Table 1.
Examples of therapeutic drugs requiring lower dosing in Npc1-/- mice.
Fig 1.
P450 enzymatic O-dealkylation activity assayed in Npc1+/+, Npc1+/-, and Npc1-/- mice at 3,6, and 9 weeks of age.
P450 O-dealkylation enzymatic activity of: MROD, methoxyresorufin-O-dealkylation; EROD, ethoxyresorufin-O-dealkylation; PROD, pentoxyresorufin-O-dealkylation, and BROD, benzoxyresorufin-O-dealkylation in Npc1+/+ (white), Npc1+/- (grey), and Npc1-/- (black) mice. Activity shown as percent of age-matched controls at 3 weeks (A), 6 weeks (B) and 9 weeks (C) of age. Data are presented as mean ± SEM, n = 6 (3 males and 3 females), * p—value < 0.05, ** p—value < 0.01, *** p—value < 0.001, **** p—value < 0.0001, calculated using two-tailed unpaired nonparametric Mann-Whitney test.
Table 2.
Enzyme activity rates, and relevant statistics of the P450 activity assays.
Fig 2.
Validation of 11 CYP genes expression by qPCR in Npc1+/+, Npc1+/-, and Npc1-/- mice at 9 weeks of age.
Validation was performed in females to recapitulate the microarray studies. White columns for Npc1+/+ samples, grey columns for Npc1+/-, and black columns for Npc1-/- mice. Data are presented as mean ± SD, n = 4–6. * p—value < 0.05, ** p—value < 0.01, *** p—value < 0.001, **** p—value < 0.0001, calculated using two-tailed unpaired t test, with Welch’s correction as necessary.
Fig 3.
In vivo assay of Midazolam metabolism Npc1+/+, Npc1+/-, and Npc1-/- mice.
(A) Midazolam plasma concentration (in ng/ml) as a function of time. (B) Midazolam metabolites plasma concentration (in ng/ml) as a function of time. (C) and (D) Differences in exposure between genotypes. Data are presented as mean ± SD for each genotype, n = 3. * p—value <0.05, ** p—value < 0.01, calculated using two-tailed, unpaired t test with Welch’s correction as necessary and one-way ANOVA.
Fig 4.
Effect of UDCA bile acid supplementation.
(A) and (B) effects of UDCA bile acid supplementation on the activity of CYP450 catalysed reactions in the Npc1 mouse model of plus or minus 0.5% ursodeoxycholic acid (UDCA, w/w) measured at (A) 6 weeks and (B) 9 weeks of age, shown as a function of percent of age-matched control littermates. Methoxyresorufin-O-dealkylation (MROD); ethoxyresorufin-O-dealkylation (EROD); pentoxyresorufin-O-dealkylation (PROD); benzoxyresorufin-O-dealkylation (BROD). Data are presented as mean ± SEM, n = 6 (3 males and 3 females), * p—value < 0.05, ** p—value < 0.01, *** p—value < 0.001, **** p—value < 0.0001 calculated using an unpaired nonparametric test Mann-Whitney test. (C) Relative protein quantitation of Cyp3a isolated from liver of 6-week old male mice found a significant decrease in Npc1+/- and Npc1-/- untreated mice compared Npc1+/+ untreated mice. UDCA treatment restored Npc1-/- Cyp3a levels to that of Npc1+/+ mice while have no effect on the Npc1+/- or Npc1+/+ Cyp3a values. (D) Relative protein quantitation of Cyp1a2 isolated from liver of 6-week old male mice found a significant decrease in Npc1+/- and Npc1-/- untreated mice compared Npc1+/+ untreated mice. UDCA treatment restored Npc1-/- Cyp1a2 levels to that of Npc1+/+ mice while decreasing the levels for Npc1+/- and Npc1+/+ Cyp1a2 values. Data are presented as mean ± SD, n = 3. * p—value < 0.05, ** p—value < 0.01, *** p—value < 0.001 calculated using two-tailed, unpaired t test. (F) Representative western blot analysis of Cyp3a and Cyp1a2 in the mouse model of Npc1 plus or minus UDCA treatment.
Table 3.
Enzyme activity rates, and relevant statistics of the P450 activity assays after treatment with UDCA as compared to untreated in Npc1+/+ mice.
Table 4.
Enzyme activity rates, and relevant statistics of the P450 activity assays after treatment with UDCA intergenotypic comparisons.
Fig 5.
Effect of UDCA on body weight, rearing and tremor of Npc1 mice.
(A) Effects of UDCA on weight of Npc1 mice. Body weight over time shown as mean ± SD, n = 8–19 for all time points except for Npc1-/- mice on UDCA at 13 weeks where n = 4 and 14 weeks where n = 1, # denotes comparison between Npc1+/+ mice untreated and Npc1+/+ mice on UDCA, * denotes comparison between Npc1-/- mice untreated and Npc1-/- mice on UDCA, # or * p—value < 0.05, ## or ** p—value < 0.01, calculated using two-way ANOVA with Bonferroni’s multiple comparison test. (B) Effects of UDCA on motor function in Npc1-/- mice. Total rearing events counted for a period of 5 minutes in Npc1+/+ and Npc1-/- mice plus or minus UDCA treatment. Data are presented as mean ± SD, n = 8–19. * p—value < 0.05, ** p—value <0.01, *** p—value <0.001, calculated using an unpaired nonparametric test Mann-Whitney test. (C-F) Effects of UDCA on tremor in Npc1-/- mice. Tremor was monitored between 0 and 63 Hz in panels (C) and (D), and quantified at 43 Hz in (E) early symptomatic (9 weeks), (F) late symptomatic (11 weeks) time points. 43 Hz is an arbitrary point selected in the high frequency tremor range to allow a simple comparison to be made between treatment groups. The low frequency tremor range overlaps at the low end with grooming activity frequencies and so was not analysed to avoid this as a potential confounder. In both early symptomatic and late symptomatic groups, mice treated with 0.5% UDCA had significantly less tremor than untreated Npc1-/-. Data are presented as mean ± SD, n = 7–19 animals/group, * p—value < 0.05, ** p—value < 0.01, *** p—value < 0.001, **** p—value < 0.0001 calculated using one-way Anova with Bonferroni's multiple comparison test.
Fig 6.
Gene expression analysis pre and post UDCA therapy.
Gene expression analysis of 4 CYP genes expression by qPCR in Npc1+/+, Npc1+/- and Npc-/- mice (untreated and treated with UDCA) at 3, 6 and 9 weeks old in females and males. White columns indicate Npc1+/+ samples, grey columns indicate Npc1+/-, black columns indicate Npc1-/-, blue columns indicate Npc1+/+ treated with UDCA, green columns indicate Npc1+/- treated with UDCA and red columns indicate Npc1-/- mice treated with UDCA. (A-B) Cyp1a2 gene expression. (C-D) Cyp2b10 gene expression. (E-F) Cyp2b13 gene expression. (G-H) Cyp2a4 gene expression. Data are presented as mean ± SEM, n = 3–6. * p—value < 0.05, ** p—value < 0.01, *** p—value < 0.001, **** p—value < 0.0001, calculated using an unpaired nonparametric Mann-Whitney test.
Fig 7.
Effect of HPBCD treatment in 7-week old male Npc1-/- mice.
(A-D) Effects of HPBCD treatment on the activity of CYP450 catalysed reactions in the Npc1 mouse presented as a function of percent of age-matched control littermates. (A) Methoxyresorufin-O-dealkylation (MROD); (B) ethoxyresorufin-O-dealkylation (EROD); (C) pentoxyresorufin-O-dealkylation (PROD); (D) benzoxyresorufin-O-dealkylation (BROD). Data are presented as mean ± SEM, n = 4 (males), * p—value < 0.05, *** p—value < 0.001, **** p—value < 0.0001 calculated using an unpaired nonparametric test Mann-Whitney test. (E) Relative protein quantitation of Cyp3a found a significant decrease in Npc1-/- untreated mice compared Npc1+/+ untreated mice similar to what was seen in Fig 4C. HPBCD treatment increased Cyp3a protein levels in Npc1-/- mice, both in comparison to the untreated Npc1+/+ or Npc1-/- mice. (F) Relative protein quantitation of Cyp1a2 revealed a significant decrease in Npc1-/- untreated mice compared Npc1+/+ untreated mice. HPBCD treatment increased Npc1-/- mice Cyp1a2 protein levels in comparison to untreated Npc1-/- mice, but did not restore to Npc1+/+ levels. Data are presented as mean ± SD, n = 4. * p—value < 0.05, **** p—value < 0.0001 calculated using two-tailed, unpaired t test. (G) Representative western blot analysis of Cyp3a and Cyp1a2 isolated from 7 week old male Npc1+/+ or Npc1-/- mouse livers plus or minus HPBCD treatment.
Fig 8.
P450 O-dealkylation enzymatic activity in NPC1+/+, NPC1+/-, and NPC1-/- cats.
(A-D) Activity of CYP450 catalysed reactions in feline model of NPC1, shown as percent of age-matched controls. Age-matched NPC1+/+ (white), NPC1+/- (grey) and NPC1-/- (black) cats were included in addition to HPBCD treated NPC1-/- cats (striped). (E) CYP3A relative protein quantitation from western blot analysis was significantly lower in NPC1+/-, and NPC1-/- compared to age-matched NPC1+/+ cats when analysed by densitometry. (F) Representative western blot analysis of CYP3A in the feline model of NPC1. Age-matched NPC1+/+, NPC1+/- and NPC1-/- cats were included in addition to HPBCD treated NPC1-/- cats. Methoxyresorufin-O-dealkylation (MROD); ethoxyresorufin-O-dealkylation (EROD); pentoxyresorufin-O-dealkylation (PROD); benzoxyresorufin-O-dealkylation (BROD). Data are presented as mean ± SEM, n = 3. Note due to small sample numbers all 3 ages (21, 38, and 59 weeks) HPBCD treated animals were combined for this figure. * p—value <0.05, ** p—value < 0.01, *** p—value < 0.001 calculated using one-way ANOVA test with Tukey’s post-hoc test.
Fig 9.
(A) Activity of cytochrome C reductase in individual NPC1 patients at various disease stages and age- and gender-matched controls. Data are expressed as pmoles cytochrome C reduced per minute, normalised for microsomal protein content (mg). (B) Combined patient samples from panel A, Activity of cytochrome C reductase in liver microsomes from NPC1 patients and age-and gender-matched controls. (C) Activity of CYP450 catalysed reactions in NPC1 patients and age-and gender-matched controls. Data are expressed as percentage of age-matched controls. Each individual sample was run in triplicate. (D) Relative CYP3A protein quantification from Western Blot analysis in livers from NPC1 patients and controls. The circles indicate the 20 year old NPC1 and control individuals, the triangles the 12–13 year old NPC1 and control individuals, and the squares represent the data from the 6 to 7 year old NPC1 and control individuals. (E) Representative western blot analysis of CYP3A in livers of NPC1 patients and their age- and gender-matched controls. Data are presented as mean ± SEM, n = 3. ** p—value < 0.01, calculated using two-tailed, unpaired t test.
Fig 10.
Absolute quantitation of mRNA expression of CYP1A2, CYP2B6, and CYP3A4.
Absolute quantitation of mRNA expression of the CYP isozymes in three NPC1 patients (black) and age-matched control (white) livers. (A) CYP1A2. (B) CYP2B6. (C) CYP3A4.