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ABSTRACT Tuberculosis (TB) remains a significant global health challenge, exacerbated by the emergence of drug-resistant strains, such as those resistant to pyrazinamide (PZA). The current scarcity of affordable and precise quantitative diagnostic tests for PZA resistance underscores the urgent need for more accessible diagnostic tools. We evaluated PZA susceptibility in 264 TB-positive samples by quantifying pyrazinoic acid (POA) production, using both the MODS-Wayne qualitative assay and our newly developed quantitative approach (MODS-WQ). The MODS-WQ was assessed in 7H9 medium (MODS-WQ7H9) or citrate buffer (MODS-WQCB), with POA levels measured via spectrophotometry against a calibration curve. PZA susceptibility determinations were based on a composite reference standard. Associations between POA levels and pyrazinamidase mutations were explored. The composite standard detected PZA resistance in 23.5% of the samples, which accounts for 62.8% of the multidrug-resistant (MDR) samples. The MODS-WQ established specific POA cutoffs of 123.25 µM for MODS-WQ7H9 and 664.7 µM for MODS-WQCB, with sensitivities of 81.3% and 92.3% and specificities of 77.2% and 95.9%, respectively. Notably, samples with mutations in the pyrazinamidase metal-binding site exhibited significantly lower POA levels compared with mutations in the enzyme periphery. Furthermore, a significant correlation was found between POA production and PZA resistance, Bactec Growth Index, and minimum inhibitory concentration (MIC) values. This study presents a novel, direct, and accessible susceptibility test for PZA resistance that quantifies POA, enhancing the detection capabilities for this condition. The citrate-buffered MODS-WQ assay demonstrated high sensitivity and specificity for quantifying POA, confirming that POA production is a reliable indicator of PZA resistance.IMPORTANCEPZA susceptibility testing continues to be a challenge, particularly in countries with high TB incidence. In response to this pressing need, we have developed a quantitative MODS-Wayne (MODS-WQ) assay. This approach offers a direct and cost-effective solution representing a significant advancement in TB diagnostics, particularly benefiting resource-limited laboratories, primarily in developing regions. The MODS-WQ assay stands out for its ability to quantify pyrazinoic acid (POA) production, as a reliable indicator of PZA resistance. Unlike traditional qualitative assays, MODS-WQ eliminates the inherent subjectivity in interpretation, providing more accurate and actionable results. Moreover, the MODS-WQ approach accounts for critical factors influencing PZA resistance, including enzymatic efficiency and efflux pump activity. By integrating these factors into the detection process, our methodology offers a comprehensive understanding of PZA resistance levels, enabling tailored treatment strategies for patients.

One of the most effective approaches to discovering novel drugs for cancer treatment involves the exploration of new synthetic compounds. The pyrazinoic acid or pyrazine‐2‐carboxylic acid (PA)‐derivative compounds can be explored as a new anticancer agent due to their nitrogenous heteroaromatic ring. In this study, ten novels PA derivatives were synthesized by Ugi multicomponent reaction and characterized using IR, NMR, and mass spectroscopy. The cytotoxic activity was assayed in three different cancer cell lines, including colon (HT‐29), lung (A549), and breast (MCF‐7). The U10 was the most potent compound, exhibiting moderate cytotoxicity with IC50 of 8.26 μM, 8.23 μM, and 22.58 μM against HT‐29, A549, and MCF‐7 cell lines, respectively. In addition, the effect of U10 exposure in the MRC‐5 cell line as a non‐tumoral lung cell line showed a selectivity index of 3.76. The apoptotic activity and intracellular ROS level induction of U10 were assayed in MCF‐7 cells. The results demonstrated that apoptosis increases from 23.94 % at 10 μM to 36.8 % at 25 μM. Intracellular ROS level assay showed that U10 was able to significantly increase intracellular ROS by increasing the concentration. Molecular docking was utilized to predict the binding sites and interactions between the synthesized compound and DNA, as well as the Bcl‐2 apoptosis regulator. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) remains a significant global health challenge, exacerbated by the emergence of drug-resistant strains, such as those resistant to pyrazinamide (PZA). The current scarcity of affordable and precise quantitative diagnostic tests for PZA resistance underscores the urgent need for more accessible diagnostic tools. We evaluated PZA susceptibility in 264 TB-positive samples by quantifying pyrazinoic acid (POA) production, using both the MODS-Wayne qualitative assay and our newly developed quantitative approach (MODS-WQ). The MODS-WQ was assessed in 7H9 medium (MODS-WQ 7H9 ) or citrate buffer (MODS-WQ CB ), with POA levels measured via spectrophotometry against a calibration curve. PZA susceptibility determinations were based on a composite reference standard. Associations between POA levels and pyrazinamidase mutations were explored. The composite standard detected PZA resistance in 23.5% of the samples, which accounts for 62.8% of the multidrug-resistant (MDR) samples. The MODS-WQ established specific POA cutoffs of 123.25 µM for MODS-WQ 7H9 and 664.7 µM for MODS-WQ CB , with sensitivities of 81.3% and 92.3% and specificities of 77.2% and 95.9%, respectively. Notably, samples with mutations in the pyrazinamidase metal-binding site exhibited significantly lower POA levels compared with mutations in the enzyme periphery. Furthermore, a significant correlation was found between POA production and PZA resistance, Bactec Growth Index, and minimum inhibitory concentration (MIC) values. This study presents a novel, direct, and accessible susceptibility test for PZA resistance that quantifies POA, enhancing the detection capabilities for this condition. The citrate-buffered MODS-WQ assay demonstrated high sensitivity and specificity for quantifying POA, confirming that POA production is a reliable indicator of PZA resistance., Importance: PZA susceptibility testing continues to be a challenge, particularly in countries with high TB incidence. In response to this pressing need, we have developed a quantitative MODS-Wayne (MODS-WQ) assay. This approach offers a direct and cost-effective solution representing a significant advancement in TB diagnostics, particularly benefiting resource-limited laboratories, primarily in developing regions. The MODS-WQ assay stands out for its ability to quantify pyrazinoic acid (POA) production, as a reliable indicator of PZA resistance. Unlike traditional qualitative assays, MODS-WQ eliminates the inherent subjectivity in interpretation, providing more accurate and actionable results. Moreover, the MODS-WQ approach accounts for critical factors influencing PZA resistance, including enzymatic efficiency and efflux pump activity. By integrating these factors into the detection process, our methodology offers a comprehensive understanding of PZA resistance levels, enabling tailored treatment strategies for patients.

Pyrazinamide has played a critical role in shortening therapy against drug-sensitive, drug-resistant, active, and latent tuberculosis (TB). Despite widespread recognition of its therapeutic importance, the sterilizing properties of this 60-year-old drug remain an enigma given its rather poor activity in vitro. Here we revisit longstanding paradigms and offer pharmacokinetic explanations for the apparent disconnect between in vitro activity and clinical impact. We show substantial host-mediated conversion of prodrug pyrazinamide (PZA) to the active form, pyrazinoic acid (POA), in TB patients and in animal models. We demonstrate favorable penetration of this pool of circulating POA from plasma into lung tissue and granulomas, where the pathogen resides. In standardized growth inhibition experiments, we show that POA exhibits superior in vitro potency compared to PZA, indicating that the vascular supply of host-derived POA may contribute to the in vivo efficacy of PZA, thereby reducing the apparent discrepancy between in vitro and in vivo activity. However, the results also raise the possibility that subinhibitory concentrations of POA generated by the host could fuel the emergence of resistance to both PZA and POA. In contrast to widespread expectations, we demonstrate good oral bioavailability and exposure in preclinical species in pharmacokinetic studies of oral POA. Baseline exposure of oral POA can be further increased by the xanthine oxidase inhibitor and approved gout drug allopurinol. These promising results pave the way for clinical investigations of oral POA as a therapeutic alternative or an add-on to overcome PZA resistance and salvage this essential TB drug.

The title 1:1 salt, C7H8NO2+·C5H3N2O2− (systematic name: 4-carboxyanilinium pyrazine-2-carboxylate), was synthesized successfully by slow evaporation of a saturated solution from water–ethanol (1:1 v/v) mixture and characterized by X-ray diffraction (SCXRD, PXRD) and calorimetry (DSC). The crystal structure of the salt was solved and refined at 150 and 293 K. The salt crystallizes with one molecule of 4-aminobenzoic acid (PABA) and one molecule of pyrazinoic acid (POA) in the asymmetric unit. In the crystal, the PABA and POA molecules are associated via COOH...Narom heterosynthons, which are connected by N—H...O hydrogen bonds, creating zigzag chains. The chains are further linked by N—H...O hydrogen bonds and π–π stacking interactions along the b axis [centroid-to-centroid distances = 3.7377 (13) and 3.8034 (13) Å at 150 and 293 K, respectively] to form a layered three-dimensional structure.

Abstract. Pyrazinamide (PZA) is unique in that it is a component of the first line therapy for drug sensitive tuberculosis and in most current and experimental treatments also for multi drug resistant tuberculosis. Furthermore, PZA has been shown to help to ensure lasting cure and prevent relapse in shorter multi drug regimens. PZA is a prodrug. Mycobacterial tuberculosis (MTB) PncA enzyme activates the anti-mycobacterial prodrug PZA by transforming it into pyrazinoic acid (POA). The majority of clinical PZA resistant isolates contain mutations within the pncA gene and therefore remain sensitive to POA as they no longer activate PZA. Resistance to the active compound POA requires an alternative resistance mechanism and in vitro selected spontaneous MTB POA resistant mutants typically acquire a range of mutations in panD or mutations in one of a series of genes most of which are associated with the regulation of the bacterial stringent response. Clinically isolated PZA resistant MTB strains resistant to PZA and POA with mutations in any of these genes are unusual. Thus, it is likely the stringent response is critical for MTB in vivo and a damaged stringent response results in at least a reduction in fitness. Various lead compounds that disrupt the MTB stringent response have been identified that might form the basis for drugs with activity against latent mycobacteria with the potential to shorten tuberculosis treatment. Here we discuss the role of latency in the lifecycle of MTB and possible links to the activity PZA with a focus on potential new targets and drugs.

Introduction: Tuberculosis (TB) is a significant cause of morbidity and mortality worldwide. The patient compliance with the long treatment regimens is essential for successful eradication. Pyrazinamide (PZA) shortens these regimens from 9 to 6 months, and therefore, improves treatment completion rates. Although PZA is a first-line medication for the treatment of TB, no simple or reliable assay to determine PZA resistance is yet available. In the presence of PZA, only susceptible Mycobacterium tuberculosis strains release pyrazinoic acid (POA). Therefore, the measurement and quantification of released POA is an indicator of PZA resistance. Methods: Two electrochemical sensors were constructed and tested with alternative working electrodes in conjunction with a portable potentiostat to measure the current produced when a potential difference of 2 V is applied to varying concentrations of POA in controlled solutions. Results: The large (13.2 mm) electrochemical sensor was able to detect POA at a minimum concentration of 40 μM to a statistically significant level (P = 0.0190). Similar graphical trends were obtained when testing the electrochemical sensor in the supernatant of a negative microscopic observation drug susceptibility assay culture, irrespective of the presence of PZA. Conclusion: Inexpensive and reusable electrochemical sensors with a portable potentiostat are a promising tool for the detection of POA, a biomarker of PZA susceptible M. Tuberculosis.

Abstract Benzotriazole-activated pyrazinoic acid was utilized as a versatile building block for the efficient and convenient synthesis of novel hybrid conjugates of pyrazinoic acid with secondary amines via amino acid linkers in high yields.

Introduction: Mycobacterium tuberculosis has been extensively studied for mutations leading to drug resistance. Pyrazinamide is a drug acting on the semi-dormant bacteria that is responsible for relapse of tuberculosis. This drug helped reduce the treatment duration of tuberculosis from nine to six months. However, this drug is not being screened for resistance along with Rifampicin and Isoniazid., Aims and Objectives: This study aimed to estimate the proportion of pncA gene mutation among tuberculosis patients and its association between treatment outcomes, clinical characteristics, and phenotypic drug resistance., Method: ology: A total of 154 samples included 73 drug-resistant and 81 drug-susceptible isolates. The isolates were subjected to DNA extraction and amplification using conventional PCR. The PCR product was sequenced by the Sanger sequencing method, and phenotypic drug susceptibility testing was done using the broth dilution method. The association of this gene with the treatment outcome was done by following up with the patients till the end of the regimen., Results: None of the drug susceptible tuberculosis patients showed significant non-synonymous mutations. Among the drug-resistant TB patients, seven unique significant mutations out of 73 isolates (9.6%) were distributed among Isoniazid-resistant tuberculosis and Multi-Drug Resistant Tuberculosis isolates. No association was found between the mutations and the clinical characteristics of the subjects harboring these isolates., Conclusion: This study estimated seven unique mutations in drug-resistant tuberculosis and none in drug-sensitive tuberculosis. Isolates harboring was not significantly associated with the participant's treatment outcome and other clinical characteristics. The pyrazinamide resistance testing by the phenotypic and genotypic methods was found to be in concordance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.)

The pro-drug pyrazinamide is hydrolyzed to pyrazinoic acid (POA) in its use for the treatment of tuberculosis. As a molecule with bactericidal activity, POA binds to the C-terminal S1 domain of ribosomal protein S1 from Mycobacterium tuberculosis (MtRpsACTD_S1) to inhibit trans-translation. Trans-translation is a critical component of protein synthesis quality control, and is mediated by transfer-messenger RNA. Here, we have determined the solution structure of MtRpsACTD_S1(280–368), and analyzed its structural dynamics by NMR spectroscopy. The solution structure of MtRpsACTD_S1(280–368) mainly consists of five anti-parallel β strands, two α helices, and two 310 helices. Backbone dynamics reveals that the overall structure of MtRpsACTD_S1(280–368) is rigid, but segment L326–V333 undergoes large amplitude fluctuations on picosecond to nanosecond time scales. In addition, residues V321, H322, V331 and D335 with large Rex values exhibit significant chemical or conformational exchange on microsecond to millisecond time scale. Titration of the truncated MtRpsACTD_S1(280–368) with POA shows similar characteristics to titration of MtRpsACTD_S1(280–438) with POA. In addition, diverse length fragments of MtRpsACTD_S1 show various HN resonance signals, and we find that the interaction of MtRpsA(369–481) with MtRpsACTD_S1(280–368) [Kd = (4.25 ± 0.15) mM] is responsible for the structural difference between MtRpsACTD_S1(280–368) and MtRpsACTD_S1. This work may shed light on the underlying molecular mechanism of MtRpsACTD recognizing and binding POA or mRNA, as well as the detailed mechanism of interactions between MtRpsACTD_S1(280–368) and the additional C-terminal MtRpsA(369–481). [ABSTRACT FROM AUTHOR]

The title 1:1 salt, C7H8NO2+·C5H3N2O2- (systematic name: 4-carboxyanilinium pyrazine-2-carboxylate), was synthesized successfully by slow evaporation of a saturated solution from water–ethanol (1:1 v/v) mixture and characterized by X-ray diffraction (SCXRD, PXRD) and calorimetry (DSC). The crystal structure of the salt was solved and refined at 150 and 293 K. The salt crystallizes with one molecule of 4-aminobenzoic acid (PABA) and one molecule of pyrazinoic acid (POA) in the asymmetric unit. In the crystal, the PABA and POA molecules are associated via COOH···Narom heterosynthons, which are connected by N—H···O hydrogen bonds, creating zigzag chains. The chains are further linked by N—H···O hydrogen bonds and π-π stacking interactions along the b axis [centroid-to-centroid distances = 3.7377 (13) and 3.8034 (13) Å at 150 and 293 K, respectively] to form a layered threedimensional structure. [ABSTRACT FROM AUTHOR]

Introduction: Tuberculosis (TB) is a significant cause of morbidity and mortality worldwide. The patient compliance with the long treatment regimens is essential for successful eradication. Pyrazinamide (PZA) shortens these regimens from 9 to 6 months, and therefore, improves treatment completion rates. Although PZA is a first-line medication for the treatment of TB, no simple or reliable assay to determine PZA resistance is yet available. In the presence of PZA, only susceptible Mycobacterium tuberculosis strains release pyrazinoic acid (POA). Therefore, the measurement and quantification of released POA is an indicator of PZA resistance. Methods: Two electrochemical sensors were constructed and tested with alternative working electrodes in conjunction with a portable potentiostat to measure the current produced when a potential difference of 2 V is applied to varying concentrations of POA in controlled solutions. Results: The large (13.2 mm) electrochemical sensor was able to detect POA at a minimum concentration of 40 μM to a statistically significant level (P = 0.0190). Similar graphical trends were obtained when testing the electrochemical sensor in the supernatant of a negative microscopic observation drug susceptibility assay culture, irrespective of the presence of PZA. Conclusion: Inexpensive and reusable electrochemical sensors with a portable potentiostat are a promising tool for the detection of POA, a biomarker of PZA susceptible M. Tuberculosis. [ABSTRACT FROM AUTHOR]

• Piperidine and acetonitrile react to amidine in the presence of zinc(II). • Zinc(II) oxide, zinc(II) chloride or zinc(II) acetate were used. • Additionally, picolinate or pyrazinoate was used as an N,O -chelating agent. • Products were various amidinium salts. 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 ]·0.5H 2 O (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. [Display omitted] [ABSTRACT FROM AUTHOR]

The propensity of monocytes to migrate into sites of mycobacterium tuberculosis (TB) infection and then become infected themselves makes them potential targets for delivery of drugs intracellularly to the tubercle bacilli reservoir. Conventional TB drugs are less effective because of poor intracellular delivery to this bacterial sanctuary. This study highlights the potential of using semicrystalline delta inulin particles that are readily internalised by monocytes for a monocyte-based drug delivery system. Pyrazinoic acid was successfully attached covalently to the delta inulin particles via a labile linker. The formation of new conjugate and amide bond was confirmed using zeta potential, Proton Nuclear Magnetic Resonance (1HNMR) and Fourier transform infrared spectroscopy (FTIR). Scanning electron microscopy (SEM) confirmed that no significant change in size after conjugation which is an important parameter for monocyte targeting. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were used to establish the change in thermal properties. The analysis of in-vitro release demonstrated pH-triggered drug cleavage off the delta inulin particles that followed a first-order kinetic process. The efficient targeting ability of the conjugate for RAW 264.7 monocytic cells was supported by cellular uptake studies. Overall, our finding confirmed that semicrystalline delta inulin particles (MPI) can be modified covalently with drugs and such conjugates allow intracellular drug delivery and uptake into monocytes, making this system potentially useful for the treatment of TB.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) has become a frequently deadly infection due to increasing antimicrobial resistance. This serious issue has driven efforts worldwide to discover new drugs effective against Mtb. One research area is the synthesis and evaluation of pyrazinamide derivatives as potential anti-TB drugs. In this paper we report the synthesis and biological evaluations of a series of ureidopyrazines. Compounds were synthesized by reacting alkyl/aryl isocyanates with aminopyrazine or with propyl 5-aminopyrazine-2-carboxylate. Reactions were performed in pressurized vials using a CEM Discover microwave reactor with a focused field. Purity and chemical structures of products were assessed, and the final compounds were tested in vitro for their antimycobacterial, antibacterial, and antifungal activities. Propyl 5- (3-phenylureido)pyrazine-2-carboxylate (compound 4, MICMtb = 1.56 μg/mL, 5.19 μM) and propyl 5-(3-(4-methoxyphenyl)ureido)pyrazine-2-carboxylate (compound 6, MICMtb = 6.25 μg/mL, 18.91 μM) had high antimycobacterial activity against Mtb H37Rv with no in vitro cytotoxicity on HepG2 cell line. Therefore 4 and 6 are suitable for further structural modifications that might improve their biological activity and physicochemical properties. Based on the structural similarity to 1-(2-chloropyridin-4-yl)-3-phenylurea, a known plant growth regulator, two selected compounds were evaluated for similar activity as abiotic elicitors. [ABSTRACT FROM AUTHOR]

Pyrazinamide, the first-line antitubercular drug, has been regarded the basic component of tuberculosis treatment for over sixty years. Researchers have investigated its effect on Mycobacterium tuberculosis for this long time, and as a result, new potential targets of pyrazinamide or its active form, pyrazinoic acid, have been found. We have designed and prepared 3-(phenyl-carbamoyl)pyrazine-2-carboxylic acids as more lipophilic derivatives of pyrazinoic acid. We also prepared methyl and propyl derivatives as prodrugs with further increased lipophilicity. Antimycobacterial, antibacterial and antifungal growth inhibiting activity was investigated in all prepared compounds. 3-[(4-Nitrophenyl)carbamoyl]pyrazine-2-carboxylic acid (16) exerted high antimycobacterial activity against Mycobacterium tuberculosis H37Rv with MIC = 1.56 μg•mL-1 (5 μM). Propyl 3-{[4-(trifluoromethyl)phenyl]carbamoyl}pyrazine-2-carboxylate (18a) showed also high antimycobacterial activity against Mycobacterium tuberculosis H37Rv with MIC = 3.13 μg•mL-1. In vitro cytotoxicity of the active compounds was investigated and no significant cytotoxic effect was observed. Based to structural similarity to known inhibitors of decaprenylphosphoryl-β-D-ribose oxidase, DprE1, we performed molecular docking of the prepared acids to DprE1. These in silico experiments indicate that modification of the linker connecting aromatic parts of molecule does not have any negative influence on the binding. [ABSTRACT FROM AUTHOR]

Pyrazinoic acid (PA) is structurally similar to nicotinic acid which acts on G-protein-coupled receptor (GPR109A). GPR109A expresses in colonic and intestinal epithelial sites, and involves in DNA methylation and cellular apoptosis. Therefore, it may be assumed that PA has similar action like nicotinic acid and may be effective against colorectal carcinoma (CRC). CRC was produced via subcutaneous injection of dimethylhydrazine (DMH) at 40 mg/kg body weight once in a week for 4 weeks. After that, PA was administered orally at 2 doses of 10 and 25 mg/kg daily for 15 d to observe the antiproliferative effect. Various physiologic, oxidative stress, molecular parameters, histopathology, RT-PCR and NMR based metabolomics were performed to evaluate the antiproliferative potential of PA. Our results collectively suggested that PA reduced body weight, tumor volume and incidence no. to normal. It restored various oxidative stress parameters and normalized IL-2, IL-6, and COX-2 as compared with carcinogen control. In molecular level, overexpressed IL-6 and COX-2 genes became normal after PA administration. Again, normal tissue architecture was prominent after PA administration. Score plots of PLS-DA models exhibited that PA treated groups were significantly different from CRC group. We found that CRC rat sera have increased levels of acetate, glutamine, o-acetyl-glycoprotein, succinate, citrulline, choline, o-acetyl choline, tryptophan, glycerol, creatinine, lactate, citrate and decreased levels of 3-hydroxy butyrate, dimethyl amine, glucose, maltose, myoinositol. Further the PA therapy has ameliorated the CRC-induced metabolic alterations, signifying its antiproliferative properties. In conclusion, our study provided the evidence that PA demonstrated good antiproliferative effect on DMH induced CRC and thus demonstrated the potential of PA as a useful drug for future anticancer therapy. [ABSTRACT FROM PUBLISHER]

Tuberculosis (TB), caused by Mycobacterium tuberculosis ( Mtb ), remains a leading cause of death with 1.6 million deaths worldwide reported in 2021. Oral pyrazinamide (PZA) is an integral part of anti-TB regimens, but its prolonged use has the potential to drive the development of PZA-resistant Mtb . PZA is converted to the active moiety pyrazinoic acid (POA) by the Mtb pyrazinamidase encoded by pncA , and mutations in pncA are associated with the majority of PZA resistance. Conventional oral and parenteral therapies may result in subtherapeutic exposure in the lung; hence, direct pulmonary administration of POA may provide an approach to rescue PZA efficacy for treating pncA -mutant PZA-resistant Mtb . The objectives of the current study were to (i) develop novel dry powder POA formulations, (ii) assess their feasibility for pulmonary delivery using physicochemical characterization, (iii) evaluate their pharmacokinetics (PK) in the guinea pig model, and (iv) develop a mechanism-based pharmacokinetic model (MBM) using in vivo PK data to select a formulation providing adequate exposure in epithelial lining fluid (ELF) and lung tissue. We developed three POA formulations for pulmonary delivery and characterized their PK in plasma, ELF, and lung tissue following passive inhalation in guinea pigs. Additionally, the PK of POA following oral, intravenous, and intratracheal administration was characterized in guinea pigs. The MBM was used to simultaneously model PK data following administration of POA and its formulations via the different routes. The MBM described POA PK well in plasma, ELF, and lung tissue. Physicochemical analyses and MBM predictions suggested that POA maltodextrin was the best among the three formulations and an excellent candidate for further development as it has: (i) the highest ELF-to-plasma exposure ratio (203) and lung tissue-to-plasma exposure ratio (30.4) compared with POA maltodextrin and leucine (75.7/16.2) and POA leucine salt (64.2/19.3) and (ii) the highest concentration in ELF ( C maxELF : 171 nM) within 15.5 min, correlating with a fast transfer into ELF after pulmonary administration ( K PM : 22.6 1/h). The data from the guinea pig allowed scaling, using the MBM to a human dose of POA maltodextrin powder demonstrating the potential feasibility of an inhaled product.

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. [ABSTRACT FROM AUTHOR]

Purpose: Analog development of existing drugs and direct drug delivery to the lungs by inhalation as treatments for multiple and extensively drug resistant (MDR and XDR) tuberculosis (TB) represent new therapeutic strategies. Pyrazinamide (PZA) is critical to drug sensitive TB therapy and is included in regimens for MDR TB. However, PZA-resistant Mycobacterium tuberculosis ( Mtb) strains threaten its use. Pyrazinoic acid esters (PAEs) are PZA analogs effective against Mtb in vitro, including against the most common PZA resistant strains. However, PAEs require testing for TB efficacy in animal models. Methods: PAEs were delivered daily as aqueous dispersions from a vibrating mesh nebulizer to Mtb infected guinea pigs for 4 weeks in a regimen including orally administered first-line TB drugs. Results: PAEs tested as a supplement to oral therapy significantly reduced the organ bacterial burden in comparison to infected, untreated control animals. Thus, PAE aerosol therapy is a potentially significant addition to the regimen for PZA resistant MDR-TB and XDR-TB treatment. Interestingly, low dose oral PZA treatment combined with standard therapy also reduced bacterial burden. This observation may be important for PZA susceptible disease treatment. Conclusion: The present study justifies further evaluation of PZA analogs and their lung delivery to treat TB. [ABSTRACT FROM AUTHOR]

Summary Background Pyrazinamide (PZA) is the most important drug against the latent stage of tuberculosis (TB) and is used in both first and second line treatment regimens. The continued increase in multi-drug resistant TB and the prevalence of PZA resistance makes the development of alternative assays for prompt identification of PZA resistance all the more important. Methods We standardized and evaluated a quantitative variant of the Wayne assay (QW) for determining PZA resistance in Mycobacterium tuberculosis strains. This assay quantifies M. tuberculosis metabolism of PZA and production of pyrazinoic acid (POA) using visible spectrophotometry. We evaluated this method using PZA concentrations of 400 μg/ml and 800 μg/ml at incubation periods of 3, 5 and 7 days. M. tuberculosis strains from 68 sputum samples were also tested with the standard Wayne assay, Tetrazolium Microplate Assay (TEMA), Bactec 460TB and pncA sequencing. We compared QW and standard Wayne assay against a dichotomous reference classification using concordant Bactec 460TB and pncA sequencing. Secondarily, we determined the quantitative correlation between both QW values and TEMA's minimum inhibitory concentration (MIC) against Bactec 460TB percentage growth. Results The standard Wayne showed sensitivity of 88% and specificity of 97.5%, giving a Youden Index (YI) of 0.855 against reference tests. The QW showed maximum YI of 0.934 on day 7 at 400 μg/ml PZA with 96% sensitivity and 97.4% specificity. Absorbance OD values for 400 μg/ml PZA were more accurate than 800 μg/ml PZA. Although QW showed high accuracy for PZA susceptibility, it did not correlate quantitatively with Bactec percentage growth. TEMA testing was unreliable and did not correlate with Bactec results. Conclusions The proposed QW assay is an inexpensive method capable of providing standardization and automation of colorimetric PZA resistance testing, with better discriminatory than the standard Wayne assay. [ABSTRACT FROM AUTHOR]

N-Phenylpyrazine-2-carboxamides (anilides of pyrazinoic acids with simple substituents in various positions) were previously shown to possess significant biological activities in vitro, markedly anti-mycobacterial and photosynthesis-inhibiting activity. Based on structure-activity relationships (SAR) extracted from previously published series, 25 new anilides of non-substituted pyrazinoic acid (POA), 5-CH3-POA, 6-Cl-POA, 5- tert-butyl-POA and 5- tert-butyl-6-Cl-POA were designed and synthesised. The phenyl part was substituted with simple hydrophobic substituents chosen from methyl and halogens. 5- tert-Butyl-N-(5-fluoro-2-methylphenyl)pyrazine-2- carboxamide ( 9), N-(3-chloro-4-methylphenyl)-5-methylpyrazine-2-carboxamide ( 12), 6-chloro-N- (3-chloro-4-methylphenyl)pyrazine-2-carboxamide ( 13) and 6-chloro-N-(5-iodo-2-methylphenyl)pyrazine-2-carboxamide ( 18) possessed whole cell anti-mycobacterial activity in vitro against Mycobacterium tuberculosis H37Rv with minimum inhibitory concentration (MIC) of around 10 μM. Importantly, no cytotoxicity in the HepG2 model was detected in vitro at the concentrations tested and the estimated IC50 values were in hundreds of μM, indicating promising selectivity. N-(3-Chloro-4-methylphenyl)pyrazine-2-carboxamide ( 11) and N-(4-chloro-2-iodophenyl)pyrazine- 2-carboxamide ( 21) exerted significant activity against Mycobacterium kansasii with MIC 12.6 μM and 8.7 μM, respectively. No activity was detected against Mycobacterium avium. SAR were in accordance with those observed for the derivatives previously published. [ABSTRACT FROM AUTHOR]

In the present work the plasma levels of PZA and its two active metabolites, pyrazinoic acid (PA) and 5-hydroxy pyrazinoic acid (5-OH PA) were determined by a sensitive and rapid LC–MS/MS method. The analytes and their labeled internal standards were extracted from 200 μL plasma samples by liquid-liquid extraction with methyl tert -butyl ether: diethyl ether (90:10, v/v ) under acidic conditions. Their separation was achieved on a Zorbax Eclipse XDB C18 (100 × 4.6 mm, 3.5 μm) column using methanol and 0.1% acetic acid (65:35, v/v ) as the mobile phase within 4.0 min. Detection and quantitation were done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions, m / z 124.1 → 81.1, m / z 125.0 → 80.9 and m / z 141.0 → 81.0 for PZA, PA and 5-OH PA respectively in the positive ionization mode. All the analytes were baseline resolved with a resolution factor of 3.3 and 6.4 between PZA and its metabolites, PA and 5-OH PA respectively. The calibration curves were linear from 0.100–30.0 μg/mL, 0.03–9.00 μg/mL and 0.002–0.600 μg/mL for PZA, PA and 5-OH PA respectively with r 2 ≥ 0.9980 for all the analytes. The intra-batch and inter-batch accuracy and precision (% CV) across quality controls varied from 93.5–106.7% and 1.10–4.57 respectively for all the analytes. The mean extraction recovery of PZA, PA and 5-OH PA was 83.7%, 89.2% and 80.8% respectively, which was consistent at higher as well as lower concentration levels. The% change in the stability of analytes under different storage conditions ranged −6.7 to 7.1 for all the analytes. The method was applied to assess the comparative bioavailability of a 500 mg PZA test and reference formulation in healthy subjects. The assay reproducibility was also tested by reanalysis of 22 incurred subject samples. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) has become a frequently deadly infection due to increasing antimicrobial resistance. This serious issue has driven efforts worldwide to discover new drugs effective against Mtb. One research area is the synthesis and evaluation of pyrazinamide derivatives as potential anti-TB drugs. In this paper we report the synthesis and biological evaluations of a series of ureidopyrazines. Compounds were synthesized by reacting alkyl/aryl isocyanates with aminopyrazine or with propyl 5-aminopyrazine-2-carboxylate. Reactions were performed in pressurized vials using a CEM Discover microwave reactor with a focused field. Purity and chemical structures of products were assessed, and the final compounds were tested in vitro for their antimycobacterial, antibacterial, and antifungal activities. Propyl 5-(3-phenylureido)pyrazine-2-carboxylate (compound 4, MICMtb = 1.56 μg/mL, 5.19 μM) and propyl 5-(3-(4-methoxyphenyl)ureido)pyrazine-2-carboxylate (compound 6, MICMtb = 6.25 μg/mL, 18.91 μM) had high antimycobacterial activity against Mtb H37Rv with no in vitro cytotoxicity on HepG2 cell line. Therefore 4 and 6 are suitable for further structural modifications that might improve their biological activity and physicochemical properties. Based on the structural similarity to 1-(2-chloropyridin-4-yl)-3-phenylurea, a known plant growth regulator, two selected compounds were evaluated for similar activity as abiotic elicitors.

Pyrazinamide, the first-line antitubercular drug, has been regarded the basic component of tuberculosis treatment for over sixty years. Researchers have investigated its effect on Mycobacterium tuberculosis for this long time, and as a result, new potential targets of pyrazinamide or its active form, pyrazinoic acid, have been found. We have designed and prepared 3-(phenyl-carbamoyl)pyrazine-2-carboxylic acids as more lipophilic derivatives of pyrazinoic acid. We also prepared methyl and propyl derivatives as prodrugs with further increased lipophilicity. Antimycobacterial, antibacterial and antifungal growth inhibiting activity was investigated in all prepared compounds. 3-[(4-Nitrophenyl)carbamoyl]pyrazine-2-carboxylic acid (16) exerted high antimycobacterial activity against Mycobacterium tuberculosis H37Rv with MIC = 1.56 μg·mL−1 (5 μM). Propyl 3-{[4-(trifluoromethyl)phenyl]carbamoyl}pyrazine-2-carboxylate (18a) showed also high antimycobacterial activity against Mycobacterium tuberculosis H37Rv with MIC = 3.13 μg·mL−1. In vitro cytotoxicity of the active compounds was investigated and no significant cytotoxic effect was observed. Based to structural similarity to known inhibitors of decaprenylphosphoryl-β-d-ribose oxidase, DprE1, we performed molecular docking of the prepared acids to DprE1. These in silico experiments indicate that modification of the linker connecting aromatic parts of molecule does not have any negative influence on the binding.

[Display omitted] 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. [ABSTRACT FROM AUTHOR]