-haematin at acidic pH. Both molecules effectively inhibited the reaction, with haematoporphyrin 6 times as active as protoporphyrin IX. We postulated that the interaction between the pi electron system of porphyrin rings leads to the formation of pi-pi adducts, which inhibit polymer elongation in the same way as antimalarial drugs (e.g., chloroquine); the presence of hydroxyl groups able to bind haem iron enhances activity.

Deferoxamine: stimulation of hematin polymerization and antagonism of its inhibition by chloroquine.

The iron chelator deferoxamine enhances the clearance of Plasmodium falciparum parasitemia and may be useful in drug combinations for the treatment of cerebral malaria. However, the deferoxamine-chloroquine drug combination is antagonistic, or at best additive, against P. falciparum in vitro. As chloroquine is thought to exert its antimalarial activity by interacting with hematin released from the proteolytic degradation of hemoglobin in the parasite food vacuole, we hypothesized that deferoxamine might interfere with the ability of chloroquine to inhibit hematin polymerization, since it was reported that deferoxamine interacts with hematin. Therefore, we assessed deferoxamine-hematin binding in more detail and investigated the effect of deferoxamine on hematin polymerization in the presence and absence of chloroquine. Isothermal titration calorimetry (ITC) experiments demonstrated an enthalpy-driven deferoxamine:hematin mu-oxo dimer binding with an association constant of 2.8 x 10(4) M(-1) at pH 6.5, a binding affinity 14-fold lower than that measured for chloroquine. At least two of the three hydroxamic acid functional groups of deferoxamine must be unionized for effective binding. We also discovered that deferoxamine antagonized chloroquine-mediated inhibition of hematin polymerization. Unexpectedly, deferoxamine increased the concentration of soluble forms of hematin and enhanced the rate of hematin polymerization. Deferoxamine also could initiate hematin polymerization. In contrast, chloroquine decreased the concentration of soluble forms of hematin and inhibited hematin polymerization. This work supports the postulate that initiation of hematin polymerization requires a higher concentration of soluble hematin monomer than does the elongation phase of polymerization and provides one possible explanation for the observed antagonism between deferoxamine and chloroquine against parasites in culture.

Protective cardiovascular effects of diazepam in experimental acute chloroquine poisoning.

To assess the effects of diazepam in chloroquine poisoning, we studied pentobarbital anesthetized and mechanically ventilated pigs. All the pigs received 50 mg.kg-1 chloroquine given intravenously for 25 min. Eight pigs acted as control (group C). Another 7 were treated with diazepam given intravenously 5 min after the end of chloroquine infusion: 2 mg.kg-1 of diazepam for 2 min, then 1 mg.kg.h-1 for 25 min (group D). Thereafter, all pigs were sacrificed. In both groups the chloroquine infusion induced a large fall in arterial pressure, a decrease in heart rate, and an increase in QRS duration. No difference was observed between the 2 groups for weight, systolic and diastolic arterial pressures, heart rate, QRS and QT durations before diazepam. After diazepam, systolic and diastolic arterial pressures, heart rate, urine volume, urinary excretion of chloroquine, plasma and blood cell chloroquine levels were higher, whereas QRS duration was lower, in group D compared to group C. No difference was observed between the 2 groups for urinary concentration of chloroquine, the ratio between plasma and blood cell chloroquine levels, hepatic, cardiac, and skeletal muscle chloroquine levels, and QT duration. After diazepam, the slope of the regression curve between QRS duration and plasma chloroquine levels was reversed in group D compared to group C. We conclude that diazepam counteracts some haemodynamic and electrocardiographic changes, and increases urinary excretion of chloroquine, in acute experimental chloroquine poisoning.

Diazepam does not improve the mechanical performance of rat cardiac papillary muscle exposed to chloroquine in vitro.

Diazepam has been reported to decrease the cardiac toxicity of chloroquine but the precise mechanism involved remains unknown. Left ventricular papillary muscles from adult Wistar rats were exposed to 10(-4) M chloroquine and assigned to three groups: group I (n = 10) exposed to chloroquine alone; group II (n = 8) exposed to chloroquine and 10(-5) M diazepam; group III (n = 8) exposed to chloroquine and 10(-4) M diazepam. The main mechanical parameters measured were: maximum unloaded shortening velocity (Vmax), maximum lengthening velocity (maxVr), active force normalized per cross-sectional area (AF/s), contraction-relaxation coupling under low load (R1), load sensitivity of relaxation (Isot.A/Isom.A), and peak power output (Emax) determined from Hill's equation of the force-velocity curve. Data are expressed as mean percent of control values +/- SD, for groups I, II, III respectively. No differences between groups I, II, and III were noted for Vmax (87 +/- 13, 82 +/- 9, 86 +/- 7), maxVr (47 +/- 6, 48 +/- 11, 52 +/- 11), AF/s (87 +/- 16, 91 +/- 10, 83 +/- 11), Isot. A/Isom. A (113 +/- 9, 108 +/- 3, 109 +/- 7), or Emax (75 +/- 10, 81 +/- 12, 72 +/- 16). Chloroquine was shown to be a negative inotropic agent since it decreased Vmax, AF/s and Emax, but diazepam did not restore the intrinsic mechanical performance of rat cardiac papillary muscle exposed to chloroquine, therefore 1) the protective cardiovascular effects of diazepam in chloroquine poisoning are not related to an improvement in intrinsic cardiac mechanical properties; 2) inotropic agents are therefore necessary in combination with diazepam for the treatment of severe chloroquine poisoning.

A rapid in vitro test for chloroquine-resistant Plasmodium falciparum.

We report a rapid (2-3 hr) in vitro test for chloroquine resistance in Plasmodium falciparum. The test is based on the inhibition of chloroquine efflux by verapamil; it is performed by diluting infected blood in culture medium and incubating the diluted blood for 60 min at 37 degrees C with 50 nM 3H-chloroquine, with and without 10 microM verapamil. The test can be performed with the ring stage parasites in the blood of infected patients and in the presence of white cells, platelets and anticoagulants (heparin, EDTA, or citrate). Although the test can be performed in triplicate with 20 microliters of blood and specimens may be kept in anticoagulants at 4 degrees C for up to 24 hr, parasitemias less than 0.1% limit the sensitivity of the assay. Inhibition of chloroquine efflux by verapamil may permit the rapid identification of chloroquine resistant P. falciparum in blood specimens from infected patients.

Depressant effects of chloroquine on the isolated guinea-pig heart.

Some anti-malarials have deleterious effects upon the heart. The actions of two of these, chloroquine and quinacrine, were compared on isolated guinea-pig atria and Langendorff preparations to assess their effects on several calcium pools. Both compounds decreased developed tension in a concentration (chloroquine 10-100 microM; quinacrine 3-100 microM) and time-dependent manner, with quinacrine being twice as potent as potent as chloroquine. Ventricular muscle was much more sensitive to chloroquine than was atrial muscle. Concentrations of chloroquine, comparable to that found in the serum of patients ingesting toxic doses, caused significant inhibition of developed tension. The effects of chloroquine were almost completely reversed on washout; quinacrine, however, was less readily reversible. Chloroquine also had a direct negative chronotropic effect, substantially reduced force-frequency relationships and developed tension in partially depolarized atrial preparations; while post-rest contraction and the positive inotropic effect of ouabain were unaffected. Increases in extracellular calcium antagonized the negative inotropic effect. Quinacrine had a marked effect on post-rest contraction and attenuated the positive inotropic action of ouabain. It is concluded that the action of chloroquine may involve a superficial calcium pool, while quinacrine may act upon several calcium pools.

Rifampicin antagonizes the effect of choloroquine on chloroquine-resistant Plasmodium berghei in mice.

Chloroquine (CQ)-resistant Plasmodium falciparum appears to decrease CQ accumulation in its food vacuole by enhancing its efflux via an active membrane pump, which has been reported to be a P-glycoprotein-like transporter. Rifampicin (RIF) is a P-glycoprotein inhibitor and also has some antimalarial activity. It is hoped that a combination of choloroquine-rifampicin (CQ + RIF) would be advantageous in the treatment of CQ-resistant malaria. Swiss albino mice were inoculated with CQ-resistant P. berghei intraperitoneally, and studied for the effect of CQ versus the combination of CQ + RIF at various doses on the clearance of parasitemia, the survival of the mice, and the recrudescence of malaria. Paradoxically, RIF decreased the survival rate and rate of clearance of parasitemia and increased the rate of recrudescence significantly when combined with various doses of CQ. Our results indicated that RIF worsened the course of the disease, and we concluded that RIF should not be combined with CQ in the treatment of malaria.

A randomized clinical trial with high dose of chloroquine for treatment of Plasmodium falciparum malaria in Brazil.

This clinical trial compared parasitological efficacy, levels of in vivo resistance and side effects of oral chloroquine 25 mg/Kg and 50 mg/Kg in 3 days treatment in Plasmodium falciparum malaria with an extended followed-up of 30 days. The study enrolled 58 patients in the 25 mg/Kg group and 66 in the 50 mg/Kg group. All eligible subjects were over 14 years of age and came from Amazon Basin and Central Brazil during the period of August 1989 to April 1991. The cure rate in the 50 mg/Kg group was 89.4% on day 7 and 71.2% on day 14 compared to 44.8% and 24.1% in the 25 mg/Kg group. 74.1% of the patients in the 25 mg/Kg group and 48.4% of the patients in the 50 mg/Kg group had detectable parasitaemia at the day 30. However, there was a decrease of the geometric mean parasite density in both groups specially in the 50 mg/Kg group. There was 24.1% of RIII and 13.8% of RII in the 25 mg/Kg group. Side effects were found to be minimum in both groups. The present data support that there was a high level resistance to chloroquine in both groups, and the high dose regimen only delayed the development of resistance and its administration should not be recommended as first choice in malaria P. falciparum therapy in Brazil.

Clinical trials of mefloquine with tetracycline.

A comparative trial of the combination of mefloquine or MSP with tetracycline was carried out in fifty-one adult Thai male patients with acute falciparum malaria. The patients were randomized to receive either the combination of tetracycline (250 mg qid for 7 days) with mefloquine 4 tablets (1,000 mg) or with MSP 4 tablets (one tablet contains 250 mg mefloquine, 500 mg sulfadoxine and 25 mg pyrimethamine). Fifty patients had a complete 28-day follow-up period. Both regimens produced similar efficacy with no difference in adverse effects. In the mefloquine plus tetracycline group, the cure rate was 72% (18/25). One patient had an RIII response, the others showed initial response to the treatment with FCT and PCT of 40.7 +/- 27.4 and 76.2 +/- 34.2 hours (mean +/- SD) respectively. However, 6 patients developed recrudescence between days 17 and 29 (RI), 3 of these had vomiting. In the MSP plus tetracycline group, the cure rate was 76% (19/25). The means (+/- SD) of FCT and PCT were 44.7 +/- 38.0 and 80.6 +/- 25.0 hours, respectively. Six patients had recrudescence between days 17 and 31 (RI), 2 of these had vomiting. Although the addition of tetracycline improved the cure rate of mefloquine when compared with standard dose of mefloquine alone (3 tablets), these combinations seem to be useful in areas where alternative drugs are not available.

Malariometric survey in Keoudom District, Laos: sensitivity of Plasmodium falciparum to anti-malarials and automedication with chloroquine.

A malariometric survey was conducted in the Keoudom District, in the northern part of Vientiane Province, Laos, where an artificial dam-lake on the Nam River is located. The parasite rate of the whole cohort representing 1,105 subjects was 2.44% with the predominance of Plasmodium vivax (70%), while P. falciparum represented 30% with the average parasite density index 3. The low spleen rate (2.3%) characterized the study area as a hypoendemic zone. IFAT antibodies were examined in 419 subjects. The seropositivity of 195 persons < 15 years was 13.7% while in > 15 year old subjects seropositivity was 61.6% with a low GMRT in both groups (140:148). Automedication with aminoquinoline was assayed by urinary analysis in 125 outpatients. Of these, 36 (28.8%) were positive, 89 (71.2%) negative. The frequency of positive blood films for P. falciparum was higher in subjects with aminoquinoline in the urine (36.1%) than in those without (10.1%). Chloroquine sensitivity assay of 15 strains of P. falciparum displayed resistance in 39.3%.

Drug resistance in malaria.

Half of the world's population live in regions where malaria is still endemic and about 2 million are killed by the disease every year. There is an exponential increase in the number of non-immune travellers who visit such areas and thousands develop malaria after they return home. The few drugs currently available for the prevention or treatment of malaria are discussed and the history of the emergence of multiple drug resistance in the malignant tertian parasite, Plasmodium falciparum, is traced from its origins in the late 1950s to the present time when it is found in the endemic regions of all continents. These are indications that chloroquine resistance is beginning to appear now also in P. vivax in the Southwest Pacific. The few new drugs under development and the uncertain future that they face are discussed.

[Effect of enkad on the development of experimental chloroquine retinopathy]. / Vliianie énkada na razvitie éksperimental'noi khlorokhinovoi retinopatii.

The effect of enkad on the development of chloroquine retinopathy in rabbits was studied by electron microscopy and electrophysiological methods. It is shown that subconjunctival enkade injection inhibits the development of a dystrophic process in the retina induced by intravitreous chloroquine administration.

[A comparative restriction analysis of the DNA of strains of the malarial parasite sensitive and resistant to chloroquine]. / Sravnitel'nyi restriktsionnyi analiz DNK shtammov maliariinogo parazita chuvstvitel'nogo i rezistentnykh k khlorokhinu.

A comparative restriction analysis was made for DNA in malaria parasites, strain H sensitive to chloroquinone, strain LNK-65 with spontaneously occurred resistance to the agent, and breeding strain LNK-65 ChlR highly resistant to it. DNA hydrolysis with EcoR1, HindIII, and BamH1 endonucleases revealed permanent differences in the DNA restriction pattern of malaria parasites. There were additional restriction bands as part of DNA restricts in the strain LNK-65 Chl bred from LNK-65 for high resistance to chloroquine on EcoR1-, HindIII-, and BamH1-hydrolysis. Great differences in the DNA restriction pattern in the strains H and LNK-65 are likely to be due to their belonging to various strains, such as P.berghei and P.yoelii, respectively. Comparison of the DNA restriction pattern of the host (murine leukocytes) and the malaria parasite suggests the plasmodium DNA is adequately removed from the host DNA.

[The resistance of Plasmodium falciparum strains to preparations of the 4-aminoquinoline series in the Republic of Guinea]. / Rezistentnost' shtammov Plasmodium falciparum k preparatam 4-aminokhinolinovogo riada v Gvineiskoi Respublike.

In vivo measurements of P. falciparum sensitivity to chloroquine diphosphate, carried out in the Republic of Guinea, resulted in isolation of strains with degrees I and II resistance from 5 of the 25 examined patients, that may be due to a wide prophylactic administration of the drug to all patients with fevers without completing the course of therapy in all the patients infected. Such courses are obligatory to prevent the dissemination of chloroquine-resistant strains of the parasite and augmentation of the resistance. Further use of chloroquine and its analogs for the prevention and chemoprophylaxis of malaria in the Republic of Guinea appears still desirable, because the majority of P. falciparum strains are still sensitive to these agents. If no favorable changes are detectable in the clinical parameters of malaria patients in 4-5 days of chloroquine treatment or the condition recurs in 1.5-2 months, assessment of P. falciparum sensitivity to chloroquine is advisable, followed by a rational choice of the drug.

[The modelling of the multiple drug resistance of the malarial parasites. 1. The combined resistance of Plasmodium berghei to chloroquine and fansidar]. / Modelirovanie mnozhestvennoi lekarstvennoi ustoichivosti maliariinykh parazitov. Soobshchenie 1. Sochetannaia ustoichivost' Plasmodium berghei k khlorokhinu i fansidaru.

Using a recurrent technique, P. berghei isolate resistant to chloroquine-fansidar combination is formed in golden hamsters. The isolate resistant to chloroquine-fansidar combination was 4 times less sensitive to chloroquine, 2 times less sensitive to fansidar and its combinations, 2 times less sensitive to sulfadoxine, 31 times less sensitive to pyrimethamine, as compared to the baseline isolate LNK65 P. berghei characterized by naturally reduced sensitivity to chloroquine.

[Drug-resistant tropical malaria in Angola]. / Lekarstvenno-ustoichivaia tropicheskaia malariia v Angole.

Three antimalarial treatment regimens by the complete standard WHO tests were examined in 105 Plasmodium falciparum-infected patients who were nonimmune newcomers treated at the Russian hospital in Luanda in 1991-1992, 61% showed chloroquine resistance and 40% fansidar resistance. All 59 patients with high rates of parasitemia were successfully cured with quinine in combination with tetracycline. Thick, if required thin, blood smears were microscopically examined. The findings suggest that Fansidar should be a drug of first-line therapy in Angola, though in the neighbouring countries quinine continues preserving its efficacy, but there is a delayed elimination of the parasites within 7 days of initiation of the therapy, making it necessary to prolong therapy with this drug up to 10 days.

[Ca++ ion transport blockers as reversants of the drug resistance of malarial parasites. 1. The effect of verapamil on the resistance to chloroquine in vivo of Plasmodium berghei and in vitro of Plasmodium falciparum]. / Blokatory transporta ionov Ca++, kak reversanty lekarstvennoi ustoichivosti maliariinykh parazitov. Soobshchenie 1. Vliianie verapamila na ustoichivost' k khlorokhinu in vivo u Plasmodium berghei i in vitro u Plasmodium falciparum.

The reversing action of verapamil on the effect of chloroquine was found in in vivo experiments by using a model P. berghei resistant to chloroquine, an LNK65 isolate having a naturally lower resistance to the agent, and its polyresistant strain with the acquired resistance to chloroquine and fansidar, as well as by employing the chlorine-resistant P. falciparum isolates from the south of the Socialist Republic of Vietnam. The magnitude of this effect was related to the dose of verapamil, the frequency of administration of a combination of the agents in vivo, while that was associated to the concentration of verapamil and the level of isolate resistance to chloroquine in vitro which was the most pronounced. Taking into account the dose-dependent effect of verapamil, it can be suggested that increasing its concentration in combination with chloroquine can provide a more marked reversing action with lower chloroquine concentrations. The parameters accepted by the authors in evaluating the combined effect enable the effect of the verapamil/chloroquine concentration to be regarded as potentiation.

[Ca++ ion transport blockers as reversants of the drug resistance of malarial parasites. 2. The effect of praziquantel on the resistance to chloroquine and compound R-70-Zh of Plasmodium berghei]. / Blokatory transporta ionov Ca++ kak reversanty lekarstvennoi ustoichivosti maliariinykh parazitov. Soobshchenie 2. Vliianie prazikvantelia na ustoichivost' k khlorokhinu i soedineniiu R-70-Zh u Plasmodium berghei.

The reversing action of anthelminthic praziquantel (P) on the effect of chloroquine (C) and compound R-70-Zh (styrylquinazoline) was revealed on a Plasmodium berghei model (white inbred mice), using a LNK65 isolate with naturally reduced sensitivity to chloroquine and its polyresistant line LNK65CHLFR with acquired resistance to chloroquine/fansidar (selected in our laboratory). P (125 mg/kg) in combination with C showed a potentiating effect not only on the LNK65 isolate, but also on the LNK65CHLFR line, while investigated separately on this line, both drugs were not effective in tested doses. Moreover, the similar effect of C on the LNK65CHLFR line was achieved in the dose that was 4 times higher than that of P/C combination. P in a standard dose on the LNK65 isolate showed a more marked activation of compound R-70-Zh that on C. The potentiating effect was manifested in combination with R-70-Zh in the dose half as high as that of C; this phenomenon was also reflected by the efficiency index (5.0 against the 4.0) accepted in our laboratory and may be associated with the higher sensitivity of the LNK65 isolate to R-70-Zh. P showed some antimalarial action which manifested itself only by morphological changes on P. berghei parasites similar to those observed under the action of some dihydropholate reductase inhibitors, such as pyrimethamine.

The inhibition of ferrochelatase enhances 5-aminolevulinic acid-based photodynamic action for prostate cancer.

BACKGROUND: The aim of this study was to clarify the mechanism of accumulation of 5-aminolevulinic acid (ALA)-dependent protoporphyrin IX (PpIX), ALA-photodynamic therapy (PDT)-induced cell death and enhanced efficiency by a ferrochelatase inhibitor in prostate cancer PC-3 cells. METHODS: The accumulation of ALA-induced PpIX in PC-3 cells was observed by fluorescence microscopy and measured by flow cytometry analysis. The efficiency of ALA-PDT was analyzed by flow cytometry and assessed by cell death, caspase-3 activity and mitochondrial membrane potential. The ALA-PDT-promoting effects of ferrochelatase inhibitors, such as deferoxamine and NOC-18, were also analyzed. We confirmed the results obtained in vivo with an animal model using nude mice. RESULTS: ALA-induced PpIX accumulation increased in time- and ALA concentration-dependent manners. ALA-PDT decreased the levels of mitochondrial membrane potential, and induced cell death occurred by both apoptosis and necrosis. Inhibition of ferrochelatase by deferoxamine and NOC-18 led to increase of PpIX accumulation and enhanced effect of ALA-PDT in PC-3 cells. In vivo, the degeneration of tumor tissue by ALA-PDT was observed within a broader range and led to apoptosis and necrosis. CONCLUSION: This study demonstrated ALA-PDT induced PC-3 cell death by the mechanisms of both necrosis and apoptosis through a caspase-independent mitochondrial pathway. Inhibition of ferrochelatase enhanced these effects, suggesting that ferrochelatase played an important role in ALA-PDT. ALA-PDT could be a new modality for focal therapy of prostate cancer.

A series of structurally simple chloroquine chemosensitizing dibemethin derivatives that inhibit chloroquine transport by PfCRT.

A series of 12 new dibemethin (N-benzyl-N-methyl-1-phenylmethanamine) derivatives bearing an N-aminomethyl group attached to the one phenyl ring and an H, Cl, OCH3 or N(CH3)2 group on the other have been synthesized. These compounds all showed strong chloroquine chemosensitizing activity, comparable to verapamil, when present at 1 µM in an in vitro culture of the chloroquine-resistant W2 strain of the human malaria parasite, Plasmodium falciparum. Their N-formylated derivatives also exhibited resistance-reversing activity, but only at substantially higher IC10 concentrations. A number of the dibemethin derivatives were shown to inhibit chloroquine transport via the parasite's 'chloroquine resistance transporter' (PfCRT) in a Xenopus laevis oocyte expression system. The reduced resistance-reversing activity of the formylated compounds relative to their free amine counterparts can probably be ascribed to two factors: decreased accumulation of the formylated dibemethins within the parasite's internal digestive vacuole (believed to be the site of action of chloroquine), and a reduced ability to inhibit PfCRT. The resistance-reversing activity of the compounds described here demonstrates that the amino group need not be attached to the two aromatic rings via a three or four carbon chain as has been suggested by previous QSAR studies. These compounds may be useful as potential side chains for attaching to a 4,7-dichloroquinoline group in order to generate new resistance-reversing chloroquine analogues with inherent antimalarial activity.