Title of Invention

"ISOTRETINOIN NANOPARTICULATE COMPOSITIONS "

Abstract The present invention relates to the preparation of a nanoparticulate isotretinoin composition having enhanced bioavailability.
Full Text FIELD OF THE INVENTION
The present invention relates to the preparation of a nanoparticulate isotretinoin composition having enhanced bioavailability.
BACKGROUND OF THE INVENTION
Isotretinoin (13-cis Vitamin A) is the drug of choice for the treatment of severe, recalcitrant cystic acne and is presently marketed by Hoffman La Roche as Accutane® which is a suspension of isotretinoin filled in soft gelatin capsules.
US Patent No. 4,464,394 discloses compositions and methods of using 13-cis vitamin A acid against the development of epithelial carcinomas of the skin, gastrointestinal tract, respiratory tract and genito-urinary tract. Although this patent describes pharmaceutical compositions, no data on the bioavailability of the active ingredient is disclosed.
WO 00/25772 discloses that the currently marketed "Accutane®' formulation of isotretinoin has a mean particle size of 100 µm and has a bioavailability of only about 20%. In this patent application the inventors describe a process for the micronization of isotretinoin to a mean particle size in the range from about 5µm to about 30µm. However, no data is presented to show the effect, if any, of this particle size reduction on the bioavailability of isotretinoin.
Isotretinoin is a relatively water insoluble compound which degrades when exposed to light and atmospheric oxygen. It is also highly teratogenic and is contra-indicated in pregnant and lactating women. Due to the low bioavailability of isotretinoin, higher doses have to be administered. It would therefore be highly desirable to develop dosage forms which are more bioavailable.
In another PCT application WO 99/52504, a process for the manufacture of (sub)micron sized particles by dissolving in compressed gas and surfactants is described. Isotretinoin is listed as one of the several drugs whose particle size can be reduced by the process disclosed in this application. No specific data on the

critical particle size or its effect on the bioavailability has, however, been given in this application. Of course, if the dramatic increase in bioavailability as a function of its particle size was known, the specifics of the critical particle sizes and their effect on the increase in bioavailability would likely have been described.
European Patent Number, EP 184942 discloses pharmaceutical compositions having not more than 22% wax content which according to the inventors is critical in determining the bioavailability. Higher wax content tends to diminish the bioavailability. The median particle size of the drug is also reduced to around 12 µm with a decade particle ratio of less than 25 µm. The said objective of enhanced bioavailability is achieved by controlling both the particle size of isotretinoin and the wax content in the formulation. An increase in the bioavailability of only about 1.6-1.9 times that of commercially available 'Accutane®' formulation was reported in this patent.
We have, through extensive experimentation, surprisingly found that the particle size of isotretinoin used in formulating the final dosage form is critical for determining its bioavailability.
SUMMARY OF THE INVENTION
The present invention provides a pharmaceutical composition of isotretinoin comprising isotretinoin having a mean particle size (d50) of less than about 1 µm (1000 nm).
The term "about" is employed in its normal terminology in the art and would mean "approximately".
The present invention also provides a process for the reduction of the particle size of isotretinoin to less than about 1000 nm, without any loss in potency.

The present invention further provides a method of using isotretinoin having a mean particle size (d50) of less than 1000 nm for the treatment of severe recalcitrant cystic acne.
Finally, the present invention provides a pharmaceutical composition comprising isotretinoin wherein the AUC and Cmax values obtained by the composition were at least three times increased as compared to the commercially available formulation of isotretinoin sold under the tradename of Accutane®
The production of isotretinoin with reduced particle size and thereby increased bioavailability would have obvious benefits of achieving the desired therapeutic effects by the administration of lower amounts of drug.
It is known that conventional techniques of micronization such as hammer mill, ball mill or air attrition mill result in a significant loss in potency of isotretinoin as it is very sensitive to oxidation. It is also known that isotretinoin powder can be micronized with negligible loss in potency by suspending it in an oily vehicle and milling it. Heretofore, the particle size reduction only to particle size between 5µm-30 µm has been reported by this wet milling method.
In "The Theory and Practice of Industrial Pharmacy" Lachman et al note that although wet milling is beneficial for reducing the particle size to about 10µ (10,000nm), flocculation prevents any further reduction in the particle size by this method. Further reduction of the particle size to sub (micron) levels often requires highly sophisticated techniques that require building up often expensive and specialized infrastructure. Surprisingly, we have found that it is possible to reduce the particle size of isotretinoin to submicron levels by utilizing the simple process of wet milling.
Therefore, in accordance with the present invention, isotretinoin obtained in the conventional coarse form preferably having a particle size of less than about 250µm is mixed with a carrier to form a medicated suspension. Isotretinoin may be present from a concentration of about 0.1 gm to 60 gm per 100 ml of the medicated

suspension. Preferably it is present from about 5-30 gm per 100 ml of the medicated suspension.
In one aspect the present invention provides a pharmaceutical composition
comprising:
(i) about 0.1% to about 60% by weight of isotretinoin having a mean particle size
(d50) of less than about 1000 nm (1 µm);
(ii) about 30% to about 40% by weight of a suspending agent; and
(iii) one or more pharmaceutically acceptable excipients selected from chelating
agents, antioxidants and surfactants.
The term "carrier" is employed in its normal terminology in the art and encompasses an inert substance which allows or facilitates the active compound to be absorbed by and act upon the body.
The medicated suspension is then subjected to impact, shear and cavitation forces produced by high sheer homogenization or wet milling to get nanoparticulate drug having a mean particle size (d50) of less than about 1000 nm. Particle size reduction is preferably carried out using wet mills. Examples of such mills include Dispermat SL (VMA_Getzmann GMBH, Germany) and bead mills such as the Dyno-Mill Type KDL (Willy Bachofen AG, Maschinenfabrik, Switzerland).
Bead mills such as the Dyno mill work on the principle of transmitting the energy for dispersion and grinding to the grinding beads via an early exchangeable agitator shaft. While processing, the material to be milled is constantly fed into the mill by a separate product pump. The grinding media for particle size reduction can be selected from spherical or particulate rigid media, less than about 3 mm in diameter and more preferably less than about 1mm in diameter. The selection of material for the grinding media is not believed to be critical to our process. We have found that zirconium oxide such as 95% ZrO stabilized with magnesia, zirconium silicate and glass grinding media provide good particle size reduction. However, other media such as stainless steel, titania, alumina and 95% ZrO stabilized with yttrium are also expected to be useful.

The time taken for the reduction in particle depends upon the mechanical means and the processing conditions selected. Processing times of less than one day have provided the desired results in high sheer milling such as the dyano mill. The entire process is preferably carried out under controlled ambient temperature conditions.
The mean particle size (d50) is reduced to less then 1000 nm. Preferably the d50 is less than 800 nm. More preferably, the d50 is less than 500 nm. The d90 of this isotretinoin is less than 4000 nm. More preferably, the d90 is less than 3000 nm. Most preferably the d90 is less than 1500 nm. Analysis of the particle size of isotretinoin is carried out using any conventional particle size analyzer (e.g. the Malvern Master Sizer™).
The nanonized isotretinoin thus obtained showed no loss in potency as a result of particle size reduction. The isotretinoin composition thus produced was storage stable.
The carrier material used to form the medicated suspension is selected from the group consisting of peanut oil, fractionated coconut oil marketed under the trade name of "Miglyol®" soyabean oil, sesame oil, mineral oil, cotton seed oil, polyethylene glycol and mixtures thereof.
Subsequent to the reduction in the particle size, the nanoparticulate isotretinoin-
carrier was mixed with a suspending agent, and optionally with other
pharmaceutically acceptable excipients, before being encapsulated into a soft
gelatin capsule dosage form.
The suspending agent used in accordance with the present invention is a standard wax mixture as described by JP Stanley, in The Theory and Practice of Industrial Pharmacy, L. Lackman, H.A. Lieberman and J.L. Klanig, eds; 2nd ed; Lea & Febiger, Phila (1976) comprising 1 part hydrogenated soyabean oil, 1.2 parts while wax and 4.2 parts hydrogenated vegetable oil. In addition to the standard wax mixture beeswax, paraffin wax and glyceryl monostearate may also be employed. The suspending agent is used in amounts of between 30-40% w/w of the formulation.

The formulation of the present invention may further contain other suitable pharmaceutical excipients such as antioxidants, chelating agents and surfactants.
The anti-oxidant employed in the present invention may be selected from the group consisting of α-tocopherol, butylated hydroxy anisole (BHA), butylated hydroxytoluene (BHT), ascorbyl palmitate and propyl gallate. Chelating agents may be chosen from amongst those conventionally known in the art such as disodium edetate and calcium disodium edetate.
Suitable surfactants which can be employed include lecithin, sorbitan monostearate, polysorbates prepared from lauric, palmitic, stearic and oleic acid; mononylphenyl ethers of polyethyleneglycols such as nanoxynols; polyoxyethylene monoesters such as polyoxyethylethylene monostearate, polyoxyethylene, monolaurate, polyoxyethylene monooleate, dioctyl sodium sulfosuccinate, sodium lauryl sulfate and poloxamers.
The composition of the invention is prepared by the process comprising the steps of: (i) dispersing coarse isotretinoin in an oil carrier to form a suspension
containing about 0.1% to about 60% by weight of isotretinoin; (ii) subjecting the suspension of step (i) to shear, impact, cavitation or attrition
so as to reduce the mean particle size (d50) of isotretinoin to less than
about 1000 nm (1 µm); (iii) mixing the material of step (ii) with a suspending agent and one or more
pharmaceutically acceptable excipients selected from chelating agents,
antioxidants and surfactants; and (iv) encapsulating the material of step (iii) into capsules.

DETAILED DESCRIPTION OF THE INVENTION
The present invention is illustrated by, but not limited to, the following examples:
Example 1
A nanoparticulate dispersion of isotretinoin was prepared using the DYNO mill (model KDL, manufactured by Willy A Bachoffen AG Maschinefabrik). 250g Isotretinoin was mixed with 1500 ml soya oil and homogenized. The mill grinding chamber was partially (80%) filled with zirconium silicate beads and the medicated suspension was continuously circulated through the media mill at a agitator disc speed of 10 m/sec for one hour at a product feed rate of 10-15 kg per hour. The final dispersion was analyzed for particle size reduction and the values obtained are given in Table1
Table 1 : Representative particle size data
(Table Removed)
Investigations were conducted in order to dertermine the effect of particle size on the bioavailability of isotretinoin in the formulations of the invention. The blood levels of the drug were compared with that of the commercially available formulation sold as "Accutane™".
The mean particle size of isotretinoin was reduced to d50 values of 100|am, 3.5|nm, 25.7|am and 0.395 |im (395 nm) The isotretinoin of different particle sizes was formulated as described in Table 2 and encapsulated in a soft gelatin capsule.

Table 2 : Isotretinoin Formulation


(Table Removed)
Nanoparticulate isotretinoin was prepared as described in Example 1. The hydrogenated soyabean oil, white wax and hydrogenated vegetable oil were melted in a separate vessel and mixed with the milled isotretinoin mixture, edetate disodium and butylated hydroxyanisole. The resulting blend was filled into soft gelatin capsules.
Pharmacokinetic data
The effect of particle size on the bioavailability of isotretinoin was studied and
compared with that of the commercially available formulation of isotretinoin sold under the brand name "Accutane®". The area under the plasma concentration-time curve (AUC) and the maximum concentration (Cmax) for orally administered isotretinoin of different particle sizes formulated as soft gelatin capsules was compared.
As isotretinoin behaves in a dose linear fashion, the Pk data from the above studies were dose normalised to the same dosage strength of 22.5 mg for all particle sizes and compared (Table 3).

Table 3: Comparision of the AUC and Cmax values of different particle sizes of isotretinoin. Dose normalised to 22.5 mg


(Table Removed)
The commercially marketed Accutane formulation and our isotretinoin capsules having a mean particle size (d50) of about 100 µm had similar AUC and Cmax values. Reduction of particle size to 25.7 µm increased the bioavailability by around 1.9 times that of Accutane®. On further reduction of the particle size to the nanoparticulate range (about 0.395 µm), there was a substantial increase in the bioavailability of isotretinoin to about four times that of Accutane®.
While the invention has been described by reference to specific examples, this was for purpose of illustration only. Numerous alternative embodiments will be apparent to those skilled in the art and are considered to be within the scope of the invention.






WE CLAIM:
1. A pharmaceutical composition comprising:
(i) about 0.1% to about 60% by weight of isotretinoin having a mean
particle size (d50) of less than about 1000 nm (1µm); (ii) about 30% to about 40% by weight of a suspending agent; and (iii) one or more pharmaceutically acceptable excipients selected from chelating agents, antioxidants and surfactants.
2. The pharmaceutical composition according to claim 1 wherein the mean particle size (d50) of isotretinoin is less than about 500nm.
3. The pharmaceutical composition according to claim 1 wherein the suspending agent is a wax mixture comprising 1 part hydrogenated soyabean oil, 1.2 parts white wax and 4.2 parts hydrogenated vegetable oil.
4. The pharmaceutical composition according to claim 1 wherein the chelating agent is selected from amongst disodium edetate and calcium disodium edetate.
5. The pharmaceutical composition according to claim 1 wherein the antioxidant is selected from the group consisting of -tocopherols, butylated hydroxyanisole, butylated hydroxytoluene, ascorbyl palmitate and propyl gallate.
6. The pharmaceutical composition according to claim 1 wherein the surfactant is selected from the group consisting of lecithin, sorbitan monostearate, polysorbates, monononyl ethers of polyethylene glycols, polyoxyethylene monostearate, polyoxyethylene monolaurate, diocyl sodium succinate, sodium lauryl sulfate and poloxamers.

7. A process for the manufacture of pharmaceutical composition comprising:
(i) dispersing coarse isotretinoin in an oil carrier to form a suspension
containing about 0.1% to about 60% by weight of isotretinoin; (ii) subjecting the suspension of step (i) to shear, impact, cavitation or attrition
so as to reduce the mean particle size (d50) of isotretinoin to less than
about 1000 nm (1 µm); (iii) mixing the material of step (ii) with a suspending agent and one or more
pharmaceutically acceptable excipients selected from chelating agents,
antioxidants and surfactants; and (iv) encapsulating the material of step (iii) into capsules.
8. The pharmaceutical composition according to claim 7 wherein the oily carrier comprises soyabean oil, peanut oil, fractionated coconut oil, mineral oil, cotton seed oil, polyethylene glycol and mixtures thereof.
9. A pharmaceutical composition comprising isotretinoin substantially as described herein.

Documents:

1693-DELNP-2004-Abstract-(18-03-2010).pdf

1693-DELNP-2004-Abstract-(25-11-2008).pdf

1693-delnp-2004-abstract.pdf

1693-DELNP-2004-Claims-(18-03-2010).pdf

1693-DELNP-2004-Claims-(25-11-2008).pdf

1693-delnp-2004-claims.pdf

1693-DELNP-2004-Correspondence-Others-(18-03-2010).pdf

1693-DELNP-2004-Correspondence-Others-(23-04-2010).pdf

1693-DELNP-2004-Correspondence-Others-(25-11-2008).pdf

1693-delnp-2004-correspondence-others.pdf

1693-DELNP-2004-Description (Complete)-(18-03-2010).pdf

1693-DELNP-2004-Description (Complete)-(25-11-2008).pdf

1693-delnp-2004-description (complete).pdf

1693-delnp-2004-form-1.pdf

1693-delnp-2004-form-18.pdf

1693-DELNP-2004-Form-2-(18-03-2010).pdf

1693-DELNP-2004-Form-2-(25-11-2008).pdf

1693-delnp-2004-form-2.pdf

1693-DELNP-2004-GPA-(23-04-2010).pdf

1693-DELNP-2004-Others-Document-(25-11-2008).pdf

1693-delnp-2004-pct-210.pdf


Patent Number 240817
Indian Patent Application Number 1693/DELNP/2004
PG Journal Number 23/2010
Publication Date 04-Jun-2010
Grant Date 02-Jun-2010
Date of Filing 15-Jun-2004
Name of Patentee RANBAXY LABORATORIES LIMITED
Applicant Address 19, NEHRU PLACE, NEW DELHI - 110019, INDIA
Inventors:
# Inventor's Name Inventor's Address
1 INDERDEEP BHATIA RANBAXY LABORATORIES LIMITED, PLOT NO. 20, SECTOR-18, UDYOG VIHAR INDUSTRIAL AREA, GRUGAON -122001 (HARYANA), INDIA
2 RAJIV MALIK RANBAXY LABORATORIES LIMITED, PLOT NO. 20, SECTOR-18, UDYOG VIHAR INDUSTRIAL AREA, GRUGAON -122001 (HARYANA), INDIA
PCT International Classification Number A61K
PCT International Application Number PCT/IB01/02329
PCT International Filing date 2001-12-06
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 PCT/IB01/02329 2001-12-06 PCT