AQUEOUS ORAL SOLUTIONS OF STEROID HORMONES AND HYDROXYPROPYL-BETA-CYCLODEXTRIN WITH OPTIMISED BIOAVAILABILITY

Abstract

The present invention concerns the preparation and the therapeutic use of aqueous solutions of Progesterone or Testosterone, complexed with hydroxypropyl--cyclodextrin (HPCD) that are suitable for oral administration. The solutions are characterised by a specific molar ratio between HPCD and the hormones such as to ensure high hormone plasma levels, following oral administration, thanks to the optimisation of their solubility, permeability, metabolic stability and ultimately, bioavailability. The formulation object of the present patent makes it possible to achieve effective plasma concentrations following oral administration of lower doses of hormone with respect to oral formulations currently on the market (e.g. Prometrium, Andriol) to the advantage of greater safety and compliance of the patients.

Claims

1. A method of treating a disease requiring Progesterone or Testosterone treatment, comprising orally administering to a patient in need thereof, a progesterone or testosterone complex of hydroxypropyl--cyclodextrin (HPCD) in which: said Progesterone (Prg) is present at a molar ratio HPCD:Prg ranging from 1.7:1 to 2.4:1, or said Testosterone (Tst) is present at a molar ratio HPCD:Tst ranging from 1.7:1 to 3.0:1; wherein said HPCD contains less than 0.3% by weight of unsubstituted (-cyclodextrin, and wherein said Prg or Tst is present at a concentration ranging from 5 to 100 mg/g.

2. The method of claim 1, wherein said molar ratio between the HPCD and Prg or Tst ranges from 2.0:1 to 2.2:1.

3. The method of claim 1, wherein said molar ratio between the HPCD and Prg or Tst is about 2:1.

4. The method of claim 1, wherein said molar ratio between the HPCD and the Progesterone ranges from 1.9:1 to 2.1:1.

5. The method of claim 1, wherein said molar ratio between the HPCD and the Progesterone is about 2.1:1.

6. The method of claim 1, wherein said molar ratio between the HPCD and the Testosterone ranges from 1.9:1 to 2.1:1.

7. The method of claim 1, wherein said molar ratio between the HPCD and the Testosterone is about 2.1:1.

8. The method of claim 1, wherein the formulation is in form of powder or granules.

Description

DESCRIPTION OF THE FIGURES

[0024] FIG. 1: Percentage of unmodified Progesterone (not metabolised) over time after incubation in human hepatic microsomes at 37 C. of solutions with different molar ratios between HPCD and Prg.

[0025] FIG. 2: Permeation profile of Progesterone through artificial silicone membranes. Different solutions have been tested characterised by different molar ratios between HPCD and Prg at the concentration of Progesterone of 32 mg/g

[0026] FIG. 3: Permeation profile of Progesterone through artificial silicone membranes. Different solutions have been tested characterised by different molar ratios between HPCD and Prg at the concentration of Progesterone of 20 mg/g.

[0027] FIG. 4: Percentage of unmodified Testosterone (not metabolised) over time after incubation in human hepatic microsomes at 37 C. of solutions with different molar ratios between HPCD and Tst

[0028] FIG. 5: Permeation profile of Testosterone through artificial silicone membranes. Different solutions have been tested characterised by different molar ratios between HPCD and Tst at the concentration of Testosterone of 20 mg/g.

[0029] FIG. 6: Permeation profile of Testosterone through artificial silicone membranes. Different solutions have been tested characterised by different molar ratios between HPCD and Tst at the concentration of Testosterone of 35 mg/g

[0030] FIG. 7: Comparative plasma pharmacokinetic profiles of Progesterone in a healthy patient after oral administration of the solution of hydroxypropyl--cyclodextrin with Prg in molar ratio 2:1 and of the oral formulation present on the market (Prometrium).

[0031] FIG. 8: Comparative plasma pharmacokinetic profiles of Testosterone in a healthy subject after oral administration of the solution of hydroxypropyl--cyclodextrin with Testosterone in a molar ratio 2:1 or 3:1, and of a Testosterone ester formulation present on the market (Andriol).

[0032] FIG. 9: reproduction of the data of FIG. 2, shown mean permeation flow rate of Progesterone 32 mg/g formulated as aqueous solutions of HPCD:Progesterone as a function of the growing molar ratio.

[0033] FIG. 10: reproduction of the data of FIG. 3, shown mean permeation flow rate of Progesterone 20 mg/g formulated as aqueous solutions of HPCD:Progesterone as a function of the growing molar ratio.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The steroid hormones used in the present invention are Progesterone (Prg), Testosterone (Tst), and derivatives thereof.

[0035] The hydroxypropyl--cyclodextrin (HPCD) used contains a residue of unsubstituted -cyclodextrin that is lower than 0.3% by weight, with respect to the HPCD. Methods for obtaining HPCD with this low impurity level are described for example in US 2006/0058262. The complexes of the invention, obtained by means of the same, lead to stable solutions at room temperature for at least 24 months.

[0036] In the complexes of the invention, the molar ratio between HPCD and Progesterone or Testosterone, can vary as follows:

[0037] a) (HPCD:Progesterone): comprised between 1.7:1 and 2.4:1, preferably between 1.9:1 and 2.1:1, most preferably about 2:1; or

[0038] b) (HPCD:Testosterone): comprised between 1.7:1 and 3.0:1, preferably between 1.9:1 and 2.1:1, most preferably about 2:1.

[0039] (i) The invention therefore concerns pharmaceutical compositions for use in the oral administration of steroid hormones, comprising a complex of hydroxypropyl--cyclodextrin (HPCD) as described in paragraphs (a) or (b) above,

[0040] (ii) The invention further concerns oral pharmaceutical compositions comprising a complex of hydroxypropyl--cyclodextrin (HPCD) as described in paragraphs (a) or (B) above, for use in. the treatment of diseases requiring progesterone or testosterone treatment,

[0041] (iii) The invention further concerns the use of a complex of hydroxypropyl--cyclodextrin (HPCD) as described in paragraphs (a) or (B) above, for the manufacture of an oral pharmaceutical composition for treating diseases requiring progesterone or testosterone treatment,

[0042] (iv) The invention further includes a method to improve the bioavailability of an orally administrable/administered. progesterone or testosterone, characterized by formulating said progesterone or testosterone as a complex of hydroxypropyl--cyclodextrin (HPCD) as described in paragraphs (a) or (b) above.

[0043] The invention further includes the compositions, uses and methods listed in paragraphs (i)-(iv) above, characterized. in that the complex is rot administered by routes other than the oral route,

[0044] The invention further includes the compositions, uses and methods listed in paragraphs (i)-(iv) above, further characterized in that said oral pharmaceutical composition is formulated in one or more dosage units, each containing less than 100 m.g of hormone (progesterone or testosterone) per gram of solution, e.g. between 5 an 100 mg/g, preferably between 5 and 50 mg/g of, more preferably between 15 and 40 mg/g, calculated as non-complexed form.

[0045] The term oral administration used herein means, as usually understood in. pharmacology, the administration of a composition which is simply and directly swallowed though the oesphagus into the stomach, without permanence in the mouth cavity, whereby the absorption of the drug takes place naturally in the gastro-intestinal tract, a.s opposed to e.g. the boccal/sublingual route, in which the gastro-intestinal absorption is undesired, and absorption takes place via the mouth cavity.

[0046] Examples of diseases requiring progesterone treatment are, without limitation endometrial hyperplasia, premenstrual syndrome, treatment of the symptoms of men.opause, treatment of infertile women needing luteal phase support as part of an Assisted Reproductive Technology (ART) treatment program, secondary amenhorrea, progesterone deficiency symptoms, pre-term birth, benign mastopathy, repeated abortion.

[0047] Examples of diseases requiring testosterone treatment are, without limitation: testosterone replacement therapy in male hypogonadal disorders, for example: eunuchoidism; hypopituitarism; endocrine impotence; male climacteric symptoms like decreased libido and decreased mental and physical activity; certain types of infertility due to disorders of spermatogenesis, post-castration disorders. Testosterone therapy may also be indicated in osteoporosis due to androgenic deficiency.

[0048] As observed in the experimental part, the ratios between HPCD and progesterone/testosterone are important in order to obtain a high absorption level of the hormone in the gastrointestinal duct and a limited degree of metabolic inactivation. The complexes of progesterone or testosterone, typically those with a HPCD:hormone molar ratio 2:1, were found to be absorbed more easily with respect to those with ratio 1:1, used as a reference. This is particularly unexpected since, as known from Zoppetti et al., J Incl Phenom.Macrocycl Chem, 2007, 57:283-288) the complex 2:1 is much more stable with respect to the complex 1:1 (formation constant=111473 m.sup.1 and 3478 m.sup.1, respectively) and therefore considered less prone to make the hormone available for absorption. The data is further unexpected, due to the fact that it goes against other publications (see Dahan et al., J Pharm Sci, 99(6), 2010), according to which the increase of cyclodextrin leads to a corresponding reduction of the permeation of the drug through the membrane. On the other hand, it has been found here that the extent of permeation of the steroid hormone through membranes is not linear when the concentration of HPCD varies, but, on the contrary, a bell-shape curve can be observed with a permeation peak at an intermediate HPCD:hormone 2:1 molar ratio. The aforementioned molar ratios also identify complexes that are sufficiently resistant to the metabolic inactivation in vitro.

[0049] The formation of the aforementioned complexes occurs according to per se known modalities. In general, HPCD can be dissolved at room temperature and under stirring in a suitable amount of water, for example in a weight ratio in water comprised between 1:2 and 2:2; then the steroid hormone is added to the solution thus obtained, again under stirring, in a molar ratio with the HPCD comprised in the ranges defined above; optionally, it is possible to add further water so as to obtain the desired volume/concentration of the final solution. In a non-limiting manner, the final solution can have a concentration of hormones present that is comprised between 5 and 100 mg/g, preferably between 5 and 50 mg/g, more preferably between 15 and 40 mg/g of solution. Other concentrations can be selected as a function of the final use.

[0050] The compositions of the invention thus obtained are typically in the liquid form, or rather aqueous solutions, comprising the complexes described above dissolved or substantially dissolved in the aqueous phase. The compositions contain, in addition to the aforementioned characteristic components, further additives as a function of the type of formulation desired. Among these additives, it is worth mentioning aromas, sweeteners, co-solvents, stabilisers, preservatives, emulsifiers, etc., underlining that such additives are merely optional, or rather, they are not essential for ensuring the stability and bioavailability of the complexes in solution, which is typical of the complexes as such.

[0051] Because of their high stability, the HPCD/hormone solutions can be provided to the user already in the liquid and ready-to-use form; it is however also possible to prepare and store the formulation in a suitable concentrated solid or liquid form, to be added with a suitable volume of water at the moment of use. For example, a kit can be foreseen comprising a pre-formulation, for example in the form of powder, granules, or a concentrated solution, that is associated with a container containing the necessary volume of aqueous solution for an extemporaneous reconstitution of the formulation. The solution object of the invention can be dispensed in normal single-dose or multidose containers made from glass or plastic material and can be safely stored at room temperature for at least 24 months.

Experimental Part

Example 1APreparation of Aqueous Solutions of Progesterone and HPCD with Different Molar Ratios at the Nominal Concentration of Progesterone of 32 mg/g

Solution 1: Solution with Molar Ratio HPCD:Progesterone 1:1

[0052] 2.3684 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 4.0070 g of water under magnetic stirring and, again under stirring, 0.5018 g of Progesterone were added. When the dissolution was obtained, 8.5028 g of water were added. The final concentration of Progesterone was 0.0326 g/g.

Solution 2: Solution with Molar Ratio HPCD:Progesterone 2:1

[0053] 4.7371 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 6.0048 g of water under magnetic stirring and, again under stirring, 0.5019 g of Progesterone were added. When the dissolution was obtained 4.0082 g of water were added. The final concentration of Progesterone was of 0.0329 g/g.

Solution 3: Solution with Molar Ratio HPCD:Progesterone 2.2:1

[0054] 5.2053 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 6.5008 g of water under magnetic stirring and, again under stirring, 0.5017 g of Progesterone were added. When the dissolution was obtained 3.0014 g of water were added. The final concentration of Progesterone was 0.0330 g/g.

Solution 4: Solution with Molar Ratio HPCD:Progesterone 2.5:1

[0055] 5.9154 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 8.0026 g of water under magnetic stirring and, again under stirring, 0.5018 g of Progesterone were added. When the dissolution was obtained 1.0081 g of water were added. The final concentration of Progesterone was 0.0325 g/g.

Solution 5: Solution with Molar Ratio HPCD:Progesterone 3:1

[0056] 7.1015 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 8.0082 g of water under magnetic stirring and, subsequently under stirring 0.5020 g of Progesterone were added.

[0057] The concentration of Progesterone was 0.0322 g/g.

Solution 6: Solution of Progesterone in Water

[0058] Under magnetic stirring 0.5018 g of Progesterone were dispersed in 15.0016 g of water.

[0059] The final concentration of Progesterone was 0.0324 g/g.

Example 1B: Microsomal Degradation Tests

[0060] The solutions of progesterone (1, 2, 3, 4, 5, 6) shown above in example 1A were diluted with water until a working concentration of 397.5 M was obtained.

[0061] The microsomes used in the study have an initial protein concentration of 20 mg/mL in a Sucrose solution with a concentration of 250 mM.

[0062] The sample solution was prepared by adding, in a plastic test tube, in the following order: 2 L of magnesium chloride 165 mM (MgCl.sub.2), 10 L of the solution object of the study (1, 2, 3, 4, 5, 6) with a theoretical concentration of Progesterone of 39.75 M, 73 L of phosphate buffer pH 7.4 and 10 L of solution NADPH 13 mM. The final concentration of the progesterone was of 39.75 M, whereas that of the cofactor NADPH was of 1.3 mM.

[0063] The reaction started at the moment in which 5 L of preincubated microsomes (20 mg/mL) were added, at the temperature of 37 C. for 3 minutes, to the sample solution in the test tube. The final concentration of protein was of 1 mg/mL.

[0064] The reaction was kept at a controlled temperature of 37 C. and under bland stirring.

[0065] The metabolic degradation reaction was stopped after 0, 10, 30, 60, 90 minutes adding 300 L of Acetonitrile to 100 L of the sample solution. The solutions thus obtained were centrifuged at 14000 rpm for 3 minutes and the supernatant underwent chromatographic analysis.

[0066] The HPLC-UV chromatographic analysis was carried out in isocratic flow at room temperature using a RP 18 column 5 m 3.9150 mm with mobile phase of Acetonitrile and Water in ratio 55 and 45. The operating wavelength was 241 nm, the flow 1 mL/min and the injection volume 20 L.

[0067] FIG. 1 shows the percentage of unmodified Progesterone (not degraded) over time after incubation at 37 C. with microsomes of human origin with the variation of the molar ratios between HPCD and Progesterone. The detailed description of the experiment is shown in the example 1. From such results it can be seen that when the concentration of HPCD is increased, with consequent variation of the molar ratio between Progesterone and HPCD, the active substance is less degraded. It can be observed that the increase in concentration of HPCD protects the Progesterone from metabolic activities. In an analogous manner the experiment was carried out on the solutions 1-6 of the example 4 relative to Testosterone leading to the result shown in FIG. 4.

Example 2Preparation of Aqueous Solutions of Progesterone and HPCD with Different Molar Ratios at the Nominal Progesterone Concentration of 20 mg/g

Solution 1: Solution with Molar Ratio HPCD:Progesterone 1:1

[0068] 1.4252 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 3.0053 g of water under magnetic stirring and, again under stirring, 0.3028 g of Progesterone were added. When the dissolution was obtained, 10.2039 g of water were added. The final concentration of Progesterone was of 0.0203 g/g.

Solution 2: Solution with Molar Ratio HPCD:Progesterone 2:1

[0069] 2.8445 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 4.2073 g of water under magnetic stirring and, again under stirring, 0.3026 g of Progesterone were added. When the dissolution was obtained 7.6073 g of water were added. The final concentration of progesterone was of 0.0202 g/g.

Solution 3: Solution with Molar Ratio HPCD:Progesterone 2.2:1

[0070] 3.1255 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 5.0073 g of water under magnetic stirring and, again under stirring, 0.3049 g of Progesterone were added. When the dissolution was obtained 6.5028 g of water were added. The final concentration of Progesterone was of 0.0204 g/g.

Solution 4: Solution with Molar Ratio HPCD:Progesterone 2.5:1

[0071] 3.5548 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 6.0029 g of water under magnetic stirring and, again under stirring, 0.3031 g of Progesterone were added. When the dissolution was obtained 5.1053 g of water were added. The final concentration of Progesterone was of 0.0203 g/g.

Solution 5: Solution with Molar Ratio HPCD:Progesterone 3:1

[0072] 4.2670 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 7.0053 g of water under magnetic stirring and, subsequently, under stirring 0.3064 g of Progesterone were added. When the dissolution was obtained 3.4019 g of water were added.

[0073] The concentration of Progesterone was of 0.0205 g/g.

Solution 6: Solution of Progesterone in Water

[0074] Under magnetic stirring 0.3013 g of progesterone in 14.6575g of water were dispersed.

[0075] The final concentration of Progesterone was of 0.0201 g/g.

Example 3Preparation of Aqueous Solutions of Testosterone and HPCD with Different Molar Ratios at the Nominal Concentration of Testosterone of 20 mg/g

Solution 1: Solution with Molar Ratio HPCD:Testosterone 1:1

[0076] 1.5600 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 6.0062 g of water under magnetic stirring and, subsequently under stirring 0.3021 g of Testosterone were added. When the dissolution was obtained 6.6003 g of water were added.

[0077] The concentration of Testosterone was of 0.0209 g/g.

Solution 2: Solution with Molar Ratio HPCD:Testosterone 2:1

[0078] 3.1395 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 6.0060 g of water under magnetic stirring and, subsequently 0.3041 g of Testosterone were added under stirring. When the dissolution was obtained 5.0020 g of water were added.

[0079] The concentration of Testosterone was of 0.0210 g/g.

Solution 3: Solution with Molar Ratio HPCD:Testosterone 2.2:1

[0080] 3.4206 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 6.0077 g of water under magnetic stirring and, subsequently 0.3012 g of Testosterone were added under stirring. When the dissolution was obtained 4.8045 g of water were added.

[0081] The concentration of Testosterone was of 0.0207 g/g.

Solution 4: Solution with Molar Ratio HPCD:Testosterone 2.5:1

[0082] 3.8857 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 6.0007 g of water under magnetic stirring and, subsequently 0.3011 g of Testosterone were added under stirring. When the dissolution was obtained, 4.2050 g of water were added.

[0083] The concentration of Testosterone was of 0.0209 g/g.

Solution 5: Solution with Molar Ratio HPCD:Testosterone 3:1

[0084] 4.6613 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 6.0070 g of water under magnetic stirring and, subsequently 0.3010 g of Testosterone were added under stirring. When the dissolution was obtained 3.2041 g of water were added.

[0085] The concentration of Testosterone was of 0.0212 g/g.

Solution 6: Solution of Testosterone in Water

[0086] Under magnetic stirring 0.3025 g of Testosterone were dispersed in 14.4962 g of water.

[0087] The final concentration of Testosterone was of 0.0204 g/g.

Example 4Preparation of Aqueous Solutions of Testosterone and HPCD with Different Molar Ratios at the Nominal Concentration of Testosterone of 35 mg/g

Solution 1: Solution with Molar Ratio HPCD:P Testosterone 1:1

[0088] 3.2091 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 7.0070 g of water under magnetic stirring and, subsequently under stirring 0.6218 g of Testosterone were added. When the dissolution was obtained 6.9264 g of water were added.

[0089] The concentration of Testosterone was of 0.0350 g/g.

Solution 2: Solution with Molar Ratio HPCD:Testosterone 2:1

[0090] 6.4156 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 7.0059 g of water under magnetic stirring and, subsequently under stirring 0.6218 g of Testosterone were added. When the dissolution was obtained 3.7234 g of water were added.

[0091] The concentration of Testosterone was of 0.0350 g/g.

Solution 3: Solution with Molar Ratio HPCD:Testosterone 2.2:1

[0092] 7.0702 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 7.0010 g of water under magnetic stirring and, subsequently under stirring 0.6226 g of

[0093] Testosterone were added. When the dissolution was obtained 3.0966 g of water were added.

[0094] The concentration of Testosterone was of 0.0350 g/g.

Solution 4: Solution with Molar Ratio HPCD:Testosterone 2.5:1

[0095] 8.0255 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 9.0089 g of water under magnetic stirring and, subsequently under stirring 0.6219 g of Testosterone were added. When the dissolution was obtained 0.1241 g of water were added.

[0096] The concentration of Testosterone was of 0.0350 g/g.

Solution 5: Solution with Molar Ratio HPCD:Testosterone 3:1

[0097] 9.6645 g of HPCD (content of unsubstituted cyclodextrin present in the HPCD lower than 0.3%) were dissolved in 10.4679 g of water under magnetic stirring and, subsequently under stirring 0.6325 g of

[0098] Testosterone were added. The concentration of Testosterone was of 0.0305 g/g.

Solution 6: Solution of Testosterone in Water

[0099] Under magnetic stirring 0.6213 g of Testosterone were dispersed in 17.1358 g of water.

[0100] The final concentration of Testosterone was of 0.0350 g/g.

Example 5Permeation Study of the Solutions with Different Molar Ratios Between HPCD and Progesterone and Between HPCD and Testosterone

[0101] The solutions (1, 2, 3, 4, 5, 6), the preparation of which is shown in Examples 1A, 2 , 3 and 4 underwent permeation analysis through suitable membranes such as to emulate the gastrointestinal mucous.

[0102] The study was carried out using the Franz Cell diffusion system.

[0103] The receptor has a volume of 7 mL and made up of a solution of 77% Ethanol and 23% water. The available permeation area of each of the 6 cells of the system was of 1.767 cm.sup.2. The stirring velocity of the system was of 400 rpm and the temperature was kept constant at 37 C.

[0104] The preparation of the Franz Cell system was completed by arranging the membrane between the receptor and the donor. In the donor of each cell 1 mL of the studied solutions, drawn under stirring, was introduced.

[0105] During the permeation analysis, not only the solution of the receptor, but also the solutions 16 present above the membrane were kept under stirring.

[0106] At withdrawal times of 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 20, 24 hours, permeate aliquots were collected from every cell (total volume withdrawn by the system from the receptor solution 2.5 mL, the sampling volume of which is 1.0 mL) and directly analysed with liquid chromatography at high pressure (HPLC) with a UV detector. In the Franz Cell system used, after sampling, the receptor solution was restored by the amount withdrawn.

[0107] For each aliquot collected, the amount of permeated hormone was analysed with the validated HPLC-UV method.

[0108] The amount of permeated progesterone was calculated starting from a calibration curve in the concentration range of 0.0378503.896 g/mL whereas for Testosterone the calibration curve was in the concentration range of 0.0223514.925 g/mL.

[0109] FIGS. 2, 3, 4 and 5 show the performance of the permeated hormone by unit area as a function of the drawing times considering the amount of hormone present by unit weight of solution.

[0110] The results, shown in FIG. 2, (also reproduced in FIG. 9 as a function of the growing ratios between HPCD and Pgr) highlight the obtaining of the best permeation values within an intermediate range of molar ratios between HPCD and Pgr, with the best values between 2:1 and 2.2:1. Therefore the permeability does not vary in a manner that is proportional to the amount of HPCD, but is maximised at the values of the intermediate ratio levels between HPCD and hormone, highlighted here.

[0111] The best permeation values found here correspond to molar ratios between HPCD and steroid hormone at which the degradative metabolism is already considerably inhibited by the presence of cyclodextrin (see the data of the example 1B); therefore, the optimal permeation effect, found here at molar ratios between HPCD and hormone around 2:1, is added to that of a limited degradative metabolism at the gastrointestinal mucous level.

[0112] In FIG. 2 the concentration of Progesterone used in the 6 solutions with different molar ratios between HPCD and Prg is of 32 mg of Progesterone per gram of solution. Moreover FIGS. 9 highlights that the comparative 1:1 complexes (theoretically more prone to hormone permeation) permeated worse than the complexes in accordance with the invention. An analogous result, shown in FIG. 3 (cf. also FIG. 10) was obtained by repeating such an experiment with a different concentration of Progesterone, 20 mg/g. These results confirm that the permeability of Progesterone is influenced by the molar ratio between HPCD and Prg and not by the concentration of the complex in solution.

[0113] Similar results were found in the case of Testosterone: in particular, FIGS. 5 and 6 highlight the obtaining of optimal permeability values for molar ratios HPCD:Testosterone of around 2:1.

Example 6 Stability Tests

[0114] A solution with molar ratio HPCD:Prg=2:1 was obtained by dissolving, in a suitable dissolver, 2720 g of HPCD (content of unsubstituted cyclodextrin present in HPCD lower than 0.3%) in 5000 g of water and then adding 272 g of Progesterone. When the dissolution was obtained 5000 g of water were added. The final concentration of Progesterone determined through HPLC/UV analysis was of 20.61 mg/g. The solution thus obtained undergoes filtration in series through filters of 0.45 and 0.22 m and is subsequently separated in vials filled with a volume such as to ensure a dose of Progesterone for vials of 25 mg. The solution has a density of 1.0675 g/mL. The vials were closed hermetically and underwent a stability study in ICH conditions at the temperature of 25 C./60% R.H.

[0115] As illustrated in table 1, the solution was stable for at least 24 months without undergoing considerable variations in the amount of Progesterone.

TABLE-US-00001 TABLE 1 Stability data of the aqueous solution of Progesterone and HPCD with a molar complexation ratio HPCD:Prg 2:1 25 C. 2 C./60% 5% R. H Analysis Specification 0 3 m 6 m 9 m 12 m 18 m 24 m Progesterone Positive Positive Positive Positive Positive Positive Positive Positive Identification (HPLC) Progesterone 95.0-105.0% of the 100.3% 99.5% 102.1% 101.3% 100.5% 101.0% 99.5% Assay theoretical value at release 90.0-105.0% of the theoretical value during stability study Each individual 0.5% <0.1% <0.1% <0.1% 0.1% 0.1% 0.1% 0.1% unknown impurity Total 0.8% <0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 0.2% Impurities Endotox <1.0 EU/mg <0.2 EU/mg NA <0.2 EU/mg NA <0.2 EU/mg NA <0.2 EU/mg (LAL Test)

Example 7 Comparative Pharmacokinetic Tests

[0116] The solution described in example 6 was used in a comparative clinical pharmacokinetic study (FIG. 7, Tables 2 and 3). Four female volunteers in post-menopausal age, non- or ex-smokers, with an age of between 18 and 75 years received, according to a crossover design, a single dose of 100 mg of Progesterone dissolved in the aforementioned solution or of 200 mg of Progesterone in soft capsules (reference formulation on the market, Prometrium). Tables 2 and 3 show the results of the pharmacokinetic parameters obtained from the clinical study: Table 2 refers to the formulation object of the present patent whereas Table 3 refers to the commercial reference formulation (Prometrium).

[0117] The results in Table 2 prove that the present composition generates an overall exposure per unit dose (AUC/dose) approximately 9 times higher than that obtained with the Reference formulation (Table 3) and an average peak concentration (C.sub.max) about 30-fold higher; moreover, the peak time (t.sub.max) achieved with the progesterone solution is substantially lower.

[0118] Moreover, plasma levels of Progesterone observed for the formulation object of the present invention (average C.sub.max 60 ng/mL ca. for a dose of 100 mg Progesterone) are in line with those which were reported in the already mentioned patent of Pitha et at, in which a dose of 100 mg Progesterone administered in form of a HPCD:Prg 1:1 complex via buccal routetherefore without hepatic first-pass effectgave a C.sub.max of around 50 ng/mL. This confirms that the complexes of the invention maintain a remarkable bioavailability, despite being likely subject to gastro-intestinal and hepatic first-pass metabolism. A dose-effective treatment of progesterone/testosterone complexes with HPCD via the oral route (more patient-friendly than e.g. the sublingual/buccal/nasal route) is therefore obtained. As a further advantage, the composition of the invention makes it possible to significantly reduce the inter-individual variability of the plasma levels, expressed in the table by the CV % value of AUC and C.sub.max, with respect to the commercial formulation of Table 3.

TABLE-US-00002 TABLE 2 PK parameters of Progesterone administered as an oral solutioncomplexed with HPfCD, ratio 2:1 C.sub.max t.sub.max AUC t.sub.1/2, z Subject (pg/mL) (h) (pg .Math. h/mL) (h) C.sub.max/Dose AUC/Dose 1 57325.1 0.5 77021.2 5.21 11465.02 15404.24 2 63841.2 0.5 91656.5 4.79 12768.24 18331.30 3 63192.8 0.5 62893.8 3.85 12638.56 12578.76 4 59081.4 0.5 56407.4 5.11 11816.28 11281.48 Mean 60860.1 0.5 71994.7 4.7 12172.0 14398.9 min 57325.1 0.5 56407.4 3.9 11465.0 11281.5 max 63841.2 0.5 91656.5 5.2 12768.2 18331.3 SD 3161.7 0.0 15680.6 0.6 632.3 3136.1 CV % 5.2 0.0 21.8 13.1 5.2 21.8

TABLE-US-00003 TABLE 3 PK parameters of Progesterone, reference formulation (Prometrium) C.sub.max t.sub.max AUC t.sub.1/2 ,z Subject (pg/mL) (h) (pg .Math. h/mL) (h) C.sub.max/Dose AUC/Dose 1 1912.6 0.75 6686.4 1.22 191.26 668.64 2 3925.4 1 13518.3 2.53 392.54 1351.83 3 8065.4 2 34464.2 1.83 806.54 3446.42 4 2323.5 1 9737.2 2.42 232.35 973.72 Mean 4056.7 1.2 16101.5 2.0 405.7 1610.2 min 1912.6 0.8 6686.4 1.2 191.3 668.6 max 8065.4 2.0 34464.2 2.5 806.5 3446.4 SD 2810.0 0.6 12556.7 0.6 281.0 1255.7 CV % 69.3 46.7 78.0 30.2 69.3 78.0

[0119] Therefore the formulation object of the present patent, characterised by an excellent bioavailability of the hormones contained in it, makes it possible to achieve high and effective plasma concentrations after oral administration of doses that are lower with respect to oral formulations currently on the market (e.g. Prometrium, Andriol), and with a greater reproducibility of the relative plasma curves, thus leading to a clear advantage in terms of compliance of the patient and of effectiveness and safety of the treatment.

Example 8Preparation of Aqueous Solutions of Testosterone and HPBCD with Different Molar Ratios at the Nominal Concentration of Testosterone of 26 mg/g

Solution 1: Solution with Molar Ratio HPCD:Testosterone 2:1

[0120] In a suitable dissolver, 4.13 g of HPCD (content of unsubstituted cyclodextrin present in HPCD lower than 0.3%) were dissolved in 4.48 g of water, subsequently 0.4 g of Testosterone were added. When the dissolution was obtained 6.38 g of water was added. The solution obtained was filtered through filter of 0.45 m and subsequently separated in vials filled with a volume such as to ensure a dose of Testosterone for vials of 26 mg. The final concentration of Testosterone in the vial determined trough HPLC/UV analysis was of 26,0 mg/g.

[0121] The vials were closed hermetically and underwent a stability study in ICH conditions at the temperature of 25 C./60% R.H.

[0122] As illustrated in table 4, the solution was stable for at least 24 months without undergoing considerable variations in the amount of Testosterone.

TABLE-US-00004 TABLE 4 Stability data of the aqueous solution of Testosterone and HPCD with a molar complexation ratio HPCD:Tst 2:1 25 C. 2 C./60% R. H. Analysis Specification 0 3 m 6 m 9 m 12 m 18 m 24 m Testosterone Positive Positive Positive Positive Positive Positive Positive Positive Identification (HPLC) Testosterone 95.0-105.0% 101.00 101.10 100.67 100.27 100.31 100.14 99.95 Assay of the theoretical value at release 90-105% of the theoretical value during stability study Each individual 0.2% <0.1% <0.1% <0.1% 0.1% 0.1% 0.1% 0.1% unknown impurity Total 2.0% 0.5% 0.5% 0.5% 0.7% 0.7% 0.7% 0.8% Impurities TAMC 100 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g TYMC 100 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g Escherichia Absent/g Absent/g Absent/g Absent/g Absent/g Absent/g Absent/g Absent/g Coli

Solution 2: Solution with Molar Ratio HPCD:Testosterone 3:1

[0123] In a suitable dissolver, 6.195 g of HPCD (content of unsubstituted cyclodextrin present in HPCD lower than 0.3%) were dissolved in 6.75 g of water, subsequently 0.4 g of Testosterone were added. When the dissolution was obtained 2.041 g of water was added. The solution obtained was filtered through filter of 0.45 m and subsequently separated in vials filled with a volume such as to ensure a dose of Testosterone for vials of 26 mg. The final concentration of Testosterone in the vial determined trough HPLC/UV analysis was of 26.0 mg/g.

[0124] The vials were closed hermetically and underwent a stability study in ICH conditions at the temperature of 25 C./60% R.H.

[0125] As illustrated in table 5, the solution was stable for at least 24 months without undergoing considerable variations in the amount of Testosterone.

TABLE-US-00005 TABLE 5 Stability data of the aqueous solution of Testosterone and HPCD with a molar complexation ratio HPCD:Tst 3:1 25 C. 2 C./60% R. H. Analysis Specification 0 3 m 6 m 9 m 12 m 18 m 24 m Testosterone Positive Positive Positive Positive Positive Positive Positive Positive Identification (HPLC) Testosterone 95.0-105.0% 100.43 100.89 100.28 99.89 99.95 100.02 100.12 Assay of the theoretical value at release 90-105% of the theoretical value during stability study Each 0.2% <0.1% <0.1% <0.1% <0.1% <0.1% 0.1% 0.1% individual unknown impurity Total 2.0% 0.5% 0.7% 0.7% 0.8% 0.8% 0.9% 0.9% Impurities TAMC 100 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g TYMC 100 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g <1 CFU/g Escherichia Absent/g Absent/g Absent/g Absent/g Absent/g Absent/g Absent/g Absent/g Coli

Example 9Comparative Pharmacokinetic Tests (Testosterone)

[0126] The solutions described in example 8 were used in a comparative clinical study of pharmacokinetics (FIG. 8 Tables 6-8). Three healthy female subjects, non or ex-smokers, with an age of between 18 and 75 years received, according to a crossover design, a single dose of 26 mg of Testosterone dissolved in the aforementioned solution or 40 mg of Testosterone undecanoate corresponding to 25.26 mg of Testosterone, in soft capsules (reference formulation on the market, Andriol). Tables 6-8 shows the results of the pharmacokinetics parameters obtained by the clinical study: Table 6 and 7 refer to the Testosterone formulations, object of the present patent, with respectively a molar ratio between HPCD and Testosterone of 2:1 and 3:1, whereas Table 8 refers to the commercial reference formulation (Andriol).

TABLE-US-00006 TABLE 6 PK parameters of Testosterone administered as an oral solution complexed with HPCD, ratio 2:1 C.sub.max t.sub.max AUC t.sub.1/2 Subject (pg/mL) (h) (pg * h/mL) (h) C.sub.max/Dose AUC/Dose 1 30655.7 0.3 35709.2 0.7 1179.1 1373.4 2 25301.7 0.3 23498.6 0.6 973.1 903.8 3 15939.8 0.3 22365.9 0.2 613.1 860.2 average 23965.8 0.3 27191.2 0.5 921.8 1045.8 min 15939.8 0.3 22365.9 0.2 613.1 860.2 max 30655.7 0.3 35709.2 0.7 1179.1 1373.4 SD 7448.4 0.0 7398.5 0.3 286.5 284.6 CV % 31.1 0.0 27.2 54.7 31.1 27.2

TABLE-US-00007 TABLE 7 PK parameters of Testosterone administered as an oral solution complexed with HPCD, ratio 3:1 C.sub.max t.sub.max AUC t.sub.1/2 Subject (pg/mL) (h) (pg * h/mL) (h) C.sub.max/Dose AUC/Dose 1 14185.4 0.3 19142.9 0.9 545.6 736.3 2 10021.3 0.3 16948.4 0.8 385.4 651.9 3 9015.7 0.3 14753.8 0.6 346.8 567.5 average 11074.1 0.3 16948.4 0.7 425.9 651.9 min 9015.7 0.3 14753.8 0.6 346.8 567.5 max 14185.4 0.3 19142.9 0.9 545.6 736.3 SD 2741.0 0.0 2194.5 0.1 105.4 84.4 CV % 24.8 0.0 12.9 18.1 24.8 12.9

TABLE-US-00008 TABLE 8 PK parameters of Testosterone, reference formulation (Andriol) Cmax tmax AUC t Subject (pg/mL) (h) (pg * h/mL) (h) Cmax/Dose AUC/Dose 1 225.6 3.5 886.0 1.3 8.9 35.1 2 453.7 3.5 3297.7 3.4 18.0 130.6 3 329.1 3.5 1789.2 2.3 13.0 70.8 average 336.1 3.5 1991.0 2.3 13.3 78.8 min 225.6 3.5 886.0 1.3 8.9 35.1 max 453.7 3.5 3297.7 3.4 18.0 130.6 SD 114.2 0.0 1218.5 1.1 4.5 48.2 CV % 34.0 0.0 61.2 45.5 34.0 61.2

[0127] The results reported in Tables 6 and 7 show that the present composition generates an overall exposure per unit dose (AUC/dose) 13-fold ca. higher than that obtained with the Reference formulation (Table 8) in the case of a complex with molar ratio HPBCD:Tst 2:1 and of 8 times higher than that obtained with the Reference formulation (Table 8) in the case of a complex with molar ratio HPBCD:Tst 3:1. Similarly the peak concentrations per unit dose (C.sub.max/Dose) are, for both complex solutions, higher than that obtained with the reference formulation. Moreover, the peak time (t.sub.max) achieved with the HPBCD:Tst solutions is substantially lower as compared to that of the Reference formulation. The results confirm those obtained in Experiment 5 (FIGS. 5 and 6) and shown that the HPBCD:Tst complexes maintain a remarkable bioavailability despite being likely subject to the hepatic first-pass effect. A dose-effective treatment of Testosterone complexes with HPCD via the oral route (more patient-friendly than e.g. the sublingual/buccal/nasal route) is therefore obtained. As a further advantage, the composition of the invention makes it possible to significantly reduce the inter-individual variability of the plasma levels, expressed in the table by the CV % of AUC and C.sub.max, with respect to the commercial formulation of Table 8.