Clofazimine, but Not Isoniazid or Rifampicin, Augments Platelet Activation in vitro.

Ronald Anderson,Chrisna Durandt,Charles Feldman,Moloko C Cholo,Annette J Theron,Jan G Nel,Gregory R Tintinger

doi:10.3389/fphar.2018.01335

Abstract

Although the inclusion of the cationic amphiphilic, anti-mycobacterial agent, clofazimine, in the chemotherapeutic regimens of patients with multidrug-resistant tuberculosis (TB) has contributed to improved outcomes, concerns remain about the cardiotoxic potential of this agent. Accordingly, the current study was undertaken with the primary objective of investigating the effects of clofazimine, on the reactivity of human platelets in vitro, a seemingly unexplored, mechanism of cardiotoxicity. Platelet-rich plasma (PRP) prepared from the blood of healthy, adult humans was treated with clofazimine (0.625–10 mg/L), or the primary anti-TB agents, isoniazid and rifampicin (at final concentrations of 5 and 10 mg/L), followed by addition of either adenosine 5′-diphosphate (ADP) or thrombin and measurement of platelet activation according to the magnitude of expression of CD62P (P-selectin), as well as the CD62P-mediated formation of heterotypic neutrophil:platelet (NP) aggregates, using flow cytometry. Clofazimine, but neither isoniazid nor rifampicin, caused dose-related potentiation of both ADP- and thrombin-activated expression of CD62P by platelets, achieving statistical significance at threshold concentrations of 0.625 and 2.5 mg/L, respectively, as well as significant formation of N:P aggregates. These stimulatory effects of clofazimine on platelet activation were partly attenuated by pre-treatment of PRP with the membrane-stabilizing agent, α-tocopherol, possibly consistent with a membrane-disruptive mechanism. In conclusion, clofazimine, at concentrations within the therapeutic range, augments platelet activation in vitro, probably by a mechanism linked to membrane destabilization. If operative in vivo, these pro-thrombotic activities of clofazimine may predispose for development of microvascular occlusion, exacerbating an already existing high risk for development of TB-associated cardiovascular disease.

Highlights

Recent developments in the antimicrobial chemotherapy of multidrug-resistant and extensively drug-resistant (MDR/XDR)tuberculosis (TB) include recognition of the therapeutic efficacy of the “repurposed” riminophenazine agent, clofazimine (Aung et al, 2014; Tang et al, 2015; Trébucq et al, 2018) which appears to target both replicating and non-replicating Mycobacterium tuberculosis bacilli (Cholo et al, 2017)
With respect to isoniazid and rifampicin, the results for the unstimulated, and adenosine -diphosphate (ADP)-activated control system and systems treated with 5 and 10 mg/L of isoniazid are shown in Figure 1B, demonstrating lack of activity of these agents
The findings of the current study demonstrate that clofazimine, but not isoniazid or rifampicin, potentiates ADP- and thrombinmediated activation of human platelets in vitro, measured according to the magnitude of upregulation of expression of the α-granule-derived adhesion molecule, CD62P, as well as ADPactivated formation of NP heterotypic aggregates

Summary

Introduction

Recent developments in the antimicrobial chemotherapy of multidrug-resistant and extensively drug-resistant (MDR/XDR)tuberculosis (TB) include recognition of the therapeutic efficacy of the “repurposed” riminophenazine agent, clofazimine (Aung et al, 2014; Tang et al, 2015; Trébucq et al, 2018) which appears to target both replicating and non-replicating Mycobacterium tuberculosis bacilli (Cholo et al, 2017). The risk of drug-related cardiotoxicity in patients with TB may be further exacerbated by the fact that the infection per se is associated with systemic inflammation and a pro-thrombotic state, characterized by an increased risk of acute coronary events (Chung et al, 2014; Huaman et al, 2017) This risk may be intensified firstly by prolonged disease and extended chemotherapy, in those patients with comorbid disease (Pepper et al, 2010) and, secondly, by the fact that clofazimine is often used in combination with other antimycobacterial agents known to possess cardiotoxic potential, bedaquiline and delaminid (Tadolini et al, 2016; Wallis, 2016). The current study has been undertaken with the primary objective of investigating the effects of clofazimine, as well as those of the first-line anti-TB drugs, isoniazid and rifampicin, on expression of the key mediator, CD62P, by agonist-activated platelets in vitro, as well as the modulatory potential of the membrane-stabilizing agent, α-tocopherol

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Discussion

Conclusion