Comparison of Toxicities among Different Bumped Kinase Inhibitor Analogs for Treatment of Cryptosporidiosis

Abstract

Recent advances on the development of bumped kinase inhibitors for treatment of cryptosporidiosis have focused on the 5-aminopyrazole-4-carboxamide scaffold, due to analogs that have less hERG inhibition, superior efficacy, and strong in vitro safety profiles. Three compounds, BKI-1770, -1841, and -1708, showed strong efficacy in C. parvum infected mice. Both BKI-1770 and BKI-1841 had efficacy in the C. parvum newborn calf model, reducing diarrhea and oocyst excretion. However, both compounds caused hyperflexion of the limbs seen as dropped pasterns. Toxicity experiments in rats and calves dosed with BKI-1770 showed enlargement of the epiphyseal growth plate at doses only slightly higher than the efficacious dose. Mice were used as a screen to check for bone toxicity, by changes to the tibia epiphyseal growth plate, or neurological causes, by use of a locomotor activity box. These results showed neurological effects from both BKI-1770 and BKI-1841 and bone toxicity in mice from BKI-1770, indicating one or both effects may be contributing to toxicity. However, BKI-1708 remains a viable treatment candidate for further evaluation as it showed no signs of bone toxicity or neurological effects in mice.

Keywords: BKI-1708; BKI-1770; BKI-1841; Cryptosporidium; bumped kinase inhibitors; calcium-dependent protein kinases; cryptosporidiosis; epiphyseal growth plate.

FIG 1

(A) Structures of probable metabolites of BKI-1770 identified by hepatocyte metabolism in vitro. For BKI-1770, corresponding ions for three metabolites were observed when in vivo mouse plasma samples were extracted and scanned on LC/MS-MS. Metabolite 1 (M1) was synthesized for quantification purposes. This stock of M1 was estimated to be a 50:50 mix of the hemiaminal (m/z: 380.4) and its dehydrated product (m/z: 362.4) labeled M1a. Metabolite 3 (M3) was synthesized as 1770-O-glucuronide. Metabolite 5 (M5) was synthesized for quantification purposes. No corresponding m/z signals were observed in mouse plasma on the LC/MS-MS scan for metabolite 2 (M2), metabolite 4 (M4), or metabolite 6 (M6) or 6a (M6a). (B) Structures of probable metabolites of BKI-1841 identified by hepatic S9 fraction metabolism in vitro. For BKI-1841, no corresponding ions for metabolite 1 (M1), metabolite M1a (M1a), or metabolite M1b (M1b) were observed when in vivo mouse plasma samples were extracted and scanned on LC/MS-MS. m/z = mass to ion ratio.

FIG 2

Pharmacokinetics of BKI-1841 in mouse plasma and brain tissue after a single 25 mg/kg oral dose. n = 3.

FIG 3

Pharmacokinetics of BKI-1770 and its metabolites in mouse plasma, brain tissue, and gastrointestinal tissues after a single 30 mg/kg oral dose. n = 3.

FIG 4

Efficacy of BKI-1841 in interferon-γ knockout mice infected with nanoluciferase expressing C. parvum. Treatments were separated into two separate experiments for BKI-1841. Untreated control groups for each experiment are grouped with their associated treatment in the figure keys. Dotted line denotes the limit of detection. Treatments were dosed on days 6 through 10 postinfection. Significant differences were observed between the control and 60 mg/kg and 30 mg/kg groups (P < 0.05) but no significant difference was observed for the 15 mg/kg or 5 mg/kg dose. n = 3. QD = once daily.

FIG 5

Efficacy of BKI-1770 and BKI-1841 in neonatal calves infected with C. parvum. Total oocyst excretion determined by RT-PCR concentration and total by fecal weight shed per day. (A) Oocyst excretion per day after treatment with BKI-1770. (B) Cumulative total oocyst excretion for days 3 through 10 after treatment with BKI-1770. (C) Oocyst excretion per day after treatment with BKI-1841. (D) Cumulative total oocyst excretion for days 3 through 10 after treatment with BKI-1841. BKI-1770 treatments were administered on days 2 through 6 postinfection. BKI-1841 BID treatments were administered on days 2 through 4 and QD treatments were administered on days 2 through 5. For BKI-1770, significant differences in total oocyst excretion per day were observed for all dosed groups compared to controls (P < 0.05). For BKI-1841, significant differences in total oocyst excretion per day were observed for the 5 mg/kg and both 2 mg/kg groups (P < 0.05) but no significant difference was observed for the 1 mg/kg group compared to controls. QD = once daily, BID = twice daily.

FIG 6

Efficacy of BKI-1770 in neonatal piglets infected with C. hominis. Piglets were inoculated orally with C. hominis oocysts 2 days after birth and treatment with BKI-1770 at 5 mg/kg BID began 3 days postchallenge (day 0 post treatment). Rectal swabs were processed for oocyst count and DNA measurement. The plot represents mean ± SEM. Wilcoxon matched-pairs signed rank test was conducted using GraphPad Prism 7.03. Significant differences were observed between controls and the 5 day-dose (P = 0.001) and the 8 day-dose (P = 0.01). The 5 day doses were given on days 0–4, the 8 day doses were given on days 0–7.

FIG 7

Bone toxicity of BKI-1770, BKI-1745, BKI-1708, and BKI-1841 as observed in changes to the width between the borders of the epiphyseal growth plate of the left and right tibia in 3–4 week old mice. Mice were dosed by oral gavage with either 100 mg/kg (BKI-1745, -1708) or 150 mg/kg (BKI-1770, -1841) QD for 7 days and euthanized 24 h after the final dose. Tibia were removed and processed for staining immediately after euthanasia. Statistical significant differences (ANOVA; P < 0.001) were observed for BKI-1770 and BKI-1745. n = 3. QD = once daily, NS = not significant.