Efficacy of Clarithromycin for Treatment of Experimental

Transcription

1 ANTIMICROBLAL AGENTS AND CHEMOTHERAPY, June 1993, p /93/ $02.00/0 Copyright X) 1993, Americn Society for Microbiology Vol. 37, No. 6 Efficcy of for Tretment of Experimentl Lyme Disese In Vivo JEFFREY ALDER,* MICHAEL MIT1TEN, KEN JARVIS, PRAMOD GUPTA, AND JACOB CLEMENT Anti-Infective Reserch Division, Abbott Lbortories, Abbott Prk; Illinois Received 24 September 1992/Accepted 18 Mrch 1993 provided effective therpy ginst rthritis induced by Borreli burgdorferi infection in the hmster. In vitro, clrithromycin ws t lest 1 log more potent thn tetrcycline ginst two isoltes of B. burgdorfieri from humn sources, s mesured by MICs nd 50%Y inhibitory concentrtions. ws effective in preventing the onset of B. burgdoferi-induced rthritis, s determined by severl prmeters of pw swelling. When dministered fter the onset of rthritis, clrithromycin therpy reduced the degree of swelling nd decresed recovery time. These results suggest tht clrithromycin hs potentil s n effective therpy for Lyme disese. Lyme borreliosis is the most commonly reported tickborne disese in Europe nd the United Sttes (4). In the first hlf of 1992, the Centers for Disese Control reported over 2,000 confirmed cses of Lyme borreliosis (5). However, becuse of difficulties in dignosis (17, 19), the ctul incidence of the disese is unknown. Borreli burgdorferi is the custive gent of Lyme disese. Lyme disese presents wide vriety of clinicl mnifesttions, which re often divided into stges. Lyme disese is initilly chrcterized by flu-like illness nd migrting rsh termed erythem migrns. Crdic or neurologic involvement my lso occur. Arthritis is the most common serious nd chronic mnifesttion of Lyme disese (8, 10, 23). The knee joint is frequent trget of this rthritis, which my pper severl months fter erythem migrns. The progression nd ppernce of clinicl signs my vry considerbly mong individuls. Tretment efficcy ppers to vry with the severity of the rthritis nd progression of the disese (3). Commonly used orl ntimicrobil gents for the tretment of Lyme disese include tetrcycline, doxycycline, erythromycin, nd penicillin V. Orl ntimicrobil gents hve ppered to be reltively ineffective in treting Lyme disese rthritis (4, 6, 7, 22). This sitution, long with the long period required for ntibiotics to kill B. burgdorferi in vitro (1, 16) nd the chronic nture of the rthritis, suggests need for effective orl ntibiotics which re sfe to dminister on long-term bsis. The limited clinicl trils support the need to investigte dditionl ntimicrobil gents for their efficcy nd sfety in tretment of Lyme disese. Severl different niml models re used to simulte Lyme disese. Some lbortory nimls do not mnifest Lyme disese s rthritic joint swelling but insted suffer infections of vrious internl orgns (17). Recovery of the pthogen from internl orgns cn be difficult nd is usully judged on plus-or-minus bsis rther thn on quntittive scle. The hmster model llows ssessment of rthritic pw swelling s mesure of pthology (21). The pw enlrgement is esily quntitted nd my be monitored over severl weeks to produce time course nlysis of infection nd response to ntibiotic tretment. Borreli-induced pw swelling in the * Corresponding uthor hmster hs been chrcterized histologiclly nd includes mny fetures of rthritis. There is destructive degrdtion of crtilge nd n infusion of monocytes nd lymphocytes (21). Spirochetes re lso present within the dmged joints of infected hmsters (21). In ddition, hmsters produce significnt nd mesurble titers of ntibody to B. burgdorferi. This investigtion demonstrtes tht clrithromycin hs significnt nti-b. burgdorferi ctivity in the hmster model of Lyme rthritis. This ctivity ws mesured on the bsis of protection from joint swelling nd in vitro potency. MATERIALS AND METHODS Origin nd cultivtion of Borreli isoltes. Two humn Lyme disese B. burgdorferi isoltes were used in this study. One isolte, B. burgdorferi 297, ws obtined from humn cerebrospinl fluid (24). This hmster-virulent isolte ws subcultured once or twice before use. A second isolte, B. burgdorferi P/Bi, ws obtined from Germn humn skin specimen (obtined from Ronld Schell, University of Wisconsin) nd ws not virulent in hmsters. All cultures were grown t 33 C in Brbour-Stoenner-Kelly (BSK II) medium (2) Ȧntimicrobil gents. The ntimicrobil gents tested were tetrcycline hydrochloride (Squibb, Princeton, N.J.), doxycycline (Genev, Broomfield, Colo.), nd clrithromycin nd 14-hydroxy-clrithromycin (Abbott Lbortories, Abbott Prk, Ill.). In vitro susceptibility procedures. The 50% inhibitory concentrtions (IC50s) of the study gents were determined by cultivting B. burgdorferi in BSK II medium both with nd without the gents. Concentrtions of the test ntimicrobil gents rnged from 0.01 to 100,ug/ml. Tubes contining 1.0 ml of BSK II medium with the ppropritely diluted ntimicrobil gents nd control tubes (no ntimicrobil gent) were inoculted to finl density of 5 x 106 orgnisms per ml. The spirochetes were in the erly log phse of growth. After incubtion t 33 C for 48 h, the tubes were exmined by drk-field microscopy for the presence of vible spirochetes. A totl of 100 spirochetes from ech tube were exmined for the presence of motility. IC50s were clculted by liner regression. MICs were clculted s the lowest dilution yielding less thn 5% vibility. In vivo susceptibility procedures (12). Golden Syrin hm-

2 1330 ALDER ET AL. sters, 5 to 6 weeks old nd weighing 80 to 100 g, were immunosuppressed by single intrperitonel injection of cyclophosphmide (Sigm, St. Louis, Mo.) (100 mg/kg of body weight) 1 dy prior to inocultion. Hmsters were then inoculted (dy 0) by intrderml injection of 107 B. burgdorfen 297 orgnisms into ech hind pw (0.1 ml of culture contining 108 orgnisms per ml). The test nimls received dily subcutneous injections of clrithromycin, the 14- hydroxy humn metbolite of clrithromycin, doxycycline, or tetrcycline for 7 consecutive dys (dys 1 to 7). In ddition, uninfected nimls nd infected but untreted nimls were included s controls in ech experiment. There were four hmsters per tretment group. In one tril, tretment ws delyed until swelling becme pprent (dy 6). Efficcy ws quntitted by mesuring the degree of pw swelling, s determined by mesuring the width nd thickness of the section of hind pw between the proximl two toe pds with electronic digitl clipers (Mitutoyo, Toyko, Jpn). The mss of the section ws clculted s length x width2/2, nd trpezoidl estimtion ws used to clculte the re under the curve (AUC) of pw mss versus time. Antibody titers. Antibody titers were determined by using fluorescent-ntibody ssy for hmster ntibody to B. burgdorferi. Slides coted with B. burgdorferi (Wmpole/ Zeus, Crnbury, N.J.) were incubted with 1:320 dilution of test hmster serum in moist chmber t room temperture for 30 min. The slides were rinsed ccording to the mnufcturer's instructions nd incubted with 1:100 dilution of got nti-hmster fluorescent conjugte ntiserum (Boehringer Mnnheim, Indinpolis, Ind.) t room temperture for 30 min, s before. The slides were then rinsed nd mounted in buffered glycerol nd scored on scle of 0 to 4+ ccording to the mnufcturer's instructions (Wmpole/ Zeus). The fluorescence ws scored s follows: 0, negtive; 1+, wek; 2+, moderte; 3+, strong; nd 4+, very strong. Recovery ofb. burgdorferi from orgns. At the conclusions of trils, bldders, spleens, kidneys, nd/or hert tissues were septiclly removed from the hmsters. The tissues were corsely minced nd individully incubted in 15 ml of BSK II medium per orgn. The tubes were incubted t 33 C for up to 3 weeks. The cultures were exmined by drk-field microscopy t weekly intervls for the presence of spirochetes. Phrmcokinetics. The concentrtions of clrithromycin, 14-hydroxy-clrithromycin, tetrcycline, nd doxycycline in hmster ser following single 20-mg/kg subcutneous dose ws determined by biossy (9). Ser were collected 0.5, 1, 2, 3, 6, 8, 12, 16, nd 24 h following dministrtion of the dose. There were four hmsters tested per time point. TABLE 1. Compound IC50s nd MICs of therpeutic gents for two B. burgdorferi strins 297 strin P/Bi strin IC50 MIC IC50 MIC Hydroxy-clrithromycin NDb Tetrcycline Doxycycline ND IC50s nd MICs both given in microgrms per milliliter. bnd, not determined ' 40 30' 20' 10',\-.001 ANTIMICROB. AGENTS CHEMOTHER. -U IC50 = pg/ml -0--O Tetrcycline IC50 = 1.96 pg/ml drug concentrtion (pg/ml) FIG. 1. Vibility of B. burgdorfeni 297 following 48 h of incubtion with clrithromycin or tetrcycline. IC50s were clculted by liner regression nlysis. RESULTS In vitro susceptibility of the B. burgdorferi isoltes to ntimicrobil gents. The IC50s of clrithromycin (0.044,ug/ml) nd the 14-hydroxy metbolite of clrithromycin (0.042,ug/ml) were lower in vitro thn those of either tetrcycline (1.960,ug/ml) or doxycycline (0.811 p,g/ml) ginst the pthogenic 297 strin (Tble 1). The IC50s were bsed upon vibility curves of B. burgdorfen exposed to known concentrtions of drugs. These curves represent typicl dose susceptibility profiles for in vitro exposure to bctericidl compounds (Fig. 1). MICs were pproximtely 1 log greter thn the corresponding IC50s. The Germn strin, which produces no pthology in the hmster, ws lso more susceptible to clrithromycin nd the 14-hydroxy metbolite thn to tetrcycline or doxycycline. The Germn strin ws more resistnt to ll four ntibiotics nd higher IC50s were obtined for ech compound. Antimicrobil tretment of B. burgdorferi-infected hmsters. The effect of erly therpy on the increse in pw mss of hmsters infected with B. burgdorferi 297 is illustrted in Fig. 2. The swelling peked on dy 7 in untreted hmsters nd grdully declined over the next two weeks. By contrst, there ws complete prevention of pw swelling in * Doxycycline 20 mg/kg 10 mg/kg 150- Doxycycine 140, - 20 mg/kg mg/kg l 120 / \ * Untreted Non- ioo o ~~~~~~ I Dy FIG. 2. Increse in pw mss of hmsters infected with B. burgdorfieri 297 nd treted with clrithromycin or doxycycline. Test drugs were dministered intrperitonelly once dy on dys 1 to 7.

3 VOL. 37, 1993 TABLE 2. Efficcy of clrithromycin nd doxycycline in tretment of B. burgdorferi 297 infection in hmsters Group, drug, nd AUCO-18 Pw mss T d dose (mg/kg) (mg. dys)b (mg)c mx (1.06) 19.2 (1.14) (1.65) 38.1 (2.26) 7 Doxycycline (1.06) 17.2 (1.02) (1.10) 21.0 (1.25) 4 None 928 (3.29) (7.76) 7 Noninfected All tretments were once dy on dys 1 to 7. There were four nimls b AUC vlues were clculted for dys 0 to 18 of the tril. Test/control c Mximum men pw mss during dys 0 to 18. Test/control (noninfected) dys 0, 3, 4, 7 to 9, 11, 14, 16, nd 18 postinfection. d Tmx, time in dys to mximum men pw mss. hmsters treted on dys 1 to 7 with clrithromycin or doxycycline t 20 mg/kg. The numericl results of this tril re presented in Tble 2. The AUC vlues, clculted s milligrms dys, indicted tht clrithromycin or doxycycline t 20 mg/kg dily effectively prevented pw swelling in infected hmsters. Over the course of the tril, the AUC vlues for untreted infected hmsters were pproximtely three times greter thn those for infected hmsters treted with clrithromycin t 20 mg/kg. Hmsters treted with clrithromycin or doxycycline t 10 mg/kg dily suffered slight nd trnsient swelling of the hind pws. The onset of this miniml swelling ws not delyed, but there ws more rpid recovery (return to bseline vlues for pw mss) in the treted hmsters. nd 14-hydroxy-clrithromycin yielded similr efficcies ginst B. burgdorferi 297 infection (Tble TABLE 3. Efficcy of clrithromycin, the 14-hydroxyclrithromycin, nd tetrcycline in tretment of B. burgdorfen 297 infection in hmsters Group, drug, nd dose AUCO20 Pw mss T d (mg/kg) (mg- dys)" (mg)c mx (1.19) 29.6 (1.63) (2.04) 66.1 (3.65) Hydroxy-clrithromycin (1.36) 44.4 (2.45) (2.17) 62.8 (3.47) 13 Tetrcycline, (1.02) 19.4 (1.07) 20 None 1,508 (4.37) (11.20) 7 Noninfected All tretments were once dy on dys 1 to 7. There were eight nimls bauc vlues were clculted for dys 0 to 20 of the tril. Test/control c Mximum men pw mss during dys 0 to 20. Test/control (noninfected) dys 0, 3, 6 to 10, 13 to 15, 17, nd 20 postinfection. On dy 20, the pw mss of untreted hmsters hd stbilized t pproximtely twice tht of noninfected hmsters. d Tm., time in dys to mximum men pw mss. ANTIBORRELIAL ACTIVITY OF CLARITHROMYCIN 1331 TABLE 4. Efficcy of clrithromycin nd doxycycline on b.i.d. schedule in tretment of B. burgdorferi 297 infection in hmsters Group, drug, nd AUCG-u Pw mss T d dose (mg/kg) (mg- dys)b (mg)c mx (1.00) 22.4 (1.20) (1.19) 34.6 (1.86) 13 Doxycycline (0.99) 22.7 (1.22) (1.23) 30.3 (1.63) 14 None 1,866 (4.52) (8.09) 6 Noninfected All tretments were b.i.d. on dys 1 to 7 (totl dily dose, 50 or 25 mg/kg). There were eight hmsters per tretment group. b AUC vlues were clculted for dys 0 to 24 of the tril. Test/control c Mximum men pw mss during dys 0 to 24. Test/control (noninfected) dys 0, 6, 10 to 14, 17, 18, 20, nd 24 postinfection. d Tmx, time in dys to mximum men pw mss. 3). There ws some trnsient swelling in both groups of treted hmsters. There ws dely in time to mximl pw swelling from 7 dys in untreted hmsters to 15 to 17 dys in treted hmsters. Untreted hmsters hd men pw mss (AUC) nerly five times tht of hmsters treted with clrithromycin t 25 mg/kg. By dy 20, the men pw msses of ll treted hmsters hd returned to bseline (noninfected) vlues. Untreted hmsters hd men pw mss tht stbilized t pproximtely twice tht of noninfected hmsters. ws lso effective when given on twicedily (b.i.d.) schedule (Tble 4). At 25 mg/kg b.i.d. (50-mg/kg totl dily dose), clrithromycin prevented the onset of swelling due to B. burgdorferi infection. Similr efficcy ws obtined with doxycycline. Untreted hmsters hd men AUC vlue over four times tht of noninfected hmsters. By the conclusion of the tril, the pw msses of ll treted hmsters hd returned to bseline vlues. therpy ws lso effective in reducing the severity of swelling nd decresing recovery time when given fter the onset of swelling (Fig. 3 nd Tble 5). 0~ Dy FIG. 3. Efficcies of clrithromycin nd tetrcycline therpies on delyed schedule for tretment of B. burgdorferi infection in hmsters. Test drugs were dministered intrperitonelly once dy on dys 6 to 12. The tretment period is indicted (Rx).

4 1332 ALDER ET AL. TABLE 5. Efficcy of clrithromycin nd tetrcycline on delyed-tretment schedule for therpy of B. burgdorferi 297 infection in hmsters Group, drug, nd AUCo-6 AUC7_17 Pw mss T d dose (mg/kg) (mg. dys)b (mg dys)' (mg)c m (1.75) 81.5 (2.92) (1.67) 54.2 (1.94) 8 Tetrcycline (1.61) 51.7 (1.85) (1.96) 99.8 (3.57) 7 None 110 b655 (3.08) (4.23) 8 Noninfected All tretments were once dy on dys 6 to 12. There were four nimls b AUC vlues were clculted for dys 0 to 6 (before tretment) nd dys 7 to 17 (during nd fter tretment) of the tril. Test/control (noninfected) rtios re given in prentheses. c Mximum men pw mss during dys 0 to 20. Test/control (noninfected) rtios re given in prentheses. Mesurements were tken on dys 0, 3, 6 to 8, 10, 13, nd 15 to 17 postinfection. d Tm., time in dys to mximum men pw mss. Hmsters treted with clrithromycin beginning on dy 6 (the first dy on which swelling ws pprent) hd men AUC vlue during nd fter therpy (AUC from dys 7 to 17 [AUC-717]) pproximtely 50% tht of untreted hmsters. Prior to the strt of therpy (AUCO-,6), the pw msses of ll groups of infected hmsters were similr. There ws similr efficcy in reducing pek pw swelling nd overll men pw mss with 25 nd 12.5 mg of clrithromycin per kg dily. By the conclusion of the tril, the pw msses of treted hmsters hd returned to vlues similr to those for noninfected hmsters, while untreted hmsters hd men pw mss pproximtely twice tht of noninfected hmsters (Fig. 3). Recovery of borrelie from treted hmsters. The recovery nd cultivtion of borrelie from hmster bldders, kidneys, herts, or spleens t the conclusion of the trils (dys 20 to 24 fter inocultion) ws not useful indictor of drug efficcy. Untreted hmsters often did not yield detectble numbers of spirochetes. The recovery of B. burgdorferi ws not used s n indictor of drug efficcy. Titers of ntibody to B. burgdorfieri in hmster ser. Hmster ser collected t the conclusion of the tril were ssyed for nti-borreli ntibody by using n immunofluorescentntibody test. There ws no pprent difference between treted nd untreted hmsters in the mount of nti-borreli ntibody present t the tril's conclusion. Both drugtreted nd untreted infected hmsters yielded immunofluorescent-ntibody men scores of 3.0 or greter, while noninfected hmsters yielded men score of 0. Phrmcokinetic evlution of nti-borreli compounds. A single 20-mg/kg dose of clrithromycin in hmsters yielded mximum concentrtion of drug in serum (Cm,) vlue of 2.098,ug/ml (Tble 6), which ws well in excess of the concentrtion required to kill B. burgdorferi in vitro (IC ,ug/ml). The Cm,, AUCO-24, nd hlf-life (t4/2) = vlues were similr to those chieved cliniclly in humns (9). A 20-mg/kg dose of the 14-hydroxy metbolite of clrithromycin, which is not produced in rodents, yielded Cm., AUCO_ 24, nd t1/2 vlues less thn those for clrithromycin t equivlent doses. The Cm, of doxycycline ws ner the in vitro IC50 nd less thn the MIC of the drug. TABLE 6. ANTIMICROB. AGENTS CHEMOTHER. Phrmcokinetic evlution of nti-borreli drugs in hmsters Compound t1i2 C.m. Tmh) AUC() Hydroxy-clrithromycin Tetrcycline Doxycycline A single 20-mg/kg subcutneous dose ws given. There were 12 hmsters per tretment group. Drug concentrtions in serum were determined 0, 0.5, 1 to 3, 6, 8, 12, 16, nd 24 h fter dosing. b Tn., time to mximum concentrtion of drug in serum. DISCUSSION There is need for systemtic preclinicl testing of potentil nti-borreli compounds. In vitro, B. burgdorferi is susceptible to number of gents, including mcrolides, tetrcyclines, nd 1-lctm ntibiotics (11, 13, 16-18, 20). However, comprison of in vitro potencies from different studies is difficult becuse of the lck of stndrd protocols for inoculum size, growth phse of B. burgdorferi, or time of incubtion. A long period of ntibiotic exposure is required to kill B. burgdorferi, which complictes the correltion of in vitro potency to potentil in vivo efficcy (1, 16). Tretment filures with both tetrcyclines nd 3-lctm ntibiotics hve been reported, especilly in tretment of rthritis ssocited with lte-stge Lyme disese (15, 16, 25). Mcrolides re not yet in widespred use ginst B. burgdorferi. Other mcrolides with in vitro ctivities hve not shown promise in preclinicl development tests (12, 14). The long exposure period required for ntibiotics to kill B. burgdorferi in vitro suggests tht drugs with long t1/2 in serum my be effective therpeutic gents. The recommended tetrcycline therpy of 250 mg four times dily (17) produces serum drug concentrtions tht re less thn the MICs reported by severl lbortories (15)., with both superior in vitro potency ginst B. burgdorferi nd promising in vivo phrmcokinetic prmeters, including biologiclly ctive metbolite in humns, merited preclinicl testing. nd the 14-hydroxy metbolite of clrithromycin were over 1 log more potent in vitro thn either tetrcycline or doxycycline ginst B. burgdorferi 297. The use of IC50s rther thn trditionl MICs is pproprite for compring the in vitro potencies of nti-b. burgdorferi compounds. The in vitro ssy depends upon microscopic observtion for the determintion of spirochete vibility. The most ccurte clcultion of drug potency occurs t the 50% point of the vibility curve (Fig. 1). The high potency of clrithromycin nd the 14-hydroxy metbolite of clrithromycin suggests tht effective serum drug concentrtions could be mintined for longer periods in humns, since hmsters do not produce the metbolite. Erly tretment with clrithromycin or doxycycline prevented rthritic pw swelling (Fig. 2). This effect ws mesured s both decrese in mximl pw swelling nd reduction in the pw swelling over the course of the tril (Tble 2). Therpy with 14-hydroxy-clrithromycin, mjor metbolite in humns but not in hmsters, showed in vivo efficcy similr to tht of clrithromycin. Additionlly, compred with untreted controls, there ws 6- to 10-dy dely in the time required to produce mximl pw swelling (Tble 3).

5 VOL. 37, 1993 The ctivity of 14-hydroxy-clrithromycin ws tested seprtely, since this metbolite is not produced in rodents. efficcy incresed with b.i.d. dosing (Tble 4). Hmsters treted with clrithromycin t 12.5 mg/kg b.i.d. suffered less mximl nd overll pw swelling thn hmsters treted on once-dily schedule (Tbles 2 nd 3). hd t1/2 in serum of 4.11 h nd Cm of 2.098,ug/ml in hmsters (Tble 6). Thus, b.i.d. dosing incresed the durtion of clrithromycin concentrtions in excess of the MIC in plsm. In humns, clrithromycin is usully dministered on b.i.d. schedule, resulting in prolonged concentrtions of the prent drug nd 14-hydroxyclrithromycin in serum (9). therpy initited fter the onset of rthritis rpidly reduced both the mximl swelling nd the durtion of rthritis (Fig. 3 nd Tble 5). After initition of therpy, nimls treted with clrithromycin experienced pw swelling only 50% s gret s tht of untreted, infected hmsters. This result demonstrted the potentil utility of clrithromycin in tretment of estblished rthritic Lyme disese. Treted nd untreted hmsters produced similr titers of ntibody ginst B. burgdorfen. This result my be due to the prolonged exposure time required for ntibiotics to kill B. burgdorfen, which hs low growth rte. Without rpid killing of the pthogen, the hmster immune system hs n opportunity for ctivtion nd production of specific ntibody. The high ntibody titers produced by treted nimls indicted tht infection ws not prevented. The efficcy of the drug tretments ws bsed upon tretment of infection rther thn prevention of infection. The hmster model of Lyme rthritis llows tretment efficcy to be judged by t lest three criteri: mximl pw swelling, time to mximl pw swelling, nd AUC vlue for pw swelling (milligrms dys)., especilly on b.i.d. schedule, ws effective tretment for B. burgdorferi infection by ll three criteri. When initited fter the onset of rthritic pw swelling, clrithromycin therpy ws ssocited with reduction in mximl pw swelling nd shorter recovery time. The concentrtions in serum produced by these doses of clrithromycin in hmsters re similr to those chieved in humns. The successful therpy with clrithromycin nd the 14-hydroxy metbolite of clrithromycin in this model suggests tht efficcy ginst estblished or incubting rthritic Lyme disese my be chievble in humns. ACKNOWLEDGMENTS The excellent work of Lenette Pige, Tom Hutch, nd Nte Shipkowitz (Infectious Disese), Nncy Rmer, Donn McDniel, nd Yu-hu Hui (Phrmcokinetics), nd John Freemn nd the lte Igncio Cervntes (Animl Cre) is grtefully cknowledged. REFERENCES 1. Agger, W. A., S. M. Cllister, nd D. A. Jobe In vitro susceptibilities of Borreli burgdorferi to five orl cephlosporins nd ceftrixone. Antimicrob. Agents Chemother. 36: Brbour, A. G. Isoltion nd cultivtion of Lyme disese spirochetes Yle J. Biol. Med. 57: Berger, B. 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