Cure of hookworm infection with a cysteine protease inhibitor

Abstract

Background: Hookworm disease is a major global health problem and principal among a number of soil-transmitted helminthiases (STHs) for the chronic disability inflicted that impacts both personal and societal productivity. Mass drug administration most often employs single-dose therapy with just two drugs of the same chemical class to which resistance is a growing concern. New chemical entities with the appropriate single-dose efficacy are needed.

Methods and findings: Using various life-cycle stages of the hookworm Ancylostoma ceylanicum in vitro and a hamster model of infection, we report the potent, dose-dependent cidal activities of the peptidyl cysteine protease inhibitors (CPIs) K11002 (4-mopholino-carbonyl-phenylalanyl-homophenylalanyl- vinyl sulfone phenyl) and K11777 (N-methylpiperazine-phenylalanyl-homophenylalanyl-vinylsulfone phenyl). The latter is in late pre-clinical testing for submission as an Investigational New Drug (IND) with the US Federal Drug Administration as an anti-chagasic. In vitro, K11002 killed hookworm eggs but was without activity against first-stage larvae. The reverse was true for K11777 with a larvicidal potency equal to that of the current anti-hookworm drug, albendazole (ABZ). Both CPIs produced morbidity in ex vivo adult hookworms with the activity of K11777 again being at least the equivalent of ABZ. Combinations of either CPI with ABZ enhanced morbidity compared to single compounds. Strikingly, oral treatment of infected hamsters with 100 mg/kg K11777 b.i.d. (i.e., a total daily dose of 200 mg/kg) for one day cured infection: a single 100 mg/kg treatment removed >90% of worms. Treatment also reversed the otherwise fatal decrease in blood hemoglobin levels and body weights of hosts. Consistent with its mechanism of action, K11777 decreased by >95% the resident CP activity in parasites harvested from hamsters 8 h post-treatment with a single 100 mg/kg oral dose.

Conclusion: A new, oral single-dose anthelmintic that is active in an animal model of hookworm infection and that possesses a distinct mechanism of action from current anthelmintics is discovered. The data highlight both the possibility of repurposing the anti-chagasic K11777 as a treatment for hookworm infection and the opportunity to further develop CPIs as a novel anthelmintic class to target hookworms and, possibly, other helminths.

Figure 1. K11777 inhibits A. ceylanicum egg hatching whereas K11002 decreases larval viability.

Eggs were recovered from hookworm-infected hamster feces and used in an egg hatch assay to measure the effect of K11002, K11777 and ABZ on egg hatching and larval viability. Data are expressed as the percentage of hatched larvae (Panels A–C) or viable larvae (Panel D) relative to controls that contained equivalent volumes of DMSO alone. NS = no survival. The results presented are representative of three separate experiments.

Figure 2. K11002 and K11777 cause morbidity in ex vivo cultured A. ceylanicum adult worms.

Adult hookworms were recovered from infected hamsters and used in an adult worm killing assay to measure the effect of K11002 (Panel A) and K11777 (Panel B) on adult worm viability. Worms were scored individually using the 5 point morbidity scale (Figure S1) at 120 hours post-treatment (HPT). Control wells were treated with ABZ (50 µM) or equivalent volumes of DMSO carrier alone. Data are expressed as means ± SD of triplicate measurements. The results presented are representative of two separate experiments.

Figure 3. ABZ combined with K11002 or K11777 enhances morbidity in ex vivo cultured A. ceylanicum adult worms.

Adult hookworms were recovered from infected hamsters and used in an adult worm killing assay to measure the effect of combining ABZ with K11002 (Panel A) or K11777 (Panel B) on adult worm survival and morbidity. Worms were scored individually using the 5 point morbidity scale (Fig S1) at different time points post-treatment. Hookworms were incubated with each compound at 25 µM. Control wells were treated with equivalent volumes of DMSO vehicle alone. Data are expressed as means ± SD of triplicate measurements. The results presented are representative of two separate experiments.

Figure 4. Oral administration of K11777 cures A. ceylanicum infection and improves blood hemoglobin levels.

Groups of Syrian hamsters (n = 6) were infected with 75 third stage A. ceylanicum larvae and followed for 24 days to monitor blood hemoglobin levels and weight gain. At 17 days post-infection (DPI) hamsters were treated orally with K11777 (100 mg/kg q.d. or b.i.d.), ABZ (10 mg/kg q.d.) or vehicle alone as indicated. At 18 DPI, one group of hamsters was treated again with K11777 (100 mg/kg b.i.d.) and one group was treated with vehicle alone. At 24 DPI, all hamsters were sacrificed and intestinal worms counted. Compared to infected vehicle controls, hamsters treated q.d. or b.i.d. with K11777 did not show significantly improved weight gain (Panel A), but levels of blood hemoglobin were significantly higher (Panel B; P<0.001 and P<0.001 at 21 and 24 DPI, respectively). Dosing q.d. significantly decreased worm burdens by 90.1% (P<0.001) whereas dosing b.i.d. resulted in cure of infection (Panel C).

Figure 5. K11777 targets cysteine protease activity in A. ceylanicum in vivo.

Soluble extracts of hookworms taken from hamsters 8 h post-treatment with single doses of ABZ (10 mg/kg), K11777 (100 mg/kg) or vehicle alone were prepared. Specific cysteine protease activity (relative fluorescence units/min/µg extract) was measured in 0.05 M sodium acetate, 2 mM DTT, pH 5.5 containing 10 µM of the dipeptidyl fluorogenic substrate Z-Phe-Arg-AMC. All activity could be inhibited by 1 µM K11777 (not shown) indicating that only cysteine protease activity was being measured. Data points are expressed as means ± SD of three separate experiments each performed in duplicate. Values for CP activity between the three experimental groups were statistically different using paired t-tests: P = 0.0024 for vehicle vs. K11777; P = 0.0074 for ABZ vs. K11777 and P = 0.0054 for vehicle vs. ABZ.