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The influence of the zinc(II) starting material in the nucleophilic addition of piperidine (abbreviated as pipe) to acetonitrile to form amidine, piperidinoacetamidine (pipeam), was assessed. Thus, zinc(II) oxide, zinc(II) chloride or zinc(II) acetate were reacted with picolinic acid or pyrazinoic acid, piperidine and acetonitrile. The acid was used as a source of an N,O-donor ligand. The reactions with picolinic acid (picH) gave pipeamH[Zn(pic)3]·0.5H2O (1), an amidinium salt of a homoleptic complex [Zn(pic)3]–. The same product was obtained when [Zn(pic)(picH)Cl], an already known picolinate complex, was reacted under similar conditions. The chloride-containing mixtures produced another amidine compound, (pipeamH)Cl. The reactions with pyrazinoic acid (pyrazH) gave pipeamH[Zn(pyraz)3] (3), the amidinium salt of a homoleptic complex [Zn(pyraz)3]–. The acetate-containing mixture yielded acetamidinium acetate. The latter product indicates the presence of ammonia in the reaction. Notably, no amidine species was isolated when the coordinatively saturated pyrazinoate complex, [Zn(pyraz)2]n (2), was reacted with piperidine and acetonitrile. A detailed structural characterization of the new compounds and a discussion of their formation are presented.

Graphical abstract, Pyrazinamide (PZA) is a component of first-line drugs, active against latent Mycobacterium tuberculosis (MTB) isolates. The prodrug is activated into the active form, pyrazinoic acid (POA) via pncA gene-encoded pyrazinamidase (PZase). Mutations in pncA have been reported, most commonly responsible for PZA-resistance in more than 70% of the resistant cases. In our previous study, we detected many mutations in PZase among PZA-resistance MTB isolates including A46V, H71Y, and D129N. The current study was aimed to investigate the molecular mechanism of PZA-resistance behind mutants (MTs) A46V, H71Y, and D129N in comparison with the wild type (WT) through molecular dynamic (MD) simulation. MTB positive samples were subjected to PZA drug susceptibility testing (DST) against critical concentration (100ug/ml). The resistant samples were subjected to pncA sequencing. Thirty-six various mutations have been observed in the coding region of pncA of PZA-resistant isolates (GenBank accession No. MH461111) including A46V, H71Y, and D129N. The post-simulation analysis revealed a significant variation in MTs structural dynamics as compared to the WT. Root means square deviations (RMSD) and Root means square fluctuation (RMSF) has been found in variation between WT and MTs. Folding effect and pocket volume were altered in MTs when compared with WT. Geometric matching supports the effect of mutation A46V, H71Y, and D129N on PZase structure that may have an insight effect on PZase dynamics, making them vulnerable to convert pro-PZA into active form, POA. In conclusion, the current analyses will provide useful information behind PZA-resistance for better management of drug-resistant TB.

Pyrazinamide (PZA) is the only first-line antitubercular drug active against latent Mycobacterium tuberculosis (Mtb). It is activated to pyrazinoic acid by the pncA-encoded pyrazinamidase enzyme (PZase). Despite the emergence of PZA drug resistance, the underlying mechanisms of resistance remain unclear. This study investigated part of these mechanisms by modelling a PZA-bound wild type and 82 mutant PZase structures before applying molecular dynamics (MD) with an accurate Fe2+ cofactor coordination geometry. After observing nanosecond-scale PZA unbinding from several PZase mutants, an algorithm was developed to systematically detect ligand release via centre of mass distances (COM) and ligand average speed calculations, before applying the statistically guided network analysis (SGNA) method to investigate conserved protein motions associated with ligand unbinding. Ligand and cofactor perspectives were also investigated. A conserved pair of lid-destabilising motions was found. These consisted of (1) antiparallel lid and side flap motions; (2) the contractions of a flanking region within the same flap and residue 74 towards the core. Mutations affecting the hinge residues (H51 and H71), nearby residues or L19 were found to destabilise the lid. Additionally, other metal binding site (MBS) mutations delocalised the Fe2+ cofactor, also facilitating lid opening. In the early stages of unbinding, a wider variety of PZA poses were observed, suggesting multiple exit pathways. These findings provide insights into the late events preceding PZA unbinding, which we found to occur in some resistant PZase mutants. Further, the algorithm developed here to identify unbinding events coupled with SGNA can be applicable to other similar problems.

Tuberculosis (TB) remains a leading cause of infectious disease-related mortality and morbidity. Pyrazinamide (PZA) is a critical component of the first-line TB treatment regimen because of its sterilizing activity against non-replicating Mycobacterium tuberculosis (Mtb), but its mechanism of action has remained enigmatic. PZA is a prodrug converted by pyrazinamidase encoded by pncA within Mtb to the active moiety, pyrazinoic acid (POA) and PZA resistance is caused by loss-of-function mutations to pyrazinamidase. We have recently shown that POA induces targeted protein degradation of the enzyme PanD, a crucial component of the coenzyme A biosynthetic pathway essential in Mtb. Based on the newly identified mechanism of action of POA, along with the crystal structure of PanD bound to POA, we designed several POA analogs using structure for interpretation to improve potency and overcome PZA resistance. We prepared and tested ring and carboxylic acid bioisosteres as well as 3, 5, 6 substitutions on the ring to study the structure activity relationships of the POA scaffold. All the analogs were evaluated for their whole cell antimycobacterial activity, and a few representative molecules were evaluated for their binding affinity, towards PanD, through isothermal titration calorimetry. We report that analogs with ring and carboxylic acid bioisosteres did not significantly enhance the antimicrobial activity, whereas the alkylamino-group substitutions at the 3 and 5 position of POA were found to be up to 5 to 10-fold more potent than POA. Further development and mechanistic analysis of these analogs may lead to a next generation POA analog for treating TB. Ministry of Education (MOE) Research reported in this work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R01 AI106398 and the Singapore Ministry of Education (MoE) Academic Research Fund Tier 1 (RG107/20).

In this work, we present a simple, two-stage method for the preparation of new and efficient antimicrobial hybrid systems, based on isoniazid and pyrazine-2-carbohydrazide tuberculostatic agents covalently linked to graphene oxide. In the first step, the carboxyl groups of graphene oxide are activated by transforming them into the corresponding acid chloride groups, which, in the second step, will react with the amino groups of the two drugs. Pyrazine-2-carbohydrazide was obtained from pyrazinoic acid and hydrazine hydrate and was confirmed by nuclear magnetic resonance spectroscopy. The materials have been characterized by elemental analysis, infrared spectroscopy, Raman spectroscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. Their antimicrobial activity was tested on mycobacterial (Mycobacterium terrae), as well as on Gram-negative bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853), Gram-positive bacteria (Staphylococcus aureus ATCC 6538, Staphylococcus aureus ATCC 25923) and fungal (Candida albicans ATCC 10231), in planktonic and biofilm growth state. The biocompatibility of the tested compounds was assessed in vitro by live-dead fluorescence staining and flow cytometry. The results of this study have shown that the functionalization of graphene oxide with isoniazid and pyrazine-2-carbohydrazide both enhanced the anti-mycobacterial activity of the respective drugs and extended their antimicrobial spectrum towards other microbial strains, in planktonic and biofilm growth state, showing a promising potential for mitigating the impact of antimicrobial resistance and the deleterious effects of microbial biofilms. At the active tuberculostatic concentrations, the tested compounds exhibit very low cytotoxicity and do not interfere with the cellular cycle of Hep-2 cells. The flow cytometry and live/dead fluorescence staining proved that the tested compounds exhibit a microbicidal effect through induction of cellular lesions, consecutive to membrane depolarization.

We modified Wayne's pyrazinamidase test against Mycobacterium tuberculosis to indirectly measure pyrazinamidase activity via pyrazinoic acid in liquid medium. The modified pyrazinamidase test was easy to perform and its results were in complete agreement with those of the conventional Wayne's method, highlighting its potential application in phenotypic pyrazinamide susceptibility testing.

Combining the advantage of higher efficacy due to local pulmonary administration of pyrazinoic acid (POA) and potent effect of pyrazinoic acid ester (PAE) delivered as an aerosol would aid in tuberculosis therapy. A combination spray dried dry powder, composed of POA, PAE (n-propyl POA), maltodextrin and leucine, was prepared for aerosol delivery to animals. Solid-state characteristics of morphology (scanning electron microscopy) crystallinity (X-ray powder diffraction), thermal properties (thermogravimetric analysis and differential scanning calorimetry) and moisture content (Karl Fisher) were evaluated. Particle size distributions, by volume (laser diffraction) for the dispersed powder and by mass (inertial impaction) were determined. Efficient delivery of the powder to a nose only animal exposure chamber employed a novel rotating brush/micro-fan apparatus. Spherical, crystalline particles were prepared. The volume median diameter, ~1.5 μm, was smaller than the mass median aerodynamic diameter, ~3.0 μm, indicating modest aggregation. Drug content variations were observed across the particle size distribution and may be explained by PAE evaporative losses. Delivery to the nose-only exposure chamber indicated that boluses could be administered at approximately 3 min intervals to avoid aerosol accumulation and effect uniform dose delivery with successive doses suitable for future pharmacokinetic and pharmacodynamic studies.

Pyrazinamide (PZA) is one of the most potent bacteriostatic drug against tuberculosis, a deadliest disease with high mortality and morbidity rate. PZA metabolizes into its active form pyrazinoic acid (POA) with the help of a metalloenzyme, pyrazinamidase (PZase). Mutagenicity and metal substitution in PZase weakens the binding of PZA with PZase and increases the drug resistance in Mycobacterium tuberculosis. The present study aims at the quantum mechanistic analysis of mutant-metal substituted PZase complexes by studying the mechanics of metals and PZA binding at MCS and catalytic site, respectively. A total of 66 complexes are scrutinised in this study to elucidate the effect of mutations on the enzymatic function of PZase. Among the 10 mutations considered in this study, 7 different mutations i.e. Asp49 → Asn, His51 → Arg, Gly78 → Cys, Asp12 → Gly, Asp12 → Ala, Thr135 → Pro and Asp136 → Gly cause a detrimental effect on the activity of PZase. In addition to this, the substitution of iron with cobalt enhances the enzymatic activity of both wild type and mutant PZase while zinc, magnesium and copper reduce it. Based on these results, it is concluded that upon substitution of iron with zinc, magnesium and copper, PZase cannot function properly. Due to mutations, the reactivity of the drug also reduces as its binding with PZase weakens and this phenomenon enhances the resistance of Mycobacterium tuberculosis against drug.

Summary Mutations in the pyrazinamidase (PZAse) coding gene, pncA , have been considered as the main cause of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis . However, recent studies suggest there is no single mechanism of resistance to PZA. The pyrazinoic acid (POA) efflux rate is the basis of the PZA susceptibility Wayne test, and its quantitative measurement has been found to be a highly sensitive and specific predictor of PZA resistance. Based on biological considerations, the POA efflux rate is directly determined by the PZAse activity, the level of pncA expression, and the efficiency of the POA efflux pump system. This study analyzes the individual and the adjusted contribution of PZAse activity, pncA expression and POA efflux rate on PZA resistance. Thirty M. tuberculosis strains with known microbiological PZA susceptibility or resistance were analyzed. For each strain, PZAse was recombinantly produced and its enzymatic activity measured. The level of pncA mRNA was estimated by quantitative RT-PCR, and the POA efflux rate was determined. Mutations in the pncA promoter were detected by DNA sequencing. All factors were evaluated by multiple regression analysis to determine their adjusted effects on the level of PZA resistance. Low level of pncA expression associated to mutations in the pncA promoter region was observed in pncA wild type resistant strains. POA efflux rate was the best predictor after adjusting for the other factors, followed by PZAse activity. These results suggest that tests which rely on pncA mutations or PZAse activity are likely to be less predictive of real PZA resistance than tests which measure the rate of POA efflux. This should be further analyzed in light of the development of alternate assays to determine PZA resistance.

Objective/background Mycobacterium tuberculosis pyrazinamidase (PZase) is known an enzyme that is involved in degradation of pyrazinamide to ammonia and pyrazinoic acid. Pyrazinamide is an important first-line drug used in the short-course treatment of tuberculosis. Previous investigations have indicated that the pyrazinamide (PZA)-resistant M. tuberculosis strains are caused by point mutations in the PZase enzyme which is the activator of the prodrug PZA. Although the general fold of PZase was determined, the structural and functional properties of the enzyme in solution were not understood very well. In this study, the PZase enzyme was overexpressed and purified. In addition, two polyols, namely sorbitol and glycerol, were chosen to study their effects on the structure, dynamics, and stability of the enzyme. To gain a deeper insight, molecular dynamics simulation and spectroscopic methods, such as fluorescence spectroscopy and circular dichroism (CD), were used. Methods The genes were cloned in Escherichia coli BL21 (DE3), harboring the recombinant pET-28a (+) plasmid, overexpressed and purified by Ni-NTA Sepharose. The far UV–visible CD spectra were measured by a Jasco-810 spectropolarimeter. The intrinsic fluorescence spectra were measured on a Cary Varian Eclipse spectrofluorometer. For molecular dynamics (MD) simulations, we have applied GROMACS4.6.5. Results The results showed that glycerol and sorbitol increased the enzyme activity up to 130% and 110%, respectively, at 37 °C. The stability of PZase was decreased and the half-life was 20 min. Glycerol and sorbitol increased the PZase half-life to 99 min and 23 min, respectively. The far UV CD measurements of PZase indicated that the CD spectra in glycerol and sorbitol give rise to an increase in the content of α-helix and β-sheets elements. The average enzyme root mean square deviation (RMSD) in sorbitol solution was about 0.416 nm, a value that is higher than the enzyme RMSD in the pure water (0.316). In dictionary of protein secondary structure (DSSP) results, we observed that the secondary structures of the protein are partially increased as compared to the native state in water. The experimental and simulation data clearly indicated that the polyols increased the PZase stabilization in the order: glycerol > sorbitol. Conclusion It can be concluded that the native conformation of the enzyme was stabilized in the sorbitol and glycerol and tend to exclude from the PZase surface, forcing the enzyme to keep it in the compactly folded conformation. The glycerol molecules stabilized PZase by decreasing the loops flexibility and then compacting the enzyme structure. It appears that more stability of PZase in glycerol solution correlates with its amphiphilic orientation, which decreases the unfavorable interactions of hydrophobic regions.

Resistance to pyrazinamide in Mycobacterium tuberculosis is usually associated with a reduction of pyrazinamidase activity caused by mutations in pncA, the pyrazinamidase coding gene. Pyrazinamidase is a hydrolase that converts pyrazinamide, the antituberculous drug against the latent stage, to the active compound, pyrazinoic acid. To better understand the relationship between pncA mutations and pyrazinamide resistance, it is necessary to analyze the distribution of pncA mutations from pyrazinamide resistant strains. We determined the distribution of Peruvian and globally reported pncA missense mutations from M. tuberculosis clinical isolates resistant to pyrazinamide. The distributions of the single amino acid substitutions were compared at the secondary structure domains level. The distribution of the Peruvian mutations followed a similar pattern as the mutations reported globally. A consensus clustering of mutations was observed in hot-spot regions located in the metal coordination site and to a lesser extent in the active site of the enzyme. The data was not able to reject the null hypothesis that both distributions are similar, suggesting that pncA mutations associated to pyrazinamide resistance in M. tuberculosis, follow a conserved pattern responsible to impair the pyrazinamidase activity.

Pyrazinamide (PZA) is active against M. tuberculosis and is a first line agent for the treatment of human tuberculosis. PZA is itself a prodrug that requires activation by a pyrazinamidase to form its active metabolite pyrazinoic acid (POA). Since the specificity of cleavage is dependent on a single bacterial enzyme, resistance to PZA is often found in tuberculosis patients. Esters of POA have been proposed in the past as alternatives to PZA however the most promising compounds were rapidly degraded in the presence of serum. In order to obtain compounds that could survive during the transport phase, we synthesized lipophilic ester and amide POA derivatives, studied their activity against M. tuberculosis, their stability in plasma and rat liver homogenate and also their activation by a mycobacterial homogenate. The new lipophilic ester prodrugs were found to be active in concentrations 10-fold lower than those needed for PZA to kill sensitive M. tuberculosis and also have a suitable stability in the presence of plasma. Amides of POA although more stable in plasma have lower activity. The reason can probably be found in the rate of activation of both types of prodrugs; while esters are easily activated by mycobacterial esterases, amides are resistant to activation and are not transformed into POA at a suitable rate.

Pyrazinamide, the amide of pyrazinoic acid, is one of the basic therapeutic agents currently used in combination for chemotherapy of tuberculosis. A reversed-phase high-performance liquid chromatography method based on ionic pair chromatography, was developed after solid-phase extraction of the analytes from plasma with prior addition of internal standard. The main metabolites, pyrazinoic acid, 5-hydroxypyrazinoic acid and 5-hydroxypyrazinamide, were included as well as uric acid and other purine derivatives to allow detailed study of the pharmacokinetics of the drug, especially in patients with impaired kidney function. Some interesting features of the chromatographic system giving some insight in the retention mechanism and of the solid-phase extraction are discussed in detail.

A new high-performance liquid chromatograhic procedure for simultaneous determination of pyrazinamide (PZA) and its three metabolites 5-hydroxypyrazinamide (5-OH-PZA), pyrazinoic acid (PA), and 5-hydroxypyrazinoic acid (5-OH-PA), in rat urine was developed. 5-OH-PZA and 5-OH-PA standards were obtained by enzymatic synthesis (xanthine oxidase) and checked by HPLC and GC-MS. Chromatographic separation was achieved in 0.01 M KH2PO4 (pH 5.2), circulating at 0.9 ml/min, on a C18 silica column, at 22 degrees C. The limits of detection were 300 microg/l for PZA, 125 microg/l for PA, 90 microg/l for 5-OH-PZA and 70 microg/l for 5-OH-PA. Good linearity (r2>0.99) was observed within the calibration ranges studied: 0.375-7.50 mg/l for PZA, 0.416-3.33 mg/l for PA, 0.830-6.64 mg/l for 5-OH-PZA and 2.83-22.6 mg/l for 5-OHPA. Accuracy was always lower than +/- 10.8%. Precision was in the range 0.33-5.7%. The method will constitute a useful tool for studies on the influence of drug interactions in tuberculosis treatment.

Aldehyde oxidase was purified about 120-fold from rat liver cytosol by sequential column chromatography using diethylaminoethyl (DEAE) cellulose, Benzamidine-Sepharose 6B and gel filtration. The purified enzyme was shown as a single band with M(r) of 2.7 x 10(5) on polyacrylamide gel electrophoresis (PAGE) and M(r) of 1.35 x 10(5) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using this purified enzyme, in vitro conversion of allopurinol, pyrazinamide and pyrazinoic acid was investigated. Allopurinol and pyrazinamide were oxidized to oxypurinol and 5-hydroxy-pyrazinamide, respectively, while pyrazinoic acid, the microsomal deamidation product of pyrazinamide, was not oxidized to 5-hydroxypyrazinoic acid. The apparent Km value of the enzyme for pyrazinamide was 160 microM and that for allopurinol was 1.1 mM. On PAGE, allopurinol- or pyrazinamide-stained band was coincident with Coomassie Brilliant Blue R 250-stained band, respectively. These results suggest that aldehyde oxidase may play a role in the oxidation of allopurinol to oxypurinol and that of pyrazinamide to 5-hydroxypyrazinamide with xanthine dehydrogenase which can oxidize both allopurinol and pyrazinamide in vivo. The aldehyde oxidase may also play a major role in the oxidation of allopurinol and pyrazinamide in the subgroup of xanthinuria patients (xanthine oxidase deficiency) who can oxidize both allopurinol and pyrazinamide.

A reversed-phase high-performance liquid chromatographic method was developed for the simultaneous determination of pyrazinamide and its metabolites in urine. Study of the metabolism of pyrazinamide by this method demonstrated that (i) 5-hydroxypyrazinamide excretion was compatible with pyrazinoic acid excretion and (ii) allopurinol decreased in vivo conversion of pyrazinamide to 5-hydroxypyrazinamide and blocked that of pyrazinoic acid to 5-hydroxypyrazinoic acid.

Arthralgia during daily treatment with chemotherapy regimens containing pyrazinamide was found to be considerably less in patients who received rifampicin concomitantly. The effect of rifampicin on the biodisposition of pyrazinamide, and the pyrazinamide-induced changes in the renal elimination of uric acid was, therefore, investigated. In one investigation in tuberculous patients undergoing treatment with daily regimens containing pyrazinamide or pyrazinamide and rifampicin, serial serum concentrations of pyrazinamide and uric acid up to 11 hours, and urinary pyrazinamide, pyrazinoic acid and uric acid concentrations collected at intervals up to 11 1 2 hours after drug administration were determined. In a second investigation in healthy volunteers, the effect of single doses of rifampicin, pyrazinamide and a combination of the two drugs on the renal excretion of uric acid during a 24-hour period was determined. Results of the first investigation showed that the serum concentrations and the renal clearance of pyrazinamide were similar in patients receiving pyrazinamide or with or without rifampicin, suggesting that rifampicin had no effect on either the metabolism or excretion of pyrazinamide. Both investigations demonstrated that pyrazinamide caused a significant decrease in the renal excretion of uric acid. Rifampicin enhanced the renal excretion of uric acid, both in the presence and in the absence of pyrazinamide, and also that of pyrazinoic acid. This effect could lead to a decrease in the deposition of uric acid in joints, and consequently to a lower incidence of arthralgia.

Thirteen non-gouty and twelve gouty men to whose regular diet was added 4 gm. of ribonucleic acid (RNA) daily, developed increases in plasma uric acid, urinary excretion of uric acid and derived parameters, without significant differences in the mean increments in non-gouty and gouty subjects. Ten non-gouty and seventeen gouty men were given intravenous injections of 1.1 to 2.5 gm. uric acid to impose even greater demands upon the tubular mechanisms for transfer of uric acid, and again the mean increments in the urinary excretion of uric acid and uric acid clearance in non-gouty and gouty subjects were indistinguishable. Pyrazinoic acid, a potent inhibitor of tubular secretion of uric acid, was found to suppress the urinary elimination of rapidly infused uric acid in gouty and non-gouty subjects alike. The data indicate that gouty subjects can excrete large extraneous loads of uric acid with as much facility as normal man, and support the view that this obtains also for the usually smaller and more slowly delivered endogenous uric acid loads natural to gout. The results thus argue against any intrinsic defect, peculiar to gout, in the renal mechanisms for excretion of uric acid. Briefly reviewed is the evidence that tubular secretion of uric acid occurs in non-gouty and gouty man, indeed that virtually all the uric acid appearing in the urine ordinarily derives from tubular secretion, hence virtually all the filtered uric acid ordinarily must be reabsorbed. It is pointed out that in this version of the filtration-reabsorption-secretion hypothesis there need be no simple relationship between the filtered and excreted uric acid, a dissociation found in gout that has been interpreted by others as indicating a primary tubular defect in transfer of uric acid. The implications of the filtration-reabsorption-secretion hypothesis in respect to other aspects of the renal regulation of uric acid are discussed in relation to excretion of uric acid in normal and gouty man under natural and artificial conditions of uric acid loading. The present study, limited to over-all clearances, gives no information as to the precise mechanisms or quantitative aspects of tubular secretion of uric acid in normal or gouty man, nor does it explain just how renal excretion of large and rapidly imposed uric acid loads is regulated.

A method for the esterification of carboxylic acids with the aid of N,N′-dicyclohexylcarbodiimide is described. The time needed for complete esterification with methanol of some aliphatic, aromatic and heterocyclic carboxylic acids was determined. A series of ten determinations of the content carried out on samples of pure pyrazinoic acid was carried out in order to evaluate the reproducibility of the method. Examples of quantitative esterification are also described for other alcohols that can be used instead of methanol in difficult gas chromatographic separations, for example, of isomers.

Countercurrent distribution studies demonstrated that after oral dosage with pyrazinamide only two nitroprusside-reacting compounds are excreted in the urine: unchanged pyrazinamide and its metabolite pyrazinoic acid. Sensitive and specific chemical methods are described for the determination of pyrazinamide and pyrazinoic acid in serum and urine, based on differential solvent extraction followed by reaction with alkaline nitroprusside. These methods were used to study the pharmacology of pyrazinamide in patients participating in a clinical trial in East Africa in which pyrazinamide (with streptomycin) was given in doses of 0·5 g. thrice daily, l·5 g. daily and 3 g. on alternate days. Peak serum concentrations of pyrazinamide occurred at about two hours and were proportional to the dose of pyrazinamide. On each of these regimens, patients excreted about 4% of the dose unchanged and about 30% as pyrazinoic acid. After three hours, serum pyrazinamide concentrations fell exponentially with half-lives having a mean of about six hours. The serum concentrations of pyrazinamide expected with each of the regimens were calculated and the clinical implication of these findings are discussed. Two detailed pharmacokinetic studies were also undertaken at an interval of three years on a healthy British subject, which demonstrated the constancy of the absorption, metabolism and excretion of pyrazinamide. The fall with time in the urinary excretion of pyrazinamide and pyrazinoic acid paralleled the decreasing serum concentrations of these compounds. In the serum, the maximum concentration of pyrazinoic acid was much less than that of the unchanged drug.