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
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Abstract |
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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 SpragueDawley 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
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Introduction |
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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 hostpathogen 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.
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Materials and methods |
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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 (, cm1). 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 (
) 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. 167168°C; IR max 3212, 1662, 1637, 1597, 1528, 1406, 1302, 1285, 1245, 1214, 1139, 1036, 839, 755, 722; NMR (300 MHz)
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 SalmonellaEscherichia coli microsome plate incorporation assay
The INH Schiff base was examined for mutagenic activity in the SalmonellaEscherichia 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 SpragueDawley 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.
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Results and discussion |
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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.
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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 SalmonellaEscherichia 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).
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Acknowledgements |
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Footnotes |
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