High-affinity inhibitors of dihydrofolate reductase: antimicrobial and anticancer activities of 7,8-dialkyl-1,3-diaminopyrrolo[3,2-f]quinazolines with small molecular size.

A series of 7,8-dialkylpyrrolo[3,2-f]quinazolines were prepared as inhibitors of dihydrofolate reductase (DHFR). On the basis of an apparent inverse relationship between compound size and antifungal activity, the compounds were designed to be relatively small and compact. Inhibitor design was aided by GRID analysis of the three-dimensional structure of Candida albicans DHFR, which suggested that relatively small, branched alkyl groups at the 7- and 8-positions of the pyrroloquinazoline ring system would provide optimal interactions with a hydrophobic region of the protein. The compounds were potent inhibitors of fungal and human DHFR, with K(i) values as low as 7.1 and 0.1 pM, respectively, and were highly active against C. albicans and an array of tumor cell lines. In contrast to known lipophilic inhibitors of DHFR such as trimetrexate and piritrexim, members of this series of pyrroloquinazolines were not susceptible to P-glycoprotein-mediated multidrug resistance and also showed significant distribution into lung and brain tissue. The compounds were active in lung and brain tumor models and displayed in vivo activity against Pneumocystis carinii and C. albicans.

Antipneumocystis activity of 17C91, a prodrug of atovaquone.

The prophylactic efficacy of 17C91, a carbamate prodrug of atovaquone (ATQ), was investigated in a severe combined immunodeficient mouse model of Pneumocystis carinii pneumonia (PCP). At an oral dosage equivalent to 100 mg of ATQ per kg of body weight per day, 17C91 protected 9 of 10 mice from PCP and had a prophylactic efficacy comparable to that of co-trimoxazole (at 250 mg of sulfamethoxazole plus 50 mg of trimethoprim per kg per day orally). The intensity of P. carinii infection (infection score) of mice treated with 17C91 correlated with the concentration of ATQ in the plasma, with clearance of the infection associated with plasma ATQ levels of >35 micrograms/ml. 17C91 given orally provided enhanced levels of ATQ in the plasma compared with the conventional ATQ formulation. Additional studies reported in this paper demonstrate that the prophylactic activity of 17C91 against PCP in severe combined immunodeficient mice is comparable to that of a new oral microparticulate formulation of ATQ.

Effect of atovaquone and atovaquone drug combinations on prophylaxis of Pneumocystis carinii pneumonia in SCID mice.

The prophylactic efficacies of atovaquone (ATQ) alone and in combination with azithromycin, clarithromycin, rifabutin, proguanil, PS-15, trimethoprim, co-trimoxazole, or dapsone were investigated in a SCID mouse model of Pneumocystis carinii pneumonia (PCP). ATQ alone was shown to have a significant dose-related effect, and at 200 mg/kg of body weight per day administered orally, the efficacy of ATQ was comparable to that of Septrin (co-trimoxazole). Of the drugs investigated orally in combination with ATQ, only dapsone (25 mg/kg/day) and to a lesser extent PS-15 (5 mg/kg/day) had any noteworthy antipneumocystis activity (at the doses examined) when administered alone. ATQ drug combinations affected the prophylactic efficacy of a subcurative dosage of ATQ (50 mg/kg/day given orally) in the following ways: dapsone (25 mg/kg/day) or co-trimoxazole (25 mg of sulfamethoxazole plus 5 mg of trimethoprim per kg/day) had no significant effect on ATQ, azithromycin (200 mg/kg/day) or clarithromycin (200 mg/kg/day) had a slight additive effect with ATQ, trimethoprim (100 mg/kg/day) or PS-15 (5 mg/kg/day) had an additive effect with ATQ, and proguanil (25 mg/kg/day) or rifabutin (200 mg/kg/day) had a marked synergistic effect on ATQ. The last result was particularly noteworthy as neither proguanil nor rifabutin was effective against PCP when administered alone. None of the drugs examined antagonized the prophylactic activity of ATQ in experimental PCP in SCID mice. The results suggest that clinical trials of ATQ with synergistic drug combinations may now be justified, particularly if such drug combinations improve ATQ's efficacy and broaden its spectrum of activity.

Artificial infections of Pneumocystis carinii in the SCID mouse and their use in the in vivo evaluation of antipneumocystis drugs.

A model for the in vivo evaluation of antipneumocystis drugs has been developed in SCID mice infected intratracheally with cryopreserved mouse-derived Pneumocystis carinii. The development of a highly reproducible fatal P. carinii pneumonia occurred within 10 weeks (mean survival time +/- SEM = 72.2 +/- 1.2 days). Continuous administration of dexamethasone (2 mg/liter in the drinking water) exacerbated the rate of onset of severe P. carinii pneumonia (mean survival time +/- SEM = 63 +/- 1.3 days) in SCID mice. The number of cysts per g of lung homogenate (homogenate counts) were maximal with an inoculum of 20,000 cysts at 6 weeks post infection. Homogenate counts correlated with infection scores (graded assessments of immunofluorescent cysts on lung impression smears) suggesting that infection scoring accurately and rapidly reflects the severity of P. carinii pneumonia in SCID mice. These studies led to the development of a drug screening protocol in which Pneumocystis-free female SCID mice (20-25 g) were started on dexamethasone 7 days prior to IT inoculation with a single dose of 20,000 cysts. Drugs were evaluated either for: a) prophylaxis (continuously from day 1 post infection) or b) treatment (from day 21 post infection) until day 42 post infection, when all mice were killed and infection scores determined. Co-trimoxazole (at 250 mg sulfamethoxazole + 50 mg trimethoprim/kg/day) given in the drinking water was found to be highly effective in both the prophylaxis and treatment of mouse P. carinii pneumonia.(ABSTRACT TRUNCATED AT 250 WORDS)

Viability of Onchocerca volvulus in vitro.

The use of a selective schedule of tests to identify a viable population of isolated adult Onchocerca volvulus (Nematoda: Filarioidea) has been investigated in a large worm population. The study was initiated to develop methodology appropriate to test new candidate macrofilaricides for their in vitro activity against O. volvulus. After removal from the host the viability of isolated intact parasites was estimated by assessing the motility indices of male worms, and the colorimetric quantification of the reduction of the bioreducible tetrazolium reagent XTT and lactate output by female worms. Additionally the motility of whole females and the movement of inner organs of female worms were scored quantitatively. These response parameters were used to sort the adult worms into viability groups at the start of the in vitro culture. The adult worms were then observed for 6 days and viability was assessed regularly during the culture period. At the end of the culture period, the reduction of the water-insoluble tetrazolium reagent MTT was used to determine the formazan formed by the entire male and female worms. The response parameters used at the start of the culture proved to be highly predictive for detecting viable and non-viable adult worms. In the group of worms selected as 'viable' around 70% kept their motility and metabolic activity at a high level until the end of the culture compared to the initial level. In contrast, none of the female worms and only 13% of the male worms categorized as 'poorly viable' demonstrated a motility index or metabolic level at the end of the culture period that was comparable to that of the worms in the 'viable' groups. For female worms the lactate output correlated significantly with weight whereas no correlation was seen between MTT reduction and weight.

A radiometric method for objectively screening large numbers of compounds against Pneumocystis carinii in vitro.

A relatively simple method is reported for accurately quantitating the incorporation of [3H]para aminobenzoic acid (pABA) into the folates of Pneumocystis carinii cultured in vitro, and the subsequent development of a highly sensitive and reproducible 96-well microtitre plate drug screening system. Incorporation of [3H]pABA under optimized conditions has been utilized as a selective indicator of the in vitro viability of P. carinii against which the inhibitory effects of potential drugs were quantified. The anti-Pneumocystis agents pentamidine, sulfamethoxazole, 566C80 and piritrexim gave median inhibitory concentration values of 7.3, 0.1, 1.4 and approximately 100 microM, respectively in this assay. The results suggest that this 96-well plate P. carinii [3H]pABA-incorporation system is suitable as a rapid high throughput primary in vitro screen for detecting compounds with anti-Pneumocystis activity.

Microculture screening assay for primary in vitro evaluation of drugs against Pneumocystis carinii.

Pneumocystis carinii inoculated into 96-well filtration plate assemblies was shown to synthesize radiolabeled folates de novo from [para-3H]aminobenzoic acid ([3H]pABA). At the end of each incubation with [3H]pABA, a vacuum manifold was used to remove the medium and wash P. carinii. The membrane at the base of each well was dried and punched out, and the level of 3H retained was determined by direct scintillation counting. High-pressure liquid chromatography analysis of duplicate filters confirmed that direct counting of 3H retained on membranes (after correction for unmetabolized [3H]pABA) was an accurate reflection of total [3H]pABA incorporation by P. carinii. Greater than 95% of the 3H recovered was shown to be present as polyglutamated species. After digestion with rat plasma folic acid gamma-glutamyl hydrolase, para-aminobenzoylglutamate, N10-formyltetrahydrofolate, and tetrahydrofolate were identified as the major 3H-labeled components. para-Aminobenzoylglutamate was presumed to have arisen from folylpolyglutamates synthesized by P. carinii and was therefore included in the calculation of total [3H]pABA incorporation. P. carinii incorporation of [3H]pABA under optimal conditions was used as a selective measure of in vitro viability against which the inhibitory effects of some antipneumocystis agents (pentamidine, sulfamethoxazole, 566C80, and piritrexim) were quantitated. The concentrations of pentamidine, sulfamethoxazole, 566C80, and piritrexim required for 50% inhibition in this assay were 7.3, 0.1, 1.4, and approximately 100 microM, respectively. The results suggest that this 96-well [3H]pABA incorporation assay has considerable potential for objective in vitro drug screening against P. carinii.

Potential of a soluble tetrazolium/formazan assay for the evaluation of filarial viability.

Using female Acanthocheilonema viteae we have investigated the bioreduction of the tetrazolium reagent XTT (2,3-bis(2-methoxy-4-nitro-sulphonyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide). Unlike the formazan formed by other tetrazolium salts, that derived from XTT readily diffuses out of A. viteae in vitro. Formazan formation can therefore be quantified by direct absorbance reading of the incubation medium, eliminating the need for a DMSO solubilization step. Optimum assay conditions involved a 4 h incubation, in the presence of the electron coupling agent phenazine methosulphate (PMS). Repeat 4 h incubations with XTT-PMS were well tolerated by worms for 5 consecutive days. This confirmed the low toxicity of XTT formazan and its usefulness in the semi-continuous assessment of filarial viability. In comparison to our previously reported MTT (3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide)-reduction assay XTT-PMS reduction showed comparable drug sensitivity and accuracy, however XTT-PMS appears to be at least 10-15 times less efficiently reduced by A. viteae females. A possible application of the XTT assay using female Onchocerca volvulus is discussed.

New macrofilaricidal leads from plants?

The continuing need for a new safe and effective macrofilaricidal drug for the treatment of filarial infections in man is outlined, together with the possibility that structural leads for the identification and synthesis of such drugs might be derived from higher plants. Many of todays successful drugs were derived from plants and it is interesting to note that the majority were discovered through follow-up research on the folk or ethnomedical uses of plants. In this review an attempt has been made to compile a list of those plant species for which there is evidence supporting their usefulness in the treatment of filarial conditions in endemic areas. Perhaps surprisingly the use of such plant medicines appears quite widespread, and details of 90 plant species are summarised. The value of this information is discussed in the light of the fact that few of these citations are supported by clinical or scientific investigations into their efficacy, and the lack of knowledge regarding the antifilarial active constituents. It is concluded that in view of apparent failure of more rational approaches to yield macrofilaricidal leads there is justification for examining further medicinal plants, and that the data summarised might be a useful starting point for such a screening programme.

In vitro assessment of the activity of anthelmintic compounds on adults of Onchocerca volvulus.

The viability of adult Onchocerca volvulus and the effect of 12 known anthelmintic compounds on the parasites have been evaluated in an in vitro culture system. Three different parameters, a colorimetric assay, using NADH-dependent reduction of a tetrazolium salt to dark blue formazan by living adult worms, motility indices of male worms and lactate excretion of female worms were used to determine worm viability. The experiments showed that over a short term period of six days the viability of the worms did not decline significantly. The use of males isolated by dissection of whole nodules for the evaluation of drug effects in vitro is preferable to collagenase isolated worms. Mel W, milbemycin a and d, ivermectin, levamisole, CGP 6140 and, to a lesser extent, suramin immobilized male worms or significantly reduced the motility indices at a concentration of 10 microM. The tetrazolium reduction by male worms was not affected by levamisole, whereas the other active compounds demonstrated significant inhibitory effects. Diethylcarbamazine, mebendazole, flubendazole, metrifonate and CGP 20376 had no significant effect on male viability. Comparable activity was seen with the intact female worms isolated by collagenase digestion. Mel W, the milbemycins and ivermectin significantly inhibited tetrazolium reduction, whereas suramin and the other compounds had only slight or no inhibitory effects on female O. volvulus. Although one still has to aim at an improvement of the culture conditions, the in vitro test system using adult O. volvulus provides a basis for further research on potential antifilarial compounds.

The effect of two novel analogues of antimycin A on oxygen consumption and survival of filarial nematodes in vitro.

The effects of two novel analogues of antimycin A (BWA466C and BWA728C) on filarial oxygen consumption, energy generation and survival were investigated in vitro. For comparison, incubations were performed with a range of mitochondrial respiration inhibitors. All compounds tested (rotenone, antimycin A, KCN, oligomycin, CCCP, rafoxanide, BWA466C and BWA728C) inhibited oxygen uptake. The two analogues were less potent than antimycin A at impairing respiration of either filariae or beef heart submitochondrial particles. However, the two compounds affected motility and were lethal in vitro. Although the analogues affected oxygen uptake similarly to antimycin A itself, the levels of ATP were significantly lower than those noted in the presence of antimycin A. Glucose consumption and lactate output were markedly reduced by BWA466C and BWA728C. Glucose transport (measured as 2-deoxy-[2,6-3H]glucose) was reduced after treatment with BWA728C. It is likely that a combination of the effects on glucose transport and inhibition of oxidative pathways of carbohydrate metabolism may lead to worm death in vitro.

Structure-activity relationships of antifilarial antimycin analogues: a multivariate pattern recognition study.

The structure-activity relationships of a series of novel antifilarial antimycin A1 analogues have been investigated by using computational chemistry and multivariate statistical techniques. The physiochemical descriptors calculated in this way contained information which was useful in the classification of compounds according to their in vitro antifilarial activity. This approach generated a 53 parameter descriptor set, which was reduced with a multivariate pattern recognition package, ARTHUR. Regression analysis of the reduced set yielded several statistically significant regression equations; e.g.-log in vitro activity = 0.017 mp + 0.65 log P - 0.81ESDL10-7.33 (R = 0.9). With use of this equation, it was possible to make predictions for further untested analogues. The analysis indicated that membrane or lipid solubility is an important determinant in biological activity agreeing with the proposed primary mode of action of the compounds as disrupters of cuticular glucose uptake.

A preliminary assessment of the feasibility of evaluating promising antifilarials in vitro against adult Onchocerca volvulus.

The suitability of motility indices and tetrazolium-based colorimetric assays for the determination of the viability of adult Onchocerca volvulus after in vitro exposure to potential macrofilaricides has been examined. Experimentation showed that both techniques could be applied to adult O. volvulus, although the variability between individual worms necessitated the use of large experimental groups. The potential of using cut anterior tips of female O. volvulus for screening was also investigated. These were shown to give reasonably consistent motility indices, and drug effects were discernible even after 72 h in vitro culture. Application of these viability criteria to studies on the short-term in vitro survival of intact male and female O. volvulus incubated in Eagles MEM plus serum, under 5% CO2 in air, showed this medium to be suboptimal with a greater than 50% loss of worm viability within 144 h of nodulectomy. Males isolated by the collagenase technique were shown to be significantly less viable than dissected males, by both motility indices and tetrazolium reduction. The results highlight the need to use either dissected males, or in the case of females, the need to minimize exposure to collagenase solution. A possible mechanism for selecting a more uniformly viable female worm population is discussed. Examination of the in vitro effects of CGP 20376 using these viability criteria/assay systems showed some delayed suppression of worm motility, but after 120 h in vitro CGP 20376 was not macrofilaricidal against male or female O. volvulus. Male worms were also implanted subcutaneously into gerbils.(ABSTRACT TRUNCATED AT 250 WORDS)

The further application of MTT-formazan colorimetry to studies on filarial worm viability.

Experiments have confirmed that MTT-formazan colorimetry in its simplest form (incubation of intact worms with MTT and direct visualisation of any formazan formed) can be readily applied to several species of filariae including Onchocerca volvulus. Data is presented which will assist the development of quantitative MTT reduction viability tests for a selection of the smaller filarial species. Assays of pieces of Onchocerca gutturosa and O. volvulus females have led us to tentatively conclude that the tips of filariae, particularly the anterior ends, may well be metabolically the most active part of the worm. Selective sampling of these regions for Onchocerca might therefore be a useful indicator for the viability of the parasite. An example of how MTT-formazan colorimetry has been applied to yield additional data to support motility observations on the in vitro survival of male O. gutturosa is also given. The in vitro timecourse of worm death caused by 10 microM CGP 20376 on Acanthocheilonema viteae females has been examined by MTT reduction and compared with 6 other non-subjective parameters. The results suggests that the parameters examined could be divided into two groups according to the time taken for CGP 20376 to cause 50% inhibition (t50) of the parameter. Fast response parameters had t50's between 1 and 6 h (motility indices, 14CO2 evolution, adenine uptake and leucine uptake), they are more sensitive measures of viability and indicate possible worm damage which may or may not be reversible. Slow response parameters had t50's between 34 and 48.5 h (lactate output, MTT reduction and adenine leakage), and are probably linked with severe degenerative changes and are indicative of worm death.(ABSTRACT TRUNCATED AT 250 WORDS)

Onchocerca gutturosa and O. volvulus: studies on the viability and drug responses of cryopreserved adult worms in vitro.

The viability and drug responses of cryopreserved adult Onchocerca have been examined in vitro. Male worms were cryopreserved in liquid nitrogen (-196 degrees C) using ethanediol as a cryoprotectant in a 2-step incubation procedure. After thawing, 85-90% of O. gutturosa males were normally motile. These motile worms were evaluated for viability using 4 measurements (long-term motility/survival in culture; [U-14C]adenine uptake and leakage; glucose utilization; MTT-formazan colorimetry) and were no different from unfrozen controls. Subsequent experiments demonstrated that the motility responses of cryopreserved worms exposed to the antifilarial drugs ivermectin, CGP 6140 and levamisole were virtually identical to unfrozen controls. Some success was also obtained with this technique in cryopreserving O. volvulus males, with 2 thawed specimens surviving in culture for 93 and 106 d respectively. Following collagenase isolation, female worms were cryopreserved in medium +10% serum without protectant at -79 degrees C. A batch of 8 female O. gutturosa were all motile when thawed 14 d later, with a mean survival time (based on 5 specimens) of 71 d (range 60-90). However, a batch of worms transferred from -79 degrees C to -196 degrees C were badly damaged when thawed. Female O. volvulus were cryopreserved at -79 degrees C in Guatemala and sent by air freight on solid CO2 to the UK. Most specimens were active when thawed. Survival of motile specimens ranged from 7 to 272 d in culture. It is concluded that these techniques are of practical value for the storage and transportation of adult Onchocerca.

Radiorespirometric detection of macrofilaricidal activity in vitro.

An in vitro method for studying radiorespiration has been adapted to single macrofilariae. Using this method viable (but not heat-killed) Dipetalonema viteae and Onchocera gibsoni macrofilariae evolved measurable amounts of 14CO2 from L-[U-14C]glutamine. Nonlinear and less uniform rates of 14CO2 evolution were demonstrated with D-[U-14C]glucose, [1-14C]acetate and [1-14C]octanoate. These findings led us to develop an in vitro screen in which inhibition of 14CO2 evolution from L-[U-14C]glutamine was used as a parameter for gauging macrofilaricidal activity. Using this screen we have examined the activity of 17 'so-called' antifilarial standards and found a greater degree of sensitivity than shown by other biochemical criteria. Other data obtained suggest a role for radiorespirometry in determining the viability of fragments of Onchocerca tissue.

Colorimetric quantitation of filarial viability.

A simple three-step colorimetric assay based on the tetrazolium salt MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) has been developed for quantifying filarial viability. Living (but not dead) filariae take up MTT and rapidly reduce it to formazan, so staining themselves dark blue. This colour change which is easily seen provides a rapid qualitative test for filarial viability. Quantitative data can be obtained by solubilizing formazan out of the worm with DMSO and measuring the absorbance of the resulting solution at 510 nm. To date the technique has been demonstrated in several species of filariae including Onchocerca volvulus. MTT reduction is thought to be selective for NADH-dependent dehydrogenase activity in viable worms. The reaction occurs readily in all developmental stages of Dipetalonema viteae including fragments of filarial tissue. Enzyme activity in viable intact D. viteae appears to be primarily associated with the hypodermis/muscle cells, with minimal formazan formation in the gut and reproductive tracts. The application of this MTT assay as a parameter for quantifying in vitro drugs effects is described. Assay procedures have been developed and optimized with D. viteae and Brugia pahangi for the assessment of effects of macrofilariae and microfilarial release, and the activity of a range of antifilarial standards reported. Several potential applications of the technique to studies on filarial biology are discussed.

The application of biochemical criteria to the assessment of macrofilarial viability.

Previous attempts to assess nematode viability have been critically reviewed and the need to apply more objective biochemical criteria emphasized. The practicalities of assay development have been discussed with regard to sensitivity, selectivity and methodological considerations. The biochemical basis and assay methology for six assays (adenine leakage, adenine uptake, leucine uptake, 14CO2 evolution, lactate output and MTT reduction) that we have recently evaluated are detailed. The viability of Acanthocheilonema viteae females exposed for 120 h in vitro to 17 standard compounds (at 10 microM) has been assessed using these six assays and compared relative to motility indices from the micromotility meter. It was concluded that, despite the slightly superior sensitivity of the 14CO2 evolution assay, the MTT reduction method was most suitable for field use due to its technical and practical simplicity, and its applicability to fragments of onchocercal tissue. It was suggested that, in the absence of a better in vitro assay, the feasability of using MTT reduction together with histology should be assessed in a validation exercise with Onchocerca gibsoni.

Leakage of incorporated radiolabelled adenine--a marker for drug-induced damage of macrofilariae in vitro.

An objective in vitro assay has been developed for quantifying drug-induced damage in Dipetalonema viteae macrofilariae. The method involves radiolabelling the worms ATP pool by incubating macrofilariae with [U-14C]-adenine. As determined by HPLC 72% of the incorporated label was in ATP, 15% in ADP and about 4% in each of NAD and AMP. Macrofilariae labelled with [U-14C]-adenine show a linear efflux of [14C]-label amounting to 21.3% of the total incorporation (mainly as uncharged catabolites) over a time course of 120 h in vitro incubation. When prelabelled worms were exposed to compounds exerting macrofilaricidal effects in vitro a marked stimulation in the leakage of [14C]-label from the worms was noted. The [14C]-label leakage appears to be linked with membrane (or cuticle) damage and the reduction of macrofilarial ATP levels. Determination of the amount of [14C]-label remaining in drug-treated worms relative to appropriate control provides a simple, sensitive and quantitative measure of drug-induced damage in macrofilariae (including Onchocerca). The method has been used to describe the macrofilaricidal activity of a wide range of antifilarial standards, membrane disruptive agents, respiratory inhibitors, fasciolicides and anti-cestode compounds.