Clindamycin in a Murine Model of Toxoplasmic Encephalitis

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1987, p /87/ $02.00/0 Copyright X 1987, Americn Society for Microbiology Vol. 31, No. 4 Clindmycin in Murine Model of Toxoplsmic Encephlitis JESSE M. HOFFLIN AND JACK S. REMINGTON* Deprtment of Immunology nd Infectious Diseses, Reserch Institute, Plo Alto Medicl Foundtion, Plo Alto, Cliforni 94301, nd Division of Infectious Diseses, Deprtment of Medicine, Stnford University Medicl Center, Stnford, Cliforni Received 6 June 1986/Accepted 29 December 1986 We investigted the efficcy of clindmycin in murine model of toxoplsmic encephlitis using direct intrcerebrl inocultion. Clindmycin reduced mortlity from 40% in norml mice nd 100% in cortisonetreted mice to 0% in both groups. Although we were unble to document pprecible levels of clindmycin in the brins of infected mice, the histologicl fetures of cerebrl infection were mrkedly ltered. The formtion of lrge numbers of cysts nd the intense inflmmtory response seen in the brins of norml mice nd the unchecked infection nd tissue necrosis in the brins of cortisone-treted mice were bsent in the brins of clindmycin-treted mice. Enumertion of cysts in the brins of mice 10 weeks fter infection reveled significntly lower number in the clindmycin-treted mice. Spred of infection to other orgns ws lso decresed during clindmycin dministrtion. These observtions suggest tht clindmycin my hve role in the therpy of toxoplsmic encephlitis. The remrkble number of cses of toxoplsmic encephlitis ssocited with the cquired immunodeficiency syndrome epidemic hs served s n impetus for the serch for tretment regimens which re not ssocited with the untowrd side effects of the conventionl regimen of pyrimethmine plus sulfonmide. Becuse of its ctivity in mouse model of toxoplsmosis (1, 11), clindmycin, which does not chieve dequte levels in cerebrospinl fluid even in the presence of bcteril meningitis (6), hs been used in severl cses of toxoplsmic encephlitis in ptients with cquired immunodeficiency syndrome, usully in combintion with pyrimethmine (12, 15). Bsed on the vilble necdotl dt, no conclusion cn be mde regrding its efficcy in this disese. Recently, we developed mouse model of toxoplsmic encephlitis by direct injection of the orgnism into the brin (J. M. Hofflin, F. K. Conley, nd J. S. Remington, J. Infect. Dis., in press). The histopthologicl chnges in the brins of such mice resemble those reported in ptients with cquired immunodeficiency syndrome nd toxoplsmic encephlitis (9). These chnges, which include inflmmtion nd mrked tissue destruction, suggest tht dmge to the blood-brin brrier my llow clindmycin to rech effective levels in the involved res of the brins of such ptients. For this reson, nd becuse of the gret need for lterntive drugs for tretment of this entity, we exmined the efficcy of clindmycin in our mouse model of toxoplsmic encephlitis. MATERIALS AND METHODS Mice. Swiss Webster femle mice (Simonsen Lbortories, Gilroy, Clif.), 7 to 10 weeks old nd 17 to 25 g in weight, were used in ll experiments. Toxoplsm orgnisms. Tchyzoites of the C56 strin were hrvested from the peritonel fluid of mice infected 6 dys erlier (8). These mice hd been treted with cyclophosphmide 2 dys before infection to prevent ntibody coting of the orgnisms (7). The infected peritonel fluid ws filtered through polycrbonte membrne (pore size, 3,um; Nuclepore Corp., Plesnton, Clif.) to obtin orgnisms * Corresponding uthor. 492 free of host cells nd debris. After filtrtion, the purified tchyzoites were wshed once nd suspended t concentrtion of 3.3 x 106 orgnisms per ml in Hnks blnced slt solution (GIBCO Lbortories, Grnd Islnd, N.Y.) contining 100 U of penicillin nd 100 p.g of streptomycin per ml (HBSS). Vibility of the orgnisms ws greter thn 95%, s determined by trypn blue exclusion. Chronic infection. Chronic infection ws estblished s described previously (13). Beginning 4 dys fter mice were injected intrperitonelly with 104 tchyzoites, sulfdizine (40 mg%) ws dded to their drinking wter for 2 weeks. During this time, immunity develops such tht when sulfdizine is discontinued, the mice remin helthy. Chronic infection is considered estblished by 6 weeks fter inocultion. Intrcerebrl infection. Injections were delivered into the right frontl lobe of the brin with stereotctic unit s previously described (3). After nesthesi with methoxyflurne nd plcement in the stereotctic unit, the skull ws penetrted with 0.5-in. (1.27-cm) short-beveled 23-guge needle. A 30-guge needle ws pssed through the 23-guge needle into the brin to mesured depth of 3 mm, nd 3-,ul inoculum (104 orgnisms) of the tchyzoite suspension ws injected with 50-,ul glss syringe. Animls injected intrcerebrlly with 3,ul of HBSS were used s controls. The 30-guge needle ws checked for ptency fter ech injection, nd mice were euthnized if needle blockge hd occurred. Immunosuppression. Beginning 2 dys before intrcerebrl infection, mice received 25 mg of cortisone cette (Merck, Shrpe & Dohme, West Point, P.) per kg (body weight) or norml sline dministered dily by subcutneous injection. Tretment with clindmycin. Beginning 1 dy fter intrcerebrl infection, mice received 150 mg of clindmycin phosphte (The Upjohn Co., Klmzoo, Mich.) per kg or norml sline intrperitonelly twice dily for 14 dys. In erlier studies from this lbortory, similr dosge ws dministered orlly nd provided complete protection (100% survivl) from intrperitonel infection with the highly virulent strin Toxoplsm sp. RH (1). Clindmycin ssys. Microbiologicl ssy for clindmycin ws performed by Theodore Spiro t Upjohn.

2 VOL. 31, 1987 CLINDAMYCIN AND TOXOPLASMIC ENCEPHALITIS 493 FIG. 1. Untreted intrcerebrl Toxoplsm infection, dy 7. Intense mononucler inflmmtion t injection site. Histologicl evlution. Ech experimentl group ws divided into two cges: one with 10 mice for observtion of time to deth nd the other for scrifice by crbon dioxide sphyxition of 3 mice ech on dys 4, 7, 14, nd 21 fter infection. At utopsy, the brins were removed nd fixed in 10% buffered Formlin. Four to six prffin-embedded 5- plm-thick coronl sections of brin in the region of the injection were stined with hemtoxylin nd eosin. Isoltion studies. At 4, 7, nd 14 dys of infection, the livers, spleens, herts, nd lungs of three intrcerebrlly infected mice in ech group were hrvested. The livers nd spleens were combined in 4 ml of norml sline nd processed with tissue grinder; the herts were processed seprtely in 2 ml of norml sline, s were the lungs. Smples (0.2 ml) of 10-, 100-, nd 1,000-fold dilutions of the resulting suspensions were inoculted intrperitonelly into three mice. Toxoplsm infection of these recipient mice ws determined by exmintion of peritonel fluid of ny tht died or by serologic testing of those tht survived 6 weeks. Positive serologic results were confirmed by the exmintion of brin suspension for the presence of cysts, s described below. Enumertion of cysts. The brins of mice surviving 10 weeks fter infection were ech triturted in mortr nd mixed with 1 ml of norml sline. The suspension ws drwn up nd expressed through 22-guge needle four times. A 5O-,uJ smple of the resulting suspension ws plced on microscope slide under cover slip (20 by 50 mm), the entire slide ws scnned, nd Toxoplsm cysts were counted. Becuse erlier studies from our lbortory (14) showed insignificnt differences in the numbers of cysts in multiple smples from n individul suspension, only single preprtions were counted. Student's t test ws performed to compre the numbers of cysts. RESULTS Mortlity. Intrcerebrl inocultion with HBSS lone did not cuse mortlity or overt neurologicl dmge. Intrcerebrl infection lone resulted in mortlity of 40%; when clindmycin ws dministered, the mortlity ws 0%. Intrcerebrl infection in cortisone-treted mice resulted in mortlity rte of 100%; when clindmycin ws dministered, the mortlity ws 0% (P = by chi-squre nlysis). No mortlity occurred in mice which hd been inoculted intrcerebrlly with HBSS nd treted with cortisone during the 1-month period of observtion. Histology. Detils of the histology in norml nd cortisonetreted mice re reported elsewhere (Hofflin et l., in press). Briefly, in untreted mice t 4 dys fter infection, hemosiderin ws present in phgocytic cells, nd there were numerous intrcellulr vcuoles which contined Toxoplsm cells. There ws no ssocited inflmmtion t this time. On dy 7, the histologic fetures included meningel nd perivsculr infiltrtion of mononucler inflmmtory cells in the region of the injection site, which ws intensely inflmed nd edemtous, often with foci of necrotic debris (Fig. 1). Extrcellulr tchyzoites, s well s intrcellulr vcuoles filled with Toxoplsm orgnisms, were present. By dy 14, necrosis ws more prominent, nd extrcellulr tchyzoites were only rrely observed. Insted, remrkble number of smll cysts were present in the region of the injection site, nd scttered isolted cysts were lso observed in the contrlterl cerebrl hemisphere. In mice treted with clindmycin, exmintion on dy 7 of infection reveled much smller number of vcuoles which contined Toxoplsm orgnisms nd mrkedly less inflmmtion thn in nontreted controls (Fig. 2). Free orgnisms were not identified, nd no tissue destruction ws present. The histology on dy 14 ws essentilly unchnged from tht observed on dy 7. In mice tht were treted with 25 mg of cortisone per kg dily beginning 2 dys before intrcerebrl infection, there ws miniml meningel nd perivsculr inflmmtion observed t ny time. In mrked contrst to wht we observed in noncortisonized mice, the inocultion site contined remrkbly few inflmmtory cells. There ws n expnding centrl region of tissue destruction nd peripherl zone in which tchyzoites were present in both extrcellulr nd intrcellulr sites (Fig. 3). There ws no evidence of cyst formtion. Other foci of tchyzoites nd tissue dmge could be detected in the contrlterl hemisphere nd cudl to the inoculted region. In cortisonized mice treted with clindmycin, the histologicl ppernce on dys 7 nd 14 ws similr to tht in FIG. 2. Clindmycin-treted intrcerebrl Toxoplsm infection, dy 7. Injection site with miniml inflmmtion or tissue destruction.

3 494 HOFFLIN AND REMINGTON nonimmunosuppressed mice treted with clindmycin (Fig. 4). There ws no evidence of the overwhelming prolifertion of orgnisms nd tissue destruction tht ws seen in cortisonized mice tht were not treted with clindmycin. Isoltion studies. As cn be seen in Tble 1, during tretment of nonimmunosuppressed mice with clindmycin, the bility of tissue suspensions to trnsmit Toxoplsm gondii ws prevented in ll except the most concentrted (1:10 dilution) suspension of lung tissue. Clindmycin ws discontinued on dy 14 fter intrcerebrl infection. Seven dys lter, tissue infection ws detected in liver or spleen nd lung suspensions but not in hert tissue. In cortisonetreted mice, clindmycin mrkedly reduced the concentrtions of the prsite but did not erdicte it from ny of the tissues tested; clindmycin ppered to be lest effective in the lung. Cyst enumertion. To determine whether the numbers of cysts in the brins of clindmycin-treted mice were comprble to the numbers in norml mice more thn 1 month fter discontinution of the ntibiotic, the brins of three mice in ech of the surviving groups were hrvested 10 weeks fter infection nd were individully suspended in norml sline. The verge number of cysts in the brins of the norml mice ws 71 (rnge, 54 to 86) compred with 9 (rnge, 6 to 12) (P = 0.004) in the brins of the clindmycin-treted mice nd 51 (rnge, 41 to 58) in the brins of the clindmycin- nd cortisone-treted mice. Clindmycin levels. Simultneous levels in serum nd brin tissue were mesured in mice fter dministrtion of 150 mg of clindmycin per kg intrperitonelly twice dily for 4 dys (Tble 2). This ws done in norml mice nd in mice chroniclly infected with Toxoplsm orgnisms to determine whether the diffuse perivsculr nd meningel inflmmtion known to ccompny the chronic infection (2) might influence the entry of clindmycin into the brin prenchym. Simultneous with levels in serum comprble to those chieved with usul doses in humns, low but detectble levels of clindmycin were present in brin prenchym t 1 nd 2 h fter dministrtion. No differences were detected between the two groups by Student's t test. We lso compred levels in control mice with those in mice tht hd received n intrcerebrl injection of Toxo- FIG. 3. Cortisone-treted intrcerebrl Toxoplsm infection, dy 7. Border (rrowheds) of necrotic re with remrkble lck of inflmmtion. ANTIMICROB. AGENTS CHEMOTHER. FIG. 4. Clindmycin- nd cortisone-treted intrcerebrl infection, dy 7. Injection site with less inflmmtion thn in noncortisonized mice but without tissue destruction seen in non-clindmycintreted, cortisonized mice (c.f. with Fig. 3). plsm orgnisms or of HBSS lone 1 week erlier (Tble 3). As described bove, t this time significnt intrcerebrl inflmmtion hd developed in response to the infection. In this study, mice were treted with 100 mg of clindmycin per kg every 2 h for four doses. This schedule ws chosen to compenste for the different serum hlf-lives of clindmycin in humns nd mice (17). Levels in serum were slightly lower thn those obtined with the higher dose, nd levels in the brin could be detected by biossy only t 1 h postdministrtion. The intrcerebrl infection nd ssocited inflmmtion hd no discernble effect on clindmycin levels in the brin when infected nimls were compred with the control groups. DISCUSSION Clindmycin hd significnt protective effect in this model of toxoplsmic encephlitis. Protection ws demonstrted in terms of mortlity, histopthology, spred of infection, nd cyst formtion. Most remrkble ws the reduction in mortlity from 100o in cortisone-treted mice to 0% when clindmycin ws dded to the regimen. In previous experiments, cortisone tretment of intrcerebrlly infected mice hs been uniformly ftl (Hofflin et l., in press). Thus, this reduction in mortlity is highly significnt nd importnt sttisticlly. Whether the incresed survivl rte cn be ttributed primrily to the effect of clindmycin on intrcerebrl Toxoplsm multipliction or to n effect on the spred of the orgnism to tissues outside of the centrl nervous system is uncler. The impct of clindmycin on the prolifertion of intrcerebrl Toxoplsm orgnisms ws demonstrted by the drmtic chnge in the histologicl fetures of the infection from those of progressive destructive process to those of well-loclized, nondestructive one. Histologiclly, there were remrkbly fewer Toxoplsm orgnisms in the brins of the clindmycin-treted mice thn in the brins of the untreted controls. This observtion ws confirmed nd quntitted by evlution of the numbers of Toxoplsm cysts in the brins of the mice t 10 weeks fter intrcerebrl infection. In ddition, clindmycin mrkedly decresed the

4 VOL. 31, 1987 CLINDAMYCIN AND TOXOPLASMIC ENCEPHALITIS 495 TABLE 1. Presence of T. gondii in different dilutions of triturted tissues from mice in four tretment groups No. of mice infected t indicted tissue dilution on: Orgn(s) nd Dy 4 Dy 7 Dy 14 Dy 21 tretment regimen Liver or spleen or both Control Clindmycin Cortisone NAb Clindmycin + cortisone Hert Control Clindmycin Cortisone NA Clindmycin + cortisone Lung Control Clindmycin Cortisone NA Clindmycin + cortisone Three mice were inoculted with ech dilution (see Mterils nd Methods). b NA, Not pplicble, becuse none of the mice survived to 21 dys. inflmmtory infiltrte which occurs in response to Toxoplsm infection in nonimmunosuppressed mice. Enhnced survivl cnnot, however, be ttributed solely to reduction in inflmmtion, becuse mice which were treted with cortisone lone did not hve significnt inflmmtory response, nd 100% of them died. However, the brins of the ltter did exhibit mrked necrosis nd unimpeded multipliction of T. gondii. These histologicl findings were unexpected in light of the known poor penetrtion of norml nd inflmed meninges by clindmycin nd the low levels of clindmycin mesured in the brins of the mice in this study. We did not detect significnt difference between clindmycin levels in the brins of norml mice nd mice with cute or chronic inflmmtion of the centrl nervous system resulting from toxoplsmosis. However, our methodology did not llow for mesurement of clindmycin levels specificlly in the region of the injection site. The infection t this site my hve resulted in disruption of the blood-brin brrier, with locl ccumultion of higher mounts of clindmycin thn reflected by our mesurement of the concentrtion of ntibiotic in the entire brin. This my be importnt becuse reports of therpeutic levels (for nerobic infections) of clindmycin in humn brin bscesses hve ppered (5), nd we hve documented level of.2 p.g/ml in brin TABLE 2. Clindmycin levels in brins nd ser of norml mice nd mice chroniclly infected intrperitonelly with T. gondii Time fter Clindmycin level intrperitonel Chronic Toxoplsm Norml dministrtion of infection clindmycin (h) Brin Serum Brin Serum b Concentrtions re expressed s microgrms per grm of brin nd s microgrms per milliliter of serum detectble by microbiologicl ssy. Vlues re mens for three mice. b -, Below the lower limit of detection of the ssy. bscess in ptient 2 h fter dministrtion of 600 mg intrvenously (unpublished observtions). The spred of infection to orgns outside the centrl nervous system ws enhnced by cortisone nd mrkedly inhibited by clindmycin dministrtion. Tretment with cortisone resulted in n increse in the number of orgnisms in peripherl orgns. This difference from controls becme more pronounced with time. In contrst, the effect of clindmycin ws observble t dy 4 of infection nd persisted during the period of dministrtion. Whether the reduction in spred of infection with clindmycin resulted from its effect primrily on orgnisms in the brin, blood, or peripherl orgns ws not determined in this study. After discontinution of clindmycin, n increse in infection of ll orgns exmined ws observed in both norml nd immunosuppressed mice. This observtion suggests renewed prolifertion or prsitemi or both. As is true of other nti- Toxoplsm gents, clindmycin ws suppressive rther thn curtive. Although the studies described re the first to exmine the effect of clindmycin specificlly on intrcerebrl toxoplsmosis, severl published studies demonstrted tht clindmycin hs n in vivo effect on experimentl murine infection with T. gondii. McMster nd co-workers (11) in 1973 cured TABLE 3. Clindmycin levels in brins nd ser of norml mice nd mice infected intrcerebrlly with T. gondii Clindmycin level Time fter intrperitonel Intrcerebrl Toxoplsm Norml dministrtion of infection clindmycin (h) Brin Serum Brin Serum b Concentrtions re expressed s microgrms per grm of brin nd s microgrms per milliliter of serum detectble by microbiologicl ssy. Vlues re mens for three mice. b -, Below the lower limit of detection of the ssy.

5 496 HOFFLIN AND REMINGTON mice of infection with the virulent strin Toxoplsm sp. RH with clindmycin given by gvge or mixed into the food. Previous studies from this lbortory (1) confirmed the bility of clindmycin to decrese mortlity in mice cutely infected with Toxoplsm orgnisms nd documented reduction in the prevlence of prsitemi in chroniclly infected mice. In ddition, it ws found tht both clindmycin nd sulfdizine decresed the prsite lod in the livers nd spleens but not in the brins of chroniclly infected mice. In mice cutely infected intrperitonelly with highly virulent Toxoplsm strin, Thiermnn nd co-workers (16) found the combintion of clindmycin nd sulfmethoxypyridzine to more effectively prevent infection in the brin thn did spirmycin or trimethoprim in combintion with the sme sulfonmide. However, pyrimethmine in combintion with sulfmethoxypyridzine ws more effective. In the single report in the literture (10) nd in extensive unpublished studies from our own lbortory of in vitro ssys of clindmycin ctivity ginst T. gondii, no efficcy ws demonstrted. The reson for the discrepncy between this result nd the efficcy found in the in vivo studies my relte to the need to hydrolyze clindmycin phosphte to the ctive clindmycin compound (4). Only necdotl reports of the use of clindmycin for toxoplsmic encephlitis in humns hve ppered. Interprettion of these reports is difficult becuse the drug hs usully been used in combintion with gents with known efficcy. Nevertheless, given the urgent need for lterntive gents for the therpy of toxoplsmic encephlitis, our results suggest tht controlled trils of clindmycin in combintion with gents of known efficcy should be undertken. ACKNOWLEDGMENTS This work ws supported by Public Helth Service grnt A from the Ntionl Institutes of Helth. J.M.H. is the recipient of n Edith J. Milo Memoril Fellowship nd Ntionl Institutes of Helth Ntionl Reserch Service Awrd IF32A LITERATURE CITED 1. Arujo, F. G., nd J. S. Remington Effect of clindmycin on cute nd chronic toxoplsmosis in mice. Antimicrob. Agents Chemother. 5: Conley, F. K., nd K. A. Jenkins Immunohistologicl study of the ntomic reltionship of toxoplsm ntigens to the inflmmtory response in the brins of mice chroniclly infected with Toxoplsm gondii. Infect. Immun. 31: Conley, F. K., nd J. S. Remington Nonspecific inhibition of tumor growth in the centrl nervous system: observtions of ANTIMICROB. AGENTS CHEMOTHER. intrcerebrl ependymoblstom in mice with chronic toxoplsm infection. J. Nti. Cncer Inst. 59: DeHn, R. M., C. M. Metzler, D. Schellenberg, nd W. D. Vnden Bosch Phrmcokinetic studies of clindmycin phosphte. J. Clin. Phrmcol. 13: De Louvois, J., nd R. Hurley Antibiotic concentrtions in intrcrnil pus: study from collbortive project. Chemotherpy (Bsel) 4: Grrod, L. P., H. P. Lmbert, nd F. O'Grdy (ed.) Antibiotics nd chemotherpy, 5th ed., p Churchill Livingstone, Ltd., Edinburgh. 7. Hndmn, E., P. M. Chester, nd J. S. Remington Delyed hypersensitivity to Toxoplsm nd unrelted ntigens in Toxoplsm-infected mice: induction nd elicittion of delyed-type hypersensitivity by ntigen-pulsed mcrophges. Infect. Immun. 28: Huser, W. E., Jr., S. D. Shrm, nd J. S. Remington Nturl killer cells induced by cute nd chronic toxoplsm infection. Cell. Immunol. 69: Luft, B. J., nd J. S. Remington Toxoplsmosis of the centrl nervous system, p In J. S. Remington nd M. N. Swrtz (ed.), Current clinicl topics in infectious diseses, vol. 6. McGrw-Hill Book Co., New York. 10. Mck, D. G., nd R. McLeod New micromethod to study the effect of ntimicrobil gents on Toxoplsm gondii: comprison of sulfdoxine nd sulfdizine individully nd in combintion with pyrimethmine nd study of clindmycin, metronidzole, nd cyclosporin A. Antimicrob. Agents Chemother. 26: McMster, P. R., K. G. Powers, J. F. Finerty, nd M. N. Lunde The effect of two chlorinted lincomycin nlogues ginst cute toxoplsmosis in mice. Am. J. Trop. Med. Hyg. 22: Millign, S. A., M. S. Ktz, P. C. Crven, D. A. Strndberg, I. J. Russel, nd R. A. Becker Toxoplsmosis presenting s pnhypopituitrism in ptient with the cquired immune deficiency syndrome. Am. J. Med. 77: Ruskin, J., J. McIntosh, nd J. S. Remington Studies on the mechnisms of resistnce to phylogeneticlly diverse intrcellulr orgnisms. J. Immunol. 103: Shrm, S. D., J. M. Hofflin, nd J. S. Remington In vivo recombinnt interleukin 2 dministrtion enhnced survivl ginst lethl chllenge with Toxoplsm gondii. J. Immunol. 135: Snider, W. D., D. M. Simpson, S. Nielson, J. W. Gold, C. E. Metrok, nd J. B. Posner Neurologic complictions of cquired immune deficiency syndrome: nlysis of 50 ptients. Ann. Neurol. 14: Thiermnn, E., W. Apt, A. Atis, M. Lorc, nd J. Olguin A comprtive study of some combined tretment regimens in cute toxoplsmosis in mice. Am. J. Trop. Med. Hyg. 27: Wgner, J. G., E. Novk, N. C. Ptel, C. G. Chidester, nd W. L. Lummis Absorption, excretion nd hlf-life of clinimycin in norml dult mles. Am. J. Med. Sci. 256:25-37.