Amelioration of hepatotoxicity by biocleavable aminothiol chimeras of isoniazid: Design, synthesis, kinetics and pharmacological evaluation

ARTICLE HIGHLIGHTS

Research background

Tuberculosis (TB) is widely viewed as a disease of the developing world and death rates are often attributed to failure of overburdened public health systems to deliver appropriate care to infected individuals. The logistics of delivering the complex regimen and ensuring patient compliance with the full course of chemotherapy challenge the resources of the public health sector, even in the most developed countries. TB drug development has made substantial progress in the past decade. There are currently at least 10 drugs being evaluated in clinical trials. Some belong to chemical classes already employed in first- or second-line treatment regimens and are being explored for more optimized use at higher doses or in new drug combinations (oxazolidinones, rifamycins and fluoroquinolones), while others represent potential novel members of the TB drug arsenal, killing Mycobacterium tuberculosis through previously untried mechanisms of action (diarylquinolines, nitroimidazoles, pyrroles and ethylene diamines). The typical challenges of drug development are augmented in TB by the complexity of the disease, the requirement for multi-drug regimens, the relative lack of TB drug development for the past several decades, and inadequate resources being brought to bear despite the urgency of the global medical need. Yet, in the face of these challenges, none of the drugs have succeeded to reach the patients. The urgent need for innovation and persistent efforts to tap novel resources cannot be denied. A fine balance needs to be achieved between protecting novel drugs or modified derivatives of existing drugs so that resistance, side effects and toxicity could be minimized, ensuring that regimens are low-cost, safe, readily available, and adopted by healthcare systems and providers. The current treatment regimen has several drawbacks, including prolonged treatment time to completely eradicate the bacteria (sterilization). This increases the risk of toxicity associated with long-term use of antitubercular drug. Isoniazid (INH) is a major first-line drug used for the treatment of TB, although the metabolic and morphological aberrations that it causes and emergence of its resistance on wide scale have been a matter of great concern for future treatment schedules of TB. Therefore, a suitable molecular modification of INH in the form of codrugs was performed in order to resolve these issues. Antioxidant aminothiols were selected as carriers to minimize hepatotoxic effects of INH. The hepatoprotective potential of these prodrugs was investigated in Wistar rats to prove effectiveness in abrogating liver damage caused by INH.

Research motivation

The root cause of INH toxicity is believed to be in the metabolism of INH at the N2 centre in hydrazinic chain by the enzymes N-acetyltransferases, which are responsible for acetylation of INH. This acetylation by N-acetyltransferases in humans is under genetic control and can be divided into two categories, viz. “fast acetylators” and “slow acetylators”. The fast acetylators, in long-term treatment of INH, lead to significant lowering of drug bioavailability and consequent generation of INH resistance. Whereas in the case of slow acetylators, high levels of INH lead to serious hepatotoxicity. In both the cases, optimization of dose is a major issue. INH after metabolism in the liver produces hydrazine metabolites (nitrogen-centred free radicals). These radicals generate highly reactive oxygen species, which act as stimulators of lipid peroxidation, resulting in cell death and hepatic necrosis. Acute poisoning leads to lactic acidosis and renal failure, development of agranulocytosis, INH-induced tenosynovitis, INH-induced liver injury, and fatal INH-induced acute liver failure, to name only a few of the toxic consequences of INH. Furthermore, INH and/or its metabolites (e.g., hydrazine) are associated with causing mitochondrial injury that may lead to oxidant stress in mitochondria and destruction of energy homeostasis. This specific observation inspired us to design prodrugs of INH; transiently masking the N2 centre in the hydrazinic chain of the INH moiety by using aminothiols which could serve by protecting the liver against toxic effects of INH. Previous studies have shown that aminothiols have antioxidant potential and they attenuate liver injury induced by INH, acetaminophen, fluoride, cisplatin, carbon tetrachloride and lead overdose. But, none of the studies have been based on introducing these antioxidant promoeities [N-acetyl cysteine (NAC), N-(2-mercaptopropionyl) glycine (MPG) and L-methionine (Met)] in the INH molecule via amide linkage at the hydrazinic centre and exploring their therapeutic potential. This study is the first to explore the hepatoprotective potential of aminothiol-INH conjugates for restoration of normal hepatic physiology in INH-intoxicated rats and their possible healing mechanism. It was foreseen that these prodrugs may find utility in safer treatment of TB.

Research objectives

The chief objective of this work was to minimize hepatotoxic effects of the antitubercular drug INH and, thereby, improve its safety profile in the management of TB. As metabolism at the hydrazinic chain of INH is responsible for possible side effects, we thought of masking the N2 centre in the hydrazinic chain transiently. Hepatoprotective action was achieved by using aminothiols such as NAC, MPG and Met as antioxidant carriers, which act by scavenging free radicals. Signs of liver injury did not manifest in the prodrug-treated groups, which was one of the important objectives of the present study. Future research could be directed at investigating the in vivo antimycobacterial potential of these prodrugs.

Research methods

Facile synthesis of target mutual prodrugs was accomplished through optimization of the Schotten-Baumann reaction and Boc-anhydride to avoid complex purification procedures. Spectral analysis was used for extensive characterization of synthesized prodrugs. Novel HPLC methods were developed and validated for simultaneous estimation of INH and aminothiols in the presence of intact prodrugs in order to study their release profiles in buffers of varied pH, rat homogenates of the gastrointestinal tract, blood, urine and faeces. For in vivo study, male Wistar rats weighing 180-220 g were fasted for 24 h. The animals were given drug solution in stipulated dose, quantity depending on the body weight of each animal. At 0 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 hr and 24 h of treatment, 3 mL of blood was withdrawn by retro-orbital puncture into EDTA-coated tubes and centrifuged at 5000 rpm at 0-5 °C for 10 min. A 0.1 mL aliquot of the supernatant solution of centrifuged blood was added to an Eppendorf tube and 0.9 mL of methanol added to it for immediate plasma protein precipitation. The solution was vortexed for 2 min and then centrifuged at 5000 rpm for 10 min at 0-5 °C in order to precipitate solid matter present in the biological sample and other impurities. Then, 20 μL of the supernatant was injected into the HPLC instrument. Hepatoprotective potential was evaluated in male Wistar rats in a 21-d study. Doses calculated on equimolar basis were administered for 21 d to respective groups. All the animals were examined for liver function markers, antioxidant markers, biochemical parameters and liver histology. Livers of the sacrificed animals were removed and fixed in 10% buffered formalin and samples were sent for microscopic examination. Various markers like aminotransferases, superoxide dismutase, glutathione peroxidase and malondialdehyde, cholesterol and triglycerides were estimated at the end of study. Relevant statistical tests were used for analysing the data.

Research results

Activation of prodrugs by amidases in the small intestine, restoration of enzyme levels, re-establishment of the antioxidant defence system to avert the formation of excessive free radicals, regeneration of hepatocytes, maintenance of structural integrity of liver by released aminothiols and significantly notable reparation of INH-induced hepatotoxicity in Wistar rats were the promising outcomes of this study. This is the pioneer study to identify and describe the therapeutic potential of hepatoprotective prodrugs of INH in Wistar rats. These prodrugs could be explored further as an alternative to INH for treatment of TB.

Research conclusions

In the present work, the N2 centre of the hydrazinic chain in INH was transiently masked with aminothiols, as literature review revealed that antioxidant aminothiols play a vital role in restoring the antioxidant defence system of a host after a free radical-mediated destructive autocatalytic process which occurs in INH-induced hepatotoxicity. The novel hepatoprotective prodrug strategy proved to be advantageous in terms of lowering the biochemical parameters and liver function markers, and remarkably improving the levels of enzymes involved in the antioxidant defence system. This study emphasized the effectiveness of aminothiols to maintain integrity of the liver. These prodrugs have the potential to be screened further for their effectiveness in patients who are on long-term treatment with INH.

Research perspectives

This study proved that concept-based design of hepatoprotective prodrugs can be applied successfully in overcoming toxic effects of a drug. This work proved that an amide conjugation strategy can provide a potential synergistic effect in abrogation of INH-induced hepatotoxicity. Future research should be directed towards investigation of in vivo antimycobacterial potential and pharmacokinetic profiles in order to elucidate the exact molecular and biochemical mechanisms of these synthesized prodrugs.