Koen Andries

The challenge of new drug discovery for tuberculosis

Tuberculosis (TB) is more prevalent in the world today than at any other time in human history. Mycobacterium tuberculosis, the pathogen responsible for TB, uses diverse strategies to survive in a variety of host lesions and to evade immune surveillance. A key question is how robust are our approaches to discovering new TB drugs, and what measures could be taken to reduce the long and protracted clinical development of new drugs. The emergence of multi-drug-resistant strains of M. tuberculosis makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the re-engineering and repositioning of some old drug families to achieve effective control. Whatever the strategy used, success will depend largely on our proper understanding of the complex interactions between the pathogen and its human host. In this review, we discuss innovations in TB drug discovery and evolving strategies to bring newer agents more quickly to patients.

The Diarylquinoline TMC207 for Multidrug-Resistant Tuberculosis

BACKGROUND The diarylquinoline TMC207 offers a new mechanism of antituberculosis action by inhibiting mycobacterial ATP synthase. TMC207 potently inhibits drug-sensitive and drug-resistant Mycobacterium tuberculosis in vitro and shows bactericidal activity in patients who have drug-susceptible pulmonary tuberculosis. METHODS In the first stage of a two-stage, phase 2, randomized, controlled trial, we randomly assigned 47 patients who had newly diagnosed multidrug-resistant pulmonary tuberculosis to receive either TMC207 (400 mg daily for 2 weeks, followed by 200 mg three times a week for 6 weeks) (23 patients) or placebo (24 patients) in combination with a standard five-drug, second-line antituberculosis regimen. The primary efficacy end point was the conversion of sputum cultures, in liquid broth, from positive to negative. RESULTS The addition of TMC207 to standard therapy for multidrug-resistant tuberculosis reduced the time to conversion to a negative sputum culture, as compared with placebo (hazard ratio, 11.8; 95% confidence interval, 2.3 to 61.3; P=0.003 by Cox regression analysis) and increased the proportion of patients with conversion of sputum culture (48% vs. 9%). The mean log(10) count of colony-forming units in the sputum declined more rapidly in the TMC207 group than in the placebo group. No significant differences in average plasma TMC207 concentrations were noted between patients with and those without culture conversion. Most adverse events were mild to moderate, and only nausea occurred significantly more frequently among patients in the TMC207 group than among patients in the placebo group (26% vs. 4%, P=0.04). CONCLUSIONS The clinical activity of TMC207 validates ATP synthase as a viable target for the treatment of tuberculosis. (ClinicalTrials.gov number, NCT00449644.)

TMC125, a Novel Next-Generation Nonnucleoside Reverse Transcriptase Inhibitor Active against Nonnucleoside Reverse Transcriptase Inhibitor-Resistant Human Immunodeficiency Virus Type 1

ABSTRACT Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1); however, currently marketed NNRTIs rapidly select resistant virus, and cross-resistance within the class is extensive. A parallel screening strategy was applied to test candidates from a series of diarylpyrimidines against wild-type and resistant HIV strains carrying clinically relevant mutations. Serum protein binding and metabolic stability were addressed early in the selection process. The emerging clinical candidate, TMC125, was highly active against wild-type HIV-1 (50% effective concentration [EC50] = 1.4 to 4.8 nM) and showed some activity against HIV-2 (EC50 = 3.5 μM). TMC125 also inhibited a series of HIV-1 group M subtypes and circulating recombinant forms and a group O virus. Incubation of TMC125 with human liver microsomal fractions suggested good metabolic stability (15% decrease in drug concentration and 7% decrease in antiviral activity after 120 min). Although TMC125 is highly protein bound, its antiviral effect was not reduced by the presence of 45 mg of human serum albumin/ml, 1 mg of α1-acid glycoprotein/ml, or 50% human serum. In an initial screen for activity against a panel of 25 viruses carrying single and double reverse transcriptase amino acid substitutions associated with NNRTI resistance, the EC50 of TMC125 was <5 nM for 19 viruses, including the double mutants K101E+K103N and K103N+Y181C. TMC125 also retained activity (EC50 < 100 nM) against 97% of 1,081 recent clinically derived recombinant viruses resistant to at least one of the currently marketed NNRTIs. TMC125 is a potent next generation NNRTI, with the potential for use in individuals infected with NNRTI-resistant virus.

Multidrug-Resistant Tuberculosis and Culture Conversion with Bedaquiline

BACKGROUND Bedaquiline (Sirturo, TMC207), a diarylquinoline that inhibits mycobacterial ATP synthase, has been associated with accelerated sputum-culture conversion in patients with multidrug-resistant tuberculosis, when added to a preferred background regimen for 8 weeks. METHODS In this phase 2b trial, we randomly assigned 160 patients with newly diagnosed, smear-positive, multidrug-resistant tuberculosis to receive either 400 mg of bedaquiline once daily for 2 weeks, followed by 200 mg three times a week for 22 weeks, or placebo, both in combination with a preferred background regimen. The primary efficacy end point was the time to sputum-culture conversion in liquid broth. Patients were followed for 120 weeks from baseline. RESULTS Bedaquiline reduced the median time to culture conversion, as compared with placebo, from 125 days to 83 days (hazard ratio in the bedaquiline group, 2.44; 95% confidence interval, 1.57 to 3.80; P<0.001 by Cox regression analysis) and increased the rate of culture conversion at 24 weeks (79% vs. 58%, P=0.008) and at 120 weeks (62% vs. 44%, P=0.04). On the basis of World Health Organization outcome definitions for multidrug-resistant tuberculosis, cure rates at 120 weeks were 58% in the bedaquiline group and 32% in the placebo group (P=0.003). The overall incidence of adverse events was similar in the two groups. There were 10 deaths in the bedaquiline group and 2 in the placebo group, with no causal pattern evident. CONCLUSIONS The addition of bedaquiline to a preferred background regimen for 24 weeks resulted in faster culture conversion and significantly more culture conversions at 120 weeks, as compared with placebo. There were more deaths in the bedaquiline group than in the placebo group. (Funded by Janssen Pharmaceuticals; TMC207-C208 ClinicalTrials.gov number, NCT00449644.).

TMC125 Displays a High Genetic Barrier to the Development of Resistance: Evidence from In Vitro Selection Experiments

ABSTRACT TMC125 is a potent new investigational nonnucleoside reverse transcriptase inhibitor (NNRTI) that is active against human immunodeficiency virus type 1 (HIV-1) with resistance to currently licensed NNRTIs. Sequential passage experiments with both wild-type virus and NNRTI-resistant virus were performed to identify mutations selected by TMC125 in vitro. In addition to “classic” selection experiments at a low multiplicity of infection (MOI) with increasing concentrations of inhibitors, experiments at a high MOI with fixed concentrations of inhibitors were performed to ensure a standardized comparison between TMC125 and current NNRTIs. Both low- and high-MOI experiments demonstrated that the development of resistance to TMC125 required multiple mutations which frequently conferred cross-resistance to efavirenz and nevirapine. In high-MOI experiments, 1 μM TMC125 completely inhibited the breakthrough of resistant virus from wild-type and NNRTI-resistant HIV-1, in contrast to efavirenz and nevirapine. Furthermore, breakthrough of virus from site-directed mutant (SDM) SDM-K103N/Y181C occurred at the same time or later with TMC125 as breakthrough from wild-type HIV-1 with efavirenz or nevirapine. The selection experiments identified mutations selected by TMC125 that included known NNRTI-associated mutations L100I, Y181C, G190E, M230L, and Y318F and the novel mutations V179I and V179F. Testing the antiviral activity of TMC125 against a panel of SDMs indicated that the impact of these individual mutations on resistance was highly dependent upon the presence and identity of coexisting mutations. These results demonstrate that TMC125 has a unique profile of activity against NNRTI-resistant virus and possesses a high genetic barrier to the development of resistance in vitro.

Randomized Pilot Trial of Eight Weeks of Bedaquiline (TMC207) Treatment for Multidrug-Resistant Tuberculosis: Long-Term Outcome, Tolerability, and Effect on Emergence of Drug Resistance

ABSTRACT The 2-year follow-up results for a randomized placebo-controlled study of 47 patients with multidrug-resistant pulmonary tuberculosis treated with either the new diarylquinoline TMC207, recently renamed bedaquiline, or placebo, added to the first 8 weeks of a background regimen, are presented. Bedaquiline significantly reduced the time to culture conversion over 24 weeks (hazard ratio, 2.253; 95% confidence interval, 1.08 to 4.71; P = 0.031). With the exception of nausea reported in 26% of patients receiving bedaquiline and none receiving placebo, adverse events occurred at similar frequencies in both groups of patients: bilateral hearing impairment, extremity pain, acne, and noncardiac chest pain occurred in 13 and 21%, 17 and 13%, 9 and 17%, and 4 and 17% of patients, respectively, receiving bedaquiline or placebo. Excluding resistance to ethambutol and ethionamide, only one patient receiving bedaquiline acquired resistance to companion drugs, but five patients receiving placebo (4.8% versus 21.7%; P = 0.18) acquired resistance to companion drugs, and resistance to ofloxacin was acquired in four patients receiving placebo and none receiving bedaquiline (0% versus 22%; 0 = 0.066). In all, 23 patients (49%), including 13 receiving placebo (54%) and 10 receiving bedaquiline (44%), discontinued the study prior to its completion, 12 during the first 24 weeks of treatment. Eight subjects were withdrawn for noncompliance or default, and seven withdrew consent, citing the rigorous program of investigations for safety and pharmacokinetic monitoring. Bedaquiline may contribute to the management of multidrug-resistant tuberculosis by effecting more rapid sputum culture negativity and by preventing acquired resistance to companion drugs.

Diarylquinolines Are Bactericidal for Dormant Mycobacteria as a Result of Disturbed ATP Homeostasis

An estimated one-third of the world population is latently infected with Mycobacterium tuberculosis. These nonreplicating, dormant bacilli are tolerant to conventional anti-tuberculosis drugs, such as isoniazid. We recently identified diarylquinoline R207910 (also called TMC207) as an inhibitor of ATP synthase with a remarkable activity against replicating mycobacteria. In the present study, we show that R207910 kills dormant bacilli as effectively as aerobically grown bacilli with the same target specificity. Despite a transcriptional down-regulation of the ATP synthase operon and significantly lower cellular ATP levels, we show that dormant mycobacteria do possess residual ATP synthase enzymatic activity. This activity is blocked by nanomolar concentrations of R207910, thereby further reducing ATP levels and causing a pronounced bactericidal effect. We conclude that this residual ATP synthase activity is indispensable for the survival of dormant mycobacteria, making it a promising drug target to tackle dormant infections. The unique dual bactericidal activity of diarylquinolines on dormant as well as replicating bacterial subpopulations distinguishes them entirely from the current anti-tuberculosis drugs and underlines the potential of R207910 to shorten tuberculosis treatment.

Location of Persisting Mycobacteria in a Guinea Pig Model of Tuberculosis Revealed by R207910

ABSTRACT The lengthy chemotherapy of tuberculosis reflects the ability of a small subpopulation of Mycobacterium tuberculosis bacteria to persist in infected individuals. To date, the exact location of these persisting bacteria is not known. Lung lesions in guinea pigs infected with M. tuberculosis have striking similarities, such as necrosis, mineralization, and hypoxia, to natural infections in humans. Guinea pigs develop necrotic primary lesions after aerosol infection that differ in their morphology compared to secondary lesions resulting from hematogenous dissemination. In infected guinea pigs conventional therapy for tuberculosis during 6 weeks reduced the bacterial load by 1.7 logs in the lungs and, although this completely reversed lung inflammation associated with secondary lesions, the primary granulomas remained largely unaffected. Treatment of animals with the experimental drug R207910 (TMC207) for 6 weeks was highly effective with almost complete eradication of the bacteria throughout both the primary and the secondary lesions. Most importantly, the few remnants of acid-fast bacilli remaining after R207910 treatment were to be found extracellular, in a microenvironment of residual primary lesion necrosis with incomplete dystrophic calcification. This zone of the primary granuloma is hypoxic and is morphologically similar to what has been described for human lung lesions. These results show that this acellular rim may, therefore, be a primary location of persisting bacilli withstanding drug treatment.

Evolution of anti-HIV drug candidates. Part 3: diarylpyrimidine (DAPY) analogues

The synthesis and anti-HIV-1 activity of a series of diarylpyrimidines (DAPYs) are described. Several members of this novel class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) are extremely potent against both wild-type and a panel of clinically significant single- and double-mutant strains of HIV-1.

Comparison of the Anti-Influenza Virus Activity of RWJ-270201 with Those of Oseltamivir and Zanamivir

ABSTRACT We have recently reported an influenza virus neuraminidase inhibitor, RWJ-270201 (BCX-1812), a novel cyclopentane derivative discovered through structure-based drug design. In this paper, we compare the potency of three compounds, RWJ-270201, oseltamivir, and zanamivir, against neuraminidase enzymes from various subtypes of influenza. RWJ-270201 effectively inhibited all tested influenza A and influenza B neuraminidases in vitro, with 50% inhibitory concentrations of 0.09 to 1.4 nM for influenza A neuraminidases and 0.6 to 11 nM for influenza B neuraminidases. These values were comparable to or lower than those for oseltamivir carboxylate (GS4071) and zanamivir (GG167). RWJ-270201 demonstrated excellent selectivity (>10,000-fold) for influenza virus neuraminidase over mammalian, bacterial, or other viral neuraminidases. Oral administration of a dosage of 1 mg/kg of body weight/day of RWJ-270201 for 5 days (beginning 4 h preinfection) showed efficacy in the murine model of influenza virus infection as determined by lethality and weight loss protection. RWJ-270201 administered intranasally at 0.01 mg/kg/day in the murine influenza model demonstrated complete protection against lethality, whereas oseltamivir carboxylate and zanamivir at the same dose demonstrated only partial protection. In the delayed-treatment murine influenza model, oral administration of a 10-mg/kg/day dose of RWJ-270201 or oseltamivir (GS4104, a prodrug of GS4071) at 24 h postinfection showed significant protection against lethality (P < 0.001 versus control). However, when the treatment was delayed for 48 h, no significant protection was observed in either drug group. No drug-related toxicity was observed in mice receiving 100 mg/kg/day of RWJ-270201 for 5 days. These efficacy and safety profiles justify further consideration of RWJ-270201 for the treatment and prevention of human influenza.