WO2004062607A2

WO2004062607A2 - Use of weak acids or their precursors for the treatment of tuberculosis (tb) and drug resistant tb

Info

Publication number: WO2004062607A2
Application number: PCT/US2004/000544
Authority: WO
Inventors: Ying Zhang
Original assignee: Ying Zhang
Priority date: 2003-01-13
Filing date: 2004-01-12
Publication date: 2004-07-29
Also published as: WO2004062607A3

Abstract

The present invention is directed to the use of weak acids or weak acid precursors as prodrugs for the treatment of tuberculosis (TB). In addition, the present invention is directed to the use of weak acids or their precursors in combination with the current TB drugs such as isoniazid, rifampin, pyrazinamide, ethambutol for the treatment and prevention of TB or drug-resistant TB and for shortening the duration of the treatment.

Description

Use of Weak Acids or Their Precursors for the Treatment of Tuberculosis (TB) and Drug Resistant TB

BACKGROUND OF THE INVENTION

Field of the Invention

A method for the treatment of TB and drug-resistant TB using weak acids, or their precursors either alone or in conjunction with current TB drugs^' is claimed. Weak acids or their precursors or a cocktail of weak acids or their precursors can be incorporated into current TB drug formulations for the above purpose.

Background of the Invention

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a major infectious morbidity and mortality worldwide, especially in the developing countries (WHO Report, 2000). Each year there are 8 million new TB cases with 2 million deaths. The current TB therapy consists of an initial phase of treatment with four frontline TB drugs, isoniazid .(INH), rifampin (RIF), pyrazinamide (PZA) and ethambutol (EMB) for 2 months daily, followed by a continuation phase of treatment with INH and RIF for another 4 months, three times a week (WHO Report, 2000). This therapy, also called DOTS (directly observed treatment, short-course), is the best TB therapy and is recommended by the World Health Organization for treating every TB patient (WHO Report, 2000). DOTS has a cure rate of up to 95% given patient compliance. Although DOTS is the best treatment for TB, the therapy takes at least 6 months. The length of the therapy makes patient compliance difficult, which is a frequent source of drug-resistant strains. The need for the lengthy treatment is a consequence of the presence of a population of persistent bacilli that are not effectively eliminated by the current TB drugs. Although TB patients are rendered non-infectious after the first two weeks of chemotherapy, the remainder of the 6 month therapy is to kill a population of slowly-metabolizing persistent bacilli and to allow the host to develop protective immunity to control the residual number of bacilli not killed by the drugs, in order to prevent relapse. Current TB drugs are mainly active against growing bacilli, except for RIF and PZA. RIF is active against both actively growing and slowly-metabolizing nongrowing bacilli, whereas PZA is active against semi-dormant nongrowing bacilli in an acidic environment (Mitchison, 1985), such as in active inflammation sites in the lesions. These two agents are important sterilizing drugs that significantly reduce the number of bacilli in infected tissues and shorten the therapy from 12-18 months to 6 months.

Drug-resistant TB is becoming an increasing public health concern in recent years and poses a potential threat to the control of the disease (WHO Report, 2000). There is growing awareness that the current TB therapy is too long, which takes a lengthy period of 6 months. Failure to adhere to the lengthy 6 month therapy is a frequent cause of drug-resistant TB. There is currently a great deal of interest to develop new drugs that are not only active against drug-resistant TB but also can shorten the duration of the therapy (O'Brien and Nunn, 2001).

Weak acids are known to be food preservatives to inhibit the growth of bacteria where their antibacterial activity is enhanced at acid pH conditions (Freese et al., 1973). However, weak acids have not been purposefully tested on mycobacteria for drug development. During our study of the mode of action of the frontline TB drug pyrazinamide, which shortens the TB therapy from previously 9-12 months to 6 months, we found that M. tuberculosis is uniquely susceptible to weak acid pyrazinoic acid (pKa=2.9), the active form of pyrazinamide; whereas other mycobacteria (e.g. M. smegmatis) or bacteria (e.g. E. coli) are more resistant to pyrazinoic acid (Zhang et al., 1999; Schaller et al., 2002). In addition, it is well known that during pyrazinamide susceptibility testing, which requires acid pH for activity, the growth of M tuberculosis is inhibited if the medium pH is below 5.5 (Heifets and Iseman, 1985). M. tuberculosis appears to be quite susceptible to acid pH compared with other mycobacteria (Portaels and Pattyn, 1982). For example, in Sauton's simple salt medium the growth of M. tuberculosis was restricted at pH 6.0, whereas other mycobacterial species grew quite well (Piddington et al., 2001). SUMMARY OF THE INVENTION

M. tuberculosis is found to be uniquely susceptible to weak acids compared with other mycobacteria or bacteria The antituberculosis activity of the weak acids was higher at acid pH than at neutral pH. The antituberculous activity of the weak acids generally inversely correlates with the pKa value of the weak acids, that is, the lower the pKa value the higher the antituberculous activity. The unique susceptibility of M. tuberculosis to weak acids correlated with deficient efflux mechanism and its poor ability to maintain internal pH and membrane potential at acid pH compared with other mycobacteria or bacteria. The antituberculous activity of weak acids correlated with their ability to disrupt the membrane potential but not the internal pH. It is proposed that weak acids or their precursors as prodrugs can be used alone or in combination with the current TB drugs for the treatment and prevention of TB or drug-resistaήt TB and for shortening the TB treatment.

DESCRIPTION OF THE DRAWINGS

Figure 1. Changes in internal pH of M. tuberculosis H37Ra (Ra) and M. smegmatis mc²6 (MC2) in response to external pH and valinomycin plus nigericin. Comparison of internal pH changes in response to external pH is shown in Panel A. The changes of internal pH in M. tuberculosis and M. smegmatis in response to valinomycin (V) plus nigericin (N) are shown in Panel B and C, respectively.

Figure 2. Comparison of the membrane potential of M. tuberculosis H37Ra and M. smegmatis MC2 in response to changes in external pH.

Figure 3. Relationship between weak acid susceptibility of tuberculosis H37Ra and M. smegmatis MC2 and disruption of membrane potential (A) and internal pH (B). Membrane potential (-mV) in Y axis is in negative. The membrane potential and internal pH values represent the average in triplicate. BA, benzoic acid; SA, salicylic acid; Asp, aspirin (acetyl-salicylic acid); NBA, 4-nonyloxybenzoic acid; LOA, linoleic acid. The concentration of the weak acids used in the experiments was 4 mM. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of prevention and treatment of mycobacterial infections using weak acids or their precursors in a mammal. Weak acids in an effective amount used for treating mycobacterial infections include formula R-COOH, where R is typically from the group comprising aromatic or benzene ring, including but not limited to benzoic acid, salicylic acid, aspirin (acetyl-salicylic acid), mefenamic acid (2-[2,3-dimethylphenyl]amino-benzoic acid), nicotinic acid, 4-nonyloxybenzoic acid, 4-octylbenzoic acid, octylloxybenzoic acid, nitrobezoic acid, hexylbenzoic acid, heptylbenzoic acid, benzofurancarboxylic acid, 4-dodecyloxylbenzoic acid, 11-phenoxyundecanoic acid; undecyloxybenzoic acid, cyclohexenyloxybenzoic acid.

The weak acids typically have greater antimycobacterial activity at acid pH than at neutral pH. The antimycobacterial activity of weak acids can be in the form of growth inhibition or actual killing of mycobacteria. By growth inhibition, it means that in the presence of appropriate concentration of weak acids the growth of mycobacteria will be suppressed as demonstrated by lack of colony formation on common mycobacterial culture agar plates containing the weak acids or by a much lower optical density of a liquid culture containing weak acids compared with control media without weak acids. The relationship between pH and weak acid antimycobacterial activity as in minimum inhibitory concentration (MIC) can be expressed by the Henderson-Hasselbach equation, where pH = pKa + log[A^"] HA, where pKa refers to the dissociation constant of a given weak acid, A^" refers to anion form of the acid and HA the protonated form of the weak acid. The antimycobacterial activity of the weak acids is enhanced at acid pH (Table 2). This is consistent with the fact that at acidic pH weak acids become protonated and form uncharged species that permeates through the membrane easily compared with charged anion species (Rotenberg, 1979). Enhanced activity at acid pH is consistent with the observation that uptake and accumulation of weak acids is increased at acidic pH, as shown for pyrazinoic acid (Zhang et al., 1999). The consequence of weak acid accumulation and recycling could lead to disruption of the proton motive force that is required for the transport of many nutrient substances into bacterial cell as a mechanism of action of weak acids (Zhang et al., 2003a). The acid pH environment important for the activity of weak acids is present in vivo as in inflammatory sites and will enhance the activity of the weak acids.

The antimycobacterial activity of the weak acids typically has a pKa value between 2 and 5. The antituberculous activity of the weak acids often inversely correlates with the pKa of the weak acid (Table 2), i.e., the lower the pKa (the stronger the weak acid) the stronger the antituberculous activity. In other words, the lower the pKa value of a weak acid, the higher antimycobacterial activity. For example, salicylic acid and nicotinic acid have pKa values of 3 and 4.8 respectively, and their MICs for M. tuberculosis were 10-20 and 200 mg/L at pH 5.5, respectively. By higher antimycobacterial activity, we mean lower amount of the weak acid being able to produce a growth inhibitory or killing effect on mycobacteria. Those who are skilled in the art will recognize that weak acids or their precursors can be used for the treatment or prevention of infections caused not only by Mycobacterium tuberculosis, but also by other slow growing mycobacterial species including but are not limited to Mycobacterium bovis, Mycobacterium africanum, Mycobacterium avium, Mycobacterium scrofulaceum, Mycobacterium kansaii, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium paratuberculosis, Mycobacterium ulcerans, and Mycobacterium xenopi.

The invention is predicated on the novel finding that M. tuberculosis is uniquely susceptible to weak acids and that weak acids are much more effective against M. tuberculosis than against other mycobacteria or bacteria (Table 2). The higher susceptibility of M. tuberculosis to weak acids is likely a reflection of its poor ability to maintain pH homeostasis than the less susceptible mycobacteria such as M. smegmatis. Indeed, comparison of internal pH in response to changes in external pH indicated that M. tuberculosis has a lower ability to maintain internal pH at acid pH between pH 3 and pH 5 than M. smegmatis (Fig. 1 A). This is further strengthened by the finding that valinomycin and nigericin had a significant effect on lowering the internal pH in M. smegmatis but not in M. tuberculosis (Fig. IB and 1C). The deficiency of M. tuberculosis in maintaining the internal pH towards neutrality at very acidic pH conditions (pH 3-5) could result from an increased proton permeability of the M. tuberculosis membrane or a decreased proton extrusion by the membrane-embedded ATPase as a result of slow metabolism in M. tuberculosis compared with other mycobacteria such as M. smegmatis.

The finding that M. tuberculosis is quite susceptible to weak acids of diverse structures suggests that these weak acids do not have a specific cellular target besides their general effect on disrupting the membrane function. Failure to isolate M. tuberculosis mutants resistant to various weak acids is also in keeping with this proposition (Zhang et al., 2003b). The susceptibility of M. tuberculosis to weak acids may be a result of its inefficient ability to maintain membrane potential compared with M. smegmatis. The observation that various weak acids appeared to preferentially disrupt the membrane potential of M. tuberculosis over that of M. smegmatis (Fig.3 A) supports this notion. This differential disruption of membrane potential in M. tuberculosis by the weak acids could result from the slow metabolism and consequently slow energy production in the slow growing M. tuberculosis and a defective efflux mechanism as shown for weak acid pyrazinoic acid, the active component of TB drug pyrazinamide (Zhang et al., 1999).

Weak acids or their precursors in appropriate concentrations can be used alone to treat or prevent mycobacterial infections or prevent the reactivation of mycobacterial infections in human patients or in animals. Weak acids or their precursors can be used to treat or prevent mycobacterial infections in conjunction with antimycobacterial agents including isoniazid, rifampin, pyrazinamide and ethambutol, or immunomodulators such as cytokines. Alternatively, weak acids or their precursors can be used to eliminate residual small number of mycobacteria following the treatment with conventional TB drugs isoniazid, rifampin, pyrazinamide and ethambutol for improved therapy of TB in the form of shortening the treatment of TB and preventing the reactivation of TB. Since persistent or dormant mycobacteria have low metabolism and thus low energy reserve, and since weak acids such as pyrazinoic acid can interfere with nutrient uptake and energy metabolism (Zhang et al, 2003a), weak acids can be used to inhibit or kill persistent or dormant mycobacteria and shorten the treatment of TB and prevent the reactivation of TB due to residual small numbers of bacteria not killed by current TB drugs such as isoniazid, rifampin, pyrazinamide and ethambutol.

Weak acids or their precursors can be used topically to treat or prevent mycobacterial skin infections either alone or in conjunction with antimycobacterial agents including isoniazid, rifampin, pyrazinamide and ethambutol or with immunomodulators such as cytokines.

Weak acids or their precursors can be used internally to treat or prevent mycobacterial infections in conjunction with antimycobacterial agents including isoniazid, rifampin, pyrazinamide and ethambutol or immunomodulators such as cytokines.

Those skilled in the art will realize that weak acids may not be easily absorbed directly through the gastrointestinal tract or bind to serum proteins. To circumvent this potential problem, it may be necessary to make precursors of weak acids in the form of prodrugs for in vivo treatment or prevention of mycobacterial infections. The weak acid precursors will have to be hydrolyzed to active form by enzymes present in M. tuberculosis, which is known to contain a range of esterases and amidases in the genome (Cole et al., 1998). Those who are skilled in the art will recognize that the weak acid precursors can be in the form of amides or esters but are not limited to esters or amides of weak acids.

United States Patent 6,242,474 by Yamasaki et al. claim that aromatic ring derivatives can be used for treatment or prevention of almost all diseases, i.e., a long list of chronic diseases such as diabetes, hypertension, cardiovascular disorders, cerebral apoplexy, autoimmune disease, cancer, AIDS, including tuberculosis. However, no specific example is given for the activity of the aromatic ring derivatives against M. tuberculosis. In addition, aromatic ring derivatives are not necessarily weak acids, which is the subject of this invention. In contrast, our invention relates to the use of weak acids with R-COOH or weak acid precursors for the treatment of mycobacterial infections based on our discovery that M. tuberculosis are uniquely susceptible to weak acids because of its deficient ability to maintain internal pH and membrane potential. Therefore, our invention is distinct from US patent 6,242,474.

EXAMPLES

Methods

Mycobacterial Growth and Susceptibility to Acid pH by Mycobacteria

M. tuberculosis strain H37Ra was grown in 7H9 liquid medium (DIFCO) supplemented with 0.05% Tween 80 and 10% bovine serum albumin-dextrose- catalase enrichment (DIFCO) at 37°C for 3 weeks with occasional shaking. M. smegmatis mc²6 (MC2) was similarly cultivated in the 7H9 medium at 37 °C for 4 days. For testing the susceptibility of mycobacteria to different pH, M. tuberculosis H37Ra or M. smegmatis cells were resuspended in sodium phosphate buffer adjusted to different pH values (pH 3.0, 4.0, 5.0, 6.0, 7.0) in 1 ml to a cell density of 1.30 at ODδooand incubated at 37 °C. At 1 day, 3 day, 5 day, 7day, aliquots of the ce l suspension were removed, washed and diluted before plating on 7H11 plates. The plates were then incubated at 37 °C for 4 weeks for M. tuberculosis and for 5 days for M. smegmatis to determine the number of surviving bacteria.

Susceptibility to Weak Acids and Isolation of Weak Acid Resistant Mutants

Various weak acids were obtained from Sigma Chemical Co., and were dissolved in DMSO at appropriate concentrations. The weak acids were incorporated into 7H11 agar at various concentrations. Three week old stationary phase M. tuberculosis H37Ra culture or 4 day old M. smegmatis mc²6 culture were tested for susceptibility to weak acids on 7H11 plates at pH 6.8 and pH 5.5 as described (Sun and Zhang, 1999). For the isolation of weak acid mutants, about 10⁸ colony forming units (CFU) of M. tuberculosis H37Ra were plated on acidic 7H11 agar plates (pH 5.5) containing various concentrations of weak acids such as salicylate, benzoic acid, nonyloxybenzoic acid and mefenamic acid. The plates were incubated at 37 °C for 4 weeks before being examined for the emergence of spontaneous mutants.

Measurement of Intracellular pH and Membrane Potential

The internal pH of mycobacteria was measured as described previously (Rottenberg, 1979). Membrane potential was measured with [³H]tetraphenylphosphonium bromide (TPP⁺) using the method as described (Rottenberg, 1979). Briefly, 3 week old H37Ra or 4 day old M. smegmatis cells were resuspended in Sauton's medium at different pHto measure the change of the membrane potential in response to changes in external pH after incubating the cells at room temperature for 50 min. [³H]TPP⁺ (380 mCi/mmol) at 10 μM final concentration was then added to the cell suspension and the mixture was fully mixed before silicone oil was added and the mixture was incubated another 10 min. The mixture was spun at 12,000 rpm for 3 min, and 100 μl supernatant were taken for scintillation counting. The cell pellets were then snap-frozen in alcohol dry ice bath. The bottom of the tubes containing cell pellet were cut off for scintillation counting. To determine the effect of weak acids on membrane potential and internal pH, various weak acids were incubated with mycobacterial cells resuspended in pH5.5 Sauton's medium for 1 hr when the measurements were made as described above. Valinomycin (10 μM) and nigericin (10 μM) were used as controls for the membrane potential and internal pH measurement.

EXAMPLE 1

Susceptibility of M. tuberculosis and M. smegmatis to Acid pH. The acid sensitivity of M. tuberculosis and M. smegmatis was determined by exposing the bacilli to various acidic pH conditions using pH 7.0 as a control, and plated for survivors after the exposure for different times. The relative sensitivity of the two mycobacterial species to acidic pH was expressed as the percentage of bacterial survival by dividing the CFU obtained after exposure to acid pH over that at neutral pH. At pH 3.0, there was relatively little difference between M. tuberculosis andM smegmatis in terms of survival due to extreme acidity (Table 1). However, at pH 4.0 and 5.0, M. tuberculosis was significantly more sensitive to acid pH than M. smegmatis (Table 1).

Table 1. Comparison of relative acid sensitivity of M. tuberculosis and M. smegmatis. M. tuberculosis H37Ra and M. smegmatis MC2 were exposed to different acid pH conditions and pH 7.0. The percentage of surviving bacteria on day 7 was calculated by dividing the CFU at acidic pH conditions pH 3, 4, and 5 over that of the control at pH 7.

EXAMPLE 2

Susceptibility of M. tuberculosis to Weak Acids.

As shown in Table 2, M. tuberculosis was more susceptible than M. smegmatis to a range of weak acids. The antimycobacterial activity of the weak acids was more pronounced at acid pH than at close to neutral pH for both mycobacterial species. In addition, the activity of the weak acids appeared to correlate with their pKa values, i.e., the lower the pKa the higher the antimycobacterial activity (Table 2). It is noteworthy that M. tuberculosis was susceptible to linoleic acid (MIC = 37 mg/L at pH 5.5) but not to linoleic acid ethyl ester (MIC > 1000 mg/L at pH 5.5), indicating that the acid form COOH is active and that M. tuberculosis does not have an appropriate esterase to convert linoleic acid ethyl ester to the active acid form.

Table 2. MICs of M. tuberculosis H37Ra and M. smegmatis to weak acids (mg/L)

Weak acids H37Ra M. smegmatis pKa pH 6.8 pH 5.5 pH 6.8 pH 5.5 values*

BA 111 37 >333 >111

4.2

SA 50-100 10-20 1000 333

3.0

Asp 111 37 1000 333

3.49

MFA 33 11 1000 333

4.2

NA >500 200 >1000 >500

4.85

INA >1000 500 >1000 1000

4.96

NBA 33 11 >1000 333

NA

OBA 33 3.7 333 111

NA

BFCA 33 11 1000 111

NA

DBA 33 11 >1000 >1000

NA

PDA 33 11 333 111

NA LOA 111 37 >1000 1000

NA LAE >1000 >1000 >1000 >1000

NA * pKa values are from The Merck Index (Budavari, 1989). BA, benzoic acid; SA, salicylic acid; Asp, aspirin (acetyl-salicylic acid); MFA, mefenamic acid; NA, nicotinic acid; INA, isonicotinic acid; NBA, 4-nonyloxybenzoic acid; OBA, 4- octylbenzoic acid; BFCA, benzofurancarboxylic acid; DBA, 4-dodecyloxylbenzoic acid; PDA, 11-phenoxyundecanoic acid; LOA, linoleic acid; LAE, linoleic acid ethyl ester. NA, not available.

EXAMPLE 3

Inability to Isolate Weak Acid Resistant Mutants of M. tuberculosis.

We have shown previously that no pyrazinoic acid-resistant mutants of M. tuberculosis could be isolated (Scorpio et al., 1997). To determine if this is a more generalized phenomenon, we attempted to isolate M. tuberculosis H37Ra mutants resistant to a range of weak acids such as salicylic acid, benzoic acid and 4- nonyloxybenzoic acid. However, we were unable to isolate any mutants resistant to the weak acids even at very high density of cells (10⁹ CFU/ml) on 7H11 plates.

EXAMPLE 4

Inefficient Maintenance of Intracellular pH in M. tuberculosis.

We compared the intracellular pH of M. tuberculosis (Fig. 1 A) and M. smegmatis (Fig. IB) in response to changes in external pH. Between pH 5 and pH 7, the two organisms behaved similarly in terms of changes in internal pH. However, at more acidic conditions (pH 3-5), the internal pH of M. smegmatis remained fairly stable at values of 5.7-5.9 (Fig. IB); in contrast, the internal pH of M. tuberculosis became more acidic, reaching 5.2 at outside pH of 3.2 (Fig. 1A). This indicates that M. tuberculosis is less efficient at maintaining the internal pH than M. smegmatis. In addition, valinomycin and nigericin had a more pronounced effect on lowering the internal pH of M. smegmatis but had little effect on M. tuberculosis (Fig. IB, 1C). This finding lends further support to the idea that M. smegmatis has a more active apparatus to maintain its internal pH at acid pH conditions (pH 3-5) than M. tuberculosis.

EXAMPLE 5

Inefficient Maintenance of Membrane Potential in tuberculosis.

We compared the membrane potential of M. tuberculosis and M. smegmatis in response to changes in external pH. The membrane potential of M. tuberculosis was generally higher than that of M. smegmatis except at very acidic pH of 3.5, at which there was little difference in the membrane potential between M. tuberculosis and M. smegmatis. However, the membrane potential of M. tuberculosis was more sensitive to changes in external pH than M. smegmatis between pH 4 and pH 8.5 (Fig.2). The more responsive change in the membrane potential of M. tuberculosis compared with M. smegmatis is most likely due to a poor ability of M. tuberculosis to maintain its membrane potential at different external pH conditions.

EXAMPLE 6

Correlation between Activity of Weak Acids and Their Ability to Disrupt Membrane Potential or Lower Internal pH.

The susceptibility of M. tuberculosis and M. smegmatis to weak acids was examined in the context of membrane potential and internal pH. It was found that the susceptibility of M. tuberculosis to weak acids appeared to correlate with their ability to disrupt membrane potential (Fig. 3). In contrast, weak acids had little effect on the disruption of membrane potential in the nonsusceptible species M. smegmatis (Fig. 3A). The antituberculous activity of the weak acids did not correlate well with their ability to decrease the internal pH (Fig. 3B). REFERENCES

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It will be apparent to those skilled in the art that the examples and embodiments described herein are by way of illustration and not of limitation, and that other examples may be used without departing from the spirit and scope of the present invention, as set forth in the appended claims.

Claims

ClaimsWhat is claimed is:

1. A method of preventing or treating a mycobacterial infection comprising administering to a mammal an effective amount of at least one compound of weak acids with formula R-COOH,

wherein,

R sub 1 is typically from the group comprising aromatic or benzene ring, including but not limited to benzoic acid, salicylic acid, aspirin (acetyl-salicylic acid), mefenamic acid (2-[2,3-dimethylρhenyl]amino-benzoic acid), nicotinic acid, 4- nonyloxybenzoic acid, 4-octylbenzoic acid, octylloxybenzoic acid, nitrobezoic acid, hexylbenzoic acid, heptylbenzoic acid, benzofurancarboxylic acid, 4- dodecyloxylbenzoic acid, 11-phenoxyundecanoic acid; undecyloxybenzoic acid, cyclohexenyloxybenzoic acid,

R sub2 is fatty acyl chain with chain length of C4-C17.

2. The method of claim 1, wherein the weak acids can be in the form of weak acid precursors as in ester, amide, hydrazide or salt of weak acids.

3. The method of claim 1, wherein the weak acids typically have dissociation constant pKa values between 2 and 5.

4. The method of claim 1, wherein the antimycobacterial activity of the weak acids could inversely correlate with the pKa value of the weak acids, i.e., the lower the pKa the higher the antimycobacterial activity of the weak acids.

5. The method of claim 1, wherein the mycobacterial infection is from the group consisting of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium avium, Mycobacterium scrofulaceum, Mycobacterium gordonae, Mycobacterium kansaii, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium paratuberculosis, Mycobacterium ulcerans, and Mycobacterium xenopi.

6. The method of claim 1, wherein the mammal is patients or animals.

7. The method of claim 1, wherein the weak acids or their precursors are used to treat or prevent mycobacterial infections in conjunction with antimycobacterial agents including isoniazid, rifampin, pyrazinamide and ethambutol.

8. The method of claim 6, wherein the weak acids or their precursors are used topically to treat or prevent mycobacterial infections in conjunction with antimycobacterial agents including isoniazid, rifampin, pyrazinamide and ethambutol.

9. The method of claim 6, wherein the weak acids or their precursors are used internally to treat or prevent mycobacterial infections in conjunction with antimycobacterial agents including isoniazid, rifampin, pyrazinamide and ethambutol.

10. The method of claim 6, wherein the weak acids or their precursors are used internally to treat or prevent mycobacterial infections following the antimycobacterial agents including isoniazid, rifampin, pyrazinamide and ethambutol, to eliminate residual persisting bacteria not killed by conventional antimycobacterial agents.

11. The method of claim 6, wherein the weak acids or their precursors can be used to prevent or treat drug-resistant mycobacterial infections alone or in conjunction with antimycobacterial agents including isoniazid, rifampin, pyrazinamide and ethambutol.

12. The method of developing antimycobacterial agents based on unique physiology of M. tuberculosis.

13. The method of Claim 11, wherein the unique physiology is deficient ability of M. tuberculosis to maintain internal pH.

14. The method of Claim 11, wherein the unique physiology is deficient ability of M. tuberculosis to maintain membrane potential.

15. The method of Claim 11, wherein the unique physiology is deficient ability of M. tuberculosis to remove weak acids from the bacterial cell.