TPT sulfonate, a single, oral dose schistosomicidal prodrug: In vivo efficacy, disposition and metabolic profiling

Abstract

Treatment of schistosomiasis relies precariously on just one drug, praziquantel (PZQ). In the search for alternatives, 15 S-[2-(alkylamino)alkane] thiosulfuric acids were obtained from a previous research program and profiled in mice for efficacy against both mature (>42-day-old) and juvenile (21-day-old) Schistosoma mansoni using a screening dose of 100 mg/kg PO QDx4. One compound, S-[2-(tert-butylamino)-1-phenylethane] thiosulfuric acid (TPT sulfonate), was the most effective by decreasing female and male worm burdens by ≥ 90% and ≥46% (mature), and ≥89% and ≥79% (juvenile), respectively. In contrast, PZQ decreased mature female and male worm burdens by 95% and 94%, respectively, but was ineffective against juvenile stages. Against 7-day-old lung-stage worms, TPT sulfonate was only effective at twice the dose decreasing female and male burdens by 95 and 80%, respectively. Single oral doses at 400 and/or 600 mg/kg across various developmental time-points (1-, 7-, 15-, 21- and/or 42 day-old) were consistent with the QD x4 data; efficacy was strongest once the parasites had completed lung migration, and female and male burdens were decreased by at least 90% and 80%, respectively. In vitro, TPT sulfonate is inactive against the parasite suggesting a pro-drug mechanism of action. In mice, TPT sulfonate is fully absorbed and subject to rapid, non-CYP-mediated, first-pass metabolism that is initiated by desulfation and yields a series of metabolites. The initially-formed free thiol-containing metabolite, termed TP thiol, was chemically synthesized; it dose-dependently decreased S. mansoni and Schistosoma haematobium motility in vitro. Also, when administered as a single 50 mg/kg IP dose, TP thiol decreased 33-day-old S. mansoni female and male burdens by 35% and 44%, with less severe organomegaly. Overall, TPT sulfonate's efficacy profile is competitive with that of PZQ. Also, the characterization of a parasiticidal metabolite facilitates an understanding and improvement of the chemistry, and identification of the mechanism of action and/or target.

Keywords: Alkylaminoalkanethiosulfuric acid; Anthelmintic; Drug disposition; Drug metabolism; Schistosoma; Schistosomiasis.

Graphical abstract

Fig. 1

Efficacy of those most potent compounds of the 15 thiosulfates tested against mature S. mansoni infections in mice. Compounds 1, 2 and 8 and PZQ (A-D respectively) were administered orally by gavage at 100 mg/kg QDx4 in 150 μL 2.5% Kolliphor EL 42 days post-infection (dpi) with 150 S. mansoni cercariae. Means ± SD of female (circle) and male worms (squares) recovered by perfusion from mice (n = 3–6 per group) at 62 (A), 56 (B), (C) 59 and 60 dpi (D), respectively, are indicated. Significance relative to vehicle controls for each sex was measured using the Student's unpaired t-test with a two-tailed distribution; significance (p < 0.05) is indicated by asterisks. See Table S1 (worksheets 4, 8, 5 and 14, respectively) for further details on data per mouse.

Fig. 2

Effect of TPT sulfonate (compound 2) on worm length and mass. TPT sulfonate was administered orally by gavage to mice at 100 mg/kg QDx4 in 150 μL 2.5% Kolliphor EL 42 days post-infection (dpi) with 150 S. mansoni cercariae. Vehicle control worms (A) and those exposed to TPT sulfonate (B) were recovered by perfusion at 56 dpi. Worms exposed to TPT sulfonate are approximately 50% shorter and less massive. Males can be distinguished from females by being thicker and paler in color. Bars = 0.6 cm.

Fig. 3

Effect of worm age on efficacy of TPT sulfonate as compared to PZQ in mice infected with S. mansoni. TPT sulfonate (compound 2) was administered orally by gavage at 100 mg/kg QDx4 in 150 μL 2.5% Kolliphor EL at 7, 21 and 42 (A), and 11 days post-infection (dpi) (B) with 150 S. mansoni cercariae. PZQ was administered under the same conditions at 7 and 21 (C), and 42 dpi (D) with 150 S. mansoni cercariae. Means ± SD of female (circle) and male worms (squares) recovered from mice (n = 3–6 per group) 56 (A), 35 (B), 49 (C) and 60 dpi (D) are indicated. Significance relative to vehicle controls for each sex was measured using the Student's unpaired t-test with a two-tailed distribution; significance (p < 0.05) is indicated by asterisks. The data shown for vehicle and 42 dpi in panels A and D are the same as those shown in Fig. 1B and D, respectively, and are provided here for easier comparisons. See also Table S1 (worksheets 2, 8 and 9 for TPT sulfonate, and worksheets 14 and 15 for PZQ) for details.

Fig. 4

Dose-ranging of TPT sulfonate indicates that mature female S. mansoni are more susceptible than males. (A) TPT sulfonate was administered orally by gavage at the doses (mg/kg) indicated in 150 μL 2.5% Kolliphor EL at 42 days post-infection (dpi) with 150 S. mansoni cercariae. Means ± SD of female (circle) and male worms (squares) recovered from mice (n = 3–6 per group) 56 dpi are indicated. (B) For comparison, PZQ was administered at the doses indicated under the same conditions. Significance relative to vehicle controls for each sex was measured using the Student's unpaired t-test with a two-tailed distribution; significance (p < 0.05) is indicated by asterisks. See Table S1 (worksheets 10 and 17 for TPT sulfonate and PZQ, respectively) for details.

Fig. 5

Increasing the single oral dose of TPT sulfonate can cure mice infected with S. mansoni: again efficacy depends on worm age. TPT sulfonate was administered orally by gavage at the doses (mg/kg) indicated in 150 μL 2.5% Kolliphor EL at 7, 21 and 42 days post-infection (dpi) (A), and 1, 7 and 15 dpi (B) with 150 S. mansoni cercariae. Means ± SD of female (circle) and male worms (squares) recovered from mice (n = 3–6 per group) 52 (A) and 44 dpi (B) are indicated. Significance relative to vehicle controls for each sex was measured using the Student's unpaired t-test with a two-tailed distribution; significance (p < 0.05) is indicated by asterisks. See Table S1 (worksheets 11 and 12 for (A) and (B), respectively) for details.

Fig. 6

Increasing the dose of TPT sulfonate from 100 QDx4 to 200 QDx4 significantly improves efficacy against 7 day-old lung worms. TPT sulfonate was administered orally by gavage at the dose 200 mg/kg QDx4 in 150 μL 2.5% Kolliphor EL at 7 days post-infection (dpi) with 150 S. mansoni cercariae. Means ± SD of female (circle) and male worms (squares) recovered from mice (n = 3–5 per group) 57 dpi are indicated. Significance relative to vehicle controls for each sex was measured using the Student's unpaired t-test with a two-tailed distribution; significance (p < 0.05) is indicated by asterisks. See Table S1 (worksheet 13) for details. Also, compare these data to those shown for 100 mg/kg QDx4 in Fig. 1A.

Fig. 7

Biotransformation of TPT sulfonate by hepatocytes is required for expression of anti-schistosomal activity in vitro. (A) Supernatants (100, 200, 400 or 700 μL) from rat hepatocytes that had been incubated with 750 μM TPT sulfonate for 30 min were added to 200 μL cultures of adult S. mansoni. Worm motility was measured with WormAssay after 2 h (black bars) and 18 h (grey bars). No hep. = a control whereby TPT sulfonate was incubated in hepatocyte medium but in the absence of hepatocytes before transfer of 700 μL of the medium to the parasites. No TPT-S = a control whereby hepatocytes had been incubated in the absence of TPT sulfonate before transfer of 700 μL of the medium to the parasites. The means ± SD values from a 60 s WormAssay recording from one of two similar experiments are shown. (B) and (C) Images of worms incubated with 400 μL supernatant from rat hepatocytes that had been incubated with 750 μM TPT sulfonate for 30 min (B) A coiled female worm 18 h after incubation: the parasite's head is toward the center of view. (C) Anterior end of a male worm 18 h after a similar incubation: the oral sucker is to the left. For both images, arrows point to fraying and blebbing of the surface tegument. (D) and (E) Images of a female and male worm, respectively, incubated with 400 μL supernatant from rat hepatocytes that had been incubated in the absence of TPT sulfonate for 30 min. Bars represent 150 μm (B, D) and 50 μm (C, E), respectively.

Fig. 8

Relative ion abundance of TPT sulfonate metabolites from mouse plasma and a mouse hepatocyte digest. MS/MS peak areas are compared for metabolites observed in (A) mouse plasma 4 h after 100 mg/kg PO dosing and in (B) a mouse hepatocyte incubation after 20 min, using 400 μM TPT sulfonate and 3 × 10⁶ cells/m. Solid colors represent metabolites depicted in Fig. 9; cross-hatching indicates metabolites differing from those in the figure by two mass units (possibly representing a gain or loss of a double bond) and vertical stripes indicate metabolites of unknown structure. Data were obtained via m/z 56 neutral loss scanning from m/z 100 to 320. The m/z 208 compound is not a metabolite per se, but a decomposition product that forms in the inlet to the API 4000 MS from the m/z 417 thiol dimer.

Fig. 9

Scheme of putative reactions that generate the major early-eluting metabolites of TPT sulfonate observed in mouse plasma. Sequential desulfations form three pairs of compounds with a mass difference of 80 (see Figures S2 and S3). All of these metabolites except the m/z 270 were also identified in hepatocyte incubations (Fig. 8B). Diagonal arrows represent a possible alternate pathway in which phenyl oxidation precedes sulfhydryl oxidation.

Fig. 10

Plasma-time profiles of TPT sulfonate and three of its metabolites in mice after IV and oral dosing. Mice were administered TPT sulfonate IV (4 mice, 15 mg/kg) or PO (8 mice, 100 mg/kg), as described in the Materials and Methods. Mean TPT sulfonate plasma concentrations and metabolite MS/MS peak areas were then determined. (A) TPT sulfonate (retention time = 1.14 min). (B) The m/z 210 thiol metabolite initially formed from desulfation of TPT sulfonate (retention time = 1.10 min). (C) The m/z 256 alkenyl sulfonic acid metabolite formed by oxidation of the m/z 210 thiol (retention time = 0.82 min). (D) The m/z 176 2-phenylethenamine metabolite(s) formed by desulfation of the m/z 256 metabolite. Three retention times were observed for the m/z 176 metabolite, whereas in our original mouse PK study this m/z was represented by a single peak (see Figure S3C and Discussion). Note: these retentions are from a different LC method than those in shown in Figure S2 and Figure S3.

Scheme 1

Synthesis of m/z 210 thiol (TPT thiol) and 176 styrene metabolites.

Fig. 11

In vitro bioactivity of the chemically synthesized m/z 210 metabolite (TP thiol) against adult S. mansoni and S. haematobium, and apparent lack of cell toxicity. (A) Adult S. mansoni (circles) or S. haematobium (squares) were incubated for 8 h with different concentrations of synthesized TP thiol (as the m/z 417 dimer). Points represent the means ± SD values across three wells at each concentration and one of two experiments is shown. (B) Counter toxicity screens with various mammalian cell lines incubated in the presence of TP thiol were performed as described in the Materials and Methods. After 24 h, cells were fixed with 10% paraformaldehyde and nuclei stained with DAPI. Nuclei were counted across 5–10 fields of view/well using an inverted microscope fitted with a 20x objective lens. Nuclei counts are expressed as percentages relative to DMSO controls. Points represent mean counts across a minimum of three wells for each concentration tested and the S.D. value for each mean was ≤20%. Data from one of two experiments performed are shown.