Research ArticlePARASITIC DISEASES

Preclinical development of an oral anti-Wolbachia macrolide drug for the treatment of lymphatic filariasis and onchocerciasis

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Science Translational Medicine  13 Mar 2019:
Vol. 11, Issue 483, eaau2086
DOI: 10.1126/scitranslmed.aau2086
  • Fig. 1 Synthesis of tylosin A analogs.

    Tylosin A (the parent compound 1) was first protected on the reactive 2′-hydroxyl group through acetylation to give compound 2. Selective activation of the mycarosyl sugar was accomplished through formation of a cyclic tin complex; this was reacted with diethylcarbamoyl chloride to give compound 3 [R = –C(O)NEt2] or with 4-fluorobenzyl chloride to give compound 3 [R = –Bn(4-F)]. Warming in methanol liberated the 2′-ester protective group to provide A-1535469 [compound 4, R = –C(O)NEt2] or A-1574083 [compound 4, R = –Bn(4-F)]. Both A-1535469 and A-1574083 were tested for efficacy against the bacterial endosymbiont Wolbachia both in insect cells and microfilariae in vitro and in worms infecting mice and gerbils in vivo.

  • Fig. 2 In vivo efficacy of two oral tylosin A analogs against Wolbachia in several animal models of nematode infection.

    The efficacy of two tylosin A analogs (A-1535469 and A-1574083) against Wolbachia in mice infected with B. malayi, L. sigmodontis, or O. ochengi is shown. Efficacy of the two drugs was tested against Wolbachia in (A) B. malayi L3/L4 larvae infecting IL-4Rα−/− BALB/c immunodeficient mice (n = 3–7) or in (B) adult female B. malayi worms infecting CCR3−/− BALB/c immunodeficient mice (n = 4–5). Tylosin A (red) was given orally or intraperitoneally (ip) where indicated; the tylosin A analogs A-1535469 (yellow) and A-1574083 (blue) were given orally. Efficacy was compared against (A) doxycycline (DOX, open symbols) or (B) minocycline (MIN, open symbols). All treatments were administered once daily for 14 days (squares) or 7 days (triangles) at the indicated doses except for minocycline (B), which was administered for 28 days (circles) twice daily (mpk, mg drug per kg body weight). (C and D) The anti-Wolbachia efficacy of orally administered A-1574083 (blue) was tested in gerbils infected with adult female B. malayi [C (n = 4–7)] or adult female L. sigmodontis [D (n = 5–6)] worms. Efficacy was compared against that for doxycycline. All treatments were administered once daily for 21 days (hexagons) or 14 days (squares) at the indicated doses except (D) doxycycline, which was administered for 14 days twice daily. (E and F) Anti-Wolbachia efficacy of oral A-1535469 (yellow) or A-1574083 (blue) was tested against male adult O. ochengi in SCID immunodeficient mice [E (n = 4–8)] or gerbils [F (n = 4–5)]. Efficacy was compared to that for minocycline (E, open symbols) or doxycycline (F, open symbols). All treatments were administered once daily for either 21 days (hexagons), 14 days (squares), or 7 days (triangles) at the indicated doses except minocycline (E), which was administered for 28 days twice daily. Data on Wolbachia depletion are presented as Wolbachia surface protein (wsp) gene copy number (A and B), Wolbachia wsp/nematode glutathione-S-transferase (gst) gene copy number ratio (C, E, and F), or Wolbachia filamenting temperature-sensitive mutant-z (ftsz)/nematode actin (act) gene copy number ratio (D). Data are presented per worm, with 5 to 15 worms per group derived from four to six animals per group and normalized to the median vehicle control Wolbachia gene copy number or ratio. Magenta lines indicate the median value for vehicle control, and blue lines indicate 90% efficacy. Short horizontal black lines above plots indicate median measurements. Data points are for single experiments (B to D and F) or else pooled from two individual experiments (A, C, and E). Significant differences, ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05 (Mann-Whitney or Kruskal-Wallis with Dunn’s tests for intergroup variation).

  • Fig. 3 In vivo efficacy of oral tylosin A analogs against B. malayi or L. sigmodontis microfilariae and worm embryonic stages.

    The embryostatic efficacy of two tylosin A analogs (A-1535469 and A-1574083), resulting in blockade of microfilarial production and subsequent depletion of mature microfilariae from mice or gerbils infected with B. malayi or L. sigmodontis, is shown. (A) The number of B. malayi microfilariae in the peritoneal cavity recovered by lavage of nematode-infected immunodeficient SCID mice 24 weeks after administration of oral A-1535469 (yellow squares) is shown (n = 7–9). Results are compared to vehicle control (gray circles) or control antibiotic treatment with minocycline (open circles). Drugs were administered at indicated doses once daily for 14 days (A-1535469, yellow squares) or twice daily for 28 days (minocycline, open circles). (B) The number of B. malayi microfilariae recovered by lavage from the peritoneal cavity of infected gerbils 6 and 12 weeks after oral administration of A-1574083 (blue squares) once daily for 14 days at the indicated dose compared with vehicle control (gray circles) is shown. Data plotted (A and B) are total yields of peritoneal microfilariae per lavage of individual animals (groups of 6 to 9). Short horizontal black lines over plots are median yields of microfilariae. (C) The number of L. sigmodontis microfilariae in the peripheral blood of infected gerbils 1 week before treatment and 2 to 16 weeks after treatment with oral A-1574083 (blue squares) compared to vehicle control (gray circles) or the antibiotic control doxycycline (open squares) is shown. Drugs were administered at indicated doses once daily (A-1574083, blue squares) or twice daily (doxycycline, open squares) for 14 days. Data and error bars are mean microfilariae counts per ml of blood ±1 SE, derived from groups of six gerbils. (D) The number of worm intrauterine embryonic stages (morulae, coiled microfilariae, and stretched microfilariae) 16 weeks after oral A-1574083 treatment (blue squares) compared to vehicle control (gray circles) or doxycycline control (open squares) is shown. Drugs were administered at indicated doses once daily (A-1574083, blue squares) or twice daily (doxycycline, open squares) for 14 days. Data plotted are the total number of released embryonic stages per individual adult female worm (groups of 7 to 11). Short horizontal lines over plots are median yields of microfilariae. Addition of +1 to all continuous variables was undertaken to visualize zero data plotted on a log scale. Significant differences, ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05, were determined by Kruskal-Wallis with Dunn’s tests for intergroup Wolbachia variation (A), Mann-Whitney tests (B), or one-way ANOVA with Dunnett’s tests for intergroup variation after logarithm to the base 10 transformation (C and D).

  • Table 1 Anti-Wolbachia activity of tylosin A analogs in vitro.

    Anti-Wolbachia activity EC50 (nM)
    Insect cellsB. malayi
    microfilariae
    A-15354691.31.6
    A-15740830.020.7
    Tylosin A3066
    Doxycycline20166
  • Table 2 Plasma concentrations of tylosin A analogs after single oral dosing in mice and gerbils.

    CompoundSpeciesDose (mg/kg)AUC0-inf (ng·hr/
    ml) (SEM)
    A-1535469BALB/c mice307800 (1620)
    10036100 (2910)
    A-1574083BALB/c mice2517.9 (0.85)
    50271 (23.7)
    752153 (1089)
    Gerbils8.9260.9 (15.5)
    473646 (619)
    82.25169 (478)
    1499200 (276)
  • Table 3 Activity of A-1574083 (ABBV-4083) in preclinical safety assays.

    AssayResult
    Chloride channel (GABA-gated)
    (antagonist radioligand)
    Ki 7.5 μM
    NK1 (human) (agonist radioligand)Ki 0.1 μM
    NK1(human) (antagonist effect)KB 0.46 μM
    Motilin (human) (agonist
    radioligand)
    IC50 > 100 μM
    ERG (human) (QPatch, human
    embryonic kidney–293 cells)
    IC50 > 28 μM
    MiniAmes test (Salmonella TA98/
    TA100)
    Negative
    In vitro micronucleus test (V79
    hamster)
    Negative
    Anesthetized dog cardiovascular
    effects (intravenous infusion)
    Minimal effects* at 12.4 μg/ml
    28-day toxicology study in
    Sprague-Dawley rats
    NOAEL: 300 mg/kg per day
    Plasma AUCss: 7.4 μg·hour/ml
    28-day toxicology study in beagle
    dogs
    NOAEL: 15 mg/kg per day
    Plasma AUCss: 3.0 μg·hour/ml

    *Monitored characteristics: mean arterial pressure, heart rate, systemic vascular resistance, cardiac output, cardiac contractility, QT interval corrected using Van de Water formula, and interval from P to R on electrocardiogram.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/11/483/eaau2086/DC1

      Fig. S1. Efficacy of tylosin A in Litomosoides larval model after intraperitoneal or oral dosing.

      Fig. S2. Study design schematics for B. malayi, L. sigmodontis, and O. ochengi efficacy models.

      Fig. S3. Preclinical PK/PD model of TylAMac analogs.

      Fig. S4. In vitro counter screening against L. loa microfilariae.

      Table S1. PK parameters of tylosin A after intraperitoneal and oral dosing in BALB/c mice.

      Table S2. B. malayi larval worm burdens 2 weeks after dosing with doxycycline, tylosin A, A-1535469, or A-1574083 in IL-4Rα−/− BALB/c mice.

      Table S3. Spectrum of TylAMac analogs and tylosin A against selected bacteria.

      Table S4. B. malayi adult worm burdens 6 weeks after dosing with minocycline, tylosin A, or A-1535469 in CCR3−/− BALB/c mice.

      Table S5. B. malayi adult worm burdens 6 weeks after dosing with doxycycline or A-1574083 in gerbils.

      Table S6. L. sigmodontis adult worm burdens 16 weeks after dosing with doxycycline or A-1574083 in gerbils.

      Table S7. O. ochengi male worm burdens 6 weeks after dosing with minocycline, A-1535469, or A-1574083 in SCID mice.

      Table S8. O. ochengi male worm burdens 6 weeks after dosing with doxycycline or A-1574083 in gerbils.

      Table S9. B. malayi adult worm burdens 24 weeks after dosing with doxycycline or A-1535469 in SCID mice.

      Data file S1. Source data for figures.

    • The PDF file includes:

      • Fig. S1. Efficacy of tylosin A in Litomosoides larval model after intraperitoneal or oral dosing.
      • Fig. S2. Study design schematics for B. malayi, L. sigmodontis, and O. ochengi efficacy models.
      • Fig. S3. Preclinical PK/PD model of TylAMac analogs.
      • Fig. S4. In vitro counter screening against L. loa microfilariae.
      • Table S1. PK parameters of tylosin A after intraperitoneal and oral dosing in BALB/c mice.
      • Table S2. B. malayi larval worm burdens 2 weeks after dosing with doxycycline, tylosin A, A-1535469, or A-1574083 in IL-4Rα−/− BALB/c mice.
      • Table S3. Spectrum of TylAMac analogs and tylosin A against selected bacteria.
      • Table S4. B. malayi adult worm burdens 6 weeks after dosing with minocycline, tylosin A, or A-1535469 in CCR3−/− BALB/c mice.
      • Table S5. B. malayi adult worm burdens 6 weeks after dosing with doxycycline or A-1574083 in gerbils.
      • Table S6. L. sigmodontis adult worm burdens 16 weeks after dosing with doxycycline or A-1574083 in gerbils.
      • Table S7. O. ochengi male worm burdens 6 weeks after dosing with minocycline, A-1535469, or A-1574083 in SCID mice.
      • Table S8. O. ochengi male worm burdens 6 weeks after dosing with doxycycline or A-1574083 in gerbils.
      • Table S9. B. malayi adult worm burdens 24 weeks after dosing with doxycycline or A-1535469 in SCID mice.

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Data file S1 (Microsoft Excel format). Source data for figures.

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