Design and synthesis of antituberculars: preparation and evaluation against Mycobacterium tuberculosis of an isoniazid Schiff base

Michael J. Hearn1,* and Michael H. Cynamon2,3

1 Department of Chemistry, Wellesley College, 106 Central Street, Wellesley, MA 02481; 2 Department of Microbiology and Immunology, Upstate Medical University, State University of New York, Syracuse, New York; 3 Department of Medicine, Veterans Affairs Medical Center, Syracuse, New York, USA

Received 22 July 2003; returned 1 October 2003; revised 8 October 2003; accepted 29 October 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: Enzymatic acetylation of the antitubercular isoniazid (INH) by N-acetyltransferase represents a major metabolic pathway for INH in human beings. Acetylation greatly reduces the therapeutic activity of the drug, resulting in underdosing, decreased bioavailability and acquired INH resistance. Chemical modification of INH with a functional group that blocks acetylation, while maintaining strong antimycobacterial action, may improve clinical outcomes and help reduce the rise of INH resistance. The goal of this study was to probe activities, toxicity and bioavailability of an investigational compound prepared by this chemical modification.

Methods: The investigational compound was chosen from a cohort of lipophilic antitubercular INH Schiff bases based on its strong activity in primary assays. The compound was evaluated in vitro, in vivo in mice, in mutagenicity tests and in rats for bioavailability.

Results: The INH Schiff base acts against both intracellular and extracellular organisms in vitro, with a wide range between active and cytotoxic concentrations. The material is active against non-tubercular mycobacteria. The INH Schiff base is non-mutagenic in the Ames test and has excellent bioavailability in Sprague–Dawley rats, achieving early peak plasma concentrations approximately three orders of magnitude above its MIC when administered orally. In tuberculosis-infected mice the compound is well tolerated and in a 4 week study provides 3 log cfu reduction in spleens and 4 log cfu reduction in lungs.

Conclusion: The results demonstrate that investigational compounds in which N-acetylation of INH is blocked by chemical modification can display strong activity, low toxicity and excellent bioavailability, making them suitable for further exploration.

Keywords: N-acetyltransferase, fast acetylators, bioavailability, acquired resistance


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The designation of tuberculosis as a global public health crisis by the World Health Organization in the mid-1990s1 has underscored the severe challenges facing the antimicrobial research community.2 The occurrence of some three million new cases of tuberculosis per year worldwide and the emergence of new strains of Mycobacterium tuberculosis characterized by drug resistance or increased virulence have made clear the pressing need for the evolution of newer and more powerful drugs, the re-examination and re-evaluation of older drugs and the detailed elucidation of the mechanisms of action of antimycobacterial compounds.37 Because of the lack of activity in the area of new drug development extending over a period of approximately 35 years, there has been a significant gap in the research community’s knowledge about the design of antimycobacterial agents.810 Today, it is clear that vigorous and widespread research on drug design will be necessary to recapture lost ground and to make new advances.11 This process has been greatly fostered by the determination of the genome sequence of M. tuberculosis12 and by the publication of a scientific blueprint for drug discovery.13 To facilitate the development of new antimycobacterials, the research community has recently begun to place strong emphasis on the identification of the targets of effective drugs, such as isoniazid,1419 and on those characteristics of pathogen cell wall structure that play a role in limiting the effectiveness of antimycobacterials.2022

The important relationship between serum levels of isoniazid and the emergence of isoniazid-resistant cultures in patients with pulmonary tuberculosis became evident in careful studies done some time ago.23 These studies stressed the importance of early intensive chemotherapy, with an emphasis on initial peak serum concentrations of isoniazid (INH), rather than on concentrations measured three or more hours after the dose. Further, the improved therapeutic outcome of certain treatment regimens was attributed to the higher early peak serum concentrations of isoniazid that they permitted, rather than to the period that minimum inhibitory concentrations of isoniazid were maintained in the serum.24 Serum concentrations are influenced by a number of factors, but among the most important of these is the enzymic acetylation of isoniazid by N-acetyltransferase (NAT). This represents a major metabolic pathway for isoniazid in human beings.

Acetylation greatly reduces the therapeutic activity of the drug. It is thought that the resultant chemical modification prevents the activation of INH that is required for proper drug action. The metabolism of isoniazid is under genetic control, and the human population may be divided into two large groups depending on the rate at which they metabolize isoniazid. ‘Rapid acetylators,’ in whom this process is efficient, may be subject to deleterious effects that detract from what would otherwise be an effective therapeutic regimen. Among these are chronic underdosing, significant deficits in drug bioavailability25 and the consequent possibility of acquired INH resistance.26 The somewhat more favourable therapeutic response of slow acetylators of isoniazid is also seen as being the result of the higher peak serum concentrations of the drug among this group of patients.24 The high prevalence of tuberculosis among certain populations is said to be partially explained by their high proportions of NAT fast alleles.27 Among such populations, the incidence of acquired resistance remains high.28

NAT is also present in mycobacterial pathogens. It was recently found that recombinant NAT from M. tuberculosis acetylates isoniazid in vitro. When the corresponding nat gene is overexpressed in a suitable isoniazid-susceptible host, Mycobacterium smegmatis, the resultant organism becomes more resistant to INH. Resistance to INH in mycobacteria can thus be related to increased expression of NAT.29

Chemical modification of the hydrazine unit of isoniazid with a functional group that blocks acetylation, while maintaining strong antimycobacterial action, has the potential to improve clinical outcomes and reduce the emergence in patients of acquired isoniazid resistance. The goal of our study was to investigate such chemical modification. Given the complexity of the discovery process, any pilot drug derived from such chemical modification must show, at an early stage of exploration, strong activity in vitro and in vivo, low toxicity and good bioavailability.

In addressing the issue of activity, recent data about drug targets, cell wall structure and host–pathogen interactions provide opportunities for rational drug design strategies focused on drug lipophilicity. For example, the very low permeability of the mycobacterial cell wall toward antimicrobial agents is probably one substantive reason why many such agents are ineffective against mycobacteria. Increases in lipophilic character, however, result in changes in pathways of diffusion across the cell wall, enhancing the contribution of diffusion through the lipid domain.30 Thus increasing the lipophilicity of an antimycobacterial agent enhances its efficacy.20,3133 Isoniazid, for example, is itself hydrophilic and is expected to use predominantly the porin pathway34 to enter mycobacteria.35 However, it has been shown that the activity of INH can be enhanced against non-tuberculous mycobacteria by the addition of long hydrocarbon chains, a result emphasizing the relative importance of the lipid pathway for diffusion. Further, if the barrier properties of mycobacteria are dependent on the organization within the cell wall of mycolic acid residues, then drugs that inhibit mycolate biosynthesis may eventually increase the permeability of the pathogen cell wall and achieve even more efficacy by a ‘snowball’ effect.36 Considering effects on the host, there is evidence that some lipophilic investigational compounds, structurally related to INH, gain their activity as the result of suppression of xenobiotic transformation.3739 These materials obtain noteworthy concentration in several organs of experimental animals, most significantly in caseation bodies.40 Some of these materials also possess one of the most important design characteristics of antimycobacterials, namely, that toxicity levels should be low enough to permit administration over a period long enough to ensure a durable cure.4153

In considering the factors discussed above, and as part of our on-going studies on the chemical synthesis and biological properties of antituberculars,5456 we have selected for closer evaluation the lipophilic Schiff base N2-cyclohexylidenyl isonicotinic acid hydrazide (Figure 1), in which the hydrazine moiety is blocked toward acetylation. The compound was chosen from a much larger set of highly active isoniazid Schiff bases, a few of which we have previously described,57 because of its strong activity in primary assays in vitro. We now report our preliminary results on this investigational compound in the areas of synthesis, activity, toxicity and bioavailability.



View larger version (8K):
[in this window]
[in a new window]
 
Figure 1. Chemical structure of INH Schiff base.

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Chemical synthesis

Elemental analyses were carried out by Galbraith Laboratories, Knoxville, TN, USA. Melting points (m.p.; °C) were taken in open capillary tubes using a Mel-Temp apparatus (Laboratory Devices, Cambridge, MA, USA), and are corrected. Infrared (IR) spectra were recorded on a Perkin-Elmer Model 1600 Fourier transform spectrophotometer as Nujol mulls or on a Perkin-Elmer Spectrum One Fourier transform spectrophotometer fitted with a universal attenuated total reflectance sampling accessory and are reported in wavenumbers ({nu}, cm–1). Reactants and reagents were obtained from Aldrich Chemical Company (Milwaukee, WI, USA) and Lancaster Synthesis Incorporated (Windham, NH, USA) and were used as received. Proton nuclear magnetic resonance (NMR) spectra were taken on Bruker 200 or 300 Fourier transform instruments in dimethyl sulphoxide-d6 and are reported in parts per million delta ({delta}) downfield from internal tetramethylsilane as reference, with coupling constants measured in cycles per second (c.p.s.). High resolution mass spectra (HR-MS) and low resolution mass spectra were determined at the National Institutes of Health Mass Spectrometry Facility at Michigan State University, East Lansing, MI, USA.

Safety Notes: Gloves were worn during the chemical synthesis, and the reactions were carried out in the hood. In general, any scale-up of preparations of compounds with relatively high proportions of nitrogen was done with due caution. No specific safety problems were encountered by us with the methods given below.

The method of synthesis of the INH Schiff base from isoniazid and cyclohexanone has been described in detail.57 In brief, the INH Schiff base was prepared by a method specifically designed to form the material reliably as a dry free-flowing white solid in analytically pure form, characterized as follows: yield 81%; m.p. 167–168°C; IR {nu}max 3212, 1662, 1637, 1597, 1528, 1406, 1302, 1285, 1245, 1214, 1139, 1036, 839, 755, 722; NMR (300 MHz) {delta} 10.8 (1H, broad singlet), 8.7 (2H, d, J = 6 cps), 7.6 (2H, d, J = 6 cps), 2.4 (4H, m), 1.6 (6H, m); soluble in ethanol, pyridine and dimethyl sulphoxide at room temperature; HR-MS (fast atom bombardment, MH+) calculated for C12H16ON3 218.1293, found 218.1294.

Analysis. Calculated for C12H15ON3: C, 66.32; H, 6.97. Found: 66.18; H, 6.87.

No attempt was made to optimize the chemical yield. The INH Schiff base was stable on the shelf at room temperature for prolonged periods of time when not stored in direct light. After standing overnight in 0.4 M HCl at room temperature, the INH Schiff base appeared not to suffer serious decomposition; following such treatment and re-isolation, its infrared spectrum was identical to that of an authentic specimen. The calculated value of log P for the INH Schiff base is 0.834; for isoniazid the calculated value of log P is –0.887.58

Drugs

For biological evaluations, INH was purchased from Sigma Chemical Company (St. Louis, MO, USA). For testing, the INH Schiff base was dissolved in dimethyl sulphoxide and subsequently diluted in distilled water. INH was dissolved in distilled water. Stock solutions were filter-sterilized by passage through a 0.22 µm-pore-size membrane filter and stored at –20°C until use. The drugs were prepared each morning, before administration.

Isolate

M. tuberculosis ATCC 35801 (strain Erdman) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). With respect to testing against this isolate, the MICs of all antimicrobial agents were determined in modified 7H10 broth (7H10 agar formulation with agar and malachite green omitted; pH 6.6) supplemented with 10% Middlebrook oleic acid-albumin-dextrose-catalase (OADC) enrichment (Difco Laboratories, Detroit, MI, USA) and 0.05% Tween 80.59 The MICs of the antimicrobial agents were determined by a broth dilution method.60

Medium

The organism was grown in modified 7H10 broth with 10% OADC enrichment and 0.05% Tween 80 on a rotary shaker at 37°C for 5 days. The culture suspension was diluted in modified 7H10 broth to yield 100 Klett units/mL (Photoelectric Colorimeter, Manostat Corporation, New York, NY, USA) or approximately 5 x 107 cfu/mL. The size of the inoculum was determined by titration and counting from triplicate 7H10 agar plates (BBL Microbiology Systems, Cockeysville, MD, USA) supplemented with 10% OADC enrichment. The plates were incubated at 37°C in ambient air for 4 weeks before counting of the colonies.

Infection study

Four-week-old female outbred CD-1 mice (Charles River, Wilmington, MA, USA) were infected intravenously through a caudal vein. Each mouse received approximately 107 viable organisms suspended in 0.2 mL of modified 7H10 broth. There were eight mice per group.

Treatment began 1 week after infection. Therapy was given 5 days per week for 4 weeks. The agents were administered by gavage: the INH Schiff base and INH were dosed at 25 mg/kg of body weight. Control groups of infected but untreated mice were killed at the initiation of therapy (early controls) or at the end of the treatment period (late controls). Mice were euthanized by CO2 inhalation. The spleens and right lungs were aseptically removed and were ground in a tissue homogenizer (IdeaWorks! Laboratory Devices, Syracuse, NY, USA). The number of viable organisms was determined by titration on 7H10 agar plates. The plates were incubated at 37°C in ambient air for 4 weeks before counting of the colonies. The use of animals complied with institutional policies and federal guidelines.

Results, as specified in the Tables, in vitro and in vivo in mice were also determined according to the protocols of the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF), which have been fully documented.61

Evaluation of the INH Schiff base in the Salmonella–Escherichia coli microsome plate incorporation assay

The INH Schiff base was examined for mutagenic activity in the Salmonella–Escherichia coli microsome plate incorporation assay by Stanford Research Institute International (Menlo Park, CA, USA).62 In brief, the assay was carried out using the standard plate incorporation procedure with Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and with E. coli strain WP2 (uvrA) in both the presence and absence of an Aroclor 1254-induced rat-liver metabolic activation system. Based on the results of the range-finding experiment where doses ranged from 156.2 to 5000 µg/plate, the experiments for mutagenicity were conducted with all five tester strains at dose levels of 312.5, 625, 1250, 2500 and 5000 µg/plate (50 µL). This was done in the presence and absence of metabolic activation containing 5% S-9 for the first experiment and 10% S-9 for the second experiment, where S-9 is the activation system, derived from rat liver homogenate, that is used to simulate mammalian liver enzyme systems and that promotes detection of substances which undergo metabolic activation from non-mutagenic forms. The results with the INH Schiff base showed a slight increase in the number of revertants with strains TA 1535 and TA 1537; however, they were minmal (two-fold or less) and not considered to be attributed to mutagenic activity. Thus the INH Schiff base was judged to be non-mutagenic under the test conditions used in this study and negative in the bacterial reverse mutation assay. A full data set for this assay is available from the corresponding author upon request.

Bioavailability of the INH Schiff base in rats

The INH Schiff base was examined for its bioavailability in rats by Stanford Research Institute International (Menlo Park, CA, USA).63 In brief, the compound was administered to male Sprague–Dawley rats by both the oral and intravenous (iv) routes, plasma was collected and the concentration of the compound was determined. Six male rats were administered 20 mg/kg of the INH Schiff base in 0.9% sodium chloride (2 mL/kg) by iv injection. Another group of six male rats received 100 mg/kg of the INH Schiff base by oral gavage (10 mL/kg). Blood samples were collected after dose administration and processed to plasma. The concentration of the INH Schiff base was measured by using high pressure liquid chromatography with ultraviolet detection at 265 nm. No adverse effects were observed in the animals in either the iv or oral treatment groups. Analysis of the plasma indicated that the drug was cleared rapidly from the plasma, with the drug concentration below the detection limit by 2 and 6 h after iv and oral administration, respectively. The elimination half-life was estimated to be less than 1 h after iv treatment and less than 2 h after oral administration in the animals.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The INH Schiff base was prepared in analytical purity and good yield as a tractable solid. The material was stable on the shelf when not stored in direct sunlight and did not deteriorate significantly after standing in 0.4 M HCl at room temperature for up to 24 h. The calculated value of log P for the INH Schiff base (0.834) is greater than that of isoniazid (–0.887), consistent with the greater solubility observed for the INH Schiff base in a number of organic solvents.

In assays in vitro (Table 1), the INH Schiff base displayed good activity against M. tuberculosis strains Erdman and H37Rv with MICs of 0.03 mg/L. On a molar basis, the Schiff base was thus slightly more active than isoniazid.


View this table:
[in this window]
[in a new window]
 
Table 1. In vitro activities of INH Schiff base
 
The INH Schiff base was evaluated for cytotoxicity in VERO cells at concentrations 10 times the MIC for M. tuberculosis H37Rv.61 After 72 h exposure, viability was assessed on the basis of cellular conversion of MTT into a formazan product using the Promega CellTiter 96 Non-radioactive Cell Proliferation Assay. For the INH Schiff base, the IC50 was >1000 mg/L. The ratio of IC50 to MIC was >40 000, giving a good range between active and cytotoxic concentrations.

Efficacy in vitro was further assayed in a tuberculosis-infected macrophage model61 for killing of M. tuberculosis strain Erdman (ATCC 35801) in monolayers of mouse bone marrow macrophages. In Table 1, EC90 and EC99 represent the lowest concentrations effecting 90% and 99% reduction in colony-forming units at 7 days (compared to drug-free controls) at four-fold concentrations equivalent to 0.25, 1, 4 and 16 times the MIC. In this model, the EC90 is said to represent bacteriostatic activity, and the EC99 indicates bactericidal activity. The EC90 denotes the effective concentration to give a 90% reduction in intramacrophage bacteria relative to a drug-free control. Since this concentration maintains roughly the level of the initial inoculum, the value estimates the necessary amount of drug to maintain a static level of bacteria within the macrophage host. The EC99 value is thought to represent a further reduction in one log and thus gives a measure of the bactericidal activity of the drug. The ratio EC90/MIC provides a measure of bioavailability and metabolism of the active agent within the living host cell, since it compares the in vitro activity against the bacillus to the activity against the bacillus while it lives within the host. For isoniazid, the EC90/MIC ratio is unity. For the INH Schiff base, the ratio of EC90/MIC of 2.7 indicates effective reduction in residual mycobacterial growth, a conclusion considered justified whenever the ratio is less than 16, the activity criterion for this assay. Considered in light of the results from the broth culture assays, the data indicate that the INH Schiff base is active against extracellular and intracellular M. tuberculosis.64 Activity was maintained in rifampicin-resistant organisms (MIC 0.05 mg/L) and in ethambutol-resistant organisms (MIC 0.05 mg/L), but not in those already resistant to isoniazid (MIC > 0.75 mg/L). The compound also demonstrated good activity against the non-tuberculous mycobacteria, Mycobacterium avium and Mycobacterium kansasii. The INH Schiff base was determined to be non-mutagenic in the Salmonella–Escherichia coli microsome plate incorporation assay under the test conditions used in this study and negative in the bacterial reverse mutation assay. By comparison, an examination of the genetic effects of isoniazid and their relationship to biotransformation has reported that isoniazid has a weak direct mutagenicity but does serve as a promutagen.65

In previous studies in vivo, the maximally tolerated dose was determined to be 1000 mg/kg, using C57BL/6 in female mice.61 After administration of a one-time dose intraperitoneally, the animals (n = 3) were observed for 1 week. Following sacrifice, organs were examined for signs of overt toxicity. No ill effects or overt toxicity were noted. By comparison, in a recent evaluation the intraperitoneal dosing of isoniazid indicated an LD50 of 151 mg/kg.51 In a 4-week study using female outbred CD-1 mice dosed at 25 mg/kg, the INH Schiff base had strong activity in both organs examined, that is, 3 log cfu reduction in spleens and 4 log cfu reduction in lungs (Table 2).


View this table:
[in this window]
[in a new window]
 
Table 2. In vivo activities of INH Schiff basea
 
An estimate was made of the bioavailability of the INH Schiff base in Sprague–Dawley rats (Table 3, Figure 2).63,66 No adverse effects in the animals were observed following iv administration (20 mg/kg) or oral administration (100 mg/kg). Analysis of the plasma indicated that the drug was cleared rapidly, with the drug concentration below the detection limit by 6 h after oral administration. The elimination half-life was calculated to be less than 2 h after oral dosing. Mean peak plasma concentration occurred between 1 and 2 h after oral administration and was determined to be 24 mg/L. It was noteworthy that the chemical entity for which these plasma concentrations were determined was in fact the Schiff base, and not isoniazid.67 It will be important to understand the ultimate metabolic fate of the INH Schiff base, and future studies will be directed toward this end.


View this table:
[in this window]
[in a new window]
 
Table 3. Plasma concentration of Schiff base in rats after administration iv and oral
 


View larger version (14K):
[in this window]
[in a new window]
 
Figure 2. Mean plasma concentrations of INH Schiff base in Sprague–Dawley rats. Filled circles, iv; filled squares, oral administration.

 
In conclusion, we have shown that an active INH Schiff base may be prepared conveniently for evaluation against mycobacteria in good yield and analytical purity using inexpensive reactants. The structure represents a lipophilic modification of isoniazid in which the hydrazine moiety has been chemically blocked from the de-activating process of enzymic N2-acetylation. In experiments against M. tuberculosis the compound has shown significant activity in vitro and in vivo. In vitro, the INH Schiff base acts against both intracellular and extracellular organisms with a wide range between active and cytotoxic concentrations. The compound was found to be non-mutagenic and has excellent bioavailability in Sprague–Dawley rats, achieving early peak plasma concentrations approximately three orders of magnitude above its MIC. In tuberculosis-infected mice, the compound is well tolerated and provides a 3 log cfu reduction in spleens and 4 log cfu reduction in lungs. Since the compound has demonstrated, at an early stage of exploration, considerable activity in vitro and in vivo, low toxicity and good bioavailability, we are continuing our investigations of this material and its closely related structural congeners.


    Acknowledgements
 
We thank Sharon E. Chase for carrying out in vivo experiments. We gratefully acknowledge the staff of the TAACF, coordinated by the Southern Research Institute, Birmingham, Alabama, under a research and development contract with the National Institute of Allergy and Infectious Diseases (NIAID) of the U.S. National Institutes of Health (NIH). For valuable discussions, we thank Dr Barbara E. Laughon, Chief, and Dr Chris Lambros, of the Complications and Co-infections Research Branch; and Dr Charles L. Litterst, Chief, and Dr Steven Turk, Drug Development and Surveillance Section; all of the Division of AIDS, NIAID. Mass spectra were determined at the National Institutes of Health Mass Spectrometry Facility at Michigan State University, East Lansing, Michigan. This work was supported by the Global Alliance for Tuberculosis Drug Development and by grant 1 R15 AI48397-01 from the Division of AIDS, NIAID, NIH.


    Footnotes
 
* Corresponding author. Tel: +1-781-283-3036; Fax: +1-781-283-3642; E-mail: MHearn{at}Wellesley.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . World Health Organization. (1994). TB: A Global Emergency. WHO Report No. 14977. WHO, Geneva, Switzerland.

2 . Dye, C. (2000). Tuberculosis 2000–2010: control, but not elimination. International Journal of Tuberculosis and Lung Disease 4, S146–52.[ISI][Medline]

3 . ACET. (1989). Recommendations from advisory committee for the elimination of tuberculosis. Morbidity and Mortality Weekly Report 38, 236–50.[Medline]

4 . American Society for Microbiology. (1995). Report of the ASM task force on antibiotic resistance. Antimicrobial Agents and Chemotherapy. 39, Suppl. 1, 1–23.[Free Full Text]

5 . Davies, J. (1998). In Genetics and Tuberculosis, Novartis Foundation Symposia (Chadwick, D. J., Ed.), Vol. 217, pp. 195–208. John Wiley and Sons, Limited, Chichester, UK.

6 . Earnest, M. & Sbarbaro, J. (1993). A plague returns: TB is back. The Sciences 33, 14ff.

7 . Potera, C. (1998). Several developments in efforts to subdue tuberculosis. American Society for Microbiology News 64, 555,557.

8 . Hearn, M. (1997). Tuberculosis today: chemical perspectives on the resurgence of the white plague. The Nucleus LXXV, 7,8,10,11.

9 . Peloquin, C. (1993). Pharmacology of the antimycobacterial drugs. Medical Clinics of North America 77, 1253–62.[ISI][Medline]

10 . Peloquin, C. (1998). Serum concentrations of the antimycobacterial drugs. Chest 113, 1154.

11 . Gangadharam, P. (1997). In Mycobacteria: Chemotherapy (Gangadharam, P. & Jenkins, P., Eds), Vol. II, pp. 345–6. Chapman and Hall, New York, NY, USA.

12 . Cole, S., Brosch, R., Parkhill, J. et al. (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–44.[CrossRef][ISI][Medline]

13 . The Global Alliance for Tuberculosis Drug Development. (2000). Scientific Blueprint for TB Drug Development. [Online.] http://www.tballiance.org/pdf/TB%20Scientific%20Blueprint%20Sum.pdf (18 July 2003, last date accessed).

14 . Barry, C., Lee, R., Mduli, K. et al. (1998). Mycolic acids: structure, biosynthesis and physiological functions. Progress in Lipid Research 37, 143–79.[CrossRef][ISI][Medline]

15 . Mdluli, K., Sherman, D., Hickey, M. et al. (1996). Biochemical and genetic data suggest that InhA is not the primary target for activated isoniazid in Mycobacterium tuberculosis. Journal of Infectious Diseases 174, 1085–90.[ISI][Medline]

16 . Mdluli, K., Slayden, R., Zhu, Y. et al. (1998). Inhibition of a Mycobacterium tuberculosis ß-ketoacyl ACP synthase by isoniazid. Science 280, 1607–10.[Abstract/Free Full Text]

17 . Miesel, L., Rozwarski, D., Sacchettini, J. et al. (1998). In Genetics and Tuberculosis, Novartis Foundation Symposia (Chadwick, D. J., Ed.), Vol. 217, pp. 209–27, John Wiley and Sons, Limited, Chichester, UK.

18 . Rozwarski, D., Grant, G., Barton, D. et al. (1998). Modification of the NADH of the isoniazid target (InhA) from Mycobacterium tuberculosis. Science 279, 98–102.[Abstract/Free Full Text]

19 . Rozwarski, D., Vilcheze, C., Sugantino, M. et al. (1999). Crystal structure of the Mycobacterium tuberculosis enoyl-ACP reductase, InhA, in complex with NAD+ and a C16 fatty acyl substrate. Journal of Biological Chemistry 274, 15582–9.[Abstract/Free Full Text]

20 . Billington, D., Coleman, M., Ibiabuo, J. et al. (1998). Synthesis and antimycobacterial activity of some heteroarylcarboxamidrazone derivatives. Drug Design and Discovery 15, 269–75.[Medline]

21 . Setlow, P. (1995). Survival of dormant spores of Bacillus species for years and years and.. How do they do it? The Nucleus LXXIII, 5.

22 . Wallis, R., Patil, S., Cheon, S. et al. (1999). Drug tolerance in Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 43, 2600–6.[Abstract/Free Full Text]

23 . Selkon, J., Devadatta, S., Kulkarni, K. et al. (1964). The emergence of isoniazid-resistant cultures in patients with pulmonary tuberculosis during treatment with isoniazid alone or isoniazid plus PAS. Bulletin of the World Health Organization 31, 273–94.[ISI]

24 . Gangadharam, P., Devadatta, S., Fox, W. et al. (1961). Rate of inactivation of isoniazid in south Indian patients with pulmonary tuberculosis-3. Serum concentrations of isoniazid produced by three regimens of isoniazid alone and isoniazid plus PAS. Bulletin of the World Health Organization 25, 793–806.[ISI][Medline]

25 . Augustynowicz-Kopec, E. & Zwolska, Z. (2002). Bioavailability factors of isoniazid in fast and slow acetylators, healthy volunteers. Acta Poloniae Pharmaceutica 59, 452–7.[Medline]

26 . Augustynowicz-Kopec, E. & Zwolska, Z. (2002). The type of isoniazid acetylation in tuberculosis patients treated at national tuberculosis and lung diseases research institute. Acta Poloniae Pharmaceutica 59, 443–7.[Medline]

27 . Adams, C., Werely, C., Victor, T. et al. (2003). Allele frequencies for glutathione S-transferase and N-acetyltransferase 2 differ in African population groups and may be associated with oesophageal cancer or tuberculosis incidence. Clinical Chemistry and Laboratory Medicine 41, 600–5.[ISI][Medline]

28 . Snider, D., Raviglione, M. & Kochi, A. (1994). In Tuberculosis: Pathogenesis, Protection and Control (Bloom, B., Ed.), p. 9. ASM Press, Washington, DC, USA.

29 . Payton, M., Auty, R., Delgoda, R. et al. (1999). Cloning and characterization of arylamine N-acetyltransferase genes from Mycobacterium smegmatis and Mycobacterium tuberculosis: increased expression results in isoniazid resistance. Journal of Bacteriology 181, 1343–7.[Abstract/Free Full Text]

30 . Connell, N. & Nikaido, H. (1994). In Tuberculosis: Pathogenesis, Protection and Control (Bloom, B., Ed.), passim. ASM Press, Washington, DC, USA.

31 . Bardou, F., Raynaud, C., Ramos, C. et al. (1998). Mechanism of isoniazid uptake in Mycobacterium tuberculosis. Microbiology 144, 2539–44.[Abstract]

32 . Bardou, F., Quemard, A., DuPont, M. et al. (1996). Effects of isoniazid on ultrastructure of Mycobacterium aurum and Mycobacterium tuberculosis and on production of secreted proteins. Antimicrobial Agents and Chemotherapy 40, 2459–67.[Abstract]

33 . Christensen, H., Garton, N., Harobin, R. et al. (1999). Lipid domains of mycobacteria studied with fluorescent molecular probes. Molecular Microbiology 31, 1561–72.[CrossRef][ISI][Medline]

34 . Chang, G., Spencer, R., Lee, A. et al. (1998). Structure of the mscl homolog from Mycobacterium tuberculosis: a gated mechanoselective ion channel. Science 282, 2220–6.[Abstract/Free Full Text]

35 . Barrow, E., Winchester, G., Staas, J. et al. (1998). Use of microsphere technology for targeted delivery of rifampicin to Mycobacterium tuberculosis-infected macrophages. Antimicrobial Agents and Chemotherapy 42, 2682–9.[Abstract/Free Full Text]

36 . Rastogi, N., Moreau, B., Capmau, M. et al. (1988). Antibacterial action of amphipathic derivatives of isoniazid against the Mycobacterium avium complex. Zentralblatt fuer Bakteriologie, Mikrobiology und Hygiene [A] 268, 456–62.

37 . DeMoen, P., Janssen, P. & Keere, B. (1954). On the metabolic fate of isoniazid (inh) and its N2-D-glucuronolactone derivative (inh-g) in man. Archives Internationales de Pharmacodynamie et de Therapie 98, 427ff.

38 . Pfaffenberg, R., Iwainsky, H., Jaehler, H. et al. (1960). Ueber den stoffwechsel des isonicotinsaeurehydrazid (inh) und der alpha-ketosaeuren bei tuberkuloesen diabetikern. Beitraege zur Klinik der Tuberkulose 123, 63–71.

39 . Iwainsky, H. (1988). In Antituberculosis Drugs (Bartmann, K., Ed.), pp. 489–90. Springer-Verlag, New York, NY, USA.

40 . Krueger-Thiemer, E. (1957). In Tuberkulose-forschungsinstitut Borstel Jahresbericht 1956–1957 (Freerksen, E., Ed.), pp. 331ff. Springer-Verlag, Berlin.

41 . Antony, S., Ynares, C. & Dummer, J. (1997). Isoniazid hepatotoxicity in renal transplant recipients. Clinical Transplantation 11, 34–7.[ISI][Medline]

42 . Brost, B. & Newman, R. (1997). The maternal and fetal effects of tuberculosis therapy. Obstetrics and Gynecology Clinics of North America 24, 659–73.[ISI][Medline]

43 . Crema, A., Baroli, F. & Ferrero, E. (1955). Ricerche sul metabolismo dell’idrazide dell’acido isonicotinico. Bollettino-Societa Italiana di Biologia Sperimentale 31, 244–6.

44 . Elliott, A. & Mitchison, D. (1994). In Tuberculosis: Back to the Future (Porter, J. & McAdam, K., Eds), pp. 247–9. John Wiley and Sons, Limited, Chichester, UK.

45 . Sarich, T., Youssefi, M., Zhou, T. et al. (1996). Role of hydrazine in the mechanism of isoniazid hepatotoxicity in rabbits. Archives of Toxicology 70, 835–40.[CrossRef][ISI][Medline]

46 . Starke, J. (1996). Tuberculosis in children. Primary Care 23, 861–81.[ISI][Medline]

47 . Timbrell, J., Wright, J. & Baillie, T. (1977). Monoacetylhydrazine as a metabolite of isoniazid in man. Clinical Pharmacology and Therapeutics 22, 602–8.[ISI][Medline]

48 . Timbrell, J. & Wright, J. (1984). Urinary metabolic profile of isoniazid in patients who develop isoniazid-related liver damage. Human Toxicology 3, 485–95.[ISI][Medline]

49 . Sookvanichsilp, N. (1999). Pyridoxal isonicotinoyl hydrazone: preparation, physical properties and acute toxicity in mice. Mahidol University Journal of Pharmaceutical Sciences (Bankok) 19, 1–4.

50 . Vigorita, M., Ottana, R., Maccari, R. et al. (1998). Synthesis and in vitro antimicrobial and antitumoral screening of novel lipophilic isoniazid analogues. Bollettino Chimico Farmaceutico 137, 267–76.[Medline]

51 . Georgieva, N. & Gadjeva, V. (2002). Isonicotinoylhydrazone analogs of isoniazid: relationship between superoxide scavenging and tuberculostatic activities. Biochemistry (Moscow) 67, 588–91.[CrossRef][ISI][Medline]

52 . De Logu, A., Onnis, V., Saddi, B. et al. (2002). Activity of a new class of isonicotinoylhydrazones used alone and in combination with isoniazid, rifampicin, ethambutol, para-aminosalicylic acid and clofazimine against Mycobacterium tuberculosis. Journal of Antimicrobial Chemotherapy 49, 275–82.[Abstract/Free Full Text]

53 . Maccari, R., Ottana, R., Monforte, F. et al. (2002). In vitro antimycobacterial activities of 2'-monosubstituted isonicotinohydrazides and their cyanoborane adducts. Antimicrobial Agents and Chemotherapy 46, 294–9.[Abstract/Free Full Text]

54 . Hearn, M., Celi, P., Chanyaputhipong, P. et al. (1995). Using near infrared spectroscopy to monitor the preparation of compounds for screening as anti-tuberculosis drugs. Journal of Near Infrared Spectroscopy 3, 19–23.

55 . Hearn, M. & Chanyaputhipong, P. (1995). Preparation and spectroscopic properties of 3-acyl-1,3,4-oxadiazolines. Journal of Heterocyclic Chemistry 32, 1647–9.[ISI]

56 . Hearn, M., Kang, H. & Thai, M. (1997). A convenient method for the preparation of tuberculostatic diacylhydrazines. Bulletin des Societes Chimiques Belges 106, 109–14.[ISI]

57 . Hearn, M. J. (2001). Antimycobacterial Compounds and Method for Making the Same. International Patent Application WO 02/43668.

58 . American Chemical Society. (2002). SciFinder Scholar. American Chemical Society, Washington, DC, USA.

59 . Vestal, A. (1969). Public Health Service Publication No. 1995, pp. 113–15. Laboratory Division, National Communicable Disease Center, Atlanta, GA, USA.

60 . Wong, C. S., Palmer, G. & Cynamon, M. (1988). In vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium kansasii to amoxycillin and ticarcillin in combination with clavulanic acid. Journal of Antimicrobial Chemotherapy 22, 863–6.[Abstract]

61 . Tuberculosis Antimicrobial Acquisition and Coordinating Facility. (1999). [Online.] http://www.taacf.org (18 July 2003, date last accessed).

62 . Kirsten, N. & Riccio, E. (2001). SRI Study No. G129–01, pp. 1–24. Stanford Research Institute International, Menlo Park, CA, USA.

63 . Green, C. (2001). SRI Study No. B080–01, pp. 1–10. Stanford Research Institute International, Menlo Park, CA, USA.

64 . Grosset, J. (2002). Mycobacterium tuberculosis in the extracellular compartment: an underestimated adversary. Antimicrobial Agents and Chemotherapy 47, 833–6.[CrossRef][ISI]

65 . Braun, R., Jakel, H.-P. & Schoneich, J. (1984). Genetic effects of isoniazid and the relationship to in vivo and in vitro biotransformation. Mutation Research 137, 61–9.[ISI][Medline]

66 . Matsuki, Y., Katakuse, Y., Matsuura, H. et al. (1991). Effects of glucose and ascorbic acid on absorption and first pass metabolism of isoniazid in rats. Chemical and Pharmaceutical Bulletin 39, 445–8.

67 . Colwell, C. & Hess, A. (1956). Stability and antibacterial effect of d-glucuronoloactone isonicotinyl [sic] hydrazone and isoniazid. Annual Review of Tuberculosis and Pulmonary Disease 73, 892–906.





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
53/2/185    most recent
dkh041v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Disclaimer
Request Permissions
Google Scholar
Articles by Hearn, M. J.
Articles by Cynamon, M. H.
PubMed
PubMed Citation
Articles by Hearn, M. J.
Articles by Cynamon, M. H.