421

Progress in the chemotherapy of leprosy: Status, issues and prospects By Robert H. Gelber Kuzell Institute for Arthritis and Infectious Diseases, 2200 Webster Street, San Francisco, CA 94115-1896, USA, and G. W. Long Hansens's Disease Center, Carville, LA 70721, USA.

I 2 2.1 2.2 2.3 3

4 5 6 6.1 6.2 6.3 6.4 6.5 6.6 7 8 9 10 II 12 13 14

Introduction . . . . . . . . . Experimental chemotherapy The continuous method . . . The kinetic method . . . . . The proportional bactericidal technique . In vitro screening of drugs for activity against M. /eprae Newer antimicrobials to treat leprosy . . . . . . . . . . The optimal new chemotherapeutic agent for use in man New promising chemotherapeutic agents Fluroquinolones . Minocyc1ine . . . . . . . . . . . . . Macrolides . . . . . . . . . . . . . . Aminoglycosides . . . . . . . . . . . Dihydrofolate reductase inhibitors . Beta lactams . . . . . . . . . . . . . Monitoring of clinical trials of lepromatous leprosy Regimens to treat leprosy . . . . . . . . . . . . . . . The significance of primary dapsone resitance .. . The significance of bacterial persistance in leprosy. Chemotherapeutic studies in animal models of lepromatous leprosy . . . . . . . . . . . . . . Compliance . . . . . . . . . . . . . . . . . . . . . Serology in assessment of therapeutic outcome . Prophylaxis. References . . . . . . . . . . . . . . . . . . . . .

422 423 424 424 425 425 426 427 427 427 428 429 429 430 431 432 434 434 436 439 440 441 442 442

E. Jucker (ed.), Progress in Drug Research / Fortschritte der Arzneimittelforschung / Progrès des recherches pharmaceutiques © Birkhäuser Verlag Basel 1990

422

Robert H. Gelber

1

Introduction

For millenia leprosy was a disease for which there was no effective medical therapy. Because the causative agent, Mycobacterium ieprae, is unique amongst bacterial agents for its tropism and destruction of peripheral nerves, patients often suffered serious deformities and were consequently ostracized. In 1943 Fauget [1] introduced sulfones at Carville, Louisiana. They proved the first antimicrobial agents to reverse leprous dermal infiltrates convincingly, and, though slowly, reduced the systemic burden of acid-fast bacilli (AFB). Early in this era of effective chemotherapy, it was noted that acid-fast bacili in skin smears became beaded and slowly disappeared, skin smears becoming generally negative in four-to-five years [2]. These observations formed the basis for two useful clinical evaluations applicable to field conditions, the morphological index (percentage of solidstaining AFB) and the bacteriological index (log density of AFB). Skin smears are, however, difficult to quantitate, vary considerably from one site to another, and, more importantly, are difficult to standardize. Shepard [3] first grew M. leprae convincingly in the feet of mice in 1960 and, soon thereafter, established that when a sulfone, dapsone, was fed to mice infected with M. leprae, it reliably inhibited bacterial multiplication [4]. Later the serum and tissue minimal inhibitory concentration of dapsone for M. leprae were found to be uniquely low, the minimal inhibitory serum concentration being dO ng/ml [5]. Furthermore, dapsone is inexpensive, costing only about $ 1 U.S. per year for the usual adult daily dose of 100 mg. Leprosy being largely a disease of developing countries, this low cost for dapsone was exceedingly important and indeed critical to its worldwide acceptability and utilization. In the 1940's and 1950's clinicians were optimistic that a solution to leprosy was in hand. However, that was not to be. As late as 1980 it was estimated that only one third of the world's estimated 10-12 million patients were receiving therapy. Furthermore, dapsone resistance was recognized clinically in the 1950's and later confirmed when M. leprae obtained from patients progressing clinically despite dapsone therapy grew in mice fed diet containing dapsone. Worldwide dapsone monotherapy of leprosy was and probably remains, despite recommendations to the contrary, the most usual therapy for leprosy. As monotherapy of tuberculosis has been long

Progress in the chemotherapy ofleprosy: Status, issues and prospects 423

recognized to result frequently in drug-resistant relapse, the appearance of dapsone-resistant relapse was not unexpected. Furthermore, the numbers of bacteria in an untreated lepromatous patients are many orders of magnitude in excess (estimated at as much as (1013) of those in patients with pulmonary tuberculosis (108), and, unlike their counterparts, lepromatous patients have a specific cellular immune defect rendering them incapable of mounting an appropriate protective response to Mycobacterium leprae. Unlike dapsone, which is primarily bacteriostatic for M. leprae in mice and man [6], rifampin was first found in 1970 to be consistently bactericidal in both species [7]. Though c10fazimine and ethionamide, the other two drugs used to treat leprosy, are bactericidal in mice [8-10], they have not proven bactericidal in man [6]. Moreover, despite monotherapy with years of dapsone [11] or rifampin [12] or various combinations of the four major drugs used to treat leprosy (dapsone, rifampin, c1ofazimine, and ethionamide) [13], persisting drug-sensitive organisms, "persisters", can be found in the tissues of patients even after lengthy periods of therapy. The preceding is where we were and are. The rest of this paper will focus on the tools of experimental leprosy chemotherapy and where we are going in basic and applied drug research. There are many important parallels with pulmonary tuberculosis, unresolved issues, considerable progress to review, and many exciting newer developments that offer promise for the future. 2

Experimental chemotherapy

To date M. leprae has not been grown on artificial media or in tissue culture. When Shepard [3] described the experimental disease that followed inoculation of viable M. leprae in the mouse foot pad, the modern era of experimental therapy of leprosy began. In foot pads M. leprae multiplies locally, does not disseminate, and reaches a plateau of 106 M. leprae in about 6 months. It was determined that as few as 1 to 3 live bacilli are required to infect the mouse and that soon after the plateau of multiplication was reached viability of M. leprae fell off precipitously. The mouse foot pad has been utilized extensively to evaluate antimicrobials for their activity against M. leprae. For these purposes three fundamental techniques have been employed: the continuous

424

Robert H. Gelber

method [14-15], the kinetic method [16-17], and the proportional bactericidal technique [8]. 2.1

The continuous method

In the continuous method [14-15] drug, mixed generally in mouse chow, is administered, continuously from the time of infection. It is important in this and other drug screens that agents be administered in amounts that are near maximal but non-toxic, so that an active drug is not judged inactive merely because it was not administered in sufficiently high concentrations. Unfortunately, for newer agents sufficient mouse pharmacologic data to make a considered judgment about dosing is often not available. Activity by the continuous method is judged to be present if subsequent counts of M. leprae in treated mice are significantly different from those in untreated control mice. This technique allows for the determination of the minimal effective dietary concentration of an antimicrobial and its resultant serum and tissue levels. The continuous method has its limitations: 1) It does not distinguish amongst active drugs as to whether they are simply bacteriostatic or bactericidal. This is a most important drawback insofar as the short-course chemotherapy of tuberculosis [18] and, by obvious extension, leprosy, requires bactericidal agents. 2) It also requires drug administration for six or more months, rather than the 2 or 3 months required for the kinetic technique and the proportional bactericidal technique, respectively. This may be critical for newer agents where large amounts may not be readily available. 2.2

The kinetic method

In the kinetic method [16-17] drug is administered from day 60 to day 150 after foot pad infection, i. e. during logarithmic growth of M. leprae. Foot pad harvests from treated and control mice are performed on day 150 and every month or two subsequently until the plateau of M. leprae multiplication is reached. A delay in mUltiplication in treated mice longer than the period of treatment that can not be accounted for by drug accumulation implies that some bactericidal activity was observed. Because the demonstration that an agent

Progress in the chemotherapy of leprosy: Status, issues and prospects 425

has actual bactericidal activity for M. leprae is critical to its pursuit for clinical application and because half or less drug is required for this method as compared to the continuous method, we and others often resort to this technique in our initial screen for antimicrobial activity against M. leprae. 2.3

The proportional bactericidal technique

The proportional bactericidal technique [8], in effect, utilizes the mouse in a manner analogous to a tube dilution and results in data quantitating the actual killing of M. leprae with corresponding confidence limits. In this technique, hind foot pads of groups of mice are infected with 101, 102, 103 and 104 M. leprae, mice are treated for the initial 60 days, foot pads (generally 10) are harvested and therein M. 1eprae enumerated 1 year after the completion of therapy. This allows a sufficient period of time to detect multiplication of M. leprae from any bacilli surviving therapy. From these results the percentage of M. leprae actually killed may be quantitated by a most probable number caiculation or, as we prefer, the method of Spearman and Karber, which allows for the expression of confidence limits [19].

3

In vitro screening of drugs for activity against M. /eprae

The rapid screening of antimicrobial agents for activity against M. leprae has been hindered by the lack of a suitable in vitro system. Mouse studies are costly and require six months to a year or more for completion. For this purpose a number of macrophage culture systems were previously developed but were not generally accepted owing to only limited evidence of bacterial metabolism, as measured either by incorporation of radiolabels or ATP production [20-23]. More recently, new macrophage and cell-free assays capitalizing on a major M. leprae metabolite, the phenolic glycolipid pathway, utilized in the first instance the incorporation of radiolabelled CO 2 into phenolic glycolipid [24-26] and in the latter the radiorespirometric oxidation of palmitic acid to carbon dioxide [27]. These systems have shown especial promise owing to the fact that differences from baseline have generally been twenty- to thirty-fold, and, also, generally drugs found to be active against M. leprae are active in these systems, and those that were previously found inactive are also inactive in these systems.

426

Robert H. Gelber

Such studies, however, require very large numbers of highly viable nude mouse-derived M. leprae and thus are not widely available to laboratory investigators. They also cannot consistently distinguish the relative activity amongst active agents and also dinstinguish bacteriostasis from bacteriocide. Of concern with these newer systems is the observation that streptomycin and kanamycin, which have been found to be consistently bactericidal for M. leprae in the mouse model, are essentially inactive by these methods. Such a defect in a technique meant as a rapid screening method might lead investigators prematurely to abandon the pursuit of potentially important antimicrobials. Also certain antimicrobials, definitely inactive in mice and not easily explained away by poor pharmacokinetics, are active in these systems, notably tetracycline and clindamycin. Nonetheless, such discrepancies with the mouse model appear infrequently, and the obvious advantages of these methods for rapid screening warrant enthusiasm. 4

Newer antimicrobials to treat leprosy

While more than 13 agents can be used to treat tuberculosis, only four antimicrobials are currently utilized to treat leprosy: dapsone, rifampin, clofazimine, and ethionamide. Resistance to each has been documented and each results in significant side effects and toxicities limiting their use in certain patients. Dapsone not infrequently results in significant allergies and hemolytic anemia. Rifampin may result in a flu-like syndrome and cause hepatotoxicity. Clofazimine causes a red-black skin discoloration which is cosmetically unacceptable to many light-skinned patients and may cause gastrointestinal intolerance. Ethionamide, also, frequently results in gastrointestinal intolerance and may cause hepatotoxicity especially when combined with rifampin, such that the combination cannot be recommended unless liver function can be closely monitored. Additionally and of utmost importance, the key to successful short-term chemotherapy of pulmonary tuberculosis has been the use of two bactericidal agents [18]. Certainly, any short-course treatment of lepromatous leprosy would likely also require two bactericidal agents, and lifetime therapy has generally proved impractical. However, previously only rifampin has been demonstrated to be bactericidal in man. Hence new bactericidal agents to treat leprosy are required, and urgently.

Progress in the chemotherapy of leprosy: Status, issues and prospects 427

5

The optimal new chemotherapeutic agent for use in man

In order to evaluate newer agents for their possible application to the treatment of leprosy the following ideal criteria would be desirable: 1) The agent should be bactericidal as demonstrated in the mouse foot pad at serum and tissue levels easily obtainable in man. 2) The agent used as monotherapy in man in a short-term clinical trial should prove more active than dapsone, i. e., skin biopsies from infected patients should regularly no longer contain M. leprae that are viable in the mouse foot pad by 1 month of therapy. 3) The proposed agent should be demonstrated to be additive or synergistic, or at least not antagonistic, with established antimicrobials. 4) The new agent should cause no serious organ toxicity in animals or man. 5) The cost of the new agent should not be prohibitive. 6) The agent should be able to be given by mouth. Injectables are not practical for many field conditions. 7) The agent should have been used for other diseases for a significant number of years so that there are no untoward effects not already recognized. 6 6.1

New promissing chemotherapeutic agents Fluoroquinolones

The fluoroquinolones are a new class of antimicrobials with especial promise for the chemotherapy of leprosy. Fluoroquinolones are derivatives of nalidixic acid, that unlike the parent compound, which provides only gram negative activity and therapeutic levels in the urine, result in broad spectrum antibacterial activity and achieve systemic bioavailability. Saito [28] found that ofloxacin resulted in bactericidal activity for M. leprae both if M. leprae were treated in vitro with ofloxacin prior to foot pad infection and if M. leprae-infected mice were fed ofloxacin. In the mouse model Grosset [29] and we (unpublished studies) found the prototype quinolone, ciprofloxacin, inactive, likely because of poor gastroinestinal absorption. On the other hand, pefloxacin 150 mg/kg/ day was found by Grosset [29] by the kinetic method to result in a delay in the resumption of the multi-

428

Robert H. Gelber

plication of M. leprae for 5 months after discontinuation. While pefloxacin 50 mg/kg/ day was by these methods found to be inactive, ofloxacen 50 mg/kg/ day and 150 mg/kg/ day resulted in a delay of the resumption of M. leprae multiplication, 150 mg/kg/day for 11 months [30]. Pattyn [31] found that pefloxacin even at 300 mg/kg three times weekly is inactive, while ofloxacin 350 mg/kg three times weekly is active but not once weekly. Clinical trials in leprosy of pefloxacin and ofloaxin are currently in progress. Unfortunately, the fluoroquinolones result in severe central nervous system toxicity in some treated patients, and their safety on long-term administration has not been established. 6.2

Minocycline

Minocycline has been available for the treatment of bacterial infections for several decades and also presents promise for the treatment of leprosy. We [32] found in mice that unlike tetracycline or doxycycline, which are inactive, it is consistently bactericidal for M. leprae: 1) by the kinetic method minocycline 0.02 %-0.04 % regularly resulted in growth delay of M. leprae of over 200 days; 2) by the proportional bactericidal method minocycline 0.04 % was 99.2 ± 0.7 % bactericidal. In these studies mouse serum concentration in animals given 0.04 % minocycline in chow was 0.9 ~g/ml, a level easily attainable in man following usual conventional doses of minocycline, 100-200 mg daily. Furthermore, in mice minocycline's activity against M. leprae was found to be consistently additive with previously established active agents, dapsone, rifampin and kanamycin. We have recently found (unpublished) that minocycline is similarly active in mice for strains of M. leprae that are both partially and fully dapsone resistant. Also, in these studies we found that minocycline 0.04 % was quite active given in diet three days weekly and once weekly, and even retained some activity given in diet only once monthly. The potent activity of minocycline against M. leprae is likely the result of it's being lipid-soluble at neutral pH [33], a characteristic that allows it to penetrate the large outer lipid barrier of M. leprae, consisting of the outer capsule and cell wall. The partition coefficient of minocycline in an octanoll aqueous buffer system at pH 6 demonstrates that minocycline is 30 times more lipid soluble than is tetracycline [33]. In some patients minocycline has caused vertigo

Progress in the chemotherapy of leprosy: Status, issues and prospects 429

[34]. Fortunately, it appears dose-related and resolves upon discontinuation. Clinical trials of minocycline in lepromatous leprosy are in progress, and their results should soon be available. 6.3

Macrolides

Previously we found (unpublished) erythromycin ethyl succinate 0.06 % in mouse diet to be inactive. Recently, certain newer macrolide antibiotics have been developed that have the virtues, over erythromycin itself, of acid pH stability and broad spectrum activity and attain very high levels within macrophages, the site of M. leprae infection. Certain of these, roxrithromycin and especially clarithromycin, are active against M. leprae in the newer developed in vitro cellfree and macrophage screens [35]. Additionally, clarithromycin, 0.01 % in mouse diet when studied by a modification of the kinetic method resulted in a delay of M. leprae multiplication of 5 months [35]. We have found indeed (unpublished) that 0.1 % of both roxrithromycin and clarithromycin in mouse diet, but not azithromycin, by the kinetic method are bactericidal for M.leprae. 6.4

Aminoglycosides

Aminoglycoside antibiotics have received only limited experimental and clinical attention for their potential role in the therapy of leprosy. This is in large part owing to the fact that injectables are not practical in many areas of the world where leprosy is endemic. In the first clinical trial of streptomycin in leprosy, five of the ten dapsoneresistant patients treated with dapsone and streptomycin relapsed clinically, and new lesions appeared to contain viable bacilli after only 23-31 months of treatment with both drugs [36]. Unfortunately, in this study, streptomycin resistance, though suspected, was not proved by mouse inoculation in these cases. However, encouraging for the clinical application of certain aminoglycosides to the therapy of leprosy is the clinical trial in lepromatous leprosy in Malaysia, wherein daily intramuscular streptomycin (500 mg) resulted in clinical improvement comparable to that obtained with dapsone and a loss of mouse foot pad infectivity of skin-biopsy specimens that was somewhat faster than with dapsone [37]. In a few studies streptomy-

430

Robert H. Gelber

cin [16, 38-39J was found by others to be active against mouse leprosy. We [40] found in mice that daily kanamycin (100 mg/kg), streptomycin (150 mg/kg), and amikacin (100 mg/kg) resulted in impressive killing of M. leprae (99.7 %,97 %, and 96 % bactericidal, respectively), while gentamicin and tobramycin were essentially inactive. Furthermore, we [41J found that reducing the mouse dosage of streptomycin to as little as 12.5 mg/kg five times weekly retained significant bactericidal activity for M. leprae and that streptomycin (100 mg/kg) once monthly coadministered with rifampin 20 mg/kg resulted in synergism and profound bactericidal activity (99.96 %±0.02 % as compared to rifampin alone 94 %±4.0 %). Since the W. H. O. advocates monthly supervised rifampin, streptomycin administered on that schedule would at least theoretically be worthy of consideration. 6.5

Dihydrofolate reductase inhibitors

Dapsone, a sulfone, acts at the same step in bacterial folate synthesis as sulfonamides, the para aminobenzoic acid condensation reaction [42]. It has long been hoped that the next step in M. leprae folate synthesis, namely the reduction of dihydrofolate to tetrahydrofolate, would prove amenable to antimicrobial intervention in leprosy. Indeed, we and others have attempted to exploit this locus of activity in mice. Trimethoprim was found inactive alone and did not potentiate the activity of dapsone [15J. Unfortunately, we [43, 44J found that other dihydrofolate reductase inhibitors similarly offered little advantage, Seydel [45J found that certain dihydrofolate reductase inhibitors, particularly brodimoprim, offered promise against M. leprae because they were found synergistic with dapsone against certain cultivable mycobacteria, had very low minimal inhibitory concentrations, and preferentially bound strongly to the target mycobacterial enzyme as compared to the mammalian enzyme. Yet we found (in an unpublished study) that against M. leprae in the mouse system brodimoprim and another dihydrofolate reductase inhibitor, SE-SC60, offered no advantage when added to dapsone alone. As a caveat, mice infected with Staphylococcus aureus sensitive to trimethoprim/ sulfamethoxazole cannot be effectively treated by the combination, suggesting that peculiarities of the mouse folate system may interfere with the ability to test dihydrofolate reductase inhibitors that could be useful in man in the mouse system.

Progress in the chemotherapy of leprosy: Status, issues and prospects 431

6.6

Beta lactams

Beta lactam antibiotics are the agents of first choice for most bacterial infections, both gram-positive and gram-negative. They have not, however, found application in the therapy of mycobacterial disease to date, primarily as a result of their demonstrated lack of permeability for mycobacteria [46, 47]. The lack of activity for mycobacteria of this class of compounds, also, appears a result of the unequivocal demonstration of the presence of beta lactamases in various mycobacterial species [46-48] and probably Mycobacterium leprae itself [49]. Previously, however, Shepard [50] found that a cephalosporin, cephaloridine, was active and indeed bactericidal for M. leprae in the mouse model. Unfortunately, cephaloridine proved uniquely nephrotoxic and for this reason was removed from the commercial market. Later Shepard [51] found three beta lactam antibiotics were minimally active and none comparable in activity to cephaloridine. Previously, we [52] found by the kinetic technique that five beta lactams (cefoxitin, cephradine, cefamandole, cefotoxamine, and moxalactam), 250 mg/kg administered intraperitoneally five times weekly, were inactive against M. leprae in mice, while cephradine 0.5 % in diet was purely bacteriostatic. In this study we found that augmentin, a beta lactamase inhibitor together with a beta lactam antibiotic, amoxacillin/clavulanic acid, 100 mg/kg by gavage five times weekly, but not amoxacillin alone, resulted in some minimal bactericidal activity for M. leprae. In more current studies [52] we evaluated the activity of augmentin (four parts amoxacillin/ one part clavulanic acid) against M. leprae by the kinetic technique. In this study augmentin was administered to groups of mice five times weekly by gavage in doses of 25 mg/kg, 50 mg/kg, 100 mg/kg, 200 mg/kg, 400 mg/kg, and 600 mg/kg. Augmentin in doses of 200 mg/kg, 400 mg/kg, and 600 mg/kg entirely prevented the multiplication of Mycobacterium leprae over this sixmonth interval. These doses of augmentin are at least as active against M. leprae in mice as was found previously for cephaloridine and result in serum levels clinically achievable in man. Lower doses of augmentin were found inactive. In a second study [52], utilizing the proportional bactericidal technique, we quantitated the bactericidal activity for M. leprae in mice of five times weekly augmentin 400 mg/kg and timentin 1000 mg/kg intraperitoneally (30 parts tica-

432

Robert H. Gelber

cillin/l part clavulanic acid). In this study timentin was found to have no significant bactericidal activity for M. leprae, while aug mentin was found 80 % ± 14 % bactericidal, similar to dapsone itself [8, 53]. These results thus confirm that beta lactam antibiotics and beta lactamase inhibitors appear to offer potential for the therapy of leprosy. Further, these results suggest the need to study other beta lactamase inhibitors, possibly with superior penetration and enzyme affinity, for even greater activity against M. leprae. 7

Monitoring of clinical trials of lepromatous leprosy

Growth of M. leprae in the foot pads of mice inoculated with homogenates of skin biopsies of lepromatous patients undergoing initial chemotherapy has provided a means of assessing the relative clinical efficacy of antimicrobial agents. For these purposes generally 5 x 103 - 1 x 104 M. leprae are inoculated. It is noteworthy that only 0.1 %-6 % of those are viable, thus only 4-360 live bacilli/foot pad are actually inoculated. Both monotherapy with daily dapsone and clofazimine have been found to render such patient-derived inocula nonviable for mice generally within 3 months [10]. On the other hand, rifampin accomplished this in only a few days [54]. This rapid "sterilization" of the skin by rifampin has limited the normal mouse in its utility as a monitor of potent chemotherapeutic regimens. For these purposes we have found that the NTLR is vastly superior [55, 56]. The NTLR and the normal mouse were previously compared to monitor a clinical trial of daily dapsone and either a single initial 1500 mg dose of rifampin or 900 mg rifampin initially and once weekly. In this study patients were biopsied generally within the first few days, 1 week, 2 weeks and I month subsequently. Of these biopsies only 1 of 65 grew in mice, while 33 grew in the NTLR either directly or when they were subpassaged to normal nice. The mean number of M. leprae inoculated per specimen into NTLR was 4 x 107, while mice received generally only 2 x 104 M. leprae. It was found that 61 % of the specimens obtained between 2 and 13 days had viable bacilli as detected in that NTLR, while in 32 % of the specimens obtained between 14 and 31 days viable M. leprae could be detected by the NTLR. We have concluded that the NTLR provides the most sensitive monitor of the viability of M. leprae from patient tissue undergoing initial therapy, wherein most of the M. le-

Progress in the chemotherapy of leprosy: Status, issues and prospects 433

prae are dead, and have concluded that this is largely a function of inoculum size, the foot pad of the mouse allowing 0.03 ml and the rat 0.5 ml. As a corollary, any chemotherapeutic regimen found that more reliably sterilizes the skin as monitored in the NTLR would likely prove the best candidate for short-term chemotherapy, with discontinuation of therapy resulting in a very low relapse rate. The thymectomized irradiated mouse (TR mouse) has, also, been used to follow clinical trials and most recently those of the W. H. O. in Bamako and Chingleput [13]. Because these mice are immunologically deficient they allow for multiplication after larger numbers of AFB are inoculated, but the number of AFB that can be inoculated is limited again by the size of the foot pad to a volume of 0.03 ml [57]. Virtually all of this work with the TR mouse was carried out at the National Institute for Medical Research London as survival of these rodents in other laboratories has been poor. Rees [58] first irradiated mice with a single dose of 900 R, which destroyed bone marrow so that it was necessary to transfuse the mice with bone marrow immediately after irradiation (T900R mouse). In the early years these T900R mice survived well without special precautions, but in more recent years, because of poor survival, these mice have been replaced by mice irradiated with 5 doses of 200 R over a period of eight weeks, which do not require bone-marrow transfusion, survive better, but are not as immunologically deficient (T200X5R mouse) [57]. This TR mouse was utilized by the W.H.O. [13] to follow a total of six less intensive and more intensive regimens in Bamako and Ching1eput in order to monitor the proportion of patients harboring persisting viable M. leprae at 3, 12, and 24 months after the initiation of therapy. Regimens included at least two agents at all times and included rifampin (1500 mg in a single dose, 900 mg weekly or 600 mg daily), dapsone (100 mg daily), prothionamide (500 mg daily), and clofazimine (100 mg daily). In these studies the largest possible number of M. leprae, to a maximum of 105 per foot pad, was inoculated into the hind foot pads of a number of TR mice. Persisting M. leprae were found in 9 % of all skin biopsies [13]. Surprisingly, neither the duration of therapy nor regimen utilized materially affected the percentage of the trial patients that harbored viable M. leprae. Unfortunately, in these studies deciding whether or not M. leprae multiplied in the TR mouse was at times difficult and frequently required subpassage when multiplication was equivocal. Thus most authorities

434

Robert H. Gelber

would agree that for monitoring such clinical trials in the future the NTLR is decidedly preferable. 8

Regimens to treat leprosy

Important major decisions concerning the therapy of multibacilliferous leprosy in 1989 hinge largely upon considerations of the significance of dapsone resistance and bacterial persistance. The World Health Organization's treatment recommendations in 1982 [59], revised in 1988 [60], assume that dapsone resistance, indeed primary dapsone resistance, is a serious problem and that bacterial persistance is of little clinical consequence. Because of these decisions the W.H.O. has made two crucial and novel treatment recommendations: on the one hand they advocate triple drug therapy and on the other hand suggest that all therapy my be discontinued after a few years of treatment. Specifically, for multibacillary disease the W.H.O. recommendation is for daily unsupervised 100 mg dapsone and 50 mg clofazimine and monthly supervised 600 mg rifampin and 300 mg clofazimine. For individuals who find clofazimine totally unacceptable owing to the coloration of skin lesions that it causes, the W.H.O. in 1982 recommended its replacement by 250-375 mg daily self-administered doses of ethionamide/prothionamide. Because of frequent hepatotoxicity when ethionamide is combined with rifampin, the W.H.O. no longer advocates the use of ehtionamide and especially in areas of the world where liver function can not be closely monitored. The W.H.O. recommends that for multibacillary leprosy therapy be maintained for a minimum of two years or until bacillary negativity (generally 5 years). For paucibacillary leprosy the W.H.O. currently recommends dapsone 100 mg daily and rifampin 600 mg once monthly for a total duration of only six months. 9

The significance of primary dapsone resistance

Primary dapsone resistance has been reported in every locale in which it has been sought [61-72] and in some locales in more than 35 % of cases [61-67]. However, the vast majority of cases of primary dapsone resistance were found resistant to only 0.0001 % and 0.001 % dapsone in mouse chow and not to higher concentrations (i. e., 0.01 % dapsone, which more nearly corresponds to the usual

Progress in the chemotherapy ofleprosy: Status, issues and prospects 435

100 mg daily human dose). In fact only a few untreated fully dapsone-resistant leprosy cases have to date been described. Thus the clinical significance of such low level resistance is most unClear. Furthermore, the judgment concerning the issue of whether a case of dapsone-resistant leprosy is primary resistance or secondary resistance is based, almost exclusively, on a reliable history. Patients who are non-compliant or discontinue therapy may relapse and, upon presenting to medical care once again, vehemently deny previous therapy. This may occur because patients are embarassed at what they perceive to have brought upon themselves or because they never understood their original diagnosis or treatment. Thus patients who are considered to be suffering from primary dapsone resistance may indeed have secondary resistant relapse. This factor may seriously prejudice surveys of the prevalence of primary dapsone resistance. In 1984 we [63] assessed the prevalence of dapsone resistance in all new multibacilliferous patients treated between 1978 -1981 and found only 1/54 (2 %) to harbor resistant bacilli, and this single isolate was only resistant to 0.0001 % in mouse chow (equivalent to a human dose of 1 mg/ day of dapsone) but sensitive to higher dietary dapsone concentrations. Our assessment of the prevalance of primary dapsone resistance has not altered over the intervening 0/47 (0 %), in years 1983-1988. This low prevalence of primary dapsone resistance also has been found in the Philippines (4 %) [64]. Furthermore, the experience at Carville, Louisiana (USA), reported by Jacobson [72] is that multibacillary, partially dapsone-resistant patients respond to dapsone monotherapy. Thus we treat lepromatous patients with daily rifampin (600 mg) and dapsone (100 mg) and tuberculoid patients with daily dapsone alone. For our lighter skinned patients the discoloration of clofazimine is indeed cosmetically unacceptable. At issue, also, is whether primary dapsone resistance is a harbinger of full resistance. Indeed, Morrison [73] was able to select a fully dapsone-resistant strain of mycobacterium species by exposing a primary sensitive strain to progressively larger subinhibitory levels of dapsone. However, Mitchison et al. [74] found that in 109 bacilli of a streptomycin-sensitive strain of M. tuberculosis 1000 were resistant to 0.25-0.50 ~g streptomycin, 100 resistant to 1-2 ~g streptomycin and only 2 resiGtant to 32-64 ~g streptomycin. Meads et al. [75] found that a predominantly streptomycin-sensitive strain of Kleb-

436

Robert H. Gelber

siella pneumoniae contained a small number of organisms slightly resistant to streptomycin and a much smaller number of highly resistant organisms. In their experiments slightly resistant strains contained the same proportion of highly resistant organisms as did the parent population, namely 0.5-4.6 per 1010 organisms. Similarly, resistance to low and high levels of sulfonamides and sulfones may be due to separate bacterial mutations affecting drug permeability into bacilli or enzyme activity of the dihydropteroate synthetase. Thus "multistep resistant mutants" may be misleading term suggesting that partial resistance is a harbinger of full resistance rather than partial and complete resistance being separate mutations with partial resistant mutants being more frequent. Drug-sensitive wild populations of M. tuberculosis have resistant mutants in the allowing frequencies: 1 x 10- 3 for ethionamide, 1 x 10- 5 for isoniazid, and 1 x 10- 8 for rifampin. Because of the inability to grow M. leprae in vitro and the limitations of the experimental animal models, there is no similar information available for dapsone and M. leprae. It has been estimated that an untreated lepromatous leprosy patient harbors roughly 1011 viable bacilli. Since in Malaysia only 2.5 % of patients treated with full dosage dapsone monotherapy underwent dapsone-resistant relapse [76], fully dapsone-resistant M. leprae mutants may occur in sensitive strains as infrequently as about 1 in 10 12 _1013. In conclusion, we have reason to doubt the high prevalence of dapsone resistance, the clinical significance of most of the previously reported low-level resistance and that low-level resistance leads to high-level resistance. Two drugs for multibacilliferous leprosy may still be enough, and one may still be dapsone. 10

The significance of bacterial persistance in leprosy

"Persistance" is a commonly encountered phenomenon for many bacterial species which, though drug sensitive, are in a state of metabolic activity which precludes effective antimicrobial action. Hobby [77] described such a situation for streptococci unresponsive to penicillin in 1942; Bigger [78] popularized this term for staphyloccal survivors of penicillin in 1944, and such a situation has been commonly observed for M. tuberculosis in man and experimental animals. In murine tuberculosis most combination chemotherapy cannot elimi-

Progress in the chemotherapy of leprosy: Status, issues and prospects 437

nate all splenic bacilli except for the regime of (1) isoniazid, pyrazinamide and rifampin or (2) certain pairs of these agents, namely, (a) isoniazid and rifampin, or (b) isoniazid and pyrazinamide [79]. In these studies, even when the best combination, isoniazid and rifampin, was given for nine months, 20 % of the animals relapsed after discontinuation of therapy. On the other hand, in the nude mouse which has been treated for tuberculosis even for the most prolonged period of time with the most bactericidal regimens, relapse invariably occurs as soon as treatment is stopped. Also, in spite of one year of isoniazid and rifampin, it was possible to achieve a relapse rate of 60 % by steroid administration [80, 81]. Thus, at least in murine tuberculosis, the persister population requires particularly potent therapy in order to reduce to undetectable levels and poses the threat of initiating relapse, should therapy be discontinued or immunosuppression be superimposed. In human tuberculosis the duration of therapy may be generally successfully reduced from 18-24 months to 6-9 months by use of certain bactericidal combinations which include isoniazid, rifampin, streptomycin, and pyrazinamide [18]. The efficacy that such agents, particularly isoniazid, rifampin, and pyrazinamide, have against "persister" intracellular bacilli is central to the success of such short-course chemotherapeutic regimens. In summary then, in human and murine tuberculosis the rate of clinical relapse following discontinuation of therapy is a function of the bactericidal activity of the drugs utilized, the duration of therapy, and the cellular immunologic status. There is ample information demonstrating that persisters to M. leprae also exist. In Malaysia [11], seven of twelve lepromatous patients treated with ten to twelve years of dapsone were found to harbor viable dapsone-sensitive bacilli, "persisters", in at least one of the following four sites: skin, peripheral nerve, skeletal muscle, or dartos muscle. Even five years of rifampin (at times combined with thiambutosine) has not eliminated these persisters in twenty out of thirty-two patients [12]. Also, no combination of rifampin or dapsone has been found to eliminate persister M. leprae from the neonatally thymectomized Lewis rat (NTLR) [82, 83]. Human lepromatous leprosy with its impaired cellular immune response may be best likened to these situations where persisters can never be eliminated and relapse is inevitable if therapy is not maintained indefinitely. However, on the other hand, the anergy in leprosy appears specific and reversi-

438

Robert H. Gelber

ble in vitro [84] and in treated patients [85]. It thus may be that with therapy as the bacterial burden and certain M. leprae products, particularly phenolic glycolipid I and lipoarabinomannan, which have been demonstrated to interfere with lymphocyte [86-88] and macrophage function [89], disappear, persisting M. leprae, as in treated pulmonary tuberculosis, may pose no serious threat to initiate clinical relapse unless immunosuppression should supervene. Most experienced clinicians have seen lepromatous patients treated with single or multiple agents for years who relapse after therapy is interrupted, but little information is available concerning the magnitude of that risk. Thus the potential that these persisters have for resulting in clinical relapse is unclear. However, in a study of 362 LL and BL patients treated for 18.5-22 years in Malaysia, it was found that 25 patients (8.8 %) relapsed over the next 8-9 years, the resultant annual relapse rate being 1 % [59]. In a trial in Malta [90] rifampin, dapsone, prothionamide, and isoniazid therapy for a total of 18-24 months prior to discontinuation was claimed to result in no clinical relapse over a 4.5 year follow-up. These two studies have been used to support the efficacy of discontinuing therapy in multibacillary disease. On the other hand, if the usual lepromatous patient, a 20-year-old male, were treated for the recommended 5 years, by the time he was 65, extrapolating from the Malaysia experience, he would run a 40 % chance of relapse. Fortunately, in Malaysia these were results of dapsone monotherapy, and more bactericidal regimens might be expected to be even more efficacious in preventing clinical relapse. The Malta trial is difficult to interpret because of a history of previous dapsone therapy in the patients, the lack of definitive clinicopathologic classifications, bacteriologic indices, morphologic indices, or foot pad inoculations. Furthermore, 4.5 years follow-up is simply not long enough; insofar as the incubation period of leprosy is believed to be an average of 5-7 years, the more bacericidal regimens might require a considerable time before clinical relapse becomes evident. Indeed Grosset [91] found that relapse with rifampin resistance averaged at least 8 years after discontinuation of therapy. There is little doubt that the W.H.O. recommendations were based on important economic considerations. Cost of the agents and problems of obtaining compliance were given high priority. If developing countries are to be convinced to utilize combination chemotherapy,

Progress in the chemotherapy of leprosy: Status, issues and prospects 439

it must be affordable. For this reason monthly supervised rifampin has been advocated by the W.H.O. since daily therapy is too costly for most endemic locales. We have found by NTLR inoculation of approximately 106 M. leprae from biopsies that 29 % of patients treated with daily dapsone plus either a single 1500 mg dose of rifampin or weekly 900 mg rifampin after one month still harbor viable M. leprae [55, 56]. Whether daily rifampin might be superior has not yet been similarly studied. Monthly therapy with rifampin, as alluded to earlier, may cause certain rare and fatal disorders (thrombocytopenia and renal failure) that have been observed with intermittent rifampin (to date this has not occurred except rarely). Also, monthly rifampin may result in sufficiently intermittent therapeutic levels so as to select resistant mutants in the same manner that low dosage and non-compliance to dapsone have in the past. In conclusion, monthly rifampin and the decision to discontinue therapy in lepromatous leprosy clearly make the practical delivery of therapy for lepromatous leprosy within the reach of countries where lifelong daily combination therapy is not feasible. However, it may be decades until the optimal treatment of lepromatous leprosy can be placed on a firm scientific and operational basis. In the meantime, guidelines are necessary. The W.H.O. guidelines appear reasonable, but only time will tell whether they are efficacious for individual patients and in disease control. 11

Chemotherapeutic studies in animal models of lepromatous leprosy

Other animal models of leprosy have not, to date, been seriously exploited in chemotherapy studies. Such models present the advantage of more nearly approximating the lepromatous patient in numbers of M. leprae, systemic dissemination, and/or being immunosuppressed. Potential candidate models include the T /R mouse, nude mouse, neonatally thymectomized Lewis rat, Mangabey monkey, and armadillo. All of these animals are expensive to house and maintain, especially in the numbers required for meaningful studies. For chemotherapeutic studies, the NTLR has perhaps been the most studied of these models of lepromatous leprosy; it has the advantages of developing a relatively large population of M. leprae, averaging in our experience 2 x 108 M. leprae per foot pad, is immuno-

440

Robert H. Gelber

suppresed, is easily maintained without serious precautions, and is long lived. In the first chemotherapy studies in the NTLR Fieldsteel [82, 92-93] found that neither dapsone or rifampin alone nor the combination were capable of totally eliminating viable M. leprae from heavily infected NTLR. Our current studies [83] are meant to examine which, if any, regimens are fully effective in this respect with the view that such combinations would be the most applicable to short course chemotherapy of lepromatous leprosy. Our preliminary results suggest that the use of 2 bactericidal agents, either rifampin plus clofazimine or rifampin plus ethionamide, eliminates all measurable viable bacilli [83]. On the other, hand dapsone plus 2 different regimens of rifampin, rifampin alone, and ethionamide plus dapsone were not so effective [83]. In fact, the results of treatment with the two combinations of rifampin and dapsone as compared to rifampin alone suggests antagonism for this bactericidal/bacteriostatic combination [83]. Because the congenitally athymic or nude mouse allows for foot pad growth of M. leprae well above the peak of multiplication in normal mice (l06) and to a level of 109 to 1010 AFB, it presents a model of the lepromatous patient for direct chemotherapy and a large enough bacillary population to determine at least, theoretically, the percentage of bacilli resistant to antimicrobials [94]. To date, limited results of such studies are available, but chemotherapy studies in nude mice are currently in progress in a number of laboratories. 12

Compliance

It has been widely appreciated that the treatment of many chronic diseases whether hypertension, diabetes mellitus, or leprosy, is fraught with serious problems of drug compliance. Non-compliance in leprosy is owing in part to stigma, denial of diagnosis, and the social and cultural implications of the diagnosis being revealed even to other family members. Furthermore, on therapy skin lesions resolve in only months to years, neuropathy may be irreversible, and intervening reactional states, which may be very severe, such as erythema nodosum leprosum, are often perceived by patients as secondary to treatment itself; thus patients often become discouraged. Medical practitioners might best succeed with patient compliance by patient

Progress in the chemotherapy of leprosy: Status, issues and prospects 441

education as to the ravages of untreated disease, the natural history of treated disease, and the implications of interrupted therapy. Thus patient education should begin from the onset and be reinforced frequently. Encouragingly, Ellard [95, 96] has found by monitoring drug levels in leprosy patients in several locales that drug ingestion of 75 % of prescribed therapy occurs. Thus failure internationally in the treatment of leprosy appears more to result from the lack of sufficient medical infrastructures in many endemic countries than from non-compliance. 13

Serology in assessment of therapeutic outcome

In 1981 Hunter and Brennan [97] discovered a unique M. 1eprae product accounting for almost 2 % of the weight of M. 1eprae itself and a major constituent of its outer capsule, phenolic glycolipid I. The antigenic specificity of POI is owing to its specific trisaccharide structure and its unique terminal sugar. Extensive serological studies have demonstrated that only patients infected with M. leprae harbor serum antibodies to M. leprae and that 91 %-96 % of lepromatous patients harbor antibodies to the phenolic glycolipid, generally in high titers [98-101]. On the other hand, tuberculoid patients less commonly, 27 %-62 % of the time, have significant antibody to POI and at lower titers [98-101]. A number of studies have demonstrated that during effective therapy antibody titers generally decrease [98, 102-105]. However, the rate of that decrease is quite variable from patient to patient, thus limiting the utility of the monitoring of serum for clinicians [106]. We have found that rising titers and persistently high titers have been associated with clinical relapse [106]. Both in San Francisco and in Malaysia lepromatous patients who are skin smear negative after an average duratior. of therapy of 12 and 20 years respectively still commonly harbor significant serum antibodies to POI (53 % in San Francisco and 27 % in Malaysia), albeit at low titers [106]. We have wondered whether treated, antibody-negative lepromatous patients also may be those that are no longer anergic and comprise a group of patients that are reliably cured, in whom discontinuation of specific chemotherapy is safe [106].

442

Robert H. Gelber

14

Prophylaxis

Sulfone prophylaxis of household contacts, particularly of lepromatous patients and especially in non-endemic locales, was advocated previously. However, this only appears to delay the onset of lepromatous disease and decrease the eventual prevalence of tuberculoid disease marginally [107]. Thus dapsone prophylaxis is no longer advocated. BeG vaccination for leprosy in Africa [108] appeared to be 80 % effective but was ineffective or only minimumly efficacious elsewhere [109-112]. With the availability of large amounts of M. leprae from heavily infected armadillo livers and spleens and the demonstration that in mice heat-killed M. leprae and BeG given intradermally confer protection against a subsequent live M. leprae challenge [113], vaccine trials of killed M. leprae alone or together with BeG are being sponsored by the World Health Organization. However, components of M. leprae and perhaps specifically phenolic glycolipid and lipoarabinamannan are immunosuppresant for lymphocytes and macrophages [86-89]. Thus specific protein epitopes of M. leprae that confer only salutary immunologic effects are being sought. It appears that cell wall epitopes of M. leprae [114] produce promising immunologic responses, including delayed-type hypersensitivity in sensitized guinea pigs and man and in vitro systemic and dermal Iymphocyte functions which suggest their critical role in effective host recognition [115-116]. Also, a 35 kD protein associated with the pellet fraction of M. leprae appears to be a critical epitope in the salutary cellular immune response to M. leprae [117]. We have found that both purified cell walls of M. Ieprae [118] and this purified 35 kD M. leprae protein (unpublished) in amounts as low as 2 Ilg are effective intradermal vaccines in mice. Thus a new generation of vaccines for leprosy may be available in the future. . References

2 3 4 5 6 7

G. H. Faget, R. C. Pogge, F. A. Johansen, J. F. Dinan, B. M. Prejean and C. G. Eccles: Public Health Reports 58,1729 (1943). J. Lowe: Lepr. Rev. 25, 113 (1954). C. C. Shepard: J. Exp. Med. 112,445 (1960). C. C. Shepard and Y. T. Chang: Int. J. Lepr. 32,260 (1964). C. C. Shepard: Proc. Soc. Exp. BioI. Med. 122, 893 (1966). C. C. Shepard: Int. J. Lepr. 41,307 (1973). R. J. W. Rees, J. M. H. Pearson and M. F. R. Waters: Brit. Med. J. 1,89 (1970).

Progress in the chemotherapy of leprosy: Status, issues and prospects 443 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

M. J. Colston, G. R. F. Hilson and D. K. Banerjee: Lepr. Rev. 49, 7 (1978). M. J. Colston, G. A. Ellard and P. T. Gammon: Lepr. Rev. 49,115 (1978). R. H. Gelber: Lepr. Rev. 58,407 (1987). M. F. R. Waters, R. J. W. Rees, A. C. McDougall and A. G. M. Waddell: Lepr. Rev. 45,288 (1974). M. F. R. Waters, R. J. W. Rees, J. M. H. Pearson, A. B. G. Laing, H. S. Helmy and R. H. Gelber: Br. Med. J. I, 133 (1978). Subcommittee on Clinical Trials of THELEP and SWG of the UNDP/ World Bank/WHO Special Program for Research and Training in Tropical Diseases: Int. J. Lepr. 55,864 (1987). C. C. Shepard: Proc. Soc. Exp. BioI. Med. 109,636 (1962). C. C. Shepard: Int. Lepr. 39, 340 (1971). C. C. Shepard: Int. J. Lepr. 35,429 (1967). C. C. Shepard: Int. J. Lepr. 37,389 (1969). W. Fox and D. A. Mitchison: Am. Rev. Respir. Dis. Ill, 325 (1975). C. C. Shepard: Int. J. Lepr. 50,96 (1982). D. J. Drutz and M. J. Cline: J. Infect. Dis. 125,416 (1972). A. Mittal, M. Satish, P. S. Seshadri, H. K. Prasad, M. Satish and I. Nath: J. Clin. Microbiol. 17,704 (1983). A. M. Dhople and K. J. Green: IRCS Med. Sci. 13,779 (1985). J. T. Kvach, T. A. Neubert, J. C. Palomino and H. S. Heine: Int. J. Lepr. 54, I (1986). N. Ramasesh, R. C. Hastings and J. L. Krahenbuhl: Infect. Immun. 55, 1203 (1987). E. B. Harris, S. G. Franzblau and R. C. Hastings: Int. J. Lepr. 56, 588 (1988). N. Ramasesh, J. L. Krahenbuhl and R. C. Hastings: Antimicrob. Agents Chemother. 33, 657 (1989). S. G. Franzblau and R. C. Hastings: Antimicrob. Agents Chemother. 31, 780 (1987). H. Saito, H. Tomioka and K. Nagashima: Int. J. Lepr. 54, 560 (1986). c.-c. Guelpa-Lauras, E. G. Perani, A.-M. Giroir and J. H. Grosset: Int. J. Lepr. 55, 70 (1987). J. H. Grosset, c.-c. Guelpa-Lauras, E. G. Perani and C. Beoletto: Int. J. Lepr. 56, 255 (1988). S. Pattyn: Int. J. Lepr. 57,381 (1989). R. H. Gelber: J. Infect. Dis. 156,236 (1987). W. R. Fair: Urology 3,339 (1974). J. C. Allen: Ann. Intern. Med. 85,482 (1976). S. G. Franzblau and R. C. Hastings: Antimicrob Agents Chemother. 32, 1758 (1988). R. C. Hastings, J. R. Trautman and R. E. Mansfield: Int. J. Lepr. 37, 130 (1969). M. F. R. Waters in R. H. Gelber: Int. J. Lepr. 44,369 (1976). J. M. Gaugas: Lepr. Rev. 38,225 (1967). S. R. Pattyn and E. Saerens: Lepr. Rev. 49,275 (1978). R. H. Gelber, P. R. Henika and J. B. Gibson: Lepr. Rev. 55,341 (1984). R. H. Gelber: Int. J. Lepr. 55, 78 (1987). J. K. Seydel, M. Richter and E. Wempe: Int. J. Lepr. 48, 18 (1980). R. H. Gelber and L. Levy: Int. J. Lepr. 44, 124 (1976). R. H. Gelber and L. Levy: Int. J. Lepr. 46, III (1978). J. K. Seydel, E. G. Wempe and M. Rosenfeld: Chemotherapy 29, 249 (1983). J. E. Kasik and L. Peacham: Biochem. J. 107,675 (1968). R. K. Mishra and J. E. Kasik: Int. J. Clin. Pharmacol. 3,73 (1970).

444

Robert H. Gelber 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69

70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85

R. J. Wallace, D. R. Nash, T. Udou, V. A. Steingrube, L. C. Steele, J. M. Swenson and V. A. Silcox: Amer. Rev. Resp. Dis. 132, 1093 (1985). K. Prabhakaran, E. B. Harris, R. Sanchez and R. C. Hastings: A. S. M. Abstact, D-23 (1986). C. C. Shepard, L. L. Walker, R. M. Van Landingham and M. A. Redus: Amer. J. Trop. Med. Hyg. 20,616 (1971). C. C. Shepard, R. M. Van Landingham, L. L. Walker and R. C. Good: Int. J. Lepr. 55,322 (1987). R. H. Gelber: Int. J. Lepr. (in press, 1990). R. H. Gelber: Lepr. Rev. 57,347 (1986). C. C. Shepard, L. Levy and P. Fasal: Amer. J. Trop. Med. Hyg. 23, 1120 (1974). R. H. Gelber, R. C. Humphres and A. H. Fieldsteel: Int. J. Lepr. 54,273 (1986). R. H. Gelber and L. Levy: Int. J. Lepr. 55,872 (1987). M. J. Colston: Int. J. Lepr. 55,859 (1987). R. J. W. Rees: Nature 211, 657 (1966). WHO Technical Report Series, No. 675 (1982). WHO Technical Report Series, No. 768 (1988). J. G. Almenda, C. J. G. Chacko, and M. Christian: Int. J. Lepr. 51,374 (1983). J. Constant-Desportes, J. L. Cartel, C. C. Guelpa-Lauras and J. Grosset: Int. J. Lepr. 51,709 (1984). R. H. Gelber: Int. J. Lepr. 52,471 (1984). R. S. Guinto, R. V. Cellona, T. T. Fajardo and E. C. De La Cruz: Int. J. Lepr. 49, 427 (1981). R. R. Jacobson: Final Report of the WHO Regional Working Group on Drug Policy and Operational Research in the Leprosy Programme. Manila, Philippines, 10 (1981). B. Ji: Lepr. Rev. 56,265 (1985). Y. Matsuo, H. Tatsukewa, D.-I. Kim and M. H. Yong: Int. J. Lepr. 50,510 (1982). J. M. H. Pearson, G. S. Haile and R. J. W. Rees: Lepr. Rev. 48, 129 (1977). Subcommittee on Clinical Trials of the Chemotherapy of Leprosy (THELEP) Scientific Working Group of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases: Lepr. Rev. 54, 177 (1983). J. M. H. Pearson: Int. J. Lepr. 49,417 (1981). N. M. Samuel, S. Samuel, J. Loudon and R. B. Adiga: Indian J. Lepr. 56, 819 (1984). R. R. Jacobson and R. C. Hastings: Int. J. Lepr. 46, 116 (1978). N. E. Morrison: Int. J. Lepr. 36, 1652 (1968). D. A. Mitchison: J. Gen. Micr. 5,596 (1951). M. Meads and N. H. Haslam: J. Immun. 63, I (1949). J. M. H. Pearson, R. J. W. Rees and M. F. R. Waters: Lancet 11,69(1976). G. L. Hobby, K. Meyer and E. Caffee: Proc. Soc. Exp. BioI. Med. 50,281 (1942). J. W. Bigger: Lancet 247, 497 (1944). G. Brouet and G. Roussel: Rev. Fr. Mal. Respir. 5,5 (1977). J. Grosset, F. Grumbach and R. Rist: Rev. Fr. Mal. Respir. 6,515 (1978). F. Grumbach: Rev. Fr. Mal. Respir. 3,625 (1975). A. H. Fieldsteel and L. Levy: Int. J. Lepr. 48,267 (1980). R. H. Gelber: Int. J. Lepr. 55,879 (1987). N. Mohagheghpour, R. H. Gelber and E. G. Engleman: J. Immunol. 138, 570 (1987). I. A. Cree, W. C. S. Smith, R. J. W. Rees and J. Swanson Beck: Lepr. Rev. 59, 145 (1988).

Progress in the chemotherapy of leprosy: Status, issues and prospects 445 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 III 112 113 114 liS 116 117 118

G. Kaplan, R. E. Gandhi, D. E. Weinstein, W. R. Levis, M. E. Patarroyo, P. J. Brennan, and Z. A. Cohn: J. Immunol.: 138,3028 (1987). v. Mehra, P. J. Brennan, E. Rada, J. Convit and B. R. Bloom: Nature 308, 194 (1984). K. H. Prasad, R. S. Mishra and I. Nath: J. Immunol. 165,239 (1987). L. D. Sibley, S. W. Hunter, P. J. Brennan and J. L. Krahenbuhl: Infect. Immun. 56, 1232 (1988). E. Freerksen and M. Rosenfeld: Chemotherapy (Basel) 23, 356 (1977). J. H. Grosset, C. C. Guelpa-Lauras and Leprosy Study Group: 28th Interscience Conference on Antimicrob. Agents Chemother., Abstract :If: 259 (1988). A. H. Fieldsteel and L. Levy: Am. J. Trop. Med. Hyg. 25,854 (1976). A. H. Fieldsteel and L. Levy: Int. J. Lepr. 47, 108 (1979). R. D. McDermott-Lancaster, T. Ito, K. Kohsaka, C.-C. Guelpa-Lauras and J. H. Grosset: Int. J. Lepr. 55,889 (1987). G. A. Ellard, V. K. Pannikar, K. Jesudasan and M. Christian: Lepr. Rev. 59,205 (1988). J. N. A. Stanley, J. M. H. Pearson and G. A. Ellard: Lepr. Rev. 57, 9 (1986). S. W. Hunter and P. J. Brennan: J. Bacteriol. 147,728 (1981). S.-N. Cho, D. L. Yanagihara, S. W. Hunter, R. H. Gelber and P. J. Brennan: Infect. Immun. 41, 1077 (1983). S.-N. Cho, T. Fujiwara, S. W. Hunter, T. H. Rea, R. H. Gelber and P. J. Brennan: J. Infect. Dis. 150,311 (1984). D. B. Young and T. M. Buchanan: Science 221,1057 (1983). P. J. Brennan: Lepr. Rev. 57,39 (1986). M.-A. Bach, D. Wallach, B. Flageul, A. Hoffenbach and F. Cottenot: Int. J. Lepr. 54,256 (1986). R. A. Miller, D. Gorder and J. P. Harnisch: Int. J. Lepr. 55,633 (1987). W. R. Levis, H. C. Meeker, G. Schuller-Levis, E. Sersen, P. J. Brennan and P. L. Fried: J. Infect. Dis. 156,763 (1987). J. T. Douglas, L. M. Steven, T. Fajardo, R. V. Cellona, M. G. Madarang, R. M. Abalos and G. J. Steenberger: Lepr. Rev. 59, 127 (1988). R. Gelber, F. Li, R. Cho, S. Byrd, K. Rajagopalan and P. Brennan: Int. J. Lepr. 57, 744 (1989). WHO Technical Report Series, No. 607 (1977). S. J. Stanley, C. Howland, M. M. Stone and I. Sutherland: J. Hyg. 87,233 (1981). K. T. Irwin, T. Sundaresan, M. M. Gyi et al.: Bull. WHO 63, 1069 (1985). S. K. Noordeen: Lepr. Rev. 56, I (1985). J. L. Stanford: Practitioners 227, 10 (1983). S. P. Tripathy: Ann. Natl. Acad. Med. Sci. (India) 19, II (1983). C. C. Shepard, R. M. Van Landingham, L. L. Walker and S. Z. Ye: Infect. Immun. 40, 1096 (1983). S. W. Hunter, M. McNeil, R. L. Modlin, V. Mehra, B. R. Bloom and P. J. Brennan: J. Immunol. 142,2864 (1989). V. Mehra, B. R. Bloom, V. R. Torigian, D. Wandich, M. Reichel, et al.: J. Infect. Immunol. 142,2873 (1989). J. M. Melancon-Kaplan, S. W. Hunter, M. McNeil, et al.: Proc. Natl. Acad. Sci. 85,1917 (1988). N. Mohagheghpour, M. W. Munn, R. H. Gelber and E. G. Engleman: J. Infect. Immun. (in press, 1990). R. H. Gelber, P. J. Brennan, S. Hunter, M. W. Munn, J. M. Monson, L. P. Murray, P. Siu, M. Tsang, E. G. Engleman and N. Mohagheghpour: Infect. Immun. 58 (in press, 1990).