Proc. Nati. Acad. Sci. USAVol. 87, pp. 5950-5954, August 1990Medical Sciences

Treatment of Pneumocystis carinji pneumonia with 1,3-f3-glucansynthesis inhibitors

(echinocandin/papiacadn/AIDS/opporunlicinfection/Candida/antifungal drug)

D. M. SCHMATZ*t, M. A. ROMANCHECK*, L. A. PITTARELLI*, R. E. SCHWARTZt, R. A. FROMTLING§,K. H. NOLLSTADT*, F. L. VANMIDDLESWORTHf, K. E. WILSON*, AND M. J. TURNER*Departments of *Biochemical Parasitology, tNatural Products Chemistry, and §Microbiology, Merck Sharp & Dohme Research Laboratories,Rahway, NJ 07065

Communicated by Edward M. Scolnick, May 11, 1990

ABSTRACT Pneumocystis carini pneumonia is a majorcause of death in AIDS patients in the United States. Thepresently available treatments have limited use due to a highincidence of adverse reactions. Therefore, there is an urgentneed for a safer method for treatment and prevention of thisdisease. Recent evidence has suggested that P. carinn is relatedto fungi and that the wall ofthe cyst form contains 1,3-13-glucanas a major constituent. Based on this, several proposed 1,3-f3-glucan synthesis inhibitors were evaluated for their ability tocontrol P. carinu pneumonia in vivo. Compounds from twoclasses of 1,3-13-glucan synthesis inhibitors, the echinocandinsand papulacandins, were found to be effective against P.carinii.

Pneumocystis carinii pneumonia is the most prevalent op-portunistic infection and a frequent cause of death in AIDSpatients in the United States (1). The causative agent, P.carinii, is found almost exclusively in the lungs, althoughthere have been a number of cases of extrapulmonary in-volvement (2). Untreated, the organisms fill the alveolar sacs,and patients succumb to respiratory failure and related organdysfunction. Prior to the AIDS epidemic, P. carinii pneumo-nia was a major problem in childhood leukemia (3). At thattime, the condition was treated with pentamidine isethionate,a drug originally developed for the control of trypanosomi-asis. Pentamidine was effective in controlling the develop-ment of P. carinii pneumonia, but its associated toxicitymade the need for a safer treatment apparent (4). The use ofpentamidine for treating P. carinii pneumonia was virtuallyeliminated by the mid-1970s when it was discovered that acombination of trimethoprim (TMP) and sulfamethoxazole(SMZ) administered orally was equally as effective in treatingP. carinii pneumonia, with minimal adverse reactions innon-AIDS patients (5). However, a majority ofAIDS patientswith P. carinii pneumonia cannot tolerate TMP/SMZ, andthe only alternative has been to return to the use of pentam-idine, which has a slightly lower incidence of adverse reac-tions. Some of the toxicity problems associated with theintravenous use of pentamidine have been eliminated byadministering the drug as an aerosol (6, 7). Nonetheless,there remains a need for additional, more effective therapies.The debate over the taxonomic assignment of P. carinii as

a fungus or protozoan has led to several discoveries. Both theDNA sequence of the ribosomal RNA gene (8, 9) and studiesof the enzyme thymidylate synthase (10) have suggested thatP. carinii is more closely related to the fungi. Recently,indirect evidence has suggested that the wall of the cyst formof P. carinii is similar to the cell wall of the yeast Saccha-romyces cerevisiae and that it may contain high levels of

1,3-f3-glucan (11). Based on these studies, it became evidentthat inhibition of 1,3-,B-glucan synthesis might be a viabletarget for preventing the formation of P. carinji cysts. Thereare two classes of compounds that have a narrow spectrumof antifungal activity against yeasts and are known to inhibit1,3-.8-glucan synthesis in vitro. The first is a group of cyclichexapeptides with fatty acyl side chains known as the echino-candins. The second is the class of lipid-linked saccharidesknown as the papulacandins.

In the current study representative compounds from bothclasses were tested in a rat model of P. carinii pneumonia todetermine if they were effective in treating this disease. Theresults of this study indicate that compounds are potentiallyuseful for the treatment and prevention of P. carinii pneu-monia.

MATERIALS AND METHODSSource of Compounds. L-671,329 is a natural product,

which is a member of the echinocandin family, and wasproduced and isolated at Merck Sharp & Dohme ResearchLaboratories as described (12, 13). L-687,781 is a member ofthe papulacandin family, which was recently isolated duringthe screening of soil cultures for antifungal agents (F.L.V.,unpublished data). TMP and SMZ were obtained from theInterchem (Paramus, NJ).Animal Model and Evaluation ofCompounds. For the initial

probe study to determine if L-671,329 had antipneumocystisactivity, a rat model similar to that originally described byFrenkel et al. (14) and Hughes et al. (15) was used. MaleSprague-Dawley rats weighing =200 g, obtained from Sasco(Omaha, NB), were maintained on a low protein diet (8.0%6)and immunosuppressed with dexamethasone (Phoenix Phar-maceuticals, Saint Joseph, MO) in the drinking water (2mg/liter) to induce P. carinii pneumonia. Tetracycline (1g/liter) was also added to the drinking water to preventbacterial infections. At the start of the seventh week ofimmunosuppression, the rats were divided into three groupsand injected i.p. twice daily with 0.5 ml of L-671,329 at a doseof 10 mg/kg (in 10%6 dimethyl sulfoxide), treated with thestandard dose ofTMP/SMZ in the drinking water (0.2 g/literand 1 g/liter, respectively) as a positive control (15), or leftuntreated for 14 days. Immunosuppression was continuedduring this period. At the completion of treatment, all ratswere sacrificed by exposure to carbon dioxide gas; the lungswere removed, homogenized with a Brinkmann homogenizerin 10.0 ml of saline, and processed for quantitation asdescribed below.

In all subsequent studies the rats were also maintained ondexamethasone and a low protein diet for the duration of thestudy. After 6 weeks of immunosuppression, three rats were

Abbreviations: TMP, trimethoprim; SMZ, sulfamethoxazole.tTo whom reprint requests should be addressed.

5950

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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HN\CH3

H N

.N

"

0 H OHH

FIG. 1. Structure of L-671,329, a lipopeptide natural product related to the echinocandins.

randomly selected and sacrificed to confirm the presence ofP. carinji pneumonia. The remaining rats were distributedinto groups of5-7 and injected i.p. twice daily for 4 days with0.5 ml of various doses of L-671,329 or L-687,781 in vehicle(10% dimethyl sulfoxide) or vehicle alone. In cases whereanimals were treated with TMP/SMZ (0.2 g/liter and 1g/liter, respectively), the drug combination was given con-tinuously in the drinking water for 4, 7, 14, or 21 days. Controlanimals for each treatment period had been immunosup-pressed for the same length of time. At the completion oftreatments, the rats were sacrificed by exposure to carbondioxide gas; the lungs were removed, homogenized, andprocessed as described below.

Evaluation of Infected Lung Tissues. The lung homogenateswere washed once with 10.0 ml of saline by centrifugation at1000 x g; the red blood cells were lysed by resuspending thepellet in 5.0 ml of an 0.85% ammonium chloride solution andincubating for 5 min at 370C, after which the samples werewashed two additional times in saline; and the final pelletswere resuspended in 2.0 ml of saline. A 5.0-,ul aliquot wastaken from each sample and dried onto Teflon-coated micro-scope slides with a fixed surface area (11-mm circles, CarlsonScientific, Peotone, IL). The extent of disease for eachanimal was determined by microscopic analysis of stainedslides. The total number of cysts per animal lung wasdetermined by quantitating the number of cysts per 50microscope fields (x 1000) of homogenized lung tissue onslides fixed with ether/sulfuric acid and stained with toluidineblue (16). Total nuclei were determined from duplicate slidesstained with Diff-Quik (American Scientific Products, Mc-Gaw Park, IL). The total number of organisms per rat lungwas determined as a function of the surface area on the slide,the volume of the applied sample, and the total volume of theprocessed homogenate.

1,3-fi-Glucan Synthesis Assay. Protoplasts of Candida al-bicans (MY1208), isolated in midlogarithmic phase, wereprepared as described by Taft et al. (17), with the exceptionthat 1.2 M glycerol was substituted for 1.2 M sorbitol and theprotoplasts were washed three additional times after No-vozyme 234 treatment. Aliquots of washed protoplasts werestored in 1.2M glycerol/phosphate-buffered saline at -800C.The 1,3-p-glucan synthesis assay was conducted as a modi-fication of that previously described by Cabib and Kang forS. cerevisiae (18) in a total volume of 80.0 ,ul containing 125mM Tris-HCl (pH 7.0), 0.25 mM dithiothreitol, 0.15 mMphenylmethylsulfonyl fluoride, 0.40 M glycerol, 0.75 mMEDTA, 1.0% bovine serum albumin, 40.0 nM guanosine5'-[y-thio]triphosphate (tetralithium salt), and 4.0 mM lami-naribiose.

RESULTS AND DISCUSSIONThe rat model for P. carinii pneumonia is useful for evalu-ating efficacy since most control animals survive during thestudy, which allows for comparison of the extent ofP. carinjipneumonia with the experimental groups (19-21). We there-fore used it to demonstrate that L-671,329 (Fig. 1), anechinocandin analog (12, 13), was effective in eliminating thecyst form ofP. carinji from the lungs of infected rats at a doseof 10 mg/kg, administered i.p. twice daily for 14 days (Table1). Although the trophozoites (nuclei) were not as effectivelyeliminated as the cysts, the results were similar to thoseobtained for animals treated with TMP/SMZ for the sameperiod. Histological evidence further supports the activity ofL-671,329 (i.e., absence of cysts in the alveolar sacs andregression of the associated pneumonia) (Fig. 2). Furtherstudies indicated that much lower doses of L-671,329 weresufficient to eliminate cysts effectively and that shortertreatment periods were adequate to see this effect.A comparison of various doses of L-671,329 to TMP/SMZ

after only 4 days of treatment demonstrated that L-671,329 issuperior in clearing P. carinji cysts during short-term ther-apy; it eliminated >98% of the cysts at doses as low as 0.3mg/kg, whereas animals treated withTMP/SMZ for the sameperiod showed no significant reduction of cysts when com-pared to untreated control animals (Table 2). Treatment withTMP/SMZ for 1 week was also not significantly effective

Table 1. Initial probe study with L-671,329Total Total Total

Treatment Rat cysts* x 10-6 nucleit x 10-9 lung weightt, gNone 71A 320 5.8 3.16

74A 1500 14 3.3274B 750 14 3.56

L-671,329 71B ND§ 2.1 2.1372B ND§ 1.0 1.40

TMP/SMZ 73B 6.0 0.20 1.1175B 5.4 1.6 2.28

Rats with acute P. carinii pneumonia were treated for 2 weeksduring continued immunosuppression. L-671,329 was used at a doseof 10 mg/kg and TMP and SMZ were used at doses of 0.2 and 1g/liter, respectively.*Total cysts were determined by microscopic examination of 50fields (x1000) of homogenized lung tissue on slides fixed withether/sulfuric acid and stained with toluidine blue (15).

tTotal nuclei were determined from duplicate slides stained withDiff-Quick.tReduced lung weight is a possible indication of decreased orga-nisms, infiltrates, and/or reduced edema in the treated animals.§No cysts were detected; the limit of detection was 2 x 105 cysts.

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FIG. 2. Histological sections of lung tissue from a control rat (Left) with acute P. carinii pneumonia and a rat in the same study treated withL-671,329 at 10 mg/kg, twice daily, for 14 days (Right). In the control animal (Left), large masses ofP. carinii cysts can be seen filling the alveolarsacs, whereas cysts are no longer present in the animal treated with L-671,329 and the alveoli are clearly visible (Right). The lung tissue wasfixed in 1o formalin, embedded in paraffin, sectioned, stained with hematoxylin/eosin, and counterstained with methanamine silver. (x 1000.)

when compared to controls, although there was a meanreduction of 61.1% of the cysts. Treatment for 2 and 3 weekswith TMP/SMZ effectively eliminated >99% of the cysts(Table 3). Further titrations with L-671,329 indicated that theED90 for this compound is -O.15 mg/kg (Table 4). The rapidclearance ofcysts with L-671,329 may be a distinct advantageover existing therapies for treating patients in respiratoryfailure due to acute P. carinii pneumonia, since the accumu-lation of cysts in the alveolar sacs impedes respiration.Another proposed 1,3-p-glucan synthesis inhibitor, the

papulacandin analog L-687,781 (Fig. 3), was also tested in theP. carinii pneumonia rat model in parallel with L-671,329 and

Table 2. A comparative study among L-671,329, L-687,781, andTMP/SMZ in the P. carinji pneumonia rat model using a 4-daytreatment period

Dose, Total cysts, % reduction Survivors/Treatment mg/kg log (mean) of cysts total

None 7.19 ± 0.11 - 5/5DMSO control 7.46 ± 0.04 5/5L-671,329 1.2 5.54 ± 0.07 98.8* 5/5

0.6 5.56 ± 0.06 98.7* 5/50.3 5.73 ± 0.15 98.1* 5/5

L-687,781 10.0 6.77 ± 0.16 79.6* 5/55.0 6.89 ± 0.14 72.6* 5/52.5 7.48 ± 0.06 0.0 5/5

TMP/SMZ 7.38 ± 0.15 0.0 5/5The total number of cysts per animal lung is expressed as the

logarithm of the mean and the logarithm of the standard error of thegeometric mean. The lower limit of detection was 2 x 104 cysts perlung (loglo = 4.26). All groups were compared to the dimethylsulfoxide (DMSO) vehicle control with the exception of the TMP/SMZ group, which was compared to untreated immunosuppressedanimals. TMP and SMZ were used at 0.2 and 1.0 g/liter, respectively.*Significantly different from the appropriate control (P 0.05, ttest).

TMP/SMZ. The papulacandins are, for the most part, muchless active than echinocandins against yeast in vivo whilehaving comparable activity in vitro (22-24). In these studies,L-687,781 eliminated 80% of P. carinii cysts at 10.0 mg/kgand 73% at 5.0 mg/kg after 4 days (Table 2). Although theeffective dose is much higher than that required for L-671,329, it clearly demonstrates that another class of 1,3-a8-glucan synthesis inhibitor is also effective against P. carinii.To confirm that L-671,329 and L-687,781 were 1,3-(-

glucan synthesis inhibitors, both compounds were titrated ina modified 1,3-3-glucan synthesis assay (18) using membranepreparations from C. albicans protoplasts (17). The assaymeasures the incorporation of [14C]glucose (fromUDP[14C]glucose) into trichloroacetic acid-precipitable glu-can. Both inhibited glucan synthesis (Fig. 4) as has beenreported for related echinocandins and papulacandins (25-27) and had IC50 values of approximately 0.4 AM. Attemptsto inhibit the formation of trichloroacetic acid-precipitableglucans produced by P. carinii membranes using these com-

Table 3. An extended study with TMP/SMZ to demonstrateefficacy in the P. carinji pneumonia rat modelTreatment Total cysts, % reduction Survivors/period Treatment log (mean) of cysts total1 week None 7.38 ± 0.08 - 7/7

TMP/SMZ 6.97 ± 0.25 61.1 7/72 weeks None 7.65 + 0.21 6/7

TMP/SMZ 5.03 ± 0.19 99.8* 6/73 weeks None 7.22 ± 0.37 3/7

TMP/SMZ 4.62 ± 0.06 99.7* 5/7The total number of cysts per animal lung is expressed as the

logarithm of the mean and the logarithm of the standard error of thegeometric mean. The lower limit of detection was 2 x 104 cysts perlung (log1o = 4.26).*Significantly different from their corresponding controls (P < 0.05,t test).

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Table 4. Additional titrations of L-671,329 in the acute P. carinjipneumonia rat model using a 4-day treatment period

Dose, Total cysts, % reduction Survivors/Treatment mg/kg log (mean) of cysts total

Experiment 1DMSO control 7.54 ± 0.05 6/6L-671,329 1.20 4.64 ± 0.24 99.8* 4/6

0.60 5.24 + 0.22 99.2* 6/60.30 6.08 ± 0.13 94.2* 6/60.15 6.13 ± 0.22 93.5* 6/6

Experiment 2DMSO control 7.19 ± 0.21 4/5L-671,329 0.60 5.77 ± 0.25 %.2* 4/5

0.30 5.91 ± 0.30 94.8 4/50.15 6.28 ± 0.15 87.7* 5/50.08 6.67 ± 0.10 69.5 4/5

The total number of cysts per animal lung is expressed as thelogarithm of the mean and the logarithm of the standard error of thegeometric mean. The lower limit of detection was 2 x 104 cysts perlung (logl0 = 4.26). The results indicate that the ED90 for thecompound is -0.15 mg/kg. DMSO, dimethyl sulfoxide.*Significantly different from their corresponding controls (P < 0.05,t test).

pounds have shown limited inhibition (a maximum of 15-20%; K.H.N. and D.M.S., unpublished results), and it is notclear at this time if there is a small percentage of 1,3-/3-linkages in P. carinji critical to the cyst wall integrity or if themode of action of these compounds against P. carinii isdifferent from that in the yeast.

This study indicates that 1,3-/3-glucan synthesis inhibitorsmerit further attention as possible drugs for the treatment andprevention of P. carinji pneumonia. The compounds werewell tolerated by the animals and no gross toxicity wasobserved. Although the efficacy of these compounds againstP. carinii has not been demonstrated in humans, it is encour-aging that drugs currently used to treat P. carinji pneumonia,such as pentamidine and TMP/SMZ, are effective in the ratmodel (15, 28). All previous treatments for P. carinji pneu-monia have been limited by the lack of specificity, resultingin adverse reactions in the patient. In contrast, there is noknown counterpart of 1,3-,8-glucan synthesis in mammaliansystems. Therefore, inhibitors of this process have the po-tential to be well tolerated by the patient. Although theseresults add further support to the classification of P. carinjias a fungus, there are many broad-spectrum antifungal agentssuch as amphotericin B (14) and ketoconazole (29) that haveno effect on this organism, whereas several antiprotozoaldrugs, including pentamidine, have antipneumocystis activ-ity (30-32). Therefore, the organism may occupy a uniqueniche among fungi.

OH OH

HOW0

0HO

0 OH0

HOo | CH3 CH3o OH

OH

OH

FIG. 3. Structure of L-687,781, a natural product that is a memberof the papulacandin family.

0

x

CLCa0

40

Ca0

.0

CulU)BcoCL

UA-

40

30

20

lo0

.01 .1 1 1 0 1 00 1000

Drug Concentration (gM)FIG. 4. Effect of L-671,329 (e) and L-687,781 (o) on the formation

of trichloroacetic acid (TCA)-precipitable polysaccharides generatedby a membrane preparation from C. albicans protoplasts.

The rapid elimination of cysts observed with L-671,329treatment may prove beneficial in treating patients withrespiratory failure due to acute P. carinii pneumonia. Theslower clearing of the trophozoite form also can be seen withTMP/SMZ treatment, where trophozoites can be found inanimals treated for 2 weeks. The number oftrophozoites doesdecrease during treatment with L-671,329, suggesting that thecyst form is important for proliferation of the trophozoites orthat trophozoites are also susceptible to the drug. It is alsoknown that trophozoites persist even after long-term treat-ment with TMP/SMZ in the rat model and that these animalswill relapse if immunosuppression is continued after the drugis withdrawn (33). The continued treatment of AIDS patientswith aerosolized pentamidine or TMP/SMZ after an acuteinfection is standard procedure, since relapse is a commonproblem. Whether this would be required with the 1,3-,3-glucan synthesis inhibitors remains to be determined.

We acknowledge the members of the Department of Microbiologyand the Department of Chemistry at Merck Sharp & Dohme Re-search Laboratories who supported this work, particularly RobertGiacobbe, Mary Nalin, and Dr. Richard Monaghan of the Fermen-tation Microbiology Group.

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