Proc. Nati. Acad. Sci. USA Vol. 87, pp. 5950-5954, August 1990 Medical Sciences Treatment of Pneumocystis carinji pneumonia with 1,3-f3-glucan synthesis inhibitors (echinocandin/papiacadn/AIDS/opporunlicinfection/Candida/ 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 major 1,3-f3-glucan (11). Based on these studies, it became evident cause of death in AIDS patients in the United States. The that inhibition of 1,3-,B-glucan synthesis might be a viable presently available treatments have limited use due to a high target for preventing the formation of P. carinji cysts. There incidence of adverse reactions. Therefore, there is an urgent are two classes of compounds that have a narrow spectrum need for a safer method for treatment and prevention of this of antifungal activity against yeasts and are known to inhibit disease. Recent evidence has suggested that P. carinn is related 1,3-.8-glucan synthesis in vitro. The first is a group of cyclic to fungi and that the wall ofthe cyst form contains 1,3-13-glucan hexapeptides with fatty acyl side chains known as the echino- as a major constituent. Based on this, several proposed 1,3- candins. The second is the class of lipid-linked saccharides f3-glucan synthesis inhibitors were evaluated for their ability to known as the papulacandins. control P. carinu pneumonia in vivo. Compounds from two In the current study representative compounds from both classes of 1,3-13-glucan synthesis inhibitors, the echinocandins classes were tested in a rat model of P. carinii pneumonia to and papulacandins, were found to be effective against P. determine if they were effective in treating this disease. The carinii. results of this study indicate that compounds are potentially useful for the treatment and prevention of P. carinii pneu- Pneumocystis carinii pneumonia is the most prevalent op- monia. portunistic infection and a frequent cause of death in AIDS patients in the United States (1). The causative agent, P. MATERIALS AND METHODS carinii, is found almost exclusively in the lungs, although Source of Compounds. L-671,329 is a natural product, there have been a number of cases of extrapulmonary in- which is a member of the echinocandin family, and was volvement (2). Untreated, the organisms fill the alveolar sacs, produced and isolated at Merck Sharp & Dohme Research and patients succumb to respiratory failure and related organ Laboratories as described (12, 13). L-687,781 is a member of dysfunction. Prior to the AIDS epidemic, P. carinii pneumo- the papulacandin family, which was recently isolated during nia was a major problem in childhood leukemia (3). At that the screening of soil cultures for antifungal agents (F.L.V., time, the condition was treated with isethionate, unpublished data). TMP and SMZ were obtained from the a drug originally developed for the control of trypanosomi- Interchem (Paramus, NJ). asis. Pentamidine was effective in controlling the develop- Animal Model and Evaluation ofCompounds. For the initial ment of P. carinii pneumonia, but its associated toxicity probe study to determine if L-671,329 had antipneumocystis made the need for a safer treatment apparent (4). The use of activity, a rat model similar to that originally described by pentamidine for treating P. carinii pneumonia was virtually Frenkel et al. (14) and Hughes et al. (15) was used. Male eliminated by the mid-1970s when it was discovered that a Sprague-Dawley rats weighing =200 g, obtained from Sasco combination of trimethoprim (TMP) and sulfamethoxazole (Omaha, NB), were maintained on a low protein diet (8.0%6) (SMZ) administered orally was equally as effective in treating and immunosuppressed with dexamethasone (Phoenix Phar- P. carinii pneumonia, with minimal adverse reactions in maceuticals, Saint Joseph, MO) in the drinking water (2 non-AIDS patients (5). However, a majority ofAIDS patients mg/liter) to induce P. carinii pneumonia. Tetracycline (1 with P. carinii pneumonia cannot tolerate TMP/SMZ, and g/liter) was also added to the drinking water to prevent the only alternative has been to return to the use of pentam- bacterial infections. At the start of the seventh week of idine, which has a slightly lower incidence of adverse reac- immunosuppression, the rats were divided into three groups tions. Some of the toxicity problems associated with the and injected i.p. twice daily with 0.5 ml ofL-671,329 at a dose intravenous use of pentamidine have been eliminated by of 10 mg/kg (in 10%6 dimethyl sulfoxide), treated with the administering the drug as an aerosol (6, 7). Nonetheless, standard dose ofTMP/SMZ in the drinking water (0.2 g/liter there remains a need for additional, more effective therapies. and 1 g/liter, respectively) as a positive control (15), or left The debate over the taxonomic assignment ofP. carinii as untreated for 14 days. Immunosuppression was continued a fungus or protozoan has led to several discoveries. Both the during this period. At the completion of treatment, all rats DNA sequence ofthe ribosomal RNA gene (8, 9) and studies were sacrificed by exposure to carbon dioxide gas; the lungs of the enzyme thymidylate synthase (10) have suggested that were removed, homogenized with a Brinkmann homogenizer P. carinii is more closely related to the fungi. Recently, in 10.0 ml of saline, and processed for quantitation as indirect evidence has suggested that the wall ofthe cyst form described below. of P. carinii is similar to the cell wall of the yeast Saccha- In all subsequent studies the rats were also maintained on romyces cerevisiae and that it may contain high levels of dexamethasone and a low protein diet for the duration of the study. After 6 weeks of immunosuppression, three rats were The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: TMP, trimethoprim; SMZ, sulfamethoxazole. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5950 Downloaded by guest on September 25, 2021 Medical Sciences: Schmatz et al. Proc. Natl. Acad. Sci. USA 87 (1990) 5951

HN \CH3

H N

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FIG. 1. Structure of L-671,329, a lipopeptide natural product related to the echinocandins. randomly selected and sacrificed to confirm the presence of RESULTS AND DISCUSSION P. carinji pneumonia. The remaining rats were distributed The rat model for P. carinii pneumonia is useful for evalu- into groups of5-7 and injected i.p. twice daily for 4 days with the 0.5 ml of various doses of L-671,329 or L-687,781 in vehicle ating efficacy since most control animals survive during the extent ofP. carinji (10% dimethyl sulfoxide) or vehicle alone. In cases where study, which allows for comparison of the groups (19-21). We there- animals were treated with TMP/SMZ (0.2 g/liter and 1 pneumonia with experimental to an g/liter, respectively), the drug combination was given con- fore used it demonstrate that L-671,329 (Fig. 1), 13), was effective in the tinuously in the drinking water for 4, 7, 14, or 21 days. Control echinocandin analog (12, eliminating treatment period had been immunosup- cyst form ofP. carinji from the lungs ofinfected rats at a dose animals for each for 14 pressed for the same length of time. At the completion of of 10 mg/kg, administered i.p. twice daily days (Table were not as treatments, the rats were sacrificed by exposure to carbon 1). Although the trophozoites (nuclei) effectively eliminated as the results were similar to those dioxide gas; the lungs were removed, homogenized, and cysts, the obtained for animals treated with for the same processed as described below. TMP/SMZ Evaluation ofInfected Lung Tissues. The lung homogenates period. Histological evidence further supports the activity of cysts in the alveolar sacs and were washed once with 10.0 ml of saline by centrifugation at L-671,329 (i.e., absence of regression of the associated pneumonia) (Fig. 2). Further 1000 x g; the red blood cells were lysed by resuspending the studies indicated that much lower doses of were pellet in 5.0 ml of an 0.85% ammonium chloride solution and L-671,329 cysts that shorter incubating for 5 min at 370C, after which the samples were sufficient to eliminate effectively and two in the final pellets treatment periods were adequate to see this effect. washed additional times saline; and A comparison of various doses of L-671,329 to TMP/SMZ were resuspended in 2.0 ml of saline. A 5.0-,ul aliquot was after only 4 days oftreatment demonstrated that L-671,329 is taken from each sample and dried onto Teflon-coated micro- superior in clearing P. carinji cysts during short-term ther- scope slides with a fixed surface area (11-mm circles, Carlson apy; it eliminated >98% of the cysts at doses as low as 0.3 Scientific, Peotone, IL). The extent of disease for each mg/kg, whereas animals treated with TMP/SMZ for the same animal was determined by microscopic analysis of stained period showed no significant reduction of cysts when com- slides. The total number of cysts per animal lung was pared to untreated control animals (Table 2). Treatment with determined by quantitating the number of cysts per 50 TMP/SMZ for 1 week was also not significantly effective microscope fields (x 1000) of homogenized lung tissue on slides fixed with ether/sulfuric acid and stained with toluidine Table 1. Initial probe study with L-671,329 blue (16). Total nuclei were determined from duplicate slides Total Total Total stained with Diff-Quik (American Scientific Products, Mc- Treatment Rat cysts* x 10-6 nucleit x 10-9 lung weightt, g Gaw Park, IL). The total number of organisms per rat lung was determined as a function ofthe surface area on the slide, None 71A 320 5.8 3.16 the volume ofthe applied sample, and the total volume ofthe 74A 1500 14 3.32 processed homogenate. 74B 750 14 3.56 1,3-fi-Glucan Synthesis Assay. Protoplasts of Candida al- L-671,329 71B ND§ 2.1 2.13 bicans (MY1208), isolated in midlogarithmic phase, were 72B ND§ 1.0 1.40 prepared as described by Taft et al. (17), with the exception TMP/SMZ 73B 6.0 0.20 1.11 that 1.2 M glycerol was substituted for 1.2 M sorbitol and the 75B 5.4 1.6 2.28 protoplasts were washed three additional times after No- Rats with acute P. carinii pneumonia were treated for 2 weeks vozyme 234 treatment. Aliquots of washed protoplasts were during continued immunosuppression. L-671,329 was used at a dose stored in 1.2 M glycerol/phosphate-buffered saline at -800C. of 10 mg/kg and TMP and SMZ were used at doses of 0.2 and 1 The 1,3-p-glucan synthesis assay was conducted as a modi- g/liter, respectively. fication of that described by Cabib and Kang for *Total cysts were determined by microscopic examination of 50 previously fields (x1000) of homogenized lung tissue on slides fixed with S. cerevisiae (18) in a total volume of 80.0 ,ul containing 125 ether/sulfuric acid and stained with toluidine blue (15). mM Tris-HCl (pH 7.0), 0.25 mM dithiothreitol, 0.15 mM tTotal nuclei were determined from duplicate slides stained with phenylmethylsulfonyl fluoride, 0.40 M glycerol, 0.75 mM Diff-Quick. EDTA, 1.0% bovine serum albumin, 40.0 nM guanosine tReduced lung weight is a possible indication of decreased orga- 5'-[y-thio]triphosphate (tetralithium salt), and 4.0 mM lami- nisms, infiltrates, and/or reduced edema in the treated animals. naribiose. §No cysts were detected; the limit of detection was 2 x 105 cysts. Downloaded by guest on September 25, 2021 5952 Medical Sciences: Schmatz et al. Proc. NatL Acad Sci. USA 87 (1990)

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 with L-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 alveolar sacs, whereas cysts are no longer present in the animal treated with L-671,329 and the alveoli are clearly visible (Right). The lung tissue was fixed 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 mean TMP/SMZ. The papulacandins are, for the most part, much reduction of 61.1% of the cysts. Treatment for 2 and 3 weeks less active than echinocandins against yeast in vivo while with TMP/SMZ effectively eliminated >99% of the cysts having comparable activity in vitro (22-24). In these studies, (Table 3). Further titrations with L-671,329 indicated that the L-687,781 eliminated 80% of P. carinii cysts at 10.0 mg/kg ED90 for this compound is -O.15 mg/kg (Table 4). The rapid and 73% at 5.0 mg/kg after 4 days (Table 2). Although the clearance ofcysts with L-671,329 may be a distinct advantage effective dose is much higher than that required for L- over existing therapies for treating patients in respiratory 671,329, it clearly demonstrates that another class of 1,3-a8- failure due to acute P. carinii pneumonia, since the accumu- glucan synthesis inhibitor is also effective against P. carinii. lation of cysts in the alveolar sacs impedes respiration. To confirm that L-671,329 and L-687,781 were 1,3-(- Another proposed 1,3-p-glucan synthesis inhibitor, the glucan synthesis inhibitors, both compounds were titrated in papulacandin analog L-687,781 (Fig. 3), was also tested in the a modified 1,3-3-glucan synthesis assay (18) using membrane P. carinii pneumonia rat model in parallel with L-671,329 and preparations from C. albicans protoplasts (17). The assay measures the incorporation of [14C]glucose (from Table 2. A comparative study among L-671,329, L-687,781, and UDP[14C]glucose) into trichloroacetic acid-precipitable glu- TMP/SMZ in the P. carinji pneumonia rat model using a 4-day can. Both inhibited glucan synthesis (Fig. 4) as has been treatment period reported for related echinocandins and papulacandins (25- Dose, Total cysts, % reduction Survivors/ 27) and had IC50 values of approximately 0.4 AM. Attempts Treatment mg/kg log (mean) of cysts total to inhibit the formation of trichloroacetic acid-precipitable glucans produced by P. carinii membranes using these com- None 7.19 ± 0.11 - 5/5 DMSO control 7.46 ± 0.04 5/5 Table 3. An extended study with TMP/SMZ to demonstrate L-671,329 1.2 5.54 ± 0.07 98.8* 5/5 efficacy in the P. carinji pneumonia rat model 0.6 5.56 ± 0.06 98.7* 5/5 Treatment Total cysts, % reduction Survivors/ 0.3 5.73 ± 0.15 98.1* 5/5 period Treatment log (mean) of cysts total L-687,781 10.0 6.77 ± 0.16 79.6* 5/5 5.0 6.89 ± 0.14 72.6* 5/5 1 week None 7.38 ± 0.08 - 7/7 2.5 7.48 ± 0.06 0.0 5/5 TMP/SMZ 6.97 ± 0.25 61.1 7/7 TMP/SMZ 7.38 ± 0.15 0.0 5/5 2 weeks None 7.65 + 0.21 6/7 TMP/SMZ 5.03 ± 0.19 99.8* 6/7 The total number of cysts per animal lung is expressed as the 3 weeks None 7.22 ± 0.37 logarithm of the mean and the logarithm of the standard error of the 3/7 geometric mean. The lower limit of detection was 2 x 104 cysts per TMP/SMZ 4.62 ± 0.06 99.7* 5/7 lung (loglo = 4.26). All groups were compared to the dimethyl The total number of cysts per animal lung is expressed as the sulfoxide (DMSO) vehicle control with the exception of the TMP/ logarithm of the mean and the logarithm of the standard error of the SMZ group, which was compared to untreated immunosuppressed geometric mean. The lower limit of detection was 2 x 104 cysts per animals. TMP and SMZ were used at 0.2 and 1.0 g/liter, respectively. lung (log1o = 4.26). *Significantly different from the appropriate control (P 0.05, t *Significantly different from their corresponding controls (P < 0.05, test). t test). Downloaded by guest on September 25, 2021 Medical Sciences: Schmatz et al. Proc. Natl. Acad. Sci. USA 87 (1990) 5953

Table 4. Additional titrations of L-671,329 in the acute P. carinji 40 pneumonia rat model using a 4-day treatment period Dose, Total cysts, % reduction Survivors/ 0 Treatment mg/kg log (mean) of cysts total x 30 Experiment 1 CL DMSO control 7.54 ± 0.05 6/6 Ca0 L-671,329 1.20 4.64 ± 0.24 99.8* 4/6 40 0.60 5.24 + 0.22 99.2* 6/6 0.30 6.08 ± 0.13 94.2* 6/6 Ca0 20 0.15 6.13 ± 0.22 93.5* 6/6 .0 Experiment 2 Cul DMSO control 7.19 ± 0.21 4/5 U)B co lo0 L-671,329 0.60 5.77 ± 0.25 %.2* 4/5 CL 0.30 5.91 ± 0.30 94.8 4/5 0.15 6.28 ± 0.15 87.7* 5/5 A- 0.08 6.67 ± 0.10 69.5 4/5 U The total number of cysts per animal lung is expressed as the .01 .1 1 1 0 1 00 1000 logarithm of the mean and the logarithm of the standard error of the geometric mean. The lower limit of detection was 2 x 104 cysts per Drug Concentration (gM) lung (logl0 = 4.26). The results indicate that the ED90 for the compound is -0.15 mg/kg. DMSO, dimethyl sulfoxide. FIG. 4. Effect ofL-671,329 (e) and L-687,781 (o) on the formation *Significantly different from their corresponding controls (P < 0.05, oftrichloroacetic acid (TCA)-precipitable polysaccharides generated t test). by a membrane preparation from C. albicans protoplasts. pounds have shown limited inhibition (a maximum of 15- The rapid elimination of cysts observed with L-671,329 20%; K.H.N. and D.M.S., unpublished results), and it is not treatment may prove beneficial in treating patients with clear at this time if there is a small percentage of 1,3-/3- respiratory failure due to acute P. carinii pneumonia. The linkages in P. carinji critical to the cyst wall integrity or ifthe slower clearing of the trophozoite form also can be seen with mode of action of these compounds against P. carinii is TMP/SMZ treatment, where trophozoites can be found in different from that in the yeast. animals treated for 2 weeks. The number oftrophozoites does This study indicates that 1,3-/3-glucan synthesis inhibitors decrease during treatment with L-671,329, suggesting that the merit further attention as possible drugs for the treatment and cyst form is important for proliferation of the trophozoites or prevention of P. carinji pneumonia. The compounds were that trophozoites are also susceptible to the drug. It is also well tolerated by the animals and no gross toxicity was known that trophozoites persist even after long-term treat- observed. Although the efficacy of these compounds against ment with TMP/SMZ in the rat model and that these animals P. carinii has not been demonstrated in humans, it is encour- will relapse if immunosuppression is continued after the drug aging that drugs currently used to treat P. carinji pneumonia, is withdrawn (33). The continued treatment of AIDS patients such as pentamidine and TMP/SMZ, are effective in the rat with aerosolized pentamidine or TMP/SMZ after an acute model (15, 28). All previous treatments for P. carinji pneu- infection is standard procedure, since relapse is a common monia have been limited by the lack of specificity, resulting problem. Whether this would be required with the 1,3-,3- in adverse reactions in the patient. In contrast, there is no glucan synthesis inhibitors remains to be determined. known counterpart of 1,3-,8-glucan synthesis in mammalian systems. Therefore, inhibitors of this process have the po- We acknowledge the members of the Department of Microbiology and the Department of Chemistry at Merck Sharp & Dohme Re- tential to be well tolerated by the patient. Although these search Laboratories who supported this work, particularly Robert results add further support to the classification of P. carinji Giacobbe, Mary Nalin, and Dr. Richard Monaghan of the Fermen- as a fungus, there are many broad-spectrum antifungal agents tation Microbiology Group. such as (14) and (29) that have no effect on this organism, whereas several antiprotozoal 1. Kovacs, J. & Masur, H. (1988) J. Infect. Dis. 158, 254-259. drugs, including pentamidine, have antipneumocystis activ- 2. Davey, R., Margolis, D., Kleiner, D., Deyton, L. & Travis, W. ity (30-32). Therefore, the organism may occupy a unique (1989) Ann. Intern. Med. 111, 681-682. niche among fungi. 3. Wilber, R., Feldman, S., Malone, W. J., Ryan, M., Aur, R. J. A. & Hughes, W. T. (1980) Am. J. Dis. Child. 134, 643-648. OH OH 4. Walzer, P., Perl, D., Krogstad, D., Rawson, P. & Schultz, M. HOW0 (1974) Ann. Intern. Med. 80, 83-93. 5. Hughes, W. T. (1977) N. Engl. J. Med. 297, 1419-1423. 0 6. Montgomery, A. B., Luce, J. M., Turner, J., Lin, E. T., Debs, HO 0 OH R. J., Corkery, K. J., Brunette, E. N. & Hopewell, P. C. 0 (1987) Lancet U, 480-483. 7. Montgomery, A. B., Debs, R. J., Luce, J. M., Corkery, K. J., HO Turner, J. & Hopewell, P. C. (1989) Chest 95, 747-750. o | CH3 CH3 8. Edman, J. C., Kovacs, J. A., Masur, H., Santi, D. V., Elwood, o OH H. J. & Sogin, M. L. (1988) Nature (London) 334, 519-522. OH 9. Stringer, S., Stringer, J., Blase, M., Walzer, P. & Cushion, M. (1989) Exp. Parasitol. 68, 450-461. 10. Edman, U., Edman, J. C., Lundgren, B. & Santi, D. V. (1989) Proc. Natl. Acad. Sci. USA 86, 6503-6507. OH 11. Matsumoto, Y., Matsuda, S. & Tegoshi, T. (1989) J. Protozool. 36, 21S-22S. FIG. 3. Structure ofL-687,781, a natural product that is a member 12. Schwartz, R. E., Giacobbe, R., Bland, J. & Monaghan, R. of the papulacandin family. (1989) J. Antibiot. 42, 163-167. Downloaded by guest on September 25, 2021 5954 Medical Sciences: Schmatz et al. Proc. Nati. Acad. Sci. USA 87 (1990)

13. Wichmann, C., Leisch, J. & Schwartz, R. E. (1989) J. Antibiot. 24. Fromtling, R. & Abruzzo, G. (1989) J. Antibiot. 42, 174- 42, 168-173. 178. 14. Frenkel, J., Good, J. & Schultz, J. (1966) Lab. Invest. 15, 25. Baguley, B. C., Rommele, G., Gruner, J. & Wherli, W. (1979) 1559-1577. Eur. J. Biochem. 97, 345-352. 15. Hughes, W. T., McNabb, P. C., Makres, T. D. & Feldman, S. 26. Perez, P., Varona, R., Garcia-Acha, I. & Duran, A. (1981) (1974) Antimicrob. Agents Chemother. 5, 289-293. FEBS Lett. 129, 245-252. 16. Chalvardijian, A. & Grawe, L. (1963) J. Clin. Pathol. 16, 27. Quigley, D. & Selitrennikoff, C. A. (1984) Exp. Mycol. 8, 383-387. 320-325. 17. Taft, C., Stark, T. & Selitrennikoff, C. P. (1988) Antimicrob. Agents Chemother. 32, 1901-1903. 28. Debs, R. J., Blumenfield, W., Brunette, E. N., Straubinger, 18. Cabib, E. & Kang, M. (1987) Methods Enzymol. 138, 637-642. R. M., Montgomery, A. B., Lin, E., Agabain, N. & Papahad- 19. Kluge, R., Spaulding, D. M. & Spain, A. J. (1979) Antimicrob. jopoulos, D. (1987) Antimicrob. Agents Chemother. 31, 37- Agents Chemother. 13, 975-976. 41. 20. Queener, S. F., Bartlett, M. S., Richardson, J. D., Durkin, 29. Hughes, W. T. & Smith, B. (1984) Antimicrob. Agents Che- M. M. & Smith, J. W. (1988) Antimicrob. Agents Chemother. mother. 26, 436-440. 32, 807-813. 30. Pearson, R. & Hewlett, E. (1985) Ann. Intern. Med. 103, 21. Hughes, W. T., Gray, V. L., Gutteridge, W. E., Latter, V. S. 782-786. & Pudney, M. (1990) Antimicrob. Agents Chemother. 34, 31. Walzer, P., Kim, C. K., Foy, J., Linke, M. & Cushion, M. 225-228. (1988) Antimicrob. Agents Chemother. 32, 96-103. 22. Traxler, P., Tosch, W. & Zak, 0. (1987) J. Antibiot. 40, 32. Toma, E., Fournier, S., Poisson, M., Morisset, R., Phaneuf, D. 1146-1164. & Vega, C. (1989) Lancet i, 1046-1048. 23. Spitzer, E. D., Travis, S. J. & Kobayashi, G. S. (1988) Eur. J. 33. Hughes, W. T. (1979) Antimicrob. Agents Chemother. 16, Clin. Microbiol. Infect. Dis. 7, 80-81. 333-335. Downloaded by guest on September 25, 2021