# Patent

## Title
Pharmaceutical composition comprising 3-beta-hydroxy-5-alpha-pregnan-20-one with improved storage and solubility properties

## Abstract

It is provided a pharmaceutical composition comprising 3-beta-hydroxy-5-alpha-pregnan-20-one, at least one sterol or an ester thereof and a mixture of acylglycerols with a solid fat content of less than 25% at 25° C. and 0% at 37° C. In addition it is provided a method for preparing the pharmaceutical composition.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional application of U.S. application Ser. No. 13/522,081, filed on Jul. 13, 2012, which is a national phase under 35 U.S.C. §371 of PCT International Application No. PCT/SE2011/050036 which has an International Filing Date of Jan. 14, 2011, which claims priority under 35 U.S.C. §119 to U.S. Provisional Application No. 61/295,027, filed Jan. 14, 2010 and Swedish Patent Application No. 1050029-6 filed on Jan. 14, 2010. The contents of each application recited above are incorporated herein by reference in their entirety.


TECHNICAL FIELD
The present invention relates generally to an improved formulation of 3-beta-hydroxy-5-alpha-pregnan-20-one.
BACKGROUND ART
3-beta-hydroxy-5-alpha-pregnan-20-one is a steroid in the pregnane family and a modulator of GABAA-receptor activity which is indicated for the treatment of sex/stress steroid induced disorders conditions (WO99/45931). 3-beta-hydroxy-5-alpha-pregnan-20-one is poorly soluble in many therapeutically acceptable solvents, which makes it difficult to administer the compound to a patient.
In animal studies, 3-beta-hydroxy-5-alpha-pregnan-20-one has been intravenously administered to rats in a formulation containing cyclodextrin (WO99/45931).
Grant et al (JPET 326:354-362, 2008) has administered 3-beta-hydroxy-5-alpha-pregnan-20-one to monkeys by using a formulation with hydroxypropyl β-cyclodextrin.
Formulations with cyclodextrins are not suitable for administration to human patients. One reason for this is, because 3-beta-hydroxy-5-alpha-pregnan-20-one is poorly soluble, the formulation results in a large therapeutic volume that can only be administered intravenously.
Since 3-beta-hydroxy-5-alpha-pregnan-20-one is poorly soluble in water there is still no pharmaceutically acceptable formulation for this compound.
DEFINITIONS
As used in the present application, the following terms have the specified meanings unless otherwise specified.
By “acylglycerol” is meant all types and combinations of fatty acids esterified to glycerol.
By “medium-chain acylglycerol” is meant a mixture of acylglycerols where the total combined percentage of octanoic (caprylic) acid and decanoic (capric) acid is at least 95%.
By “solid fat content” is meant the percentage of solid as determined by pulse NMR (nuclear magnetic resonance).
“Room temperature” denotes a temperature of between 18° C. and 25° C.
“UC1010” denotes 3-beta-hydroxy-5-alpha-pregnan-20-one.
“Sterol or ester thereof” denotes steroids with at least one hydroxyl group and esters of said steroids where at least one hydroxyl group has been used for the synthesis of an ester.
Steroids, such as sterols, are usually described by the number of carbon atoms in the compound. Thus, for example, cholesterol is a C27 sterol, which indicates that the compound consists of 27 carbon atoms.
Unless stated otherwise, concentrations are stated as mg/g, that is, mg per gram of pharmaceutical composition.



BRIEF DESCRIPTION OF FIGURES
FIG. 1 shows mean plasma concentrations of 3-beta-hydroxy-5-alpha-pregnan-20-one (ng/mL) in rabbits following subcutaneous administration of 3-beta-hydroxy-5-alpha-pregnan-20-one in sesame oil with cholesterol (1:1) in two doses: 1 mg/kg (squares) and 5 mg/kg (circles).
FIG. 2 shows 3-beta-hydroxy-5-alpha-pregnan-20-one concentration in filtrate (0.2 μm) from a suspension of 3-beta-hydroxy-5-alpha-pregnan-20-one in relation to cholesterol:3-beta-hydroxy-5-alpha-pregnan-20-one ratio.
FIGS. 3a, 3b, 4a and 4b show photographs of suspensions of 3-beta-hydroxy-5-alpha-pregnan-20-one.



SUMMARY OF THE INVENTION
An object of the present invention is to provide an improved formulation of 3-beta-hydroxy-5-alpha-pregnan-20-one in a pharmaceutically acceptable carrier.
Another object of the present invention is to provide a formulation of 3-beta-hydroxy-5-alpha-pregnan-20-one with enhanced storage properties.
Yet another object is to provide a formulation of 3-beta-hydroxy-5-alpha-pregnan-20-one with improved pharmacokinetics.
Yet another object of the present invention is to provide a formulation of 3-beta-hydroxy-5-alpha-pregnan-20-one with increased solubility in a pharmacologically acceptable carrier.
These and other objects are met by a first general aspect of the present invention which provides a pharmaceutical formulation comprising 3-beta-hydroxy-5-alpha-pregnan-20-one, at least one sterol or an ester thereof and a mixture of acylglycerols with a solid fat content of less than about 25% at 25° C. and about 0% at 37° C.
In a second general aspect of the present invention there is provided methods for preparing a pharmaceutical composition.
In a third general aspect of the present invention there is provided a pharmaceutical composition obtainable according to a method according to the invention.
In a fourth general aspect of the present invention there is provided use of a pharmaceutical composition for the treatment of conditions of the central nervous system.
DETAILED DESCRIPTION
The inventors have found that the addition of a sterol surprisingly increases the solubility and improves the pharmacokinetics of 3-beta-hydroxy-5-alpha-pregnan-20-one in acylglycerols.
Generally, the pharmaceutical composition comprises 3-beta-hydroxy-5-alpha-pregnan-20-one, at least one sterol or an ester thereof and a mixture of acylglycerols with a solid fat content of less than about 25% at 25° C. and about 0% at 37° C.
In particular a sterol with a hydroxyl group bound to the third carbon atom of the sterol structure is useful in the invention. The sterol may be cholesterol or beta-sitosterol, but also other sterols such as stigmasterol, brassicasterol or avenasterol may be used. In particular, cholesterol may be used.
In addition, cholesteryl esters can be used. Examples of such esters are sodium cholesteryl sulphate, cholesteryl bensoate, cholesteryl acetate, cholesteryl caprylate, cholesteryl decanoate, cholestyl palmitate, cholesteryl oleate and cholesteryl stearate.
The sterol or ester thereof can be a C18-C30 sterol or an ester thereof, a C21-C27 sterol or an ester thereof, or a C27-C29 sterol or ester thereof.
The pharmaceutical composition can, in a first embodiment, be such that 3-beta-hydroxy-5-alpha-pregnan-20-one is essentially dissolved in the composition. Thus 3-beta-hydroxy-5-alpha-pregnan-20-one can be dissolved or essentially dissolved according to this embodiment of the invention.
The weight ratio of sterol (or ester thereof) to 3-beta-hydroxy-5-alpha-pregnan-20-one can, in this embodiment, be in the range of about 1:10 to 10:1. The sterol or ester thereof may be added in an amount that is similar to the amount of 3-beta-hydroxy-5-alpha-pregnan-20-one by weight. Because 3-beta-hydroxy-5-alpha-pregnan-20-one and a sterol have similar molecular weights this results in almost equimolar amounts of 3-beta-hydroxy-5-alpha-pregnan-20-one to sterol.
Thus, the weight ratio of sterol to 3-beta-hydroxy-5-alpha-pregnan-20-one can be in the range of from 1:5 to 5:1. In particular, the weight ratio of sterol to 3-beta-hydroxy-5-alpha pregnan-20-one can be from 1:3 to 3:1.
Suitable concentrations of 3-beta-hydroxy-5-alpha pregnan-20-one are between 0.1 mg/g and 75 mg/g. The concentration of 3-beta-hydroxy-5-alpha pregnan-20-one can also be between 1 mg/g and 50 mg/g, between 5 mg/g and 30 mg/g or between 10 mg/g and 25 mg/g.
Alternatively, in a second embodiment, the pharmaceutical composition comprises a suspension of 3-beta-hydroxy-5-alpha-pregnan-20-one. In this case the pharmaceutical composition will comprise 3-beta-hydroxy-5-alpha-pregnan-20-one in particles as well as 3-beta-hydroxy-5-alpha-pregnan-20-one dissolved in the composition. The sterol increases the soluble fraction of 3-beta-hydroxy-5-alpha-pregnan-20-one in such a suspension compared to a suspension without a sterol. One advantage with a suspension is that the formulation can contain a high concentration of 3-beta-hydroxy-5-alpha-pregnan-20-one. An additional advantage with a composition that comprises a suspension is that it results in slow release of 3-beta-hydroxy-5-alpha-pregnan-20-one.
When the pharmaceutical composition comprises a suspension, the particles are preferably of a range of sizes that is not engulfed by macrophages. Macrophages do primarily engulf particles of a size that is 2-3 micrometer (Champion et al, Pharm Res 2008; 25(8):1815-1821).
In this second embodiment, the weight ratio of sterol (or ester thereof) to 3-beta-hydroxy-5-alpha pregnan-20-one can be in the range of about 1:10 to 10:1. The weight ratio of sterol to 3-beta-hydroxy-5alpha pregnan-20-one can be in the range of from 1:5 to 5:1. In particular, the weight ratio of sterol to 3-beta-hydroxy-5-alpha-pregnan-20-one can be from 1:4 to 3:1 or from 1:3 to 3:1.
Suitable concentrations of 3-beta-hydroxy-5-alpha-pregnan-20-one are, in this second embodiment, between 0.1 mg/g and 750 mg/g. The concentration of 3-beta-hydroxy-5-alpha-pregnan-20-one can also be between 1 mg/g and 300 mg/g, between 1 mg/g and 100 mg/g, between 1 mg/g and 50 mg/g, between 5 mg/g and 30 mg/g or between 10 mg/g and 25 mg/g.
The following applies to the invention in general.
Generally, the mixture of acylglycerols is characterized in that it has a solid fat content of less than about 25% at 25° C. and about 0% at 37° C. Thus, the solid fat content is, for practical purposes, 0% at 37° C. The solid fat content is at most 0.01% at 37° C.
The mixture of acylglycerols can be a vegetable oil. Thus, it can be a vegetable oil selected from the group consisting of sesame oil, peanut oil, olive oil, and castor oil, or mixtures thereof.
In particular the mixture of acylglycerols can be a medium-chain acylglycerol, that is, a mixture of acylglycerols wherein the total combined percentage of fatty acids with 8 carbon atoms (octanoic acid) and 10 carbon atoms (decanoic acid) is at least 95%. The medium-chain acylglycerol can be various mixtures of monoacylglycerols, diacylglycerols and triacylglycerols.
The medium-chain acylglycerol can consist of from about 50% to about 65% of monoacylglycerols, about 25% to about 35% of diacylglycerols, less than about 5% of triacylglycerols and less than about 2.5% of glycerol. An example of such a medium chain acylglycerol is Akoline MCM.
The medium-chain acylglycerol can be such that it comprises at least about 95% triacylglycerols. Akomed R MCT is an example of such a medium-chain acylglycerol.
The mixture of acylglycerols can comprise a mixture of a vegetable oil and a medium-chain acylglycerol. The mixture of acylglycerols can comprises a mixture of castor oil and a medium-chain acylglycerol where castor oil is present in an amount of between 40% and 60% by weight. The mixture of acylglycerols can consist of about 48% by weight of castor oil and about 52% by weight of a medium-chain acylglycerol. In particular the mixture of acylglycerols can consist of about 48% by weight castor oil and about 52% by weight of a medium-chain acylglycerol.
The pharmaceutical composition may comprise additional excipients known to a person skilled in the art such as antioxidants, preservatives, surfactants, coloring, flavoring, or thickening agents.
The pharmaceutical composition can be administered to the patient by different means. Thus, it may be administered orally, parenterally or topically. Thus, the pharmacological composition may be administered subcutaneously, intramuscularly, intravenously, nasally, transdermally or vaginally.
In a second general aspect of the present invention there is provided methods for preparing a pharmaceutical composition of 3-beta-hydroxy-5-alpha-pregnan-20-one.
One method, in which 3-beta-hydroxy-5-alpha-pregnan-20-one is dissolved or essentially dissolved in the composition, comprises the steps of a) dissolving 3-beta-hydroxy-5-alpha-pregnan-20-one in ethanol, b) adding a mixture of acylglycerols with a solid fat content of less than about 25% at 25° C. and about 0% at 37° C. and a sterol or ester thereof, c) mixing until a homogeneous liquid is obtained and d) evaporating the ethanol.
When the mixture of acylglycerol is a solid or a semi-solid at room temperature, such as a medium-chain acylglycerol, the method may comprise a further step, which is the melting of the medium-chain acylglycerol before mixing it with the ethanol-drug preparation. The melting step enables the homogeneous mixing of this type of acylglycerol with other components. Once melted and mixed with the other components, the preparation remains in a liquid state for at least the time periods indicated in Table 1.
When the formulation comprises a suspension, the formulation is advantageously prepared according to a method that comprises the following steps: 1) dissolving or suspending the sterol or ester thereof in the mixture of acylglycerols, 2) suspending 3-beta-hydroxy-5-alpha-pregnan-20-one in the acylglycerol-sterol mixture, 3) gently mixing. Surprisingly, this procedure leads to suspended particles comprising 3-beta-hydroxy-5-alpha-pregnan-20-one of smaller size.
In a third general aspect of the present invention there is provided pharmaceutical compositions obtainable by the methods according to the second aspect of the invention.
In a fourth general aspect of the present invention there is provided the use of the pharmaceutical composition according to the invention for the treatment or prevention of conditions of the central nervous system.
The pharmaceutical composition can be used to treat or prevent conditions of the central nervous system. Examples of such conditions that can be treated are epilepsy, menstruation cycle dependant epilepsy, depression, stress related depression, migraine, tiredness and in particular stress related tiredness, premenstrual syndrome, premenstrual dysphoric disorder, menstrual cycle linked mood changes, stress related memory changes, menstrual cycle linked memory changes, Alzheimer's dementia, menstrual cycle linked difficulties in concentration, menstrual cycle linked sleep disorders and tiredness, substance abuse, menstrual cycle linked alcoholism, or combinations thereof.
In particular the pharmaceutical composition can be used to treat steroid induced mood disorders, in particular premenstrual dysphoric disorder.
The pharmaceutical composition can also be used for treating or preventing side effects of oral contraceptives and postmenopausal therapy.
The pharmaceutical composition can also be used for control or termination of steroid-induced anesthesia.
EXAMPLES
The invention will now be described with non-limiting examples.
In order to find a pharmaceutical composition comprising 3-beta-hydroxy-5-alpha-pregnan-20-one, several different vehicles and combinations of vehicles were evaluated. 3-beta-hydroxy-5-alpha-pregnan-20-one was dissolved in various vehicles and was evaluated by eye for physical stability at room temperature over time. A formulation that did not undergo any visible changes in appearance and remained clear on storage in room temperature without signs of haziness, precipitation, sedimentation, phase separation into two or more liquid phases or change of colour, within 30 days was considered as “stable”.
In addition, the particle size and solubility of 3-beta-hydroxy-5-alpha-pregnan-20-one in formulations comprising a suspension of 3-beta-hydroxy-5-alpha-pregnan-20-one were evaluated.
General Procedure for Examples 1-49
The following procedure was adopted for the preparation of 3-beta-hydroxy-5-alpha-pregnan-20-one-containing formulations.
The desired amount of 3-beta-hydroxy-5-alpha-pregnan-20-one and sterol (for example cholesterol) was weighed in a 100 ml or 250 ml round-bottomed flask. To every gram of the mixture of 3-beta-hydroxy-5-alpha-pregnan-20-one and sterol a volume of about 30 ml of absolute ethanol was added. The mixture was treated in an ultrasonication bath (not exceeding 50° C.) until a clear liquid was obtained. This was normally accomplished within 10 minutes. The additional lipid ingredients indicated in the “Vehicle” column in Table 1 was then added up to 20 g. The resulting mixture was gently shaken by hand until a clear, homogeneous liquid was obtained. When the lipid was a solid at room temperature it was melted in the warm ultrasonication bath to a liquid form before addition.
Compounds from the following suppliers were used (product numbers within brackets): Cholesterol from Sigma (C8503), olive oil and peanut oil from Apoteket, Sweden (26 36 16 and 26 66 01, respectively), sesame oil from Fluka (85067), and castor oil from Sigma (259853). Akomed R medium-chain triacylglycerol (MCT) and Akoline medium-chain monoacylglycerol (MCM) were both from AarhusKarlshamns Sweden AB, Karlshamn, Sweden.
The alcohol was evaporated from the liquid on a rotary evaporator at a pressure of about 25 mbar and a temperature of about 40° C. until the weight of the flask was essentially constant. The remaining ethanol content was essentially less than 1%. The aim was to obtain a liquid with the appearance of clear oil at room temperature. The oily liquid was then transferred to clear glass vials and stored at room temperature until evaluation.
The samples were evaluated by observing the samples in the glass vials and recording signs of haziness, precipitation, sedimentation, phase separation into two or more liquid phases, or change of colour after 1 or 2 days after preparation and after 30 days after preparation. In some cases, other time intervals were used (indicated in Table 1). Where indicated, the entire sample was placed in a refrigerator (2-8° C.) to provoke precipitation.
Example 1
3-beta-hydroxy-5-alpha-pregnan-20-one (UC1010) (5 mg/g) and peanut oil was mixed into an emulsion with ethanol as described above in the concentrations shown in Table 1, and the ethanol was evaporated. The final weight of the preparation was 20 g. The mixture then had the form of an oily liquid. After one day when the sample was evaluated there were signs of precipitation. At 30 days the precipitate has formed a bottom sediment. Thus, the formulation was not stable.
Example 2
Example 2 was carried out essentially as Example 1 with the difference that cholesterol (5.5 mg/g) was added. When the sample was evaluated after one day the appearance of the sample had not changed. It was still unchanged after 30 days and after four months. After five months there was a slight precipitation. Example 2 compared with Example 1 shows how the addition of 5.5 mg/g cholesterol to a solution of 5 mg/g 3-beta-hydroxy-5-alpha-pregnan-20-one in peanut oil substantially increases the solubility so that, instead of precipitating, no precipitation occurred and the sample was stable for four months. However, a slight sedimentation occurred after 5 months.
Examples 3 to 49
Examples 3 to 49 were carried out essentially as described above with the variations with regard to 3-beta-hydroxy-5-alpha-pregnan-20-one concentration, acylglycerol mixture used, sterol used and sterol concentration that are shown in Table 1.
The data from examples 1 to 49 is presented in Table 1. The effect of addition of cholesterol is, for example, evident in examples 8 and 12, where the addition of cholesterol (10 mg/g) substantially increased solubility so that the sample did not precipitate but instead was stable for 12 months.
In Table 1, “Weighed amount of UC1010 (mg/g)” is the amount of 3-beta-hydroxy-5-alpha-pregnan-20-one per gram of final total composition including sterol (when a sterol is present). “Vehicle” denotes the carrier being tested. The amount of sterol is stated as “mg/g”, that is, the weight of sterol per weight of final total composition, including 3-beta-hydroxy-5-alpha-pregnan-20-one. “Appearance at preparation” describes the change of appearance of the mixture during preparation; usually the preparation is initially an emulsion or a solution whereas it has an oily appearance after evaporation of the ethanol; “Unchanged” denotes a sample that was stable and where thus 3-beta-hydroxy-5-alpha-pregnan-20-one remained in solution, without visible signs of haziness, precipitation, sedimentation, phase separation into two or more liquid phases or change of colour. This is also indicated by an asterisk (*) in the table.














TABLE 1







Weighed


Appearance after
Appearance after





amount of


1-2 days or other
30 days or other



Batch
UC1010

Appearance at
time period that
time period that is


Example
name
(mg/g)
Vehicle
preparation
is indicated
indicated
Comments






















1
ACA09
5
Peanut oil
Emulsion to oil
Signs of
Bottom sediment




0630-1



precipitation


2
ACA09
5
Peanut oil +
Emulsion to oil
Unchanged *
Slight sedimentation



0629-6

cholesterol (5.5 mg/g)


after 5 months


3
ACA09
10
Peanut oil
Emulsion to
Precipitation in



0629-4


suspension
almost the entire







sample volume


4
ACA09
10
Peanut oil +
Emulsion to oil
Bottom sediment



0629-3

cholesterol (10 mg/g)
(precipitation






after a few






hours)


5
ACA09
20
Peanut oil +
Emulsion to oil
Bottom sediment



0629-5

cholesterol (20 mg/g)
(precipitation






after a few






hours)


6
ACA09
10
Olive oil +
Emulsion to oil
Bottom sediment



0629-8

cholesterol (10 mg/g)


7
ACA09
3
Sesame oil +
Emulsion to oil
Unchanged *
Slight sedimentation



0709-2

cholesterol (3 mg/g)


after 13 months


8
ACA09
5
Sesame oil
Emulsion to oil
Precipitation
Bottom sediment



0702-2


9
ACA09
5
Sesame oil +
Emulsion to oil
Unchanged *
Precipitation of



0822-B

cholesterol (2.5 mg/g)


crystals after 2








months


10
ACA09
5
Sesame oil +
Emulsion to oil
Unchanged *
A few particles on the



0702-1

cholesterol (5 mg/g)


bottom


11
ACA09
5
Sesame oil +
Emulsion to oil
Unchanged *
Precipitation of



0822-A

cholesterol (5 mg/g)


crystals after 2








months


12
ACA09
5
Sesame oil +
Emulsion to oil
Unchanged *
Unchanged after 12



0822-C

cholesterol (10 mg/g)


months *


13
ACA09
10
Sesame oil +
Emulsion to oil
Precipitation of



0822-E

cholesterol (5 mg/g)

crystals


14
ACA09
10
Sesame oil +
Emulsion to oil
Precipitation of



0822-D

cholesterol (10 mg/g)

crystals


15
ACA09
11
Sesame oil +
Emulsion to oil
Precipitation of



0822-F

cholesterol (20 mg/g)

crystals


16
ACA09
10
Sesame oil +
Emulsion to oil
Unchanged *
Crystals on the



0716-7

β-sitosterol (10 mg/g)


bottom an on the








walls of the vial


17
ACA09
10
Akomed R MCT
Solution to oil
Precipitation of



1024-1



crystals within an







hour


18
ACA09
10
Akomed R MCT +
Solution to oil
Slight precipitation



1024-2

cholesterol (10 mg/g)

of crystals on the







bottom after 2







days


19
ACA09
15
Akomed R MCT +
Solution to oil
Slight precipitation



1025-1

cholesterol (15 mg/g)

of crystals on the







bottom after 24







hours


20
ACA09
10
50% Akomed R
Solution to oil
Unchanged *
Unchanged after 10



1024-3

MCT, 50% Akoline


months *





MCM


21
ACA09
10
50% Akomed R
Solution to oil
Unchanged *
Unchanged after 10



1024-4

MCT, 50% Akoline


months *





MCM +





cholesterol (10 mg/g)


22
ACA09
15
50% Akomed R
Solution to oil
Slight precipitation



1025-2

MCT, 50% Akoline

of crystals on the





MCM

bottom after 1







week


23
ACA09
15
50% Akomed R
Solution to oil
Unchanged *
Unchanged after 10



1025-3

MCT, 50% Akoline


months *





MCM +





cholesterol (15 mg/g)


24
ACA09
20
50% Akomed R
Solution to oil
Slight precipitation



1026-1

MCT, 50% Akoline

of crystals on the





MCM

bottom after 24







hours


25
ACA09
20
50% Akomed R
Solution to oil
Unchanged *
Very slight



1026-2

MCT, 50% Akoline


precipitation after





MCM +


2.5 months





cholesterol (20 mg/g)


26
ACA09
25
50% Akomed R
Solution to oil
Slight precipitation



1101-1

MCT, 50% Akoline

of crystals on the





MCM +

bottom after 3





cholesterol (25 mg/g)

days


27
ACA09
30
50% Akomed R
Solution to oil
First sign of



1102-1

MCT, 50% Akoline

precipitation after





MCM +

2 days





cholesterol (30 mg/g)


28
ACA09
15
Castor oil
Solution to oil
Slight precipitation



1025-4



of crystals on the







bottom after 26







days


29
ACA09
20
Castor oil
Solution to oil
Slight precipitation



1026-3



of crystals on the







bottom after 3







days


30
ACA09
20
Castor oil +
Solution to oil
Unchanged *
Unchanged after 10



1101-2

cholesterol (20 mg/g)


months *


31
ACA09
25
Castor oil +
Solution to oil
Unchanged *
Unchanged after 1
Unchanged



1109-1

cholesterol (25 mg/g)


month, very slight
after 1 week








precipitation after 2
refrigerated *








months


32
ACA09
30
Castor oil +
Solution to oil
Slight precipitation

Small crystals



1109-2

cholesterol (31 mg/g)

of crystals on the

on the







bottom after 8

bottom after







days

1 week









refrigerated -









unchanged









after 2 days *


33
ACA09
40
Akoline MCM
Solution to oil
Massive



1122-1



precipitation







within an hour


34
ACA09
40
Akoline MCM +
Solution to oil
Unchanged after
Precipitation after 1



1122-2

cholesterol (40 mg/g)

24 hours *
week


35
ACA09
25
50% Castor oil, 50%
Solution to oil
Unchanged *
Unchanged after 10
After 1 week



1101-4

Akoline MCM +


months *
refrigerated,





cholesterol (25 mg/g)



the entire









sample was









solidified,









Slight









precipitation









after melting


36
ACA09
30
50% Castor oil, 50%
Solution to oil
Unchanged *
Unchanged after 1
After 1 week



1102-2

Akoline MCM +


month, very slight
refrigerated,





cholesterol (30 mg/g)


precipitation after
the entire








approx 2 months
sample was









solidified,









Slight









precipitation









after melting


37
ACA09
34
48% Castor oil, 52%
Solution to oil
Unchanged after
Precipitation in entire
Unchanged *



1108-1

Akoline MCM +

15 days *
sample after 1 month
after 1 week





cholesterol (34 mg/g)



refrigerated


38
ACA09
38
50% Castor oil, 50%
Solution to oil
Slight precipitation

Small crystals



1108-2

Akoline MCM +

of crystals on the

on the





cholesterol (38 mg/g)

bottom after 9

bottom after







days

1 week









refrigerated -









unchanged









after 2 days


39
ACA09
40
50% Castor oil, 50%
Solution to oil
Massive



1120-3

Akoline MCM +

precipitation after





cholesterol (20 mg/g)

24 hours


40
ACA09
40
50% Castor oil, 50%
Solution to oil
Massive



1120-1

Akoline MCM +

precipitation after





cholesterol (40 mg/g)

24 hours


41
ACA09
40
47.3% Castor oil,
Solution to oil
Unchanged after
Starts to precipitate



1122-3

47.3% Akoline

24 hours *





MCM, 5.4% benzyl





alcohol +





cholesterol (40 mg/g)


42
ACA09
40
50% Castor oil, 50%
Solution to oil
Very slight



1120-2

Akoline MCM +

precipitation of





cholesterol (60 mg/g)

crystal sin the







entire sample after







24 h


43
ACA09
45
45% Castor oil, 55%
Solution to oil
Slight precipitation

Crystals on



1108-3

Akoline MCM +

of crystals on the

the bottom





cholesterol (45 mg/g)

bottom after 9

after 1 week







days

refrigerated


44
ACA09
45
50% Castor oil, 50%
Solution to oil
Slight precipitation



1121-1

Akoline MCM +

of crystal sin the





cholesterol (67.5 mg/g)

entire sample after







24 h


45
ACA09
50
50% Castor oil, 50%
Solution to oil
Very slight



1121-2

Akoline MCM +

precipitations of





cholesterol (75 mg/g)

crystal sin the







entire sample after







24 h


46
ACA09
5
Olive oil
Emulsion to oil
Unchanged after
Crystals at the bottom



1216-2



4 days, first signs
after 9 months







of precipitation







after 5 days


47
ACA09
5
Olive oil +
Emulsion to oil
Unchanged after
Unchanged after 8



1216-1

cholesterol (10 mg/g)

3 weeks *
months


48
ACA09
7.5
Olive oil
Emulsion to oil
Precipitation after



1221-2



approx. 1 hour


49
ACA09
7.5
Olive oil +
Emulsion to oil
Unchanged after
Crystals at the bottom



1221-1

cholesterol (7.5 mg/g)

1 day, very slight
after 8 months







precipitation after







1 week









Examples 50-75
Examples 50 to 75 were carried out essentially as examples 1-49. Akoline Medium-Chain Monoglyceride (MCM) (Batch 8192270 and 8218940) and Akomed R Medium-Chain Triglyceride (MCT) (Batch 4765) were obtained from AarhusKarlshamns Sweden AB, Karlshamn, Sweden. Absolute ethanol (>99%) was obtained from VWR International.
The procedure for making and evaluating lipid-based formulations was as follows: The batch sizes were either 20 g or 100 g of final formulation. The desired amounts of 3-beta-hydroxy-5-alpha-pregnan-20-one and cholesterol were weighed in a round-bottomed flask, 250 or 1000 ml depending on the batch size.
To every gram of 3-beta-hydroxy-5-alpha-pregnan-20-one and cholesterol mixture a volume of about 15 to 30 ml of absolute ethanol was added. The smaller volume of alcohol per gram of solute was used when preparing the largest batch size of 100 g of final formulation. The mixture was treated in an ultrasonication bath (not exceeding 55° C.) until a clear solution was obtained. This was normally accomplished within a few minutes. The glycerides were then added and the resulting mixture was treated in the ultrasonication bath for a few seconds until a clear, homogeneous liquid was obtained. The alcohol was evaporated from the liquid on a rotary evaporator at a pressure of about 20 mbar and a temperature of about 40° C. until weight of the flask was more or less constant. Normally, the remaining ethanol content was 0.5% (w/w) or less. The evaporation time was 0.5-1.5 h, depending on the batch size. The aim was to obtain a practically uncolored liquid with the appearance of a clear oil at room temperature. The liquid was transferred to clear glass vials, which were stored at room temperature until evaluation. Some selected formulations were portioned and stored at 2-8° C. for limited period of time.
The evaluation comprised observation of the physical stability at room temperature over time. The samples were observed for haziness, precipitation of particles, aggregates or crystals, and subsequent sedimentation and/or phase separation into two or more liquid phases, and/or change of colour.
It was possible to dissolve up to 25 mg of 3-beta-hydroxy-5-alpha-pregnan-20-one per gram of final formulation based on 50% MCT and 50% MCM (examples 68 and 69) without any noticeable change in appearance when stored at room temperature for more than 1 month. One of the samples (example 69) also withstood storage at 2-8° C. and repeated temperature cycling.
In order to check the robustness and reproducibility of the formulations and the procedure for the preparation thereof, a scaling up of the batch size from 20 g to 100 g of final formulation was performed. The compositions corresponding to examples 60 and 69 were selected for this procedure. From the behavior during preparation and the initial observations of the resulting formulations (examples 74 and 75) it can be concluded that the adopted procedure is both robust and reproducible for making up to 100 g of formulation.














TABLE 2







Amount



Appearance after





of


Appearance after 1-2
about 1 month (if not



Batch
UC1010

Appearance at
days at room
otherwise stated) at


Example
name
(mg/g)
Vehicle
preparation
temperature
room temperature
Comments







50
ACA10
30
50% Akoline
Solution to oil
Signs of precipitation
Precipitation




0129-1

MCM, 50%

after 1 day





Akomed R





MCT +





cholesterol





(30 mg/g)


51
ACA10
30
50% Akoline
Solution to oil
Signs of precipitation
Precipitation



0129-2

MCM, 50%

after 1 day





Akomed R





MCT +





cholesterol





(45 mg/g)


52
ACA10
28
50% Akoline
Solution to oil
Unchanged
Precipitation
Signs of



0129-3

MCM, 50%



precipitation





Akomed R



after 1 day in





MCT +



the





cholesterol



refrigerator,





(56 mg/g)



substantial









precipitation









after 2 days


53
ACA10
30
67% Akoline
Solution to oil
Signs of slight
Precipitation



0130-1

MCM, 33%

precipitation after 2 days





Akomed R





MCT +





cholesterol





(30 mg/g)


54
ACA10
30
67% Akoline
Solution to oil
Unchanged
Precipitation



0130-2

MCM, 33%





Akomed R





MCT +





cholesterol





(45 mg/g)


55
ACA10
30
67% Akoline
Solution to oil
Unchanged
Precipitation



0130-3

MCM, 33%





Akomed R





MCT +





cholesterol





(60 mg/g)


56
ACA10
20
50% Akoline
Solution to oil
Unchanged
Unchanged after 29
Withstand 3



0203-B

MCM, 50%


weeks
days in the





Akomed R



refrigerator,





MCT +



but not 4 days,





cholesterol



due to





(40 mg/g)



solidification of









MCM


57
ACA10
20
50% Akoline
Solution to oil
Unchanged
Unchanged after 29
Withstand 3



0203-C

MCM, 50%


weeks
weeks in the





Akomed R



refrigerator





MCT +





cholesterol





(60 mg/g)


58
ACA10
20
50% Akoline
Solution to oil
First signs of
Precipitation



0204-1

MCM, 50%

precipitation after a few





Akomed R

hours, large star-shaped





MCT +

crystals after 2 days





cholesterol





(100 mg/g)


59
ACA10
20
30% Akoline
Solution to oil
Unchanged after 3 days
Precipitation



0204-2

MCM, 70%





Akomed R





MCT +





cholesterol





(40 mg/g)


60
ACA10
20
30% Akoline
Solution to oil
Unchanged
Unchanged after 7
Withstand 6



0206-1

MCM, 70%


weeks
days in the





Akomed R



refrigerator,





MCT +



but had





cholesterol



precipitated





(60 mg/g)



after about 2









weeks without









concomitant









solidification of









MCM









Massive









precipitation









after 28 weeks


61
ACA10
20
30% Akoline
Solution to oil
First signs of
Precipitation



0206-2

MCM, 70%

precipitation after about





Akomed R

30 min





MCT +





cholesterol





(100 mg/g)


62
ACA10
20
15% Akoline
Solution to oil
Precipitation
Precipitation



0206-3

MCM, 85%





Akomed R





MCT +





cholesterol





(40 mg/g)


63
ACA10
20
15% Akoline
Solution to oil
Unchanged after 1 days
Precipitation



0206-4

MCM, 85%





Akomed R





MCT +





cholesterol





(60 mg/g)


64
ACA10
20
15% Akoline
Solution to oil
Signs of precipitation
Precipitation



0206-5

MCM, 85%

directly after evaporation





Akomed R





MCT +





cholesterol





(100 mg/g)


65
ACA10
20
100%
Solution to oil
Precipitation at the
Precipitation



0213-A

Akomed R

bottom





MCT +





cholesterol





(40 mg/g)


66
ACA10
20
100%
Solution to oil
Precipitation in the whole
Precipitation in the



0213-C

Akomed R

sample
whole sample





MCT +





cholesterol





(60 mg/g)


67
ACA10
20
100%
Solution to oil
Precipitation directly after
Precipitation in the



0213-B

Akomed R

evaporation
whole sample





MCT +





cholesterol





(80 mg/g)


68
ACA10
25
50% Akoline
Solution to oil
Unchanged
Unchanged
A portion of



0301-1

MCM, 50%



the sample





Akomed R



was placed in





MCT +



the





cholesterol



refrigerator:





(50 mg/g)



partial









solidification









after 1 day,









but clear









solution after









melting at RT.









After another 1









day in the









fridge the









sample started









to precipitate









Massive









precipitation









after 25 weeks


69
ACA10
25
50% Akoline
Solution to oil
Unchanged
Unchanged after 25
A portion of



0301-2

MCM, 50%


weeks
the sample





Akomed R



was placed in





MCT +



the





cholesterol



refrigerator:





(75 mg/g)



partial









solidification,









but clear









solution after









melting at RT.









The sample









withstood









repeated









cycles during









several weeks









without









precipitationI


70
ACA10
25
50% Akoline
Solution to oil
Very small amount of
Precipitation
A portion of



0301-3

MCM, 50%

needle-shaped crystals

the sample





Akomed R

after 1 day

was placed in





MCT +



the





cholesterol



refrigerator:





(100 mg/g)



partial









solidification









and very slight









precipitation









after melting at









RT


71
ACA10
30
50% Akoline
Solution to oil
Unchanged after 3 days,
Precipitation
A portion of



0301-4

MCM, 50%

slight tendency to

the sample





Akomed R

precipitation after 5 days

was placed in





MCT +



the





cholesterol



refrigerator:





(60 mg/g)



partial









solidification









and slight









precipitation









after melting at









RT


72
ACA10
30
50% Akoline
Solution to oil
Unchanged after 3 days,
Precipitation
A portion of



0301-5

MCM, 50%

precipitation after 5 days

the sample





Akomed R



was placed in





MCT +



the





cholesterol



refrigerator:





(90 mg/g)



partial









solidification









and substantial









precipitation









after melting at









RT


73
ACA10
30
50% Akoline
Solution to
—
—
—



0301-6

MCM, 50%
crystals





Akomed R
(precipitated





MCT +
during





cholesterol
evaporation;





(120 mg/g)
discarded)


74
ACA10
25
50% Akoline
Solution to oil
Unchanged
Unchanged
Batch size 100 g



0320

MCM, 50%



Very slight





Akomed R



precipitation





MCT +



after 23 weeks





cholesterol





(75 mg/g)


75
ACA10
20
30% Akoline
Solution to oil
Unchanged
Unchanged
Batch size 100 g



0321

MCM, 70%



Slight





Akomed R



precipitation





MCT +



after 23 weeks





cholesterol





(60 mg/g)









Example 76
The objective of the study was to investigate the comparative pharmacokinetics in plasma of a 3-beta-hydroxy-5-alpha-pregnan-20-one formulation comprising sesame oil and cholesterol following subcutaneous administration to New Zealand White rabbits. Two groups of three female rabbits each received single doses 1 mg/kg (formulation as in example 7) or 5 mg/kg (formulation as in example 10). Following subcutaneous injection into the dorsal neck region of the animals, blood samples were taken at 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 hours post dose. Concentrations of 3-beta-hydroxy-5-alpha-pregnan-20-one in plasma were measured by a validated LC-MS/MS method. Data are presented in FIG. 1, and show mean plasma concentration (ng/mL) of 3-beta-hydroxy-5-alpha-pregnan-20-one for the two doses: 1 mg/kg (squares) and 5 mg/kg (circles).
Example 77
Suspensions of 3-beta-hydroxy-5-alpha-pregnan-20-one were prepared for investigation of solubility as follows: cholesterol was first dissolved at room temperature in sesame oil at 10 and 20 mg/ml, respectively. Suspensions of 3-beta-hydroxy-5-alpha-pregnan-20-one in sesame oil with different amounts of cholesterol were prepared on a magnetic stirrer at about 500 rpm using a conventional Teflon coated stir bar, at room temperature for several days, with occasional cycling to 2-8° C. At this time, the particles have become substantially smaller. Thereafter, samples of the respective suspensions were filtered through a 0.2 μm filter and analyzed with regard to concentration of 3-beta-hydroxy-5-alpha-pregnan-20-one. The results are presented in Table 3 and FIG. 2 where it can be seen that increasing the amounts of cholesterol increases the soluble fraction of 3-beta-hydroxy-5-alpha-pregnan-20-one. FIG. 2 shows data for a 3-beta-hydroxy-5-alpha-pregnan-20-one concentration of 10 mg/g. FIGS. 3a and 3b show microscopy pictures taken at 5× enlargement of suspensions with a 3-beta-hydroxy-5-alpha-pregnan-20-one concentration of 10 mg/g and a cholesterol:3-beta-hydroxy-5-alpha-pregnan-20-one ratio of 1:1 immediately upon mixing (3a) and after several days of stirring (3b).








TABLE 3








3-beta-hydroxy-5-alpha-pregnan-20-one


The weight ratio
concentration in filtrate (mg/ml)









cholesterol to 3-
Initial 3-beta-
Initial 3-beta-


beta-hydroxy-5-
hydroxy-5-alpha-
hydroxy-5-alpha-


alpha-pregnan-
pregnan-20-one
pregnan-20-one


20-one
concentration 10 mg/g
concentration 40 mg/g





0:1
1  
—


1:4
—
1.4


1:2
—
1.9


1:1
1.8
—


2:1
2.2
—









Example 78
Micronized 3-beta-hydroxy-5-alpha-pregnan-20-one (10 mg/g) with a mean particle size of 6 micrometer was suspended in sesame oil with cholesterol (20 mg/g) and stirred as in example 77. Photos were taken immediately upon suspension (FIG. 4a) and after 19 hours of stirring (FIG. 4b).
CONCLUSION
It has been shown that the presence of a sterol such as cholesterol improves the possibilities to formulate 3-beta-hydroxy-5-alpha-pregnan-20-one in a pharmaceutical composition comprising acylglycerols, either as an oily solution or an oily suspension.

## Claims

1. A pharmaceutical oil composition, consisting of:
3-beta-hydroxy-5-alpha-pregnan-20-one;
cholesterol or an ester thereof; and
a mixture of acylglycerols, wherein the mixture of acylglycerols is a medium-chain acylglycerol.

2. The pharmaceutical oil composition according to claim 1 wherein the mixture of acylglycerols has a total combined percentage of (i) fatty acids with eight carbon atoms and (ii) fatty acids with ten carbon atoms of at least 95%.

3. The pharmaceutical oil composition according to claim 1, wherein the 3-beta-hydroxy-5-alpha-pregnan-20-one is essentially dissolved.

4. The pharmaceutical oil composition according to claim 1, wherein the 3-beta-hydroxy-5-alpha-pregnan-20-one is in the form of an oil suspension consisting of some of the 3-beta-hydroxy-5-alpha-pregnan-20-one in the form of suspended particles and some of the 3-beta-hydroxy-5-alpha-pregnan-20-one dissolved in the oil composition.

5. The pharmaceutical oil composition according to claim 1, wherein the medium-chain acylglycerol consists of from about 50% to about 65% of monoacylglycerols, about 25% to about 35% of diacylglycerols, less than about 5% of triacylglycerols and less than about 2.5% of glycerol.

6. A pharmaceutical oil composition for parenteral administration according to claim 1.

7. A pharmaceutical oil composition for vaginal administration according to claim 1.

8. A method of terminating steroid induced anesthesia, comprising: administering the pharmaceutical composition according to claim 1 to a subject in need thereof.

9. The pharmaceutical oil composition according to claim 1, wherein said medium-chain acylglycerol is a mixture of acylglycerols wherein the total combined percentage of fatty acids with 8 carbon atoms and 10 carbon atoms is at least 95%.

10. The pharmaceutical oil composition according to claim 1, wherein said medium-chain acylglycerol comprises at least about 95% triacylglycerols.

11. The pharmaceutical oil composition according to claim 1, wherein said medium-chain acylglycerol is a mixture of acylglycerols wherein the total combined percentage of fatty acids with 8 carbon atoms and 10 carbon atoms is at least 95% and said medium-chain acylglycerol comprises at least about 95% triacylglycerols.