Pharmaceutical semi-solid composition of isotretinoin

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Methodology The formulations tested are put in solution in 250 ml of BME. Taurocholate 5 (10 mM) was added to the solutions to better mimate the in physiological conditions. The different solutions so prepared are put in contact with Caco-2 cells at the apical or basolateral side. The cells culture inserts) are incubated for 3 hours at 37°C and samples of 100 p1 are taken every hours 10 The formulations tested were the following Formulation SEDDS~ (batch number 26F97/1 ):

Isotretinoin : 10 mg 15 Gelucire~ 50/13 : 134 mg Phospholipon 90~ : 11 mg Tween 80~ : 71 mg IPP° : 24 mg Pro capsula una Formulation suspension (batch number 25F97/1 ) Isotretinoin : 20 mg Gelucire~ 50/13 : 83.7 mg Soyabean oil : 270 mg Procapsula una RESULTS

Passage of formulations from apical side --~ basolateral side Time SEDDS~ Suspension SEDDS Suspension control minutes(26F97/1 (25F97/1 + TC + TC

) ) 60 0.7721 0.6708 0.7019 0.6469 0.0718 120 2.4096 0.8749 1.4347 0.9513 0.1836 180 2.6226 1.1311 3.2419 1.5073 0.6156 Passage of formulations from basolateral side -~ apical side Time SEDDS~ Suspension SEDDS~ Suspension control minute (26F97/1 (25F97/1 + TC + TC

) ) 60 2.0496 0.3948 8.1291 0.8713 0,0650 120 3.0844 0.9068 8.3496 1.8460 0.1131 180 4.3653 1.0763 9.7110 2.0779 0.1481 The results demonstrate that the passage of isotretinoin is superior for the SEDDS~ formulation than for the suspension formulation. In order to confirm these results, a comparative pharmacokinetics study has been performed.

PK studies The bioavailability of SEDDS° (26F97/1 ) and suspension (25F97/1 ) isotretinoin formulations has been assessed and compared to the bioavailability of the reference (Roaccutane~ 20 mg, HofFman LaRoche) on six healthy volunteers in a single dose, three way, cross-over pharmacokinetic study). The drug was taken with food (standardized breakfast). The plasma concentration of isotretinoin and its active metabolite 4-oxo-isotretinoin were quantified using a fully validated LC/MS/MS method.

The figure 3 described the mean pharmacokinetic profile obtained for each formulation.

The following table gives the value of the main pharmacokinetics parameters obtained for each formulation of isotretinoin.

Formulations AUC72n CmaX Tmax (ng. hlml) (ng/ml) (h) Roaccutane~ 20 1747.89 116.63 1.83 mg (96C15315AA) Suspension 20 4308.72 230.96 5.67 mg (25F97/1 ) SEDDS~ 10 mg 1494.64 98.36 3.00 (26F97/1 ) It appears that both the SEDDS~ and the suspension formulation are able to significantly increase the bioavailability of isotretinoin in comparison to the marketed reference. Indeed the ratio between AUC72n of the supension 20 mg and Roaccutane~ 20 mg is of 2.47. The SEDDS~ 10 mg present an AUC72n similar to this of Roaccutane~ 20 mg what means an approximately 2-fold increase of bioavailability (ratio AUC7~n SEDDS~ 10mg/ AUC72n Roaccutane~ 20 mg = 0.86). Furthermore, the suspension and SEDDS°

formulations both presented a lower intraindividual variability of the bioavailability as demonstrated by the values of relative standard deviations (rsd) which are of 36.0 %, 22.72 % and 28.18 % for Roaccutane~ 20 mg, suspension 20 mg and SEDDS~ 10 mg respectively.

Nevertheless, the results obtained in vivo are not correlated with the results obtained on caco-2 cells since on this model the permeability of the SEDDS~ formulation was much higher than the permeability of the suspension formulation while in vivo the suspension formulation gives the best results.

A second pharmacokinetic study was performed on completely different formulations (6 subjects, 2-way, fed, cross-over study). Those were formulations of isotretinoin under the form of a suspension in which the ratio between Gelucire° 50/13 and soyabean oil was very different that the previous formulation of suspension The two formulations tested were the following:

F1 : suspension without surfactant (batch number H23K99/1 ) Isotretinoin : 20 mg Gelucire° 50/13 : 247 mg Soyabean oil : 133 mg F2 : suspension with surfactant (batch number H07L99/1 ) Isotretinoin : 20 mg Gelucire° 50/13 : 240 mg Soyabean oil : 130 mg Span 80~ : 20 mg The figure 4 gives the comparative pharmacokinetic profile of each formulation for isotretinoin The figure 5 gives the comparative pharmacokinetic profile of each formulation for 4-oxo-isotretinoin, the active metabolite of isotretinoin.

In order to confirm the first bioavailability data obtained with the present invention, a larger pharmacokinetic study has been performed.

The bioavailability of a capsule of isotretinoin16 mg (see the formulation herebelow) from the present invention has been assessed and compared to the bioavailability of the reference (ROACCUTANE~ 20 mg capsule, Roche) on 24 healthy subjects.

This study (SMB-(SO-SD011 ) was a single dose, two treatment, two period, two sequence, randomised, crossover and with at least 18 days wash-out 5 between the two periods.

The subjects were healthy Caucasian volunteers of both sexes (non-pregnant, non-breast-feeding), aged 18 to 50 years, non smokers or smoking less than 10 cigarettes per day.

The drugs was taken with food (a standardized breakfast).

10 Blood samples were collected according to the following sampling schedule pre-dose and 1 h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 1 Oh, 12h, 14h, 24h, 36h, 48h, 72h, 96h, 120h, 168h and 216 hours post-dose.

The plasma concentration of isotretinoin and its active metabolite 4-oxo isotretinoin were quantified using a fully validated LC/MSIMS method. The 15 continuous variables were evaluated according to an univariate ANOVA, based on log-transformed data. The Wilcoxon non-parametric ANOVA were used where appropriate. Bioequivalence was evaluated using the Shuirman two one-sided t-test (90% CI) and the westlake single sided confidence interval (95% CL) The figures 6 and 7 describe the mean pharmacokinetic profile of isotretinoin and 4-oxoisotretinoin for the two formulations (n=24 subjects) while the tables herebelow give the comparative main pharmacokinetic parameters.

Formulation of isotretinoin 16 mg (mg / capsule) isotretinoin 16 stearoyl macrogol glycerides (Gelurire 50!13~) 192 soya bean oil refined 104 sorbitane oleate (Span 80~) 16 As seen, the dose of 16 mg of the formulation corresponding to the present invention gives a bioavailability similar to 20 mg of the marketed formulation, what is the evidence of the supra-bioavailability of the formulation corresponding to the present invention.

The tables hereinbelow gives the value of the main pharmacokinetics results and statistical analysis obtained for each formulation of isotretinoin and 4-oxoisotretinoin.

This study demonstrated that ROACCUTANE~ 20 mg and isotretinoin 16 mg x are bioequivalent after a .single oral dose administration of each product in fed conditions. Indeed, the primary parameters AUC (AUC~ and AUC~~sn ) were within the predetermined confidence interval.

This study demonstrated also that Isotretinoin 16 mg has a safety profile comparable with that described in the literature for other isotretinoin preparations and similar to this of ROACCUTANE~ 20 mg.