Short-course quinazoline drug treatments are effective in the Litomosoides sigmodontis and Brugia pahangi jird models

Abstract

The quinazolines CBR417 and CBR490 were previously shown to be potent anti-wolbachials that deplete Wolbachia endosymbionts of filarial nematodes and present promising pre-clinical candidates for human filarial diseases such as onchocerciasis. In the present study we tested both candidates in two models of chronic filarial infection, namely the Litomosoides sigmodontis and Brugia pahangi jird model and assessed their long-term effect on Wolbachia depletion, microfilariae counts and filarial embryogenesis 16-18 weeks after treatment initiation (wpt). Once per day (QD) oral treatment with CBR417 (50 mg/kg) for 4 days or twice per day (BID) with CBR490 (25 mg/kg) for 7 days during patent L. sigmodontis infection reduced the Wolbachia load by >99% and completely cleared peripheral microfilaremia from 10-14 wpt. Similarly, 7 days of QD treatments (40 mg/kg) with CBR417 or CBR490 cleared >99% of Wolbachia from B. pahangi and reduced peritoneal microfilariae counts by 93% in the case of CBR417 treatment. Transmission electron microscopy analysis indicated intensive damage to the B. pahangi ovaries following CBR417 treatment and in accordance filarial embryogenesis was inhibited in both models after CBR417 or CBR490 treatment. Suboptimal treatment regimens of CBR417 or CBR490 did not lead to a maintained reduction of the microfilariae and Wolbachia load. In conclusion, CBR417 or CBR490 are pre-clinical candidates for filarial diseases, which achieve long-term clearance of Wolbachia endosymbionts of filarial nematodes, inhibit filarial embryogenesis and clear microfilaremia with treatments as short as 7 days.

Keywords: Brugia pahangi; Doxycycline; Filaria; Litomosoides sigmodontis; Macrofilaricidal; Microfilariae; Quinazoline; Wolbachia.

Graphical abstract

Fig. 1

Short course treatment of L. sigmodontis-infected jirds with CBR417 and CBR490 reduces Wolbachia levels. Wolbachia ftsZ/filarial actin ratio of female adult worms isolated from jirds infected with L. sigmodontis that have been treated with doxycycline (DOX), CBR417, CBR490, vehicle control or left untreated. A: 13-week-infected jirds were either left untreated or treated twice a day (BID) with 40 mg/kg (mpk) doxycycline for 14 days, once per day (QD) with 50 mg/kg CBR417 for 4 days or BID with 75 or 25 mg/kg CBR490 for 7 days. B: 16-week-infected jirds were either treated BID with vehicle control for 7 days, BID with 40 mg/kg doxycycline for 14 days, QD with 50, 20 or 10 mg/kg CBR417 for 7 days, BID with 25 mg/kg CBR490 for 7 days, or QD with 20 or 10 mg/kg CBR490 for 7 days. Jirds were sacrificed 16 (A) or 18 (B) weeks after treatment. N = 5–6 per group. Analysis for statistical significance was done by Kruskal-Wallis followed by Dunn's multiple comparison post-hoc test. **P < 0.01; ****P < 0.0001.

Fig. 2

Embryogenesis is inhibited by CBR417 and CBR490 treatment in the L. sigmodontisjird model. Embryonic stages (oocyte, morula, pretzel, stretched mf and degenerated embryos) per female adult worms isolated from jirds infected with L. sigmodontis that were treated with doxycycline (DOX), CBR417, CBR490, vehicle control or left untreated. A: 13-week-infected jirds were either left untreated or treated bi-daily (BID) with 40 mg/kg doxycycline for 14 days, once per day (QD) with 50 mg/kg CBR417 for 4 days or BID with 75 or 25 mg/kg CBR490 for 7 days. B: 16-week-infected jirds were either treated BID with vehicle control for 7 days, BID with 40 mg/kg doxycycline for 14 days, QD with 50 or 20 mg/kg CBR417 for 7 days, or QD with 10 mg/kg CBR490 for 7 days. Jirds were sacrificed 16 (A) or 18 (B) weeks after treatment. Analysis for statistical significance was done by Kruskal-Wallis followed by Dunn's multiple comparison post-hoc test.

Fig. 3

Microfilariae levels decline in response to CBR417 and CBR490 treatment. Mf count per 10 μL of blood drawn from jirds infected with L. sigmodontis and that have been treated with doxycycline (DOX), CBR417, CBR490, vehicle control or left untreated. A: 13-week-infected jirds were either left untreated () or treated bi-daily (BID) with 40 mg/kg doxycycline for 14 days (), once per day (QD) with 50 mg/kg CBR417 for 4 days () or BID with 75 () or 25 mg/kg () CBR490 for 7 days. B: 16-week-infected jirds were either treated BID with vehicle control for 7 days (), BID with 40 mg/kg doxycycline for 14 days (), QD with 50 (), 20 () or 10 () mg/kg CBR417 for 7 days, QD with 20 () or 10 () mg/kg CBR490 for 7 days or BID with 25 mg/kg CBR490 for 7 days (). Jirds were sacrificed 16 (A) or 18 (B) weeks after treatment. N = 5–6 per group.

Fig. 4

B. pahangi microfilariae recovered at necropsy are significantly reduced in jirds treated with 40 mg/kg CBR417 given QD for 7 days (P < 0.05). Mf were recovered from the peritoneal cavities and counted from each jird 17 wpt.

Fig. 5

Embryograms of femaleB. pahangishow that CBR417 and CBR490 affect late stages of developing mf.Both CBR417 and CBR490 (40 mg/kg QD for 7 days) significantly reduced the number of later-staged mf. Female worms recovered from jirds treated with CBR417 also had significantly more degenerated embryos in their ovaries. Embryonic stages (oocytes, embryos, pre-mf, stretched mf and degenerated embryos) per female adult worm were removed from jirds infected with B. pahangi and counted from the ovaries and uteri of individual worms. Results are shown as mean relative frequencies of embryonic stages ± SEM

Fig. 6

Transmission electron micrographs reveal extensive damage to the developing B. pahangi microfilariae in ovaries of worms from jirds treated with CBR417 QD 40 mg/kg for 7 days. N = nucleus; N* = abnormal nucleus; Mf = microfilariae developing in uteri; Mf* = abnormal microfilariae in uteri; bar = 4 μm.

Fig. 7

High content imaging of ovaries showa highly significant reduction inWolbachiafrom femaleB. pahangiworms recovered from jirds treated with 40 mg/kg QD x 7 days CBR417 (P < 0.0001) and CBR490 (P < 0.0001). Ovaries from individual females were removed and stained with Wolbachia specific 16S rRNA probes using fluorescence in situ hybridization (FISH) and DAPI. Ovaries were imaged using confocal microscopy and Wolbachia quantified using high content image analysis. Means ± SD (average signal from 1 to 2 ovaries from 5 to 7 worms per treatment) are shown, and mean elimination (%) is reported. To assess significance between the vehicle control and treatment groups a one-way ANOVA with Dunnett's multiple comparisons test was used (****P < 0.0001).

Fig. 8

CBR417is highly effective in reducingWolbachiatiters in adultB. pahangi. qPCR analysis of individual worms showed there was a >99% reduction in titers in female (A) and male (B) worms compared to worms from vehicle-treated animals (****P < 0.0001).