CLINICSIN CHESTMEDICINEELSEVIER

SAUNDERS Clin Chest Med 26 (2005) 327 -340

New Drugs for Tuberculosis: Current Status andFuture Prospects

Richard J. O'Brien, MDa,*, Me! Spigelman, MDb

Following nearly 3 decades of neglect, there isnow renewed interest in the development of newdrugs for the treatment and prevention of tuberculosis[I]. Three reasons are usually given for needing newtuberculosis drugs: (I) to improve current treatmentof active tuberculosis by shortening the total durationof treatment or by providing for more widely spacedintennittent therapy; (2) to improve the treatment ofmultidrug-resistant tuberc1:l1osis (MDR- TB), and(3) to provide more effective treatment of latent tu-berculosis infection (LTBI) in low-incidence coun-tries where this intervention is a component of thecontrol strategy. Of these, the fin!t is most compelling.

Despite the great decrease in tuberculosis inci-dence throughout the latter half of the twentieth cen-tury in industrialized countries, the disease remains asignificant global health problem, particularly amongadults in developing countries [2]. In countries af-fected by the AIDS epidemic, notably those in sub-Saharan Africa, rates of tuberculosis have increaseddramatically, overwhelming control programs [2]. TheWorld Health Organization (WHO) has recentlypromoted the directly observed treatment, short course(DOTS) strategy as an effective intervention that willlead to reduced tuberculosis transmission and decreas-ing numbers of tuberculosis cases [3]. This strategyhas been shown to be among the most cost-effectiveglobal health interventions available today [4]. Animportant component of that strategy is the provision

of high-quality drugs in standardized regimens ofshort-course, rifampin-based treatment given underdirect supervision.

The current treatment regimens, however, sufferfrom a number of drawbacks. With the combinationof available drugs, the duration of treatment requiredfor curing patients cannot be reduced below 6 monthswithout a significant increase in relapses. When givenunder suboptimal conditions, these regimens are as-sociated with high rates of patient nonadherence, withthe consequence of increased mortality and creationof chronic, infectious, drug-resistant cases [5]. It isrecommended that treatment be directly observed bya health care provider, especially during the first2 months and whenever rifampin is used. The in-frastructure required is cumbersome, labor intensive,and expensive. Thus, shorter treatment regimens orthose that could be administered once or twice a weekwould significantly improve treatment outcome.

Development of drug resistance is far more likelywhen supervised treatment is not given, when recom-mended regimens are not used, and when drugs withpoor bioavailability are used. All these factors arefrequently present in countries where DOTS has notbeen established. WHO has documented an increas-ing problem of MDR- TB that threatens to under-mine recent progress in global efforts to control thedisease [6]. The second-line drugs that are used fortreatment of MDR- TB are much more expensive,more toxic, or less effective than first-line drugs. Al-though the development of more effective therapyfor MDR-TB would not alone solve the problem,providing better treatment would be an importantpersonal health benefit for those aft1icted by MDR-

* CoITesponding author.

E-mail address: rick.obrien@finddiagnostics.org(R.J. O'Brien).

0272-5231/05/$ ~ see front matter @ 2005 Elsevier Inc. All rights reserved.

doi: 10.1016/j.ccm.2005.02.013 ch e.~tm ed. th eclin ics. com

"Foundation for Innovative New Diagnostics, Case Postale 93, 1216 CointriniGeneva, SwitzerlandbGlobal Alliance for TB Drug Development, 80 Broad Street, 31st Flool; New York, NY 10004, USA

O'BRIEN ~ SPIGELMAN328

Clinical Trials Program of the International UnionAgainst Tuberculosis and Lung Disease. With sup-port from the European Community, the Europeanand Developing Countries Clinical Trials Partnershipaims to provide ~ 600 million over 5 years to per-form clinical trials and to establish capacity for the

conduct of high-quality clinical trials, includingthose for tuberculosis, thr<I>Ughout Africa [12]. Under-pinning all this effort is the Global Alliance for TBDrug Development (TB Alliance), a recently estab-

lished organization that is forging public-private part-nerships with the objective of building a portfolio ofnew tuberculosis drugs and bringing a major newtuberculosis drug to market in the next decade [13].

This article reviews two classes of compoundsthat have advanced into phase II and III clinical trials,long-acting rifamycins and tluoroquinolones, and anumber of other drugs that have entered or it is hopedwill enter clinical development in the near future.

TB and would improve! the effectiveness of theWHO-supported MDR~TB treatment programsknown as DOTS-Plus [7]!

The resurgence of ~berculosis in the UnitedStates beginning in the late 1980s, coupled with theoutbreaks of MDR-TB largely associated with HIVinfection, led to increased federal support for bothdomestic and global tuberculosis control [8], Thatsupport has resulted in cpntinued declines in tuber-culosis in the United Statbs beginning in 1993 and arenewed call for the el' 'ation of tuberculosis as apublic health problem [9 , An important componentof the tuberculosis elim' tion strategy in the UnitedStates is the treatment of individuals who have LTBIand are at increased ris of developing active TB[10], The most widely us d LTBI treatment regimen,9 months of isoniazid, i associated with significantnonadherence, however, us, a more easily admin-istered LTBI treatment regimen is a priority in anumber of low-incidence countries,

Rifapentine: the search for widely spacedintermittent treatment

Tuberculosis drug development-a changingenvironment

Increased resources directed toward tuberculosisdrug development are now being marshaled fromboth the public and private sectors. Governmentalorganizations, such as United States National In-stiwtes of Health (NIH , are investing in basic re-search aimed at the iden 'fication of new drug targetsand a better understan .g of the phenomena of

Imycobacterial latency. F undations, such as the Billand Melinda Gates Fo dation, are supporting re-search and developmen to enhance the understand-ing of the basic bioi gy of tuberculosis and todevelop new tuberculos s drugs. A number of smallbiotech companies hav programs focused on theidentification of new hemical entities with anti-mycobacterial activities t could become lead com-pounds in the drug-d~ elopment process. Several

large pharmaceutical co panies, such as GlaxoSmith-

Kline (Brentford, Uni d Kingdom), AstraZeneca(London, United Kingdom), and Novartis (Basel,Switzerland), have launched programs directed at thediscovery and development of new tuberculosisdrugs. Other companies, notably Aventis and Bayer,have made compounds available for clinical studies.

At the same time, the clinical trials infrastructure,which had been greatly eroded beginning in the early1980s, is being reestablished with the formation ofgroups such as the UniI d States Tuberculosis Trials

Consortium (TBTC) [1 ] sponsored by the centers

lfor Disease Control an Prevention (CDC) and the

Rifampin is the comerswne of modem short-course tuberculosis treatment, but rifampin-basedregimens must be administered for at least 6 monthsfor optimal effectiveness. Although this treatment isalso highly effective when given three times per weekthroughout the course of treatment [14], more widelyspaced regimens are less effective and may be as-sociated with acquired drug resistance in HIV-infected patients, even when properly taken.

A number of rifamycin derivatives with muchlonger serum half-lives than that of rifampin (2-4 hours) have been evaluated in regimens given in-termittently. The flfSt of these compounds to undergoclinical investigation was rifabutin [15]. The initialclinical trials of the drug focused on the preventionof Mycobacterium avium complex (MAC) infectionin HIV-infected patients [16]. Although the drug wasapproved for MAC prophylaxis in the United Statesand for the treatment of tuberculosis in several othercountries, it now is used primarily as a substitute forrifampin in patients who cannot use that drug be-cause of drug-drug interactions [17]. A TBTC trialof a rifabutin-containing regimen given twice weeklyin HIV-infected patients found high rates of acquiredrifamycin resistance among patients who had moreadvanced immunosuppression, leading to CDC recom-mendations against the use of widely spaced treat-ment of tuberculosis with rifamycin regimens in such

patients [18].

NEW DRUGS FOR WBERCULOSIS 329

Another long-acting rifamr cin derivative, rifalazil,

has an even longer half-life and potent activity in

animal models suggesting that it might be used inultrashort treatment regimens [19]. One attractivefeature of the compound is its rather low potential forenzyme induction and drug interactions [20]. Initialphase I tolerability studies, however, found relativelyhigh rates of side effects manifesting as a flulikesyndrome when the drug Wa~ drninistered as a single

50-mg dose [21]. The h othesized mech:anism

causing the dose limiting s' e effect is release ofcytokines with evidence for increased interleukin-6levels in the serum. Following an early bactericidalactivity (EBA) study that did not demonstrate drugactivity of once-weekly rifal~il (at 10- and 25-mgdoses) plus isoniazid given f<lr 2 weeks [22], furtherclinical development stopped. It is believed thatclosely related compounds can be identified that arebetter tolerated and lack the propensity for enzymeinduction. Currently, there is significant interest in theuse of rifalazil for the treatment of chlamydiainfections [23].

The greatest interest and investment in long-actingrifamycins has been in rifapentine, a cyclopentyl-substituted rifampin with a half-life of 14 to 18 hoursin normal adults. Following a 600-mg dose, serumlevels in excess of the minimum inhibitory concen-tration (MIC) persist beyond 72 hours, suggestingthat the drug might be useful in intermittent regimens(Fig. 1). A series of experimental studies in micefound that a once-weekly continuation phase of rifa-pentine and isoniazid for 4 months following a stan-dard 2-month induction phase with daily isoniazid,rifampin, and pyrazinamide was as effective as stan-

Pard therapy given daily foJ! 6 months [24]. These~tudies provided the scientific underpinning for the~arge phase III trial that was begun by CDC in~995 and subsequently became known as TBTC~tudy 22.i Study 22 was an unmasked clinical trial that

randomly assigned adults who had newly diagnosed,drug-susceptible pulmonary tuberculosis to a4-month (l6-week) continuation-phase regimen ofeither once-weekly rifapentine-isoniazid or twice-weekly rifampin-isoniazid following successful com-pletion of a standard 2-month induction phase [25].The primary study end points were treatment failureand relapse and safety and tolerability of rifapentine.The rifamycins were dosed at 600 mg and isoniazid at900 mg. Although the trial focused on HIV-negativepatients, HIV-positive patients were also enrolledinitially to gain experience with this important subsetof patients. Enrollment of HIV-positive patients was~topped early in the trial, however, following thefinding of a high rate of relapse with acquired ri-fampin monoresistance among HIV-positive patientsassigned to the rifapentine arm [26].

A total of 1003 HIV-negative patients wereenrolled into the completed study. The crude rate offailure and relapse was significantly higher in therifapentine arm (9.2% versus 5.6%, P=0.04). In amultivariate analysis, the factors statistically associ-ated with an adverse outcome were the presence ofcavitary disease on chest radiograph, sputum culturepositivity at study entry (ie, at the end of the intensivephase of therapy), white race, and weight less than90% of ideal body weight at time of the diagnosis oftuberculosis. The treatment regimen was not associ-

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Fig. Rifampin and rifapentine time-concentration qjrves following 600-mg dose in nonnal adults.

330 O'BRIEN & ISPIGELMAN

Fig. 2. Tuberculosis Trials Consortium study 22. Relapse rate by arm, cavitary chest radiograph, and 2-month culture. (Adaptedfrom Tuberculosis Trials Consortium. Rifapentine and isoniazid once a week versus rifampicin and isoniazid twice a week fortreatment of drug-susceptible pulmonary tuberculosis in HN-negative patients: a randomised clinical trial. Lancet2002;360:528-34; with pennission.)

ated with an adverse outcome. Cavitary disease andculture positivity after 2 months were also predictorsof an adverse outcome among patients in the rifampinarm (Fig. 2). Among patients who had noncavitarytuberculosis and negative 2-month sputum cultures,the relapse rate was low in both arms. Rifapentinewas well tolerated, and rates of adverse events weresimilar in both treatment groups, with 3% of pa-tients in both groups discontinuing treatment be-cause of a drug-related adverse event. These resultswere similar to those from a study in Hong-Kong thatused Chinese-manufactured rifapentine of inferiorbioavailability [27] and with those from a company-sponsored trial that enrolled patients largely fromAfrica [28].

The TBTC study results led to new recommen-dations for the use of the rifapentine-isoniazidcontinuation-phase regimen for HIV-negative adultswho have drug-susceptible, noncavitary tuberculo-sis and negative acid-fast bacillus (AFB) smears at2 months [29]. This category includes approxi-mately 40% of patients in the United States whohave newly diagnosed pulmonary tuberculosis. Theregimen provides substantial cost savings for thesepatients, because encounters for directly observedtreatment during the continuation phase are reducedby 50% [30].

Rifapentine-based treatment is not recommendedfor patients who have more advanced tuberculosis orpatients who have HIV infection. Pharmacokineticstudies undertaken as part of Study 22 indicated thatlow levels of isoniazid and rapid isoniazid acetylationwere associated with relapse, suggesting that a moreeffective companion drug might improve once-weekly treatment [31]. Experimental studies havealso suggested that, in addition to a better companiondrug, higher doses of rifapentine might also result inmore effective treatment [24].

Following the completion of Study 22, the TBTCundertook a large phase n trial of higher rifapentinedoses. In Study 25, 150 HIV-negative patients whohad drug-susceptible pulmonary tuberculosis andcompleted initial-phase treatment were randomly as-signed to 600, 900, and 1200 mg rifapentine givenonce weekly with isoniazid for 16 weeks. The ri-fapentine dose was masked with the use of dummytablets of rifapentine. The primary study end pointswere adverse events and drug discontinuation. Allregimens were well tolerated, and only one patientassigned to the l200-mg dose stopped treatment be-cause of a possible drug-related adverse event [32].Because the results of Study 22 were known whenthis study began, the protocol was modified to pro-vide extended treatment for an additional 3 months(or 12 weeks) for patients who had cavitary diseaseand had positive sputum cultures at entry (ie, at2 months). Twenty such patients were enrolled,received extended treatment, and were followedprospectively for relapse. Only one patient who wasassigned to the 600-mg dose relapsed. The relapserate of 5%, when compared with historical data fromStudy 22 (22%), suggests that extended treatment andhigher rifapentine doses may provide more effectivetreatment for patients who are at increased risk ofrelapse [33]. The results also suggest that the 900-mgrifapentine dose would be appropriate to use insubsequent trials.

Experimental studies have also suggested thatonce-weekly rifapentine and isoniazid for as short aperiod as 3 months may provide effective treatmentfor LTBI, comparable to that conferred by 6 monthsof daily isoniazid or by 2 months of daily rifampinand pyrazinamide [34]. Based on these findings,the TBTC has embarked on an ambitious study ofrifapentine/isoniazid for LTBI treatment, intendingto enroll and randomly assign 8000 patients to either

331NEW DRUGS FOR TUBERCULOSIS

9 months of daily self-admffiistered isoniazid or12 doses of once-weekly rifapentine/isoniazid. Be-cause of the large sample size required and thecapacity of the TBTC sites to enroll eligible patients,study completion is not expected before 2008.

Moxifloxacin: the next treatment-shorteningdrug?

During the past decade, fluoroquinolone anti-biotics have become the most important second-linedrugs for treating patients who have MDR-TB. Untilrecently, however, these drugs have not been consid-ered for the treatment of drug-susceptible disease, inpart because the few randomized, controlled trials offluoroquinolones for drug-susceptible tuberculosisthat have been conducted have not demonstrated abenefit. This perspective began to change with thepublication of a clinical trial conducted by the Tu-berculosis Research Centre in Chennai, India. Thisstudy, which did not have a standard control group,randomly assigned patients who had newly diagnosedpulmonary tuberculosis to one of four ofloxacin-containing regimens [35]. Rates of 2-month sputumculture conversion, a marker of the sterilizing activityof tuberculosis drug regimens [36], ranged from 92%to 98%, which compares favorably to an expectedrate of approximately 80% with standard four-drugtreatment [25]. Rates of relapse during the 2 yearsfollowing completion of treatment were 2% and 4%in patients randomly assigned to 3 months of dailyisoniazid, rifampin, pyrazinamide, and ofloxacin,

I

followed by twice-weekly isoniazid and rifampinfor 1 and 2 months, respectively. These results sug-gest that fluoroquinolones might permit substantialshortening of tuberculosis treatment from the currentminimum of 6 months.

Recent experimental data also suggest that fluo-roquinolones may be potent sterilizing drugs thatcould allow shortened regimens for the treatment ofactive tuberculosis, including MDR-TB, and beeffective against LTBI. Thus, newer fluoroquinoloneshave the potential to achieve all three objectives of anew tuberculosis drug. Several fluoroquinolones withmarkedly enhanced in vitro activity against M tuber-culosis are now available. Of these, the most potentare moxifloxacin and gatifloxacin. The MICs of thesetwo agents are fourfold lower than that of levofloxa-cin, the fluoroquinolone that is currently preferred forthe treatment of drug-resistant tuberculosis [37,38].Moxifloxacin also has excellent activity against M. tu-berculosis in animal models [39,40]. A recent evalua-tion of fluoroquinolones in a model of mycobacterialpersistence found that moxifloxacin had the greateststerilizing activity [41]. The pharmacokinetic profileof moxifloxacin, with a relatively long half-life andhigh area under the time concentration curve, alsosuggests that this agent may be an ideal antimyco-bacterial drug [42].

A series of studies of moxifloxacin in mousemodels of acute tuberculosis have also contributed tothe interest in this drug. The initial study, in whichinfected mice were treated for 1 month with severalfluoroquinolones, found that moxifloxacin has thegreatest bactericidal activity, comparable to that ofisoniazid (Fig. 3) [39]. A second study suggested that

Sparfloxacin Moxifloxacin

Fig. 3. Thirty-day experimental study of isoniazid (INH), sparfloxacin, and moxifloxacin in a mouse model of acute tuberculosis.Drug doses in mgikg. (Adapted from Ii B, Lounis N, Maslo C, et al. In vitro and in vivo activities of moxifloxacin and

clinafloxacin against Mycobacterium tuberculosis. Antimicrob Agents Chemother 1998;42:2066-9; with permission.)

332

10

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2HRZM/4HRM

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+ 2RZM/4RM

cn 80)c:;,

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Duration of Treatment, Months

*H=isoniazid, R=rifampin, Z=pyrazinamide, M=moxifloxacin

Fig. 4. Experimental study of moxifloxacin-containing regill)ens in murine tuberculosis. (Adapted from Nuermberger EL,Yoshirnatsu T, Tyagi S, et al. Moxifloxacin-containing regimen greatly reduces time to culture conversion in murine tuberculosis.Am J Respir Crit Care Med 2004;169:334-5; with permission.)

one of four 2-month intensive-phase regimens: twostandard-treatment regimens given either daily orthree times weekly or similar regimens in whichmoxifloxacin replaces ethambutol, with assignmentmasked by placebo moxifloxacin and ethambutol.The primary study end points are 2 month sputumculture conversion and withdrawal because of ad-verse events. Investigators from Johns HopkinsUniversity are working with colleagues from Rio deJaneiro on a similar study that is supported by theUnited States Food and Drug Administration Officeof Orphan Products Development (R. Chaison, per-sonal communication, 2004).

A product development team supported by theUnited Nations Childrens Fund/United Nations De-velopment ProgramlWorld Bank/WHO Special Pro-gram for Research and Training in Tropical Diseasesand the European Commission is embarking onseveral studies of a gatifloxacin fixed-dose com-bination product for the treatment of drug-susceptibletuberculosis. These efforts include preclinical phar-macology and toxicology studies and a phase I studydesigned to compare the drug-drugipharmacokineticinteractions of gatifloxacin and isoniazid, rifampin,and pyrazinamide. A phase n study is being con-ducted in Durban, South African, randomly assigningnewly diagnosed patients to one of three fluoroqui-nolone-containing regimens (ofloxacin, moxifloxacin,and gatifloxacin) in combination with isoniazid,rifampin, and pyrazinamide during the first 2 monthsof treatment. A variety of bacteriologic markers arebeing evaluated as potential surrogate markers oftreatment response. A large phase ill trial of gati-floxacin included in a 4-month regimen that intends

moxifloxacin also has potent sterilizing activity andmight substantially improve the efficacy of once-weekly rifapentine treatment, replacing isoniazid thathas been shown in clinical studies to be a poor com-panion drug [43]. The most recent study found thatthe combination of rifampin, pyrazinamide, andmoxifloxacin had substantially greater sterilizingactivity than the standard regimen, again suggestingthe possibility that the drug would permit significantshortening of treatment (Fig. 4) [44].

The results of two small EBA studies have dem-onstrated that moxifloxacin has bactericidal activitysuperior to that of rifampin and perhaps comparableto that of isoniazid, the most potent bactericidal drugin EBA studies [45,46]. The only other publishedexperience with moxifloxacin treatment of tubercu-losis is a small case series that indicated good toler-ability to chronic administration of the drug [47]. Thenext step in the clinical development of moxifloxa-cin for TB is the conduct of a series of phase IIclinical trials in which moxifloxacin replaces variousdrugs in the initial 2-month phase of TB treatmentand where sputum culture conversion at 2 monthsis the primary study end point [48]. Data from suchstudies, which have historically taken 2 years tocomplete, are usually required to proceed to the largerand more costly phase III trials that commonly takemuch longer to complete.

To develop clinical data that would justify largerphase III efficacy trials of moxifloxacin, the TBTChas embarked on a phase II trial of the drug, Study27. This study randomly assigns newly diagnosed,AFB-positive, HIV-positive and -negative patientswho have suspected pulmonary tuberculosis to

NEW DRUGS FOR ~ERCULOSIS

to enroll over 2000 patients at centers in five coun-tries in sub-Saharan Africa was expected to beginin late 2004 (C. Lienhardt, personal communica-tion, 2004).

The emerging tuberculosis drug pipeline

In addition to the rifamycin derivatives and fluo-roquinolones, a variety of other compounds or classesof compounds are under investigation as potentialantimycobacterial drugs. These include a diarylquino-line (R2079 1 0), a nitroimidazopyran (PA-824), anitro-dihydroimidazo-oxazole (OPC 67,683), a pyr-role (LL3858), macrolides, oxazolidinones, and adiamine (SQI09).

spleens at 12.5 mg/kg to the same extent as isoniazid(25 mg/kg). When therapy was started on day 14 afterinoculation and continued until day 70 (establishedinfection model), 12.5 mg/kg ofR2079 10 was at leastas active in decreasing CFU count in lung as wasisoniazid (25 mg/kg) or rifampin (10 mg/kg). At adose of 25 mg/kg, R207910 was even more activethan at 12.5 mg/kg, reducing lung CFU count from6 to 0.4 log. The combination of R207910 with anytwo of the three commonly used drugs (isoniazid,rifampin, and pyrazinamide) was more effective thanthe standard regimen of isoniazid, rifampin, andpyrazinamide. In fact, the combination of R2079l0,isoniazid, and pyrazinamide and the combination ofR2079 10, rifampin, and pyrazinamide both resultedin negative spleen and lung cultures after 8 weeksof therapy.

Pending results of the phase I studies, the abilityof R2079 1 0 to shorten the therapy of active TB willbe tested.Diarylquinolines (R20791 0)

Nitroimidazopyrans (PA-824)The diarylquinolines, under investigation by

Johnson & Johnson (New Brunswick, New Jersey),have been shown to have potent in vitr~ activityagainst M tuberculosis and seem promising in ananimal model [49]. The lead compound, R2079l0, iscurrently in clinical testing in phase I studies.R20791 0 is equally active against drug-sensitiveM tuberculosis (MIC 0.03 ILg/mL) and strainsresistant to a variety of commonly used drugs suchas isoniazid, rifampin, streptomycin, ethambutol,pyrazinamide, and fluoroquinolones. Similar potencywas also found against other mycobacteria, such asM smegmatis, M bovis, M avium, andM.fortuitum,but the compound is not active against several otherbacterial species, such as Nocardia asteroides,Escherichia coli, Staphylococcus aureus, Enterococ-cus faecium, and Hemophillis influenzae. Tworesistant M smegma tis isolates were not cross-resistant to a wide range of antibiotics, includingthe fluoroquinolones. Thus, the mechanism of actionof R2079l0 seems to be unique among the com-monly used antimicrobials.

In addition to the in vitro activity of R2079l0,the compound has also shown excellent in vivoactivity in mouse models of established and nones-tablished disease. When R207910 was administeredby gavage 5 days/week from day 1 to day 28 afterintravenous inoculation of Swiss mice with 7-logcolony forming units (CFU) of strain H37Rv M. tu-berculosis (nonestablished infection model), the com-pound was able to prevent mortality at the lowestdosage used (1.5 mgikg), prevent gross lesions at6.5 mgikg, and reduce CFU counts in lungs and

The TB Alliance is developing PA-824, a novelnitroimidazopyran with a molecular weight of 359,for fIrst-line therapy of active tuberculosis and for thetreatment of MDR-TB. The history of the nitro-imidazoles goes back to the 1970s, when Ciba-Geigy(Basel, Switzerland) explored a novel series of nitro-imidazole compounds as radiosensitizing agents foruse in cancer therapy. Subsequent studies describedthese compounds' antimicrobial activity, includingactivity against M. tuberculosis. Ciba-Geigy halteddevelopment when their lead compound (CGI-17341)was found to be mutagenic in the Ames assay. In the1990s, PathoGenesis (Seattle, Washington) decidedthis class of compounds warranted further explorationfor potential tuberculosis therapy and synthesizedmore than 700 novel compounds. They determinedthat the nitroimidazopyran PA-824 was the mostactive of these compounds against M tuberculosis ina murine infection model [50].

Following Chiron's (Seattle, Washington) pur-chase of PathoGenesis in 2000, development ofPA-824 was halted because of the company'sdecision to focus on other therapeutic areas. In 2002,the TB Alliance and Chiron signed an exclusivelicense agreement granting the TB Alliance world-wide rights to PA-824 and nitroirnidazole derivatives.Since then, the TB Alliance has continued thedevelopment of PA-824.

A series of in vitro pharmacology studies indicatethat PA-824 may be efficacious against both drug-

334

sensitive and drug-resistant tuberculosis. In vitrostudies demonstrate that the MIC of PA-824 againsta variety of drug-sensitive tuberculosis isolates(:::;0.015-0.25 lJ.g/mL) is similar to that of isoniazid(0.03-0.06 lJ.g/mL). PA-824 is highly selective, withpotent activity only against bacille Calmette-Guerin(BCG) and M tuberculosis among the mycobacterialspecies tested, and without significant activity againsta broad range of gram-positive and gram-negativebacteria (with the exception of H. pylori and someanaerobes). In vitro studies using anaerobic culturemodels indicate that PA-824 has activity againstnonreplicating bacilli, whereas isoniazid does nothave activity in these models. Finally, PA-824 hasbeen shown to have activity against strains of tu-berculosis with known resistance to standard anti-tuberculosis therapies, indicating a novel mechanismof action.

To evaluate in vivo activity, PathoGenesis testedPA-824 in a mouse model of tuberculosis, employingan M tuberculosis reporter strain expressing fireflyluciferase. PA-824 was administered orally at 25, 50,and 100 mg/kg/day in mice for 10 days, withisoniazid used in the control arm. Administration ofPA-824 at all doses significantly reduced M tuber-culosis levels in both spleen and lung compared withcontrols and demonstrated a linear dose response. Inlonger-term studies, PA-824 at 50 mg/kg/day dem-onstrated reductions in bacillary burden similar toisoniazid at 25 mg/kg/day in murine lungs, and allmice treated with PA-824 survived infection,whereas all untreated control animals died by day 35.Daily oral administration ofPA-824 at 37 mg/kg/dayfor 35 days in a guinea pig aerosol infection modelalso caused statistically significant reductions ofM. tuberculosis in lungs and spleens comparedwith controls, reductions comparable to those causedby isoniazid.

The activity ofPA-824 against MDR-TB isolatesand against both replicating (aerobic) and nonrep-licating (anaerobic) M tuberculosis bacilli indicatesthis compound has a novel mechanism of action.PA-824 seems to inhibit significantly both proteinand lipid synthesis but does not affect nucleic acidsynthesis. PA-824 produces an accumulation ofhydroxymycolic acid with a concomitant reductionin ketomycolic acids, suggesting inhibition of anenzyme responsible for the oxidation of hydroxy-mycolate to ketomycolate.

Unlike the Ciba-Geigy lead compound, CGI-17341, PA-824 has not demonstrated mutagenicityin the Ames test (with or without S9 activation), andinitial toxicity studies indicated the doses needed fortherapeutic activity in murine and guinea pig infec-

tion models are below the acute and chronic toxicthresholds observed for PA-824 in mice.

More recent studies by Grosset et al [51] haveindicated that, in a murine model, the minimumeffective dose (defined as the minimum dose whichprevents the development of gross lung lesions andsplenomegaly) of PA-824 is 12.5 mgikg/day, that theabsence of lung lesions on gross inspection correlateswell with bacteriostatic activity measured by CFUcount, that the minimum bactericidal dose (defined asthe minimum dose which reduces the long colonyforming unit counts by 99%) is 100 mgikg/day, andthat the activity of PA-824 at 100 mgikg is com-parable to the activity of isoniazid at 25 mgikg.

The potential genotoxicity of PA-824 was exam-ined further with chromosomal aberration, mousemicronucleus, and mouse lymphoma tests. Theresults indicate that PA-824 is not genotoxic. Fur-thermore, in vitro studies indicate that PA-824 nei-ther inhibits nor is metabolized by major P450enzyme isoforms.

Pharmacokinetic studies have been performed inthe rat, dog, and monkey, because the systemicexposure in dogs is low for both males and femalessecondary to poor absorption and rapid metabolism.Results of the single-dose studies indicate that thehalf-life of PA-824 is approximately 2 to 5 hours inmale rats and monkeys and trends toward a longerhalf-life in female rats (8-9 hours). The half-life indogs is shorter (1-2 hours). In monkeys, singledoses of PA-824 are rapidly absorbed with a timeto maximal concentration (TmaJ of 3.33 hours orless, whereas Tmax in the rat ranges up to 8 hours.There was no significant effect of sex on rate ofabsorption in any species. There was not a sig-nificant food-effect on PA-824 pharmacokinetics inthe rat.

The pharmacokinetics of PA-824 was determinedin plasma, heart, liver, kidney, spleen, and lungfollowing a single 100-mgikg oral dose of PA-824in rats. The time to reach maximal concentrations ofPA-824 in these tissues was 4 hours as comparedwith 6 hours in plasma. Exposure (area under thecurve) in tissues was approximately three- to eight-fold higher than that in plasma. These data suggestthat, in the rat model, penetration of PA-824 intolung, spleen, and other tissues is extensive. In re-peated dose studies, there was no evidence of accu-mulation in the rat or monkey.

Two 14-day good -laboratory practice toxicologystudies, one in the rat and one in the monkey, havebeen completed. The results of these studies indicatethat toxicity is observed when exposures at or aboveapproximately 500 ~g/hour/mL are achieved. Phase I

335,W DRUGS FOR TUBERCULOSIS

antituberculous drugs, LL3858 sterilizes lungs andspleens of infected animals in a shorter timeframethan conventional therapy.

studies of PA-824 are planned for the first quarterof 2005.

Dihydroimidazo-oxazoles (OPC-67683)Macro/ides

OPC-67683 is a newly synthesizednitro-dihydro-imidazo-oxazole derivative under development byOtsuka Phannaceutical Company (Tokyo, Japan)for the treatment of tuberculosis and is currently inphase I study in normal volunteers (Otsuka Phanna-ceutical Company, personal communication, 2004).The compound has potent in vitro antimicrobialactivity against M. tuberculosis, with MICs againstH37Rv and 67 clinically isolated strains ranging from0.006 to 0.024 l1g/mL. Furthermore, OPC-67683shows no cross-resistance with any of the currentlyused first-line tuberculosis drugs, most likely indi-cating a novel mechanism of action. Therefore thecompound may be of benefit both in shorteningduration of therapy in the treatment of active diseaseand in the treatment of MDR-TB.

In vivo studies using a chronic mouse model oftuberculosis have demonstrated the efficacy of OPC-67683 to be superior to that of the currently usedtuberculosis drugs. In the mouse model, the dose thatprovided the effective plasma concentration of0.100 l1g/mL was 0.625 mgikg, confmning the re-markable in vivo potency of OPC-67683.

In other nonclinical in vitro and in vivo studies,OPC-67683 does not have any antagonistic activitywith other first-line tuberculosis drugs when used incombination. Combinations with other first-line thera-peutic drugs reveal synergistic, additive, or no appre-ciable interactions.

Pyrrole (LL3858)

Pyrrole derivatives were first described by Deiddaet al [52] as having fairly potent antimycobacterialactivities against several strains of M tuberculosis.The MICs were between 0.7 and 1.5 lJ.g/mL forthe most potent derivative, 1,5-diaryl-2-methyl-3-(4-methylpiperazin-l-yl) methyl-pyrrole (BM212).The activity ofBM212 against various drug-resistantstrains of M. tuberculosis was similar to its activityagainst sensitive strains, probably indicating a novelmechanism of action. Although some nontuberculosismycobacterial strains seemed to be sensitive, theMICs were higher than for M tuberculosis.

A novel pyrrole compound, LL3858, is currentlyin development for tuberculosis by Lupin Limited(Mumbai, India). This compound has submicromolarMICs and seems to be very active in a mouse modelof tuberculosis. In combination with currently used

The Institute for Tuberculosis Research, Collegeof Pharmacy at the University of Illinois at Chicago,in conjunction with the TB Alliance, is currentlystudying the potential for macrolide antibiotics in thetreatment of tuberculosis. Among approved antimi-crobial agents that do not include tuberculosis as anindication, the macro Ii des are one of the morepromising to yield a clinically useful tuberculosisdrug. This potential is based on their oral bioavail-ability and distribution to the lungs, low toxicity,infrequent adverse reactions, extensive intracellularconcentration and activity, anti-inflammatory activity,and, perhaps most importantly, demonstrated clinicalutility and bactericidal activity in infections causedby several pathogenic and opportunistic mycobac-teria, including M avium, M leprae, M chelonei, andM. fortuitum.

Erythromycin, the first-generation prototypicalmacrolide, is a natural product derived from Strepto-myces erythreus. The compound interferes withprotein synthesis and possesses most of the favorableproperties mentioned previously but suffers from ashort serum half-life and acid lability, which results ingastric motility-based discomfort. In addition, activ-ity is restricted to gram-positive bacteria.

Therefore, second-generation macrolides withsuperior acid stability and serum half-life were devel-oped. Clarithromycin, roxithromycin, and azithromy-cin represent the most successful second-generationmacrolides. It quickly became apparent that thesecond-generation macro Ii des were, along with rifa-butin, the most active clinical agents against theMAC. With the exception of azithromycin (an azalidethat possesses a spectrum of activity different fromthat of other macrolides), these compounds also werefound to possess potent activity against M. leprae inmacrophages and mice and were shown to be effec-tive in clinical trials. Clarithromycin is currentlyrecommended by the WHO for treatment of leprosyin cases of rifampin resistance or intolerance. Otherstudies demonstrated low MICs or clinical utility ofsecond-generation macrolides against M kansasii,M. marinum, M. xenopi, and other opportunisticmycobacterial pathogens. The impressive activity ofsecond-generation macrolides unfortunately did notinclude M. tuberculosis.

The third-generation macrolides, representedlargely by the ketolides, were developed with the

336 O'BRIEN & SPIGELMAN

intention of overcoming the ribosome-modificationand effiux-resistance mechanisms found in gram-positive cocci. Telithromycin was the first such agentto be brought to market. A comparative study of theantimycobacterial activity of clarithromycin versustelithromycin (as well as the fluorinated analogueoftelithromycin) revealed the superior activity of cla-rithromycin for both the moderately clarithromycin-susceptible mycobacteria M bovis BCG, M avium,M. ulcerans, and M. paratuberculosis, and theclarithromycin-resistant mycobacteria M tuberculo-sis, M. bovis, M. africanum, and M simiae [53].Thus, although the general resistance mechanisms tomacrolides of gram-positive cocci and mycobacteriaseem to be similar, there are significant differencesin their structure-activity relationships.

Studies conducted several years ago confirmedthat clarithromycin was the most active antimyco-bacterial macrolide among 15 first- and second-generation macro Ii des (S. Franzblau, personalcommunication, 2004). The most potent of thecommercially available macrolides, cethromycin, stillhas a MIC that is higher than the maximum plasmaconcentration (Cmax) that is obtainable in man. Furthertesting of modifications of the substituents on themacrolide structure have produced much more potentantimycobacterial compounds with low toxicity.These compounds form the basis for the ongoingwork in optimizing the macrolide structure for ac-tivity against M tuberculosis.

Linezolid is the first commercially availableoxazolidinone antibiotic. Although not approved foruse in mycobacterial disease, there are convincing invitro data that the drug is active against M tuber-culosis. A few oxazolidinones have been evaluatedfor their activity in murine in vivo systems. The mostactive compound seems to be PNU-100480, the ac-tivity of which seems to be similar to that of isoniazidor rifampicin [59].

Because of the lack of effective therapeuticoptions for patients who have MDR disease, linezolidhas been used sporadically in patients who haveMDR-TB. Although all reports are anecdotal, line-zolid does seem to have biologic activity as evi-denced by sputum culture conversion [60]. Somewhatdistressing, however, is the reported occurrence ofperipheral and optic neuropathy associated with pro-longed use of linezolid [61].

Overall, the class of oxazolidinones seems to holdpromise for the treatment of tuberculosis. Unfortu-nately, there has not yet been a truly concerted effortto optimize activity of the oxazolidinones for M. tu-berculosis. In the meantime, the evidence for po-tential neuropathies associated witb long-tenD useof linezolid will require careful use of this drug as itbecomes used more commonly in the treatment ofMDR-TB.

SQIO9

Oxazolidinones

Oxazolidinones represent a relatively new classof antimicrobial agents, initially discovered by scien-tists at DuPont (Wilmington, Delaware) in the 1970s[54,55]. They act by inhibiting protein synthesis bybinding to the 70S ribosomal initiation complex[56,57]. The spectrum of activity of the oxazolidi-nones includes anaerobic and gram-positive aerobicbacteria, such as methicillin-resistant S. aureus and S.epidermidis, the enterococci, and also mycobacteria[58,59].

N-adamantan-2-yl-N'-(3,7-dimethylocta-2,6-dienyl)-ethane-l,2-diamine (SQI09) was originallydeveloped as a second-generation antibiotic from aflfSt-line tuberculosis drug, ethambutol, to improveefficacy of the drug against M tuberculosis andlower its toxicity. Although SQ 1 09 is a diamine, itsstructural dissimilarity to ethambutol and differencesin its intracellular target(s) suggest that it is a newantimycobacterial agent, not an ethambutol analogue(Fig. 5).

In collaboration with Dr. Clifton Barry at the NIH,Sequella, Inc. (Rockville, Maryland) synthesized adiverse combinatorial library of compounds with the

R2

IR1' I""Y~ r-\

N, I R3 '-{H R4

Library of 63,238 diaminesEthambutol

Fig. 5. Chemical structures of ethambutol, compounds in the original combinatorial library, and SQlO9.

337NEW DRUGS FOR TUBERCULOSIS

1,2-diamine phannacophore of ethambutol and testedthem for activity against M tuberculosis using anMIC- and target-based (cell wall) reporter high-throughput screening assay [62]. These efforts found2796 mostly lipophilic compounds to be activeagainst M tuberculosis in vitro, and 26 demonstratedin vitro activity equal to or greater than (up to l4-fold)ethambutol. Sixty-nine of the most potent hit com-pounds were later studied in a sequential set of invitro and in vivo tests: MIC followed by cytotoxicityscreen, followed by activity in infected macrophages,followed by penneability evaluation, followed by invivo efficacy testing, followed by phannacokineticstudies. SQl09 was identified as the most potentcompound in the series and was then subjected to in-tensive phannacokineticl phannacodynamic testing.

SQ 1 09 is a lipophilic, nonsymmetric derivative of1,2-ethylenediamine with unsaturated geranyl andbulky adamantane fragments present. SQ 1 09 hasbeen synthesized as a stable dihydrochloride salt ona multikilogram scale with high chemical purity(99.7%). The fonnulation to be used in clinical deve-lopment, hard gelatin capsules, has been developed.

SQl09 has an MIC against M. tuberculosis in therange of 0.1 to 0.63 [1g/mL (broth microdilution,Alamar blue, BACTEC [Becton Dickinson, FranklinLakes, New Jersey]). The compound is bactericidalwith 99% inhibition of M. tuberculosis growth inmacrophages at its MIC. When tested in vivo (inmice), SQl09 is able to reduce infection in lungsand spleen by 2 to 2.5 log. It is active against MDRstrains of M tuberculosis in vitro. SQl09 has a lowmutational frequency in M tuberculosis in vitro(2.18 x 10~9) and demonstrates enhanced antimyco-bacterial activity in vitro and in vivo when usedin combination with rifampicin and isoniazid (rapidmouse model and chronic infection model).

The mechanism of action of SQ I 09 seems to bethat of a cell wall inhibitor because, like the cellwall- targeting antibiotics (ethambutol, isoniazid,ethionamide, and thiacetazone), it induces a pro-moter, Rv0341, that was employed in the originalluciferase high-throughput screening assay. Becausethe Rv0341 luciferase reporter responds with lightproduction to inhibition of a wide variety of enzymetargets involved in cell wall construction, the specifictarget of SQ 1 09 is not known. To address the issue,a proteomic study was initiated to identify proteinsin H37Rv M tuberculosis that are affected by thedrug in comparison with ethambutol and isoniazid.The results of this study suggest that most of the44 distinct proteins whose expression is increased(ESAT -6 and others) or decreased (MPT64 andothers) by SQI09 were similar to those affected byethambutol. Only two gene products whose functionsare unknown were regulated differently by ethambu-tol and SQI09. Similarly, two different genes wereaffected, but in opposite directions, by exposure ofM. tuberculosis to SQI09 or ethambutol [63].

The pharmacokinetic/pharmacodynamic profilesof SQI09 were evaluated in three species (mice,rats, and dogs). Single-dose pharmacokinetic studiesin mice indicate that SQI09 has 4% oral bio-availability as measured by drug concentration inplasma. The high potency of SQ I 09 in vivo at lowdoses (1 mgikg) combined with tissue distributiondata argue, however, that, despite low bioavailabil-ity, SQI09 antimicrobial effects can be attributedto effective concentrations achieved at the sites ofbacterial infection. Although blood concentrationsremain low, SQI09 distributes into lungs and spleen(target sites of the bacterial infection), greatlyexceeding the MIC (Fig. 6). Oral administrationof SQI09, 30 to 75 mg/m2 (10-25 mgikg in mice)

B

.~~" ~~ ~q, 0"'<' ..~ ~ ~~ .~~ i$o<' ~~ ~~ ~~V .b(' Vol; .q' ~o-~... ~ ~ ~,~.. ~..~ ca ~,o ,<,,~ .~~ ~ ~"

$- ~<t~~ V'"

Fig. 6. Tissue levels of SQlO9 after intravenous administration of 3 ffig/kg (A) and oral administration of 25 ffig/kg

(B) to mice.

338 O'BRIEN & SPIGELMAN

one time per day maintains drug levels above theMIC without accumulation of the drug in thetarget tissues.

The liver may have a first-pass effect on SQlO9metabolism, resulting in low content of the drug inplasma after oral dosing. P450 reaction phenotypingsuggests exclusive involvement of CYP1D6 andCYP1C19 in SQlO9 metabolism; analysis ofmetabo-lites formed upon incubation of SQlO9 with human,mouse, dog, and rat rnicrosomes suggest similarmetabolism of the drug in all tested species. SQlO9 isundergoing formal preclinical 90-day pharmacologyand toxicology studies in preparation for humanclinical trials.

In summary, SQlO9 is a novell,l-diamine-baseddrug candidate with in vitro and in vivo activityagainst M tuberculosis. It has pharmacokinetic/pharmacodynamic properties that are characterizedby a rapid and broad distribution into various tis-sues (ie, lungs) that is advantageous for tuberculo-sis infection.

Summary

During the recent decade, significant progress hasbeen made in reinvigorating the almost nonexistentpipeline of novel agents for the treatment of tuber-culosis and in reestablishing the infrastructure for theconduct of clinical trials of new tuberculosis drugsand treatment regimens. Recent studies of long-actingrifamycin derivatives and potent tluoroquinolone an-tibiotics are leading to improved regimens for thetreatment of active and latent tuberculosis. A numberof other compounds in late preclinical and earlyclinical development show great promise. The rapidincrease in knowledge of mycobacterial pathogenesisis leading to the identification of new drug targets,including those believed to playa role in latent in-fection or in the phenomenon of persistence. A majorchallenge will be to sustain and increase funding forcontinued developmental and clinical work if thepromise of tuberculosis elimination, or at least sig-nificant lessening of the global tuberculosis epi-demic, is to be achieved.

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