INTRODUCTION

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Pneumocystis carinii is an important opportunistic pathogen that remains a significant cause of lethal pneumonia in immunocompromised individuals such as patients with AIDS and patients receiving chemotherapy or immunosuppressive drugs for organ transplantation or other pathological conditions. Patients suffering from P. carinii pneumonia (PCP) are usually treated with trimethoprim-sulfamethoxazole (TMP-SMX) or pentamidine (24). However, the relatively high frequency of adverse reactions to these drugs reflects the need for new therapeutic approaches. For this reason, the pharmaceutical industry is investigating more effective and less toxic agents.

Sordarin derivatives are a new class of antifungal agents that target protein synthesis (11), with marked in vitro activity against P. carinii (13) and excellent in vivo activity in experimental PCP (E. Dei-Cas, E. M. Aliouat, C. Mullet, E. Mazars, and D. Gargallo, Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F-65, 1997).

Well-defined mouse, rat, or rabbit experimental models (2, 4, 22) can be used to describe the in vivo anti-PCP activity of new compounds. Several in vitro tests for evaluating compound activity against P. carinii have been described using axenic cultures or coculture with feeder cells (8, 10, 14). However, no universally accepted standard method is presently available for the in vitro evaluation of anti-P. carinii molecules (10). The anti-Pneumocystis activity of any given antimicrobial could be evaluated in terms of its intrinsic activity (in vitro) and serum time profile (in vivo) (12). However, the results obtained with different in vitro assays (8, 10) yield limited information on the intrinsic activity of anti-Pneumocystis compounds. Furthermore, comparisons between product activities and extrapolation to in vivo activity remain unreliable.

The Hill equation, which describes sigmoid concentration-effect relationships, has proven its utility by revealing in vitro pharmacodynamic properties of several antibiotics (17, 30). This approach offers at least three parameters which can be used to describe the in vitro activity of antimicrobial compounds (17): the maximum effect (E_max) as a measure of efficacy, the 50% effective concentration (EC₅₀) as a parameter of intrinsic activity, and the slope () of the concentration-effect relationship.

The aim of the present work was to establish the experimental conditions allowing definition of the in vitro pharmacodynamic parameters of tested drugs for defining the intrinsic activity of anti-Pneumocystis molecules, as well as the relationships between their in vitro activities and in vivo effects on microorganisms.

(This work was presented in part at the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, Calif., 24 to 27 September, 1998 [E. M. Aliouat, P. Aviles, E. Dei-Cas, E. Herreros, L. Dujardin, and D. Gargallo-Viola, abstr. J-15].)

	MATERIALS AND METHODS

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Drugs. GM 191519, GM 193663, GM 219771, and GM 237354 are new sordarin derivatives synthesized by GlaxoWellcome, S.A. (Madrid, Spain). TMP-SMX (Sigma Chemical Co., St. Louis, Mo.), pentamidine isothionate (Sigma Chemical Co.), and atovaquone (GlaxoWellcome, Greenford, United Kingdom) were also tested. Sordarin derivatives were dissolved in sterile distilled water at a starting concentration of 10 mg/ml. TMP, pentamidine, and atovaquone were dissolved in 100% dimethyl sulfoxide (DMSO) (Sigma Chemical Co.) to produce a 10-mg/ml stock solution. SMX was also dissolved in DMSO but to a concentration of 30 mg/ml. TMP and SMX solutions were mixed appropriately to obtain a final combination of 1:5. Finally, the drug stock solutions were diluted in Dulbecco's modified Eagle's medium (DMEM) (Bio-Whittaker, Boehringer Ingelheim, Brussels, Belgium) supplemented with 10% heat-inactivated fetal calf serum (FCS) (GIBCO BRL, Life Technologies Inc.) to produce the required drug concentrations. Compound solutions were prepared immediately before use.

Source of P. carinii. Corticosteroid-treated rats were used as the source of P. carinii organisms. Seven-week-old female Wistar rats (Iffa-Credo, Lyon, France) were immunosuppressed with dexamethasone (Fortecortin; Merck, Darmstadt, Germany) administered in drinking water (2 mg/liter) for approximately 10 weeks (24). Animals had access to sterile standard food (gamma-irradiated rodent maintenance diet) and water ad libitum. At the end of the immunosuppression period the rats were sacrificed and P. carinii was recovered from their lungs. The research complied with national legislation, with company policy on the care and use of animals, and with related guidelines (1).

Isolation and quantitation of P. carinii organisms. P. carinii organisms were isolated as previously described (3), with some modifications. After the immunosuppression period, rat lungs were removed aseptically and cut into small pieces in sterile DMEM. P. carinii organisms were extracted by agitation of lung pieces with a magnetic stirrer for 1 h at 4°C. To remove tissue debris, the resulting homogenate was poured through sterile gauze and centrifuged at 2,900 × g for 10 min at 4°C. After centrifugation, the pellet was resuspended in a buffered hemolytic solution (9:1 solution of 0.15 M NH₄Cl in 20 mM Tris-HCl, pH 7.4), incubated for 10 min at 4°C, and centrifuged. Then, the pellet was resuspended in DMEM and filtered successively through 250- to 63-µm-pore-size stainless steel filters. Finally, a polysucrose gradient (Histopaque-1077; Sigma Chemical Co.), to obtain purified Pneumocystis organisms with a minimum of host contamination, was performed as follows. Polysucrose solution and inoculum suspended in DMEM were prepared 1:1 (vol/vol) in a 15-ml tube (Costar Corporation, Cambridge, Mass.) and centrifuged at 1,000 × g for 15 min at 4°C. The band accumulated at the interface between DMEM and polysucrose solution was collected and washed twice with DMEM (2,900 × g for 10 min at 4°C). P. carinii was quantitated on air-dried smears stained with RAL-555 (Réactifs RAL), a rapid panoptic methanol-Giemsa stain, which stains every Pneumocystis life cycle stage. In addition, samples were used to search for putative contaminant organisms. Moreover, final suspension was plated on blood (Difco, Detroit, Mich.) and Sabouraud dextrose agar (Difco) for the detection of bacterial or fungal contamination, respectively. The total numbers of P. carinii forms (trophozoites, precysts, and cysts) were calculated as previously described (3): (n · Sa · R)/Fa, where n is the average number of microorganisms per oil immersion field (10 fields were counted for each smear), S_a is the 2-µl smear area, R is the ratio between total volume of the microorganism suspension and calibrate smear volume (2 µl), and F_a is the oil immersion field area.

Axenic in vitro culture of P. carinii. In order to determine the in vitro drug susceptibility of P. carinii, axenic cultures of the organism were produced as follows. All the experiments were carried out in 24-well plates (Nalge Nunc International, Roskilde, Denmark) with a final volume of 2 ml of DMEM supplemented with 10% FCS containing a final inoculum of 1.5 × 10⁶ organisms per ml. Plates with organisms were incubated for 5 days in an atmosphere of 5% CO₂ at 37°C, and the kinetic patterns of in vitro P. carinii development were determined. Daily for 5 consecutive days, the total volume of each well was removed and centrifuged for 10 min at 2,900 × g, and the pellet was resuspended with 200 µl of phosphate buffer solution Dulbecco (Sigma Chemical Co.). Two-microliter smears were obtained from each suspension. P. carinii organisms were stained with RAL-555 and were quantified as described above. All experiments were performed in triplicate.

In vitro susceptibility studies. The above method was validated by performing three independent experiments involving GM 237354 as reference compound. Concentration-response curves were calculated, and the corresponding E_max, EC₅₀, and slope were obtained.

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Analysis of results. The anti-Pneumocystis activity of a single concentration of compound may be expressed in terms of percent inhibition, defined as the decrease (expressed as percentage) in P. carinii forms in antifungal-treated cultures with respect to the total microorganism count in compound-free culture. Once all the differences between drug-treated and untreated wells were calculated, the concentration-effect relationship was established by means of the Hill equation (17, 30): E_R = E_R,max · C^S/[(EC₅₀)^S + C^S], where E_R is the effect of each drug concentration (C) upon percent inhibition estimated from experimental results, S is a parameter reflecting the steepness of the concentration-effect relationship curve, and EC₅₀ is the concentration of the compound at which 50% of the maximum effect (E_R,max) is obtained.

In vivo study. An in vivo pilot study which involved PCP in rats was performed to explore whether in vitro results reflect in vivo efficacy. PCP was established by using a previously described method. Briefly, animals were immunosuppressed with dexamethasone (Fortecortin; Merck) at a concentration of 2 mg/liter in the drinking water for 9 weeks. Tetracycline (Terramicine; Pfizer Laboratories) at 1 g/liter was added as antibacterial prophylactic agent. All animals remained on immunosuppressive therapy with dexamethasone throughout the study. Before treatment, PCP was microscopically verified as previously reported (28). Animals were divided into groups of six, and then, sordarin derivatives (GM 191519, GM 237354, and GM 219771) were dosed at 0.1, 1.0 and 5.0 mg/kg by subcutaneous route. The drugs were given twice a day for 10 consecutive days. Control animals were dosed with sterile water. Twenty-four hours after the last dose all animals were sacrificed by overdosing of sodium pentobarbital (Euthalender; Normon). Lungs were aseptically removed and weighted. P. carinii extractions were performed by using a previously described method (2). P. carinii cystic forms were quantitated by Toluidine Blue-0 staining (Sigma-Aldrich, S.A.). The number of cysts were determined by visual assessment by light microscopy (20 microscopic fields). All results were expressed as log₁₀ of the number of cysts per gram of lung (logQ/g). Daily dose (milligrams per kilogram per day) and logQ/g were plotted and adjusted to an E_max model by nonlinear least-squares regression techniques using commercial software (WinNonlin). Then, ED₅₀ (defined as the daily dose which reduces 50% the P. carinii burden in lungs) was calculated.

	RESULTS

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In vitro P. carinii growth. Figure 1 shows the P. carinii growth curve obtained after 5 days of incubation. The curve displays two well-defined portions: a first segment (from day 0 to 3 or 4), where P. carinii gradually increases in number, with doubling times of 85.3 h (up to day 3) and 102.7 h (up to day 4), and a second segment, reflecting P. carinii population decrements after the fourth day of incubation. Moreover, the formation of large, typical trophozoite clusters was observed throughout the incubation period, while the number of cystic forms gradually decreased in proportion, to less than 2% of total microorganisms after 3 days of incubation.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Typical growth curve of rat-derived P. carinii cultivated in DMEM supplemented with 10% FCS.

Optimal assay setup conditions. Preliminary experiments were performed using pentamidine and GM 237354. High (10-µg/ml), medium (1-µg/ml), and low (0.01-µg/ml) concentrations of compound were tested against P. carinii for 5 days of culture. Percent inhibition compared to drug-free control was determined (Fig. 2). When P. carinii was incubated for 1 to 3 days with three concentrations of GM 237354, relatively low growth inhibition was observed, even when the highest concentration (10 µg/ml) was tested. Maximum inhibition rates of 38 and 60% were observed after 2 or 3 days of culture, respectively (Fig. 2A). However, after 3 days of culture, pentamidine (10 and 1 µg/ml) reached 100% inhibition (Fig. 2B). Both concentrations, i.e., 10 and 1 µg of pentamidine and GM 237354 per ml, had the maximum effect by the fourth day of incubation. The inhibitory effect of pentamidine at 0.01 µg/ml showed similar levels (44 to 54% inhibition) after 3 to 5 days of culture, whereas the GM 237354 inhibitory effect reached 70 and 96% inhibition at 4 and 5 days postinoculation, respectively (Fig. 2A).

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Influence of incubation time on the in vitro activities of GM 237354 and pentamidine for 5 consecutive days of culture. Both compounds were tested at three different concentrations. , 10 µg/ml; , 1 µg/ml; , 0.01 µg/ml. The effect of each compound concentration on P. carinii was expressed as percent inhibition versus the drug-free control.

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In vitro susceptibility studies. The robustness of the method was assayed by performing three different experiments with GM 237354. All the three resulting concentration-response curves obtained essentially overlapped (data not shown). Mean calculated values for E_max (99.06%), EC₅₀ (0.00076 µg/ml), and the slope (0.53) showed variation coefficients of 2, 20, and 11.5%, respectively.

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View larger version (29K): [in this window] [in a new window]   FIG. 3.   In vitro activity of GM 237354 against P. carinii. (A) Culture medium with 0.5 µg of GM 237354 per ml. A dramatic reduction in the number of microorganisms was observed. (B) Culture medium with 5 × 104 µg of GM 237354 per ml. A marked decrease in the number of microorganisms was observed. (C) Drug-free control culture. Trophozoites (open arrowhead) and trophozoite clusters (solid arrowhead) were observed. RAL-555 staining was used. Magnification, ×1,100.

View larger version (22K): [in this window] [in a new window]   FIG. 4.   Concentration-in vitro activity relationships of the three commercial compounds against P. carinii. , pentamidine; , atovaquone; , TMP-SMX. Results were calculated after 4 days of incubation.

View larger version (30K): [in this window] [in a new window]   FIG. 5.   Concentration-in vitro activity relationships of the four sordarin derivatives against P. carinii. , GM 191519; , GM 237354; , GM 193663; , GM 219771. Results were calculated after 4 days of incubation.

In vivo study. The number of P. carinii cysts per gram of lung in untreated animals which develop PCP reaches 10^7.6 at the end of the treatment period (73 days after the start of dexamethasone immunosuppression). All the three sordarin derivatives caused a marked reduction in the number of P. carinii cyst forms per gram of lung. Dose-response curves obtained after sordarin derivative treatment of experimental PCP in rats are displayed in Fig. 6. Good agreement between experimentally observed and calculated values was obtained (r², 0.999, 0.980, and 0.998 for GM 191519, GM 237354, and GM 219771, respectively) for each therapeutic regimen used. Calculated ED₅₀s for sordarin derivatives demonstrated that GM 191519 was the most potent of the three compounds (ED₅₀, 0.05 mg/kg/day) followed by GM 237354 (ED₅₀, 0.30 mg/kg/day) and GM 219771 (ED₅₀, 0.49 mg/kg/day). These in vivo potencies are in the same order as those observed for in vitro results.

View larger version (9K): [in this window] [in a new window]   FIG. 6.   In vivo efficacies of GM 191519 () and GM 219771 () on the P. carinii burden in lung. The data are means with standard deviations for six animals. The dose-effect relationships were calculated according a simple Emax model.

	DISCUSSION

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A quantitative and reproducible in vitro susceptibility axenic model for evaluating pharmacodynamic parameters of anti-Pneumocystis compounds is described in the present study. The results obtained indicate that in vitro EC₅₀ could be an index of the expected anti-PCP in vivo effect, at least for sordarin derivatives. Pharmacodynamic parameters of anti-Pneumocystis drugs were calculated using the Hill equation, which has been previously used to describe in vitro concentration-effect relationships of antibacterial compounds (17, 30). Antimicrobial agents were evaluated by comparing P. carinii growth in drug-treated versus drug-free control cultures after a 4-day incubation period. Microorganism growth was assessed on dry smears stained by methanol-Giemsa-like staining (RAL-555), which allows total microorganism quantitation. Most P. carinii microorganisms developing in this system are vegetative forms. In agreement with other reports (5, 16), we found that in vitro Pneumocystis proliferation leads to the formation of clusters in which more than 98% are trophozoite forms.

In vitro susceptibility tests are a basic step in any pharmacological screening for new anti-infective drugs. Several factors influence the outcome and reproducibility of susceptibility tests (6), including medium composition, inoculum size, incubation time, and the nature of the microorganism. In the present study, P. carinii organisms were cultivated by means of an axenic culture rather than the usual coculture methods. The main reasons for adopting this approach can be summarized as follows. (i) Mammalian cell growth produces extracellular catabolic products and depletes broth nutrientsprocesses capable of affecting P. carinii growth and influencing the susceptibility patterns. (ii) Developing cocultured cells could influence the stability of tested compounds. (iii) New compounds can display toxicity against monolayer cells. This fact could affect P. carinii growth and hence the susceptibility results obtained. (iv) Microorganisms attached to cell monolayers could acquire susceptibility patterns different from those of unattached microorganisms in suspension due to several factors, such as different growth rates, metabolic processes, or drug accessibilities. (v) Interactions between unattached microorganisms and compounds are probably easier than those found when microorganisms are attached to target cells. All the above considerations could also negatively affect interlaboratory reproducibility. Furthermore, in order to minimize the presence of a biological matrix in the culture medium, FCS should be replaced by a synthetic substitute, since the presence of serum could have multiple effects in susceptibility testing. Some authors have reported compound inactivation as a result of drug binding to serum components (9), where only the free fraction shows antimicrobial activity. Moreover, considering the behavior of P. carinii in in vitro cultures, some authors have attempted to improve culture performance by using cocultured cells to evaluate susceptibility. However, the presence of mammalian cells in the system could add a poorly controlled variable and potentially result in a lack of assay reproducibility.

Another variable with specific weight in drug susceptibility test outcomes is incubation time. Our results show that this parameter must be considered to establish an effective compromise between microorganism growth in drug-free controls and microorganism inhibition in drug-treated microculture wells.

In the antibacterial field, the MIC is the most widely used parameter for determining susceptibility. For this reason, internationally accepted guidelines have been published (18) for performing the tests so as to avoid or minimize interlaboratory variability. Similar initiatives have been proposed by the international scientific community for other tests which offer complementary information on the in vitro activities of antibacterial drugs (20, 21). More recently a standard method was proposed for pathogenic yeast (19). P. carinii has recently been included in the fungal kingdom, though it may be regarded as an atypical fungus (29): it lacks ergosterol (a target for most antifungals) and refuses to grow well in vitro (25). Recent efforts have been made to improve this situation (16), however.

Antigenic and genomic host species-related differences have been reported among P. carinii isolates, yielding a strong host species specificity (4). However, it has not been proven whether Pneumocystis strains from different mammal hosts can exhibit different drug susceptibility patterns. The development of an in vitro pharmacodynamic model affords an improved tool for the evaluation of susceptibility patterns of Pneumocystis strains from different host species.

Mouton et al. have offered a pharmacological explanation for the parameters in the Hill equation (17). They also suggested that the Hill model could be very useful for understanding changes in susceptibility, relating EC₅₀ values to classical MICs. The findings described herein provide sufficient information for considering EC₅₀ to be an accurate indicator of in vitro activities of anti-Pneumocystis compounds. Such an approach will provide a new tool for selecting new compounds and establishing therapeutic protocols. By using this pharmacodynamic approach, the results obtained revealed a high in vitro anti-Pneumocystis activity on the part of sordarin derivatives which belong to a new family of antifungals targeting fungal protein synthesis (11). Moreover, EC₅₀ seems to reflect in vivo anti-Pneumocystis activity, as sordarin derivative in vivo ED₅₀s suggest. Future experimental in vivo work is warranted to further establish the performance of this new in vitro approach with other anti-Pneumocystis compounds.