ML modulation of the GABA-gated currents.

a<p>Concentration that evoked 50% of the maximal response for each ML.</p>b<p>Maximum potentiation relative to GABA alone.</p>c<p>n = 4 for calculation of concentration to reach E_max and E_max (%) for MOX, because for one egg, it was not possible to calculate the MOX E_max (%) from the fitted curve.</p>*<p>p<0.05 vs IVM;</p>***<p>p<0.001 <i>vs</i> IVM.</p

Absolute brain accumulation of MLs in Mdr1ab(−/−) and wild-type mice as a function of the administrated dose.

<p>IVM (filled squares) or MOX (empty squares) was administered to Mdr1ab(−/−) mice or to wild-type mice at increasing doses. Highest doses used for each ML were below the LD₅₀ to ensure a non-lethal effect of the administration. Mice in each group were sacrificed 24 h after treatment and drug concentrations were determined in brain and plasma. Absolute brain accumulation was plotted against the administrated dose in (<b>A</b>) Mdr1ab(−/−) or (<b>B</b>) wild-type mice. A positive and significant linear correlation was observed between brain uptake and the administered dose rate (R²>0.94 in all cases). All measurements are expressed as mean ± S.D. of six animals.</p

Drug concentration in plasma and brain 2 and 24 h after SC administration of an equivalent molar dose rate of MLs in Mdr1ab(−/−) mice.

<p>Ivermectin (IVM) or moxidectin (MOX) was administered subcutaneously in Mdr1ab(−/−) mice (6 per drug) at similar molar dose rate (0.23 µmol/kg, corresponding to 0.20 mg/kg and 0.15 mg/kg for IVM and MOX, respectively). Mice were sacrificed at 2 or 24 h after treatment. Drug concentrations were determined in plasma and brain, and the brain/plasma concentration ratios were calculated.</p>*<p>p<0.05,</p>**<p>p<0.01,</p>***<p>p<0.001 <i>vs</i> IVM.</p

Concentration-response curves of rat GABA(A) receptor expressed in <i>Xenopus</i> oocytes.

<p>(<b>A</b>) Average concentration-response curve for the reference agonist GABA alone. Data were normalized to the maximum GABA-evoked response and fitted to the Hill equation (EC₅₀ = 12.8±0.3 µM, Hill slope = 1.30±0.02. Data are given as mean ± S.E.M. from 3 independent oocytes batches (n = 4 oocytes for each batch). (<b>B</b>) Concentration-dependent potentiation of the GABA receptor, presented as the percentage of the GABA-evoked response at EC₁₀ (2 µM). To analyse the potentiation of the GABA-evoked current induced by IVM or MOX, GABA-responsive oocytes were exposed to 2 µM GABA, followed by washing and then 2 µM GABA in association with increasing concentrations of IVM (n = 8) or MOX (n = 5). Data were fitted to the Hill equation and are given as mean ± S.E.M.</p

Neurological symptoms observed after IVM or MOX administration in Mdr1ab(−/−) mice.

<p>The Mdr1ab(−/−) mice (3 per dose rate) injected subcutaneously at dose rates of 0.11, 0.40 and 0.69 µmol/kg bw for IVM and 1.27, 1.64 and 2.56 µmol/kg bw for MOX were observed and the development of symptoms evoking neurologic signs (rapid breathing, balance problems, tremor, ataxia, sleepiness, lethargy) was recorded over a period of 2 weeks; every 60 min for the first 12 hours and thence minimally twice per day. Mice were euthanized when severe tremors or ataxia or profound lethargy was noted.</p>†<p>Mice were euthanized when severe tremor or ataxia was noted.</p>a<p>Dose rate averaging the LD₅₀ for IVM.</p>b<p>Dose rate averaging the LD₅₀ for MOX.</p>c<p>Ataxia: lack of voluntary <u>coordination of muscle movements</u>, as in walking.</p>d<p>Tremor: rhythmic, <u>muscle contraction</u> and relaxation involving <u>oscillations</u> or twitching.</p

Comparison of chemical structures of ivermectin and moxidectin.

<p>Ivermectin is a mixture of B1a (substituent butyl on C25) and B1b (substituent isopropyl on C25) forms. The majority (more than 90%) of the drug is present as the B1a form.</p

IVM and MOX concentrations in brain and brain-to-plasma ratio at increasing dose rates in Mdr1ab(−/−) and wild-type mice.

<p>IVM or MOX was administered to Mdr1ab(−/−) mice (6 per dose rate) or to wild-type mice (3 per dose rate) at increasing doses below the LD₅₀ to ensure a non-lethal effect of the administration. Drug concentrations were determined in brain and plasma after animals were sacrificed at 24 h post-treatment. Absolute brain accumulation was plotted against the plasma concentration to determine brain concentration at LD₅₀, brain-to-plasma concentration ratio calculated and R².</p>a<p>LD₅₀ determined graphically from <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001883#pntd-0001883-g001" target="_blank">Figure 1</a>.</p>b<p>LD₅₀ for IVM and MOX determined from the literature <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001883#pntd.0001883-Shoop1" target="_blank">[20]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001883#pntd.0001883-WHO1" target="_blank">[21]</a>.</p>c<p>Brain concentration reached at LD₅₀, determined graphically from <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001883#pntd-0001883-g003" target="_blank">Figure 3A</a>.</p>d<p>Brain concentration reached at LD₅₀, determined graphically from <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001883#pntd-0001883-g003" target="_blank">Figure 3B</a>.</p>e<p>Brain-to-plasma concentration ratio calculated and R² obtained from the slope of the linear relationship between brain concentration and plasma concentration, which quantifies blood-brain transport.</p

Acute toxicity of IVM and MOX in Mdr1ab(−/−) mice.

<p>Acute toxicity was determined by observing survival during a 14-day period after subcutaneous administration of IVM (black square) or MOX (open square) to small groups (2–8 animals) of Mdr1ab(−/−) mice. Extrapolation from the graph yields an estimated LD₅₀ of 0.46 µmol/kg (0.40 mg/kg) and 2.3 µmol/kg (1.47 mg/kg) for IVM and MOX, respectively.</p

Lipid content in bile of wild-type and mdr1ab^-/- mice.

<p>Cholesterol and bile acids were quantified in bile in wild-type (open bars) and mdr1ab^-/- (solid bars) 35-week old male mice. Results are expressed as means ± s.e.m of 5 mice. Differences are considered significant when *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001.</p

Morphological comparison and liver lipid content in wild type and mdr1ab^-/- males.

<p>2A. Whole body. 2B. Respective livers at 35 weeks of age. 2C. Oil red O staining of liver sections. Livers were collected from 25- or 35-week old mice and kept frozen in OCT at −80°C until analysis. 2D. Lipid content of liver. Total cholesterol (upper histogram) and triglycerides (lower histogram) in liquid-frozen liver samples from 25- and 35- week old mice, were quantified by liquid chromatography in wild-type (open bars) and mdr1ab^-/- (solid bars) males. Results are expressed as means ± s.e.m of 5 to 7 mice. Differences are considered significant when <i>p<</i>0.05. ***, <i>p</i><0.001.</p