Liver stage Plasmodium in-vitro infection

M5717 showed hepatic anti-plasmodial activity when incubated with luciferase Plasmodium berghei– Luc-infected HepG2 spheroids 24–48 h post-infection (pi) [9]. The drug has shown remarkable potency against P. berghei– Luc-infected HepG2 spheroids, with an IC₅₀ of 1.0 ± 0.1 nM and an IC₉₉ 9.9 ± 1.6 nM (Fig. 1) [10]. Therefore, this latter concentration, effective in low-metabolizing HepG2 hepatic spheroids, was used for translation to hepatic-stage activity in vivo. Given the large excess of FBS-derived proteins present in the in vitro medium relative to the drug concentration, only the human blood/plasma ratio (B/P = 1.6) was used to convert the IC₉₉a 24-h mean effective concentration at the corresponding area under the drug exposure curve (AUC) of 180 ng∙h/mL in human plasma.

Liver stage Plasmodium in-vivo infection

the P. berghei-The Luc liver-stage mouse model was used to validate the IC₉₉ value obtained in vitro [9]. Our results show that single oral doses of 30, 3 and 1.5 mg/kg of M5717 were fully effective in preventing the onset of blood-stage infection in all treated mice, while 0.3 mg/kg and 0.6 mg/kg did not or only partially prevented the development of parasitaemia (Table 1).

To correlate the in vivo exposures obtained at different oral doses with the effective concentrations previously determined in our in vitro 3D liver cell model, the AUC_0−24h was converted to a mean plasma drug concentration (C_av0−24h) over 24 h and was compared to the IC of the compound₉₉ in vitro (Table 1). Importantly, both models used a similar protocol, with a 24 h exposure time, between 24 and 48 h pi (Fig. 1). All mice for which the drug was fully effective in preventing the appearance of blood-stage parasites achieved systemic plasma C_av0−24h > 10nM. With the non-preventive dose of 0.3 mg/kg, a C_av0−24h of 4 nM was observed, which corresponded to only half of the CI₉₉ and did not prevent the development of blood-stage parasites. A dose of 0.6 mg/kg produced a C_av0−24h close to CI₉₉ value, only partially preventing the development of blood-stage parasitaemia.

Therefore, the use of in vitro IC₉₉ concentration of 10 nM as a relevant parameter for the efficacy of M5717 in P. berghei-infected liver stages were supported by the mean in vivo blood concentration of M5717 over 24 h, as inferred from the mouse model. As a result, the IC₉₉ the value has been converted to a target exposure value, i.e. AUC_{0 – 24h} of 180 ng∙h/mL which can be used for a PopPK model developed based on exposure data from Phase 1 clinical trials to simulate doses that would exceed the aforementioned AUC value.

PopPK modeling

The model was derived from results obtained from the completed Phase 1 clinical trial [4, 8]. Briefly, M5717 PK was three-compartmental, with a first order elimination, transit uptake pattern in combination with a first order uptake, and a recirculation pattern to account for a secondary peak between 24 and 30 h . Body weight was included a priori on clearance and volume parameters. The model successfully regenerated the data used to build it, as evidenced by visual predictive checks; standard diagnostic plots revealed no unacceptable trends.

Dose simulations for the prevention of development of parasitaemia

Assuming human target exposure of AUC_0−24h of 180 ng∙h/mL, simulations of the dose required to prevent blood-stage infection were performed assuming (i) full and direct extrapolation of in vitro results to humans, (ii) similar sensitivity Between Plasmodium species and to M5717 due to high sequence homology (i.e. 98.2% BLAST P. falciparum PF3D7_1451100 against P. berghei PBANKA_1314800), (iii) similar relevance of IC₉₉ (VS_{a V}) parameter, and (iv) a human blood to plasma ratio of 1:6. The results suggest that a 30 mg dose would not provide sufficient exposure, whereas 80, 100, 130 and 200 mg of M5717 would be required for parasite clearance to be observed in 81.5, 96.1, 99.7 and 100 of the subjects, respectively (Fig. 2).

Exceedance probability (purple) AUC_{0 – 24} of 180 ng∙h/mL (IC₉₉) (yellow bar). The X axes represent the total dose of M5717 as free base and the Y axes indicate the area of ​​simulated M5717 under the curve.