Efficacy, Pharmacokinetics and safety evaluation of Cryptolepine-Artemisinin based combinations in the management of uncomplicated malaria.

The emergence of Plasmodium falciparum strains (multidrug resistant) resistant to commonly used antimalarial agents has motivated the research for novel drugs and drug combinations as new strategies for the prophylaxis and treatment of malaria. Cryptolepis sanguinolenta, a popular West African antimalarial plant has been used for several decades for the treatment of malaria and other infections. Cryptolepine is the major alkaloid isolated from the plant with remarkable antimalarial activity and by far the most studied. This study was conducted to characterize some drug-likeness properties of cryptolepine and determine whether a safe and novel antimalarial combination could be developed in combination with the artemisinin derivatives. The in vitro efficacy of cryptolepine and the aqueous root extract of Cryptolepis sanguinolenta against the late stage gametocytes of P. falciparum (NF54) using the PrestoBlue® assay was also ascertained in this study. In vivo pharmacokinetics in rat and the in vitro absorption, distribution and metabolism of cryptolepine were also characterised in rat and human models. Cryptolepine hydrochloride was isolated from the root of C. sanguinolenta and identified by Ultra Violet (UV) spectra, Thin Layer chromatography (TLC), High Performance Liquid chromatography (HPLC) and melting point determination studies. A SYBR Green I fluorescent-based in vitro drug sensitivity assay using a fixed ratio method was performed on the chloroquine-sensitive plasmodial strain 3D7 to build isobolograms from cryptolepine-based combinations with standard antimalarial drugs. Cryptolepine exhibited promising synergistic interactions in vitro with artesunate, artemether, dihydroartemisinin and amodiaquine. The combination of cryptolepine with chloroquine and lumefantrine showed an additive effect whereas antagonism was observed with mefloquine in the isobologram analysis. In vivo, the Rane’s test in ICR mice infected with Plasmodium berghei NK-65 strains was used to build an isobologram from cryptolepine-artesunate fixed ratio combination (1:1) and fractions of their ED50s. Cryptolepine combination with artesunate again showed synergy as the Zexp was 1.02±0.02 mg/kg which was iv significantly lesser than the Zadd of 8.3±0.31 mg/kg. The aqueous root extract of C. sanguinolenta and its major alkaloid, cryptolepine had minimal inhibitory effects on the late stage gametocytes from Plasmodium falciparum strain NF54. In the in vitro pharmacokinetic assays, cryptolepine showed a high passive permeability, a low human P-gp efflux potential, a good metabolic stability and a moderate protein binding in rat and human plasma. The preliminary incubation in human and rat hepatocyte showed a low to moderate hepatic extraction. Nine metabolites were identified in human and rat hepatocytes, resulting from metabolic pathways involving oxidation (M2-M9) and glucuronidation (M1, M2, M4, M8 and M9). Some of the metabolites were also identified in the urine (M2, M6 and M9) and plasma of rats (M6). The enzyme phenotyping assay and the metabolites identified in the hepatocytes suggests that both cytosolic and microsomal liver enzymes may be involved in the metabolism of cryptolepine in rat and human hepatocytes and among them may be aldehyde oxidase, UDP-glucuronyltransferase and the cytochrome P450 enzyme system. The in vivo rat Pharmacokinetic profile of cryptolepine showed very high clearance and volume of distribution (Vss), a moderate half-life, low oral exposure, early Tmax, and a low Cmax. Elimination was faster and systemic exposure (AUC) to cryptolepine was low to moderate in rats with unchanged excretion of cryptolepine in the urine less than 0.1% of the administered dose. This indicates metabolism, unchanged drug and/or biliary excretion as the main clearance pathway(s). The haematological, biochemical, organ/body weight ratio and histopathology indices in the rats treated with cryptolepine at all doses (25, 50, 100 mg/kg p.o) and in combination with artesunate (4 mg/kg) or artemether (50 mg/kg) showed no significant acute toxicity compared to the control group. All treatments presented no morphological changes in the kidney, spleen, stomach, and liver tissues used in the histopathology study. These findings provide a strong basis for the selection of cryptolepine as a potential lead compound in the development of combination therapy against malaria.

Source: KNUST