ES2358330T3 - PHARMACEUTICAL FORMULATIONS OF FSH AND / OR LH LIQUID OR LIOPHILIZED TOGETHER WITH POLOXÁMERO 188 NON-IONIC TENSIOACTIVE AND A BACTERIOSTATIC AGENT. - Google Patents

Abstract

A liquid pharmaceutical composition comprising follicle stimulating hormone (FSH) or one of its variants, as well as a surfactant that is poloxamer 188 and also comprises methionine and a bacteriostatic agent chosen from phenol and m-cresol.

Description

Field of the Invention

The invention relates to the field of pharmaceutical formulations of follicle stimulating hormone (generally abbreviated as FSH by its initials in English: follicle-stimulating hormone), formulations of luteinizing hormone, (generally abbreviated as LH by its initials in English: luteinising hormone) and mixtures of FSH and luteinizing hormone (LH), as well as methods to produce such formulations.

Background of the invention

Follicle stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (CG) are injectable proteins that fall into the gonadotropin class. FSH, LH and hCG are used alone and in combination in the treatment of infertility and reproductive disorders in both female and male patients. 10

In nature, FSH and LH are produced by the pituitary gland. For pharmaceutical use, FSH and LH and their variants can be produced by recombination (rFSH and rLH) or they can be produced from the urine of post-menopausal women (uFSH and uLH).

FSH is used in female patients to induce ovulation (OI) and in controlled ovarian hyperstimulation (COH) for assisted reproduction techniques (ART). In a typical treatment regimen for ovulation induction, 15 patients are given daily injections of FHS or a variant (approximately 75 to 300 IU of FSH / day) for a period of approximately 6 to approximately 12 days. In a typical treatment regimen for controlled ovarian hyperstimulation, the patient is given daily injections of FHS or a variant (approximately 150-600 IU of FSH / day) for a period of approximately 6 to approximately 12 days.

FSH is also used to induce spermatogenesis in men suffering from oligospermia. A regimen that uses 150 IU of FSH 3 times a week in combination with 2,500 IU of hCG twice a week has been used successfully to obtain an improvement in sperm count in men suffering from hypogonadotropic hypogonadism1.

LH is used in female patients in combination with FSH in OI and COH, particularly in those patients who have very low levels of endogenous LH or resistance to LH, such as women suffering from hypogonadotropic hypogonadism (HH, Group I of the WHO) or older patients (i.e., 35 years of age or older) and in 25 patients in whom embryo implantation or early abortion pose problems. LH in combination with FSH has traditionally been available in a preparation called human menopausal gonadotropins (hMG) extracted from the urine of post-menopausal women. LA hMG has an FSH: LH activity ratio of 1: 1.

The CG acts on the same receptor as the LH and produces the same responses. GC has a half-life in the circulation greater than LH and therefore is generally used as a source of long-acting LH activity. 30 GC is used in OI and COH regimens to mimic the natural peak of LH and activate ovulation. An injection of human chorionic gonadotropin (hCG) is used to activate ovulation at the end of stimulation with FSH or with a mixture of FSH and LH. GC can also be used in conjunction with FSH during stimulation for OI and COH in order to provide LH activity during stimulation in patients in whom it is desired to have LH activity, such as those mentioned above. 35

FSH, LH and CG are members of the family of glycoprotein hormones, heterodimers, which also includes thyroid stimulating hormone (usually abbreviated as TSH by its initials in English: thyroid stimulating hormone). Members of this family are heterodimers that comprise a subunit  and a . Subunits are held together by non-covalent interactions. The human FSH heterodimer (hFSH) consists of: (i) a mature alpha amino acid subunit of 92 amino acids that is also common in the other 40 members of the human family (i.e. chorionic gonadotropin (CG), the hormone luteinizing (LH) and thyroid stimulating hormone (TSH)); and (ii) a mature beta subunit of 111 amino acids that is characteristic of FSH2. The human LH heterodimer consists of (i) the mature alpha glycoprotein alpha subunit of 92 amino acids; and (ii) a mature beta subunit of 112 amino acids that is characteristic of LH3. The alpha and beta subunits of glycoproteins may have a tendency to dissociate in the formulations due to interaction with a preservative, a surfactant and other excipients. The dissociation of the subunits leads to the loss of biological power4.

FSH is formulated for intramuscular (IM) or subcutaneous (SC) injection. The FSH is supplied in lyophilized form (solid) in bottles or ampoules of 75 IU / bottle and 150 IU / bottle with a durability of one year and a half to two years if stored at 2-25ºC. The solution for injection is formed by reconstituting the lyophilized product with water for injection (API). For ovulation induction or controlled ovarian hyperstimulation, 50 daily injections with initial doses of 75 to 600 IU are recommended for up to approximately ten days. Depending on the patient's response, up to three cycles of treatment with increasing doses of FSH can be used. With lyophilized formulations, the patient is asked to reconstitute a bottle of new lyophilized material with diluent and to administer it immediately after reconstitution daily [Prospectus N1700101A, published in February 1996 by Fertinex® (urofolitropin for injection, purified) for injection subcutaneous, by Serono Laboratories, Inc., Randolph, MA]. 55

FSH has also been formulated in liquid formats either for single doses or for multiple doses, in bottles or ampoules. Single dose formats must remain stable and powerful during storage before use. Multi-dose formats must not only remain stable and potent during storage before use, but must also remain stable, potent and relatively free of bacteria during the period of administration in a multi-dose use regimen, after Blister seal 5 has been broken. For this reason, multi-dose formats often contain a bacteriostatic agent.

LH is formulated for intramuscular (IM) or subcutaneous (SC) injection. LH is supplied in lyophilized (solid) form in bottles or ampoules of 75 IU / bottle with a durability of one year and a half to two years if stored at 2-25ºC. The solution for injection is formed by reconstituting the lyophilized product with water for injection (API). For ovulation induction or controlled ovarian hyperstimulation, along with FSH, daily injections with initial doses of 75 to 600 IU of LH are recommended up to approximately ten days.

EP 0618808 (Applied Research Systems ARS Holding N.V.) describes a pharmaceutical composition comprising a solid intimate mixture of gonadotropin and a stabilizing amount of sucrose alone or in combination with glycine.

EP 0814841 (Applied Research Systems ARS Holding N.V.) describes a stable liquid pharmaceutical composition comprising recombinant human chorionic gonadotropin (hCG) and a stabilizing amount of mannitol.

EP 0448146 (AZKO N.V.) discloses a lyophilisate containing stabilized gonadotropin comprising a part by weight of gonadotropin; and 200 to 10,000 parts by weight of a stabilizer of a dicarboxylic acid salt associated with gonadotropin. twenty

EP 0853945 (Akzo Nobel N.V.) describes a liquid formulation containing gonadotropin characterized in that the formulation comprises a gonadotropin and stabilizing amounts of a polycarboxylic acid or one of its salts and a thioether compound.

WO 00/04913 (Eli Lilly and Co.) describes a formulation comprising FSH or a variant of FSH containing an alpha and a beta subunit and a preservative chosen from the group consisting of phenol, m-cresol, p -25 cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl paraben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof, in an aqueous diluent .

There is still a need for stable liquid formulations of FSH or variants of FSH, and mixtures of FSH and LH, for administration either in a single dose or in multiple doses.

Summary of the Invention 30

An object of the invention is to provide new lyophilized formulations as well as liquid FSH or variants of FSH and LH or variants of LH, to provide methods for their preparation and methods for pharmaceutical or veterinary use in the treatment of fertility disorders .

A further object of the invention is to provide new lyophilized and liquid formulations of mixtures of FSH and LH to provide methods for their preparation and methods for pharmaceutical or veterinary use in the treatment of fertility disorders.

In a first aspect, the invention provides a lyophilized and liquid pharmaceutical composition comprising FSH or one of its variants and the Pluronic® F68 surfactant.

In a second aspect, the invention provides a method for making a liquid pharmaceutical composition comprising forming a solution of FSH or one of its variants, the surfactant Pluronic® F68 and API. 40

In a third aspect, the invention provides a method for making a packaged pharmaceutical composition comprising dispensing a solution comprising FSH and the Pluronic® F68 surfactant in a container.

In a fourth aspect, the invention provides an article made for human pharmaceutical use comprising a vial comprising an FSH solution or a variant of the FSH and the Pluronic® F68 surfactant and written material indicating that said solution can be maintained for a period of time. period of or approximately twenty four hours 45 or more after the first use.

In a fifth aspect, the invention provides a lyophilized and liquid pharmaceutical composition comprising FSH and LH and the Pluronic® F68 surfactant.

In a sixth aspect, the invention provides a method for making a lyophilized or liquid pharmaceutical composition comprising forming a solution of FSH and LH and the Pluronic® F68 surfactant. fifty

In a seventh aspect, the invention provides a method for making a packaged pharmaceutical composition comprising dispersing a solution comprising FSH and LH and the Pluronic® F68 surfactant in a container.

In an eighth aspect, the invention provides an article made for human pharmaceutical use comprising a vial comprising a solution of FSH and LH and the Pluronic® F68 surfactant and written material indicating that said solution can be maintained for a period of or about twenty four hours or more after first use.

In a ninth aspect, the invention provides an article made for human pharmaceutical use comprising a first container comprising lyophilized FSH, or one of its variants and the Pluronic® F68 surfactant and a second container comprising a solvent for reconstitution, preferably an aqueous solution containing a bacteriostatic agent chosen from phenol and m-cresol, preferably m-cresol.

In a tenth aspect, the invention provides an article made for human pharmaceutical use comprising a first container comprising lyophilized LH, or one of its variants, and the Pluronic® F68 surfactant and a second container comprising a solvent for reconstitution, preferably an aqueous solution containing a bacteriostatic agent chosen from phenol and m-cresol, preferably m-cresol.

 In an eleventh aspect, the invention provides an article made for human pharmaceutical use comprising a first container comprising FSH as well as LH or a variant of the FSH or LH, lyophilized and the Pluronic® F68 surfactant and a second container comprising a solvent for reconstitution, preferably an aqueous solution with m-cresol.

Detailed description of the invention

Brief description of the drawings

Figure 1 shows the percentage of oxidized un subunit in FSH formulations containing Pluronic® F68, methionine at 10 g / ml (“Met 10 mcg / ml”) and 100 g / ml (“Met 100 mcg / ml ”) Versus a formulation without methionine (“ Without 20 methionine ”), for times of 0, 1 week and 2 weeks.

The liquid and lyophilized formulations of FSH or FSH and LH of the invention have improved and more suitable properties or stability and are useful for the treatment of infertility in women and / or men. These formulations and the elaborated articles are additionally suitable for use in injectable delivery systems and alternative systems, for example, but not limited to, nasal, pulmonary, oral, subcutaneous, intramuscular or prolonged parenteral release. In a particularly preferred embodiment, the formulations of the invention are for subcutaneous and / or intramuscular injection. The solutions of FSH or variants of FSH and LH and the formulations provided may also have increased in vivo potency over time, as compared to known commercial products, by preventing or reducing the loss of activity or of stability, or by improving any aspect of efficiency or adequacy of administration, for example at least one among the mode, frequency, dosage, comfort, ease of use, in vitro or in vivo biological activity and the like.

Follicle stimulating hormone, or FSH, as used herein, refers to FSH produced as a complete mature protein that includes, but is not limited to, human FSH or "hFSH", whether produced by recombination. or isolated from human sources, such as the urine of post-menopausal women. The protein sequence of the alpha subunit of the glycoprotein is given in SEQ. ID. No. 1, and the protein sequence of the beta subunit of human FSH is given in SEQ. ID. No. 2

  The term "FSH variant" is intended to include those molecules that differ from human FSH in the amino acid sequence, the glycosylation pattern or in a junction within the subunit, but which has FSH activity. Examples include CTP-FSH, a long-acting modified recombinant FSH, consisting of the natural  subunit and a  hybrid subunit in which the carboxy-terminal peptide of the hCG has been condensed with the C terminal of the  subunit of the FSH, as described in LaPolt et al., Endocrinology, 1992, 131, 2514-2520; or in Klein et al., Development and characterization of a long-acting recombinant hFSH agonist; Human Reprod. 2003, 18, 50-56. Also included is a single chain CTP-FSH, a single chain molecule, consisting of the following sequences (from N terminal to C terminal):

 FSH

 hCG-CTP (113-145) FSH

where FSH means the un subunit of FSH, hCG CTP (113-145) means the carboxy terminal peptide of hCG and FSH means the un subunit of FSH, as described in Klein et al.5. Other examples of FSH variants include FSH molecules that have additional glycosylation sites incorporated in the  and / or  subunit, as described in WO 01/58493 (Maxygen), particularly as described in claims 10 and 11 50 of WO 01/58493, and FSH molecules with SS bonds within the subunit, as described in WO 98/58957.

The FSH variants referred to herein include the carboxy-terminal deletions of the beta subunit that are shorter than the complete mature SEQ protein. ID. No. 2. The carboxy-terminal deletions of the human beta subunit are given in the SEQ. ID. Nº: 3, 4 and 5. It is understood that the carboxy-55 variants

beta chain terminals form dimers with a known alpha subunit to form a FSH variant heterodimer.

FSH heterodimers or FSH variant heterodimers can be produced by any suitable method, such as by recombination, by isolation or purification from natural sources as may be the case, or by chemical synthesis, or by any combination of they. 5

The use of the term "recombinant" refers to preparations of FSH, LH or variants of FSH and LH that are produced by the use of recombinant DNA technology (see eg WO 85/01958). Genomic sequences and FSH cDNA clones for the alpha and beta subunits of several species are known6. An example of a method that expresses FSH or LH using recombinant technology is by transfecting eukaryotic cells with DNA sequences encoding an alpha and beta subunit of FSH or 10 LH, either provided on a vector or on two vectors, each subunit having a separate promoter, as described in European patents No.: EP 0211894 and EP 0487512. Another example of the use of recombinant technology to produce FSH or LH is through the use of homologous recombination to insert a regulatory segment heterologous with operative connection in endogenous sequences encoding the FSH or LH subunits, as described in European patent EP 0505500 (Applied Research Systems ARS Holding NV). fifteen

The FSH or the FSH variant used according to the present invention can be produced not only by recombinant means, including from mammalian cells, but can also be purified from other biological sources, such as from urinary sources . Acceptable methodologies include those described in Hakola, K. Molecular and Cellular Endocrinology, 127: 59-69, 1997; Keene et al., J. Biol. Chem., 264: 4769-4775, 1989; Cerpa-Poljak et al., Endocrinology, 132: 351-356, 1993; Dias et al., J. Biol. Chem., 269: 25289-25294, 1994; Flack et al., 20 J. Biol. Chem., 269: 14015-14020, 1994; and Valove et al., Endocrinology, 135: 2657-2661, 1994; in United States Patent 3,119,740 and in United States Patent 5,767,067.

The luteinizing hormone, or LH, as used herein, refers to LH produced as a complete mature protein, which includes, but is not limited to, human LH or "hLH", whether produced by recombination or well isolated from human sources, such as the urine of post-menopausal women. The protein sequence of the alpha subunit of the human glycoprotein is given in SEQ. ID. No. 1, and the protein sequence of the beta subunit of human LH7 is given in SEQ. ID. No. 6. In a preferred embodiment, the LH is recombinant.

The term "LH variant" is intended to include those molecules that differ from human LH in the amino acid sequence, the glycosylation pattern or at a junction within the subunit, but which has LH activity. 30

The LH heterodimers or LH variant heterodimers can be produced by any suitable method, such as by recombination, by isolation or purification from natural sources as may be the case or by chemical synthesis, or any combination thereof.

The term "administer" or "administration" means introducing a formulation of the present invention into the body of a patient who needs it to treat a disease or disorder. 35

The term "patient" means a mammal that is treated for a disease or disorder. Patients have, but are not limited to, the following origins: human, sheep, pigs, horses, cattle, rabbits or the like.

The term "potency" with respect to FSH activity refers to the ability of an FSH formulation or a mixed formulation to produce biological responses associated with FSH, such as an ovarian weight gain in the Steelman-Pohley8 trial, or Follicular growth in a female patient. Follicular growth in a female patient can be evaluated by ultrasound, for example, depending on the number of follicles having an average diameter of or about 16 mm on day 8 of stimulation. Biological activity is assessed against an accepted pattern for FSH.

 The term "potency" with respect to LH activity refers to the ability of an LH formulation or a mixed formulation to produce biological responses associated with LH, such as the weight gain method of the seminal vesicle9. Biological activity is evaluated with respect to an accepted pattern for LH.

The term "aqueous diluent" refers to a liquid solvent that contains water. Aqueous solvent systems may consist only of water, or may consist of water plus one or more miscible solvents, and may contain dissolved solutes, such as sugars, buffers, salts or other excipients. The most commonly used non-aqueous solvents are short chain organic alcohols, such as methanol, ethanol, propanol, short chain ketones, such as acetone, and polyalcohols, such as glycerol.

An "isotonicity agent" is a compound that is physiologically tolerated and that confers an appropriate tonicity to the formulation to prevent the net flow of water through the cell membranes that are in contact with the formulation. Compounds such as glycerin with known concentrations are generally used for this purpose. Other suitable isotonicity agents include, but are not limited to, amino acids or proteins (by

for example, glycine or albumin), salts (for example, sodium chloride) and sugars (for example, dextrose, sucrose and lactose).

The term "bacteriostatic" or "bacteriostatic agent" refers to a compound or compositions added to the formulation to act as an antibacterial agent. A protected formulation of the present invention containing FSH or a variant of the FSH or FSH and LH preferably satisfies the legal or regulatory directives of the efficacy of the preservatives to be a commercially viable multipurpose product, preferably in humans. Examples of bacteriostatic agents include phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl paraben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, dehydroacetate acetate. sodium and thimerosal, phenol and m-cresol being the bacteriostatic agents used in the present invention.

The term "buffer" or the term "physiologically acceptable buffer" refers to solutions of compounds that are known to be safe for pharmaceutical or veterinary use in formulations and that have the effect of maintaining or controlling the pH of the formulation in the Desired pH range for the formulation. Suitable buffers to control the pH of a moderately acidic pH at a moderately basic pH include, but are not limited to, compounds such as phosphate, acetate, citrate, arginine, TRIS and histidine. "TRIS" refers to 2-amino-2-hydroxymethyl-1,3-propanediol and any of its pharmacologically acceptable salts. Preferred buffers are phosphate buffers with saline solution or an acceptable salt.

The term "phosphate buffer" refers to solutions containing phosphoric acid or its salts, adjusted to a desired pH. Generally phosphate buffers are prepared from phosphoric acid or a phosphoric acid salt, including, but not limited to, sodium and potassium salts. Several phosphoric acid salts are known in the art, such as sodium and potassium, monobasic, dibasic and tribasic salts of the acid. It is also known that phosphoric acid salts are presented as salt hydrates. Phosphate buffers may cover a pH range such as from about pH 4 to about pH 10, and the preferred ranges are from about pH 5 to about pH 9 and a more preferred range of from or about 6.0 to or up to about 8.0, more preferably at or about pH 7.0.

The term "jar" broadly refers to a container suitable for preserving the FSH in solid or liquid form in a sterile contained state. Examples of a vial as used herein include ampoules, cartridges, blister packs, and other similar containers suitable for delivering FSH to the patient by a syringe, pump (including osmotic), catheter, transdermal patch or pulmonary spray. or transmucosal. Suitable bottles for products packaged for parenteral, pulmonary, transmucosal or transdermal administration are well known and recognized in the art. 30

The term "stability" refers to the physical, chemical and conformational stability of FSH and LH in the formulations of the present invention (including the maintenance of biological potency). The instability of a protein formulation can be caused by chemical degradation or aggregation of the protein molecules to form higher order polymers, by dissociation of the heterodimers into monomers, deglycosylation, modification of glycosylation, oxidation (particularly of the subunit ) or any other structural modification that reduces at least one biological activity of an FSH polypeptide included in the present invention.

A "stable" solution or formulation is one in which the degree of degradation, modification, aggregation, loss of biological activity and the like, of the proteins therein is acceptably controlled and does not increase unacceptably over time. Preferably, the formulation maintains at least up to or about 80% of the FSH activity indicated on the label and at least up to or about 80% of the activity 40 of the LH indicated on the label, during a period of 6 months at a temperature of or about 2-8 ° C, more preferably of or about 2-8 ° C, more preferably of or about 4-5 ° C. FSH activity can be measured using the Steelman-Pohley ovarian weight gain bioassay5. LH activity can be measured using the seminal vesicle weight gain bioassay10.

The term "treatment" refers to the administration, monitoring, control and / or care of a patient for whom the administration of FSH and / or LH is adequate for the purpose of follicle or testicular stimulation or any other physiological response. regulated by the FSH and / or the LH. Treatment may therefore include, but is not limited to, the administration of FSH and / or LH for induction or improvement of sperm quality, stimulation of testosterone release in males or follicular development or induction of ovulation in females.

The term "use in multiple doses" is intended to include the use of a single vial, ampoule or cartridge of a formulation of FSH 50 or a formulation of FSH and LH for more than one injection, for example 2, 3, 4, 5, 6 or more injections. Injections are preferably made for a period of at least or about 12 hours, 24 hours, 48 hours, etc., preferably for a period of or about 12 days. Injections can be spaced in time, for example, over a period of 6, 12, 24, 48 or 72 hours.

A "salt" of a protein is an acid or base addition salt. Said salts are preferably formed between any one or more of the groups loaded in the protein and any or more physiologically acceptable non-toxic cations or anions. Organic and inorganic salts include, for example, those prepared from acids such as hydrochloric, sulfuric, sulfonic, tartaric, fumaric, hydrobromic, glycolic, citric, maleic, phosphoric,

succinic, acetic, nitric, benzoic, ascorbic, p-toluenesulfonic, benzenesulfonic, naphthalenesulfonic, propionic, carbonic and the like, or, for example, ammonium, sodium, potassium, calcium or magnesium.

The inventors have found that by formulating FSH and mixtures of FSH and LH with the surfactant Pluronic® F68 (BASF, Pluronic® F68 is also known as Poloxamer 188), stable formulations are obtained that minimize the loss of active ingredient (FSH or FSH and LH) produced by adsorption on the surfaces of the bottle and / or the administration device 5 (eg, syringe, pump, catheter, etc.).

The inventors have also found that by formulating the FSH and mixtures of FSH and LH with the surfactant Pluronic® F68 (BASF, Pluronic® F68 is also known as Poloxamer 188), a stable formulation is obtained that avoids the problem of precipitation in the presence of a bacteriostatic agent, such as m-cresol and phenol. Precipitation, which results in the formation of cloudy or milky solutions, occurs when TWEEN 20 is used with m-cresol or phenol. 10

Pluronic® surfactants are block copolymers of ethylene oxide (EO) and propylene oxide (PO). The propylene oxide (PO) block is embedded between two ethylene oxide (EO) blocks.

Pluronic® surfactants are synthesized by a two-stage procedure:

1. A hydrophobe of the desired molecular weight is created by the controlled addition of propylene oxide to the hydroxyl groups of propylene glycol; Y

2. Ethylene oxide is added to fit the hydrophobe between the hydrophilic groups.

In Pluronic® F77, the percentage of polyoxyethylene (hydrophilic) is 70% and the molecular weight of the hydrophobic (polyoxypropylene) is approximately 2,306 Da. twenty

In Pluronic® F87, the percentage of polyoxyethylene (hydrophilic) is 70% and the molecular weight of the hydrophobic (polyoxypropylene) is approximately 2,644 Da.

In Pluronic® F88, the percentage of polyoxyethylene (hydrophilic) is 80% and the molecular weight of the hydrophobic (polyoxypropylene) is approximately 2,644 Da.

In Pluronic® F68, the percentage of polyoxyethylene (hydrophilic) is 80% and the molecular weight of hydrophobe 25 (polyoxypropylene) is approximately 1,967 Da.

Typical properties of Pluronic® F77 are indicated below:

Average molecular weight: 6,600;

Melting point / critical pour point: 48 ° C;

Physical form at 20 ° C: solid; 30

Viscosity (Brookfield) cps (Pa · s): 480 (0.48)

[liquid at 25 ° C, paste at 60 ° C and solid at 77 ° C];

Surface tension, dynes / cm (mN / m) at 25 ° C:

- 0.1% concentration: 47.0

- 0.01% concentration: 49.3 35

- 0.001% concentration: 52.8

Interfacial tension, dynes / cm (mN / m) at 25 ° C against Nujol:

- 0.1% concentration: 17.7

- 0.01% concentration: 20.8

- 0.001% concentration: 25.5

Draves humidification, seconds at 25 ° C:

- 1.0% concentration:> 360

- 0.1% concentration:> 360

Foam Height: 5

- Ross Miles, 0.1%, mm at 50 ° C: 100

- Ross Miles, 0.1%, mm at 26 ° C: 47

- Dynamic, 0.1%, mm at 400 ml / min:> 600

Turbidity point in aqueous solution, ºC:

- 1% concentration:> 100 10

- 10% concentration:> 100

HLB (hydrophilic-lipophilic balance): 25

The typical properties of Pluronic® F87 are indicated below:

Average molecular weight: 7,700;

Melting point / critical pour point: 49 ° C; fifteen

Physical form at 20 ° C: solid;

Viscosity (Brookfield) cps (Pa · s): 700 (0.70)

[liquid at 25 ° C, paste at 60 ° C and solid at 77 ° C];

Surface tension, dynes / cm (mN / m) at 25 ° C:

- 0.1% concentration: 44.0 20

- 0.01% concentration: 47.0

- 0.001% concentration: 50.2

Interfacial tension, dynes / cm (mN / m) at 25 ° C against Nujol:

- 0.1% concentration: 17.4

- 0.01% concentration: 20.3 25

- 0.001% concentration: 23.3

Draves humidification, seconds at 25 ° C:

- 1.0% concentration:> 360

- 0.1% concentration:> 360

Foam Height: 30

- Ross Miles, 0.1%, mm at 50 ° C: 80

- Ross Miles, 0.1%, mm at 26 ° C: 37

- Dynamic, 0.1%, mm at 400 ml / min:> 600

Turbidity point in aqueous solution, ºC:

- 1% concentration:> 100 35

- 10% concentration:> 100

HLB (hydrophilic-lipophilic balance): 24

The typical properties of Pluronic® F88 are indicated below:

Average molecular weight: 11,400;

Melting point / critical pour point: 54 ° C;

Physical form at 20 ° C: solid;

Viscosity (Brookfield) cps (Pa · s): 2,300 (2,30) 5

[liquid at 25 ° C, paste at 60 ° C and solid at 77 ° C];

Surface tension, dynes / cm (mN / m) at 25 ° C:

- 0.1% concentration: 48.5

- 0.01% concentration: 52.6

- 0.001% concentration: 55.7 10

Interfacial tension, dynes / cm (mN / m) at 25 ° C against Nujol:

- 0.1% concentration: 20.5

- 0.01% concentration: 23.3

- 0.001% concentration: 27.0

Draves humidification, seconds at 25 ° C: 15

- 1.0% concentration:> 360

- 0.1% concentration:> 360

Foam height:

- Ross Miles, 0.1%, mm at 50 ° C: 80

- Ross Miles, 0.1%, mm at 26 ° C: 37 20

- Dynamic, 0.1%, mm at 400 ml / min:> 600

Turbidity point in aqueous solution, ºC:

- 1% concentration:> 100

- 10% concentration:> 100

HLB (hydrophilic-lipophilic balance): 28 25

The typical properties of Pluronic® F68 are indicated below:

Average molecular weight: 8,400;

Melting point / critical pour point: 52 ° C;

Physical form at 20 ° C: solid;

Viscosity (Brookfield) cps (Pa · s): 1000 (1.00) 30

[liquid at 25 ° C, paste at 60 ° C and solid at 77 ° C];

Surface tension, dynes / cm (mN / m) at 25 ° C:

- 0.1% concentration: 50.3

- 0.01% concentration: 51.2

- 0.001% concentration: 53.6 35

Interfacial tension, dynes / cm (mN / m) at 25 ° C against Nujol:

- 0.1% concentration: 19.8

- 0.01% concentration: 24.0

- 0.001% concentration: 26.0

Draves humidification, seconds at 25 ° C:

- 1.0% concentration:> 360

- 0.1% concentration:> 360 5

Foam height:

- Ross Miles, 0.1%, mm at 50 ° C: 35

- Ross Miles, 0.1%, mm at 26 ° C: 40

- Dynamic, 0.1%, mm at 400 ml / min:> 600

Turbidity point in aqueous solution, ºC: 10

- 1% concentration:> 100

- 10% concentration:> 100

HLB (hydrophilic-lipophilic balance): 29

The preferred surfactant is Pluronic® F68.

Pluronic® F68 is preferably present in the formulation at a concentration that is sufficient to maintain the stability of FSH and / or LH throughout the desired storage period (eg, 6 to 12 to 24 months) and also at a concentration that is sufficient to avoid protein losses due to adsorption on surfaces, such as those in the bottle, vial or cartridge or syringe.

Preferably, the concentration of Pluronic® F68, in liquid formulations, is from or from about 0.01 mg / ml to or up to about 1 mg / ml, more preferably from or from about 0.05 mg / ml to or up to about 20 0.5 mg / ml, more particularly preferred of or from about 0.2 mg / ml to or up to about 0.4 mg / ml, most preferably of or about 0.1 mg / ml.

The follicle stimulating hormone (FSH) in lyophilized formulation is preferably present at a concentration (weight / weight) of or about 0.1 to 10 µg / mg of the total formulation. In one embodiment, the follicle stimulating hormone (FSH) is present at a concentration of or about 0.3 to 5 µg / mg of the total formulation. In a further embodiment, the follicle stimulating hormone (FSH) is present at a concentration of or about 0.37 to 2 µg / mg of the total formulation.

The luteinizing hormone (LH) in lyophilized formulation is preferably present at a concentration of or about 0.1 to 3 µg / mg of the total formulation. In one embodiment, luteinizing hormone (LH) is present at a concentration of or about 0.1 to 1 µg / mg of the total formulation. In a further embodiment, luteinizing hormone (LH) is present at a concentration of or about 0.1 to 0.6 µg / mg of the total formulation.

In liquid formulations - including reconstituted formulations - comprising FSH, preferably the concentration of FSH in the formulation is from or about 150 IU / ml to or up to about 2,000 IU / ml, more preferably from or about 300 IU / ml up to or up to about 1,500 IU / ml, more particularly preferable of or from about 450 to or up to about 750, most preferably of or about 600 IU / ml.

In liquid formulations - including reconstituted formulations - comprising LH, preferably the concentration of LH in the formulation is from or about 50 IU / ml to or up to about 2,000 IU / ml, more preferably from or about 150 IU / ml up to or up to about 1,500 IU / ml, more particularly preferably of or from about 300 to or up to about 750 IU / ml, particularly preferable of or of about 625 IU / ml.

In formulations comprising both FSH and LH, the relationship between FSH and LH (FSH: LH, UI: UI, FSH measured by the ovarian weight gain test in rats and LH measured by the weight gain test of the seminal vesicle in rats) is preferably in the range of or from about 6: 1 to or up to about 1: 6, more preferably from or from about 4: 1 to or up to about 1: 2, more particularly preferred of or from about 3: 1 to or about 1: 1. Particularly preferred ratios are 1: 1 and 2: 1.

In lyophilized formulations, the Pluronic® F68 surfactant is preferably present at a concentration of or from about 0.001 to or up to about 0.1 mg per mg of the total formulation, more preferably from or from about 0.01 to or up to about 0.075 mg / mg

Preferably, the concentration of Pluronic® F68 in the reconstituted formulations is from or from about 0.01 mg / ml to or up to about 1 mg / ml, more preferably from or from about 0.05 mg / ml to 5 or up to about 0 , 5 mg / ml, more particularly preferred of or from about 0.2 mg / ml to or up to about 0.4 mg / ml, most preferably of or about 0.1 mg / ml.

Preferably, FSH and LH are produced by recombination, particularly preferably they are produced in Chinese hamster ovary cells transfected with a vector or vectors comprising DNA encoding the alpha subunit and the beta subunit of human glycoprotein of FSH or the LH. The DNA encoding the 10 alpha and beta subunits may be present in the same or in different vectors.

Recombinant FSH and LH have several advantages over their urinary counterparts. Culture and isolation techniques using recombinant cells allow consistent batches to be obtained. On the contrary, FSH and LH of urinary origin vary significantly from one batch to another in characteristics such as purity, glycosylation and sialylation pattern and oxidation of the subunits. Due to the greater consistency between lots and purity of recombinant FSH and LH 15, hormones can be easily identified and quantified using techniques such as isoelectric focusing (IEF). The ease with which recombinant FSH and LH can be identified and quantified makes it possible to fill the bottles by mass of the hormone (mass dosage) instead of filling them by bioassay.

Preferably, the FSH formulations of the present invention have a pH of from or about 6.0 to or 20 to about 8.0, more preferably of or from about 6.8 to or up to about 7.8, including a pH of approximately 7.0, 7.2 and 7.4. A preferred buffer is phosphate, with the preferred counterions being sodium or potassium ions. Phosphate buffers with saline solution are well known in the art, such as Dulbecco phosphate buffered saline solution. Buffer concentrations in the total solution may vary from or about 5mM, 9.5mM, 10mM, 50mM, 100mM, 150mM, 200mM, 250mM and 500mM. Preferably, the concentration of the buffer solution is about 10mM. A 10mM buffer solution in phosphate ions with a pH of 7.0 is particularly preferred.

Preferably, the FSH and LH formulations or mixtures of the present invention have a pH of from or about 6.0 to or up to about 9.0, more preferably from or from about 6.8 to or up to about 8.5, including a pH of about 7.0, 8.0 and 8.2, most preferably a pH of or 30 of about 8.0.

The invention relates to liquid formulations as well as freeze-dried (lyophilized) formulations that can be reconstituted, in which the solvent (also for reconstitution) is water for injection. Liquid formulations can be for a single dose or for multiple doses. Liquid formulations as well as those freeze-dried of FSH and / or LH of the invention that are intended to be used in multiple doses comprise a bacteriostatic agent chosen from phenol and m-cresol. The most preferred is m-cresol. The bacteriostatic agent is used in an amount that gives a concentration that is effective in keeping the formulation essentially free of bacteria (suitable for injection) during the multiple dose injection period, which can be for approximately 12 or 24 hours until or up to about 12 or 14 days, preferably from or about 6 to or up to about 12 days. The bacteriostatic agent is preferably present in a concentration of or about 0.1% (mass of the bacteriostatic agent / solvent mass) up to or up to about 2.0%, more preferably from or about 0.2% up to or up to approximately 1.0%. In the case of phenol, about 0.5% is particularly preferred. In the case of m-cresol, the particularly preferred concentration is about 0.3% (for example, about 3 mg / ml API). Four. Five

In a preferred embodiment, the invention provides a liquid pharmaceutical composition, preferably for use in multiple doses, comprising FSH or one of its variants, the Pluronic® F68 surfactant and a bacteriostatic agent chosen from m-cresol and phenol, preferably m -cresol.

In a further preferred embodiment, the invention provides a liquid pharmaceutical composition, preferably for use in multiple doses, comprising LH, the Pluronic® F68 surfactant and a bacteriostatic agent chosen from m-cresol and phenol, preferably m-cresol.

In still a further preferred embodiment, the invention provides a liquid pharmaceutical composition, preferably for use in multiple doses, comprising FSH and LH, the Pluronic® F68 surfactant and a bacteriostatic agent chosen from m-cresol and phenol, preferably m- cresol Preferably, FSH and LH are present in a ratio (FSH: LH) of or from about 2: 1 to or up to about 1: 1. 55

In a further preferred embodiment, the invention provides a method for making a liquid pharmaceutical composition, preferably for use in multiple doses, comprising making an aqueous solution of

FSH or one of its variants, the Pluronic® F68 surfactant and a bacteriostatic agent chosen from m-cresol and phenol, preferably m-cresol, and API.

In a further preferred embodiment, the invention provides a method for making a liquid pharmaceutical composition, preferably for use in multiple doses, comprising making an aqueous solution of LH, the Pluronic® F68 surfactant and a bacteriostatic agent chosen from m-cresol and phenol, preferably m-cresol, and 5 API.

In a further preferred embodiment, the invention provides a method for making a liquid pharmaceutical composition, preferably for use in multiple doses, comprising making an aqueous solution of FSH and LH, the Pluronic® F68 surfactant and a bacteriostatic agent chosen from m -cresol and phenol, preferably m-cresol, and API. 10

In yet another preferred embodiment, the invention provides a method for making a packaged pharmaceutical composition comprising administering a solution comprising FSH, the Pluronic® F68 surfactant and a bacteriostatic agent chosen from m-cresol and phenol, preferably m-cresol.

In yet another preferred embodiment, the invention provides a method for making a packaged pharmaceutical composition comprising administering a solution comprising FSH and LH, the Pluronic® 15 F68 surfactant and a bacteriostatic agent selected from m-cresol and phenol, preferably m -cresol.

In yet another preferred embodiment, the invention provides an article made for pharmaceutical use in humans comprising a bottle comprising a solution of FSH or a variant of FSH, the Pluronic® F68 surfactant and a bacteriostatic agent chosen from m-cresol and phenol, preferably m-cresol, and written material indicating that said solution can be maintained for a period of or about twenty-four hours or more after the first use. Preferably, the written material indicates that the solution can be maintained until or until about 12 or 14 days after the first use.

In yet another preferred embodiment, the invention provides an article made for pharmaceutical use in humans comprising a vial comprising a solution of FSH and LH, the Pluronic® F68 surfactant and a bacteriostatic agent chosen from m-cresol and phenol, preferably m-cresol, and written material indicating that said solution can be maintained for a period of or approximately twenty-four hours or more after the first use. Preferably, the written material indicates that the solution can be maintained until or until about 12 or 14 days after the first use.

In a particularly preferred embodiment, the formulation comprises m-cresol and Pluronic® F68. The inventors have surprisingly found that formulations comprising Pluronic® F68 do not precipitate in the presence of m-cresol, a problem observed with other surfactants.

Before its first use, that is, before the bottle, blister or cartridge seal has been broken, the formulations of the invention can be maintained for at least or about 6 months, 12 months or 24 months. Under preferred storage conditions, before their first use, the formulations are protected from bright light (preferably in the dark) and at temperatures of or from about 2-8 ° C, more preferably from or from about 4-5 ° C.

In a specific embodiment, the invention provides a lyophilized formulation for reconstitution, preferably for use in multiple doses, comprising FSH or one of its variants and the Pluronic® F68 surfactant.

In a further specific embodiment, the invention provides a lyophilized formulation for reconstitution, preferably for use in multiple doses, comprising LH and the Pluronic® F68 surfactant.

In a further specific embodiment, the invention provides a lyophilized formulation, preferably for use in multiple doses, comprising FSH and LH and the Pluronic® F68 surfactant. Preferably, FSH and LH are present in a ratio (FSH: LH) of or from about 2: 1 to or up to about 1: 1.

In a further specific embodiment, the invention provides a method for preparing a lyophilized formulation, preferably for use in multiple doses after reconstitution, comprising forming a mixture of FSH or one of its variants with the Pluronic® F68 surfactant. and subjecting said mixture to lyophilization.

In a further specific embodiment, the invention provides a method for preparing a lyophilized formulation, preferably for use in multiple doses after reconstitution, which comprises forming a mixture of LH with the Pluronic® F68 surfactant and subjecting said mixture to lyophilization. fifty

In a further specific embodiment, the invention provides a method for preparing a lyophilized formulation, preferably for use in multiple doses after reconstitution, comprising forming a mixture of FSH and LH, as well as the Pluronic® F68 surfactant and subjecting said lyophilization mixture.

In yet another preferred embodiment, the invention provides a method of making a packaged pharmaceutical composition comprising administering a lyophilized mixture comprising FSH and the Pluronic® F68 surfactant.

In yet another preferred embodiment, the invention provides a method for making a packaged pharmaceutical composition comprising administering a lyophilized mixture comprising LH and the Pluronic® 5 F68 surfactant in a container.

In yet another preferred embodiment, the invention provides a method for making a packaged pharmaceutical composition comprising administering a lyophilized mixture comprising FSH as well as LH and the Pluronic® F68 surfactant in a container.

In yet another preferred embodiment, the invention provides an article made for pharmaceutical use in humans comprising a first container or vial containing FSH or a lyophilized FSH variant and Pluronic® F68 surfactant. A second container or bottle contains a diluent for reconstitution, preferably water and a bacteriostatic agent chosen from m-cresol and phenol, preferably m-cresol.

In yet another preferred embodiment, the invention provides an article made for pharmaceutical use in humans comprising a first container or bottle containing LH or a lyophilized LH variant and Pluronic® F68 surfactant. A second container or bottle contains a diluent for reconstitution, preferably water and a bacteriostatic agent chosen from m-cresol and phenol, preferably m-cresol.

In yet another preferred embodiment, the invention provides an article made for pharmaceutical use in humans comprising a first container or vial containing FSH or a variant of FSH as well as LH or a lyophilized LH variant and Pluronic® surfactant F68 A second container or bottle contains a diluent 20 for reconstitution, preferably water and a bacteriostatic agent chosen from m-cresol and phenol, preferably m-cresol.

In a particularly preferred embodiment, the solvent for reconstitution comprises m-cresol. The inventors have found that lyophilized formulations comprising Pluronic® F68 do not precipitate when reconstituted with a diluent containing m-cresol, a problem observed with other surfactants, for example Tween.

The lyophilized formulations of the invention can be maintained for at least 6 months, 12 months or 24 months. Under preferred storage conditions, before their first use, the formulations are protected from bright light (preferably in the dark) and at temperatures of or from about 2-8 ° C, more preferably from or from about 4-5 ° C. 30

After the first use of a liquid or reconstituted formulation for use in multiple doses it can be maintained and used for at least 24 hours, preferably at least for or about 4, 5 or 6 days, more preferably for up to 12 or 14 days. After the first use, the formulation is preferably stored at a temperature below room temperature (ie, below about 25 ° C), more preferably below or at about 10 ° C, more preferably at or about 2-8 ° C, most preferably at or about 5-0 ° C.

The formulations of the invention contain the antioxidant methionine. The antioxidant prevents oxidation of FSH and LH (particularly of the  subunit).

The methionine in the liquid and / or reconstituted formulations is preferably present at a concentration of or from about 0.01 to or up to about 1.0 mg / ml, more preferably from or from about 0.05 to or up to about 0, 5 mg / ml, most preferably of or about 0.1 mg / ml.

Preferably, the formulations of the invention contain a mono- or di-saccharide or a sugar alcohol as a stabilizer and tonicity adjusting agent, such as sucrose, dextrose, lactose, mannitol and / or glycerol. Sucrose is most preferred, preferably at a concentration of or about 60 mg / ml.

As indicated above, the invention provides liquid formulations for single use and for use in multiple doses containing a bacteriostatic agent or to which a bacteriostatic agent is added when the formulation is reconstituted. The formulations of the invention are suitable for pharmaceutical or veterinary use.

As indicated above, in a preferred embodiment the invention provides an elaborated article comprising packaged material and a bottle comprising a solution of FSH or a variant of FSH, LH, or FSH and LH, Pluronic® F68, methionine and a bacteriostatic agent chosen from phenol and m-cresol, optionally with 50 buffers and / or other excipients, in an aqueous diluent, wherein said packaged material comprises written material indicating that said solution can be maintained for a period of twenty four hours or more after first use. The invention further comprises an elaborated article comprising packaged material, a bottle comprising a formulation of FSH or a variant of the FSH according to the invention, wherein said packaged material

It comprises written material that instructs the patient on how to reconstitute the FSH or a variant of the FSH in the aqueous diluent to form a solution that can be maintained for a period of twenty-four hours or more.

As indicated above, in a preferred embodiment the invention provides an elaborated article comprising a packaged material and a bottle comprising FSH or a variant of FSH, LH or a variant of LH, or lyophilized FSH and LH, Pluronic® F68 and methionine. The bacteriostatic agent in the second container that includes the diluent is chosen from phenol and m-cresol, optionally with additional excipients, wherein said packaged material comprises written material indicating that said solution can be maintained for a period of twenty-four hours or more after Your first use.

The range of hormone protein in the formulations of the invention includes amounts that give, after reconstitution, concentrations of about 1.0 µg / ml to about 50 mg / ml, although 10 can be operated with higher and lower concentrations and depend of the intended administration vehicle, for example the solution formulations will differ from the transdermal patches and those of pulmonary, transmucosal or osmotic or microbomb methods. The concentration of the hormone protein is preferably from or about 5.0 /g / ml to or up to about 2 mg / ml, more preferably from or about 10 /g / ml to or up to about 1 mg / ml, most preferably from or from about 50 g / ml to or up to about 200 /g / ml.

The range of hormone protein in the formulations of the invention includes amounts that give, after reconstitution, concentrations of about 1.0 µg / ml to about 50 mg / ml, although it can be operated at higher and lower concentrations and depends on the The intended administration vehicle, for example, the solution formulations will differ from the transdermal and pulmonary administration patches, transmucosal or by osmotic or microbomb methods. The concentration of the hormone protein is preferably from or about 5.0 /g / ml to or up to about 2 mg / ml, more preferably from or about 10 /g / ml to or up to about 1 mg / ml, most preferably from or from about 50 µg / ml to or up to about 200 µg / ml.

Preferably, the formulations of the invention retain at least or about 80% of the activity of the FSH 25 and / or of the activity of the LH at the time of packaging for a period of 24 months (before its first use). FSH activity can be measured using the Steelman-Pohley ovarian weight gain bioassay5. LH activity can be measured by bioassay of seminal vesicle weight gain in rats.

The liquid formulations of the present invention can be prepared by a process comprising mixing the FSH or a variant of the FSH, the LH, or a mixture of FSH and LH and the Pluronic® F68 and a bacteriostatic agent chosen from the phenol and m-cresol as solids or dissolve FSH or a variant of FSH, LH, or a mixture of FSH and LH ("protein") and Pluronic® F68 and a bacteriostatic agent chosen between phenol and m-cresol in an aqueous diluent. The mixing of the components and their dissolution in an aqueous diluent is carried out using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a measured amount of FSH or a variant of FSH, LH or a mixture of FSH and LH in a buffered solution is combined with Pluronic® F68 and a bacteriostatic agent chosen between phenol and m -cresol in a buffered solution in sufficient quantities to provide the protein, Pluronic® F68 and the bacteriostatic agent in the desired concentrations. The resulting solution is then supplied in bottles, ampoules or cartridges. Variations of this procedure will be recognized by those skilled in the art. For example, the order in which the components are added, if additional additives are used, the temperature and pH to which the formulation is prepared, are all 40 factors that can be optimized for the concentration and the means of administration used.

In a preferred embodiment, the liquid formulations of the invention are made by preparing individual stock solutions of known concentration of all components of the formulation (eg, sodium phosphate buffer solution, sucrose, TWEEN, methionine, FSH and / or LH), and taking aliquot volumetric amounts to form a "stock solution" of the same composition as the final formulation. The "stock solution" is preferably filtered through a Duropore® (Millipore) 0.22 micron PDF membrane to remove the micro-organisms and then the aliquots are administered in individual containers, such as bottles, ampoules or cartridges.

The lyophilized formulations of the present invention can be prepared by a method comprising mixing the FSH or a variant of the FSH, the LH or a variant of the LH, or a mixture of FSH and LH, and Pluronic® F68, as well as Other excipients such as the antioxidant methionine and / or a buffer and subject the mixture to lyophilization. The components are mixed and lyophilized using conventional procedures. To prepare a suitable formulation, for example, a measured amount of FSH or a variant of FSH, LH or a variant of LH or a mixture of FSH and LH is combined with Pluronic® F68 and the resulting mixture is lyophilized and then It is supplied in bottles, ampoules or cartridges. Variations of this procedure will be recognized by those skilled in the art. For example, the order in which the components are added, if additional additives are used, the temperature and pH to which the formulation is prepared, are all factors that can be optimized for the concentration and the means of administration used.

The formulations of the invention can be administered using recognized devices. Examples comprising these single bottle systems include pen-injector devices for administering a solution such as EasyJect®, Gonal-F® Pen, Humaject®, NovoPen®, B-D® Pen, AutoPen® and Optipen®.

The products claimed herein include packaged material. The packaged material provides, in addition to the information required by the regulatory agencies, the conditions under which the product should be used. The packaging material of the present invention provides instructions to patients to reconstitute the FSH or a variant of the FSH in the aqueous diluent to form a solution and to use the solution for a period of twenty-four hours or more for the product in two bottles. of wet / dry product. For the product in solution in a single bottle, the label indicates that said solution can be stored after its first use for a period of twenty-four hours or more, preferably up to 12 or 14 days. The products claimed herein are useful for use as a pharmaceutical product in humans.

Preserved stable formulations can be administered to patients as clear solutions. The solution may be for single use only or may be reused several times and may be sufficient for a single cycle or several cycles of the patient's treatment and, therefore, provides a more convenient treatment regimen than is currently available. fifteen

FSH or a variant of FSH, LH or mixtures of FSH and LH in formulations or solutions, both stable and preserved, described herein, can be administered to a patient according to the present invention by various administration methods. , including SC or IM injection; Transdermal, pulmonary, transmucosal administration, implants, by osmotic pump, cartridge, micropump, oral and other means appreciated by those skilled in the art, as is well known in the art. twenty

The following examples are provided solely to further illustrate the preparation of the formulations and compositions of the invention. The scope of the invention should not be considered as consistent only in the following examples.

Example 1

Comparative Formulations 25

materials

 Item

 Maker

 massive r-hFSH used as a candidate for formulations

 Laboratorios Serono SA

 D-Mannitol (DAB, Ph Eur, BP, FU, USP, FCC, E421)

 Merck

 Sucrose (DAB, Ph Eur, BP, NF)

 Merck

 NaCl (ACS, ISO)

 Merck

 Na2HPO42H2O (GR for analysis)

 Merck

 NaH2PO4 H2O (GR for analysis)

 Merck

 Benzyl alcohol (GR for analysis)

 Merck

 m-Cresol (for synthesis)

 Merck

 TWEEN 20 (polysorbate 20) (for synthesis)

 Merck

 Pluronic® F68 (Poloxamer 188)

 Sigma

 l-Methionine (for biochemistry)

 Merck

 85% orthophosphoric acid (Ph Eur, BP, NF)

 Merck

 1.5 ml glass cartridge

 SFAM (with Aguettant silicone)

 Type A rubbers

 West company

 Warped closure

 Aguettant

 Millex-GV Syringe filter unit - Durapore

 Millipore

 Durapore 0.22 m GV membrane filters

 Millipore

 20 ml plastic syringe Plastipak

 Becton Dickinson

 Steel support for filtration

 Sartorius

Team

 HPLC systems

 Model 486 or 490 detector Model 600S controller Pump model 626 Autosampler model 717 Waters 2

 pH meter

 Model9 654 Metrohm 1

 Osmometer

 030-D Osmomat 1

The following study evaluated the following parameters for a large number of formulations:

- Compatibility of the surfactant and the bacteriostatic agent

- Oxidation of the alpha subunit

The formulations were multi-dose formulations and were contained either in TWEEN 20 or Pluronic® F68, as well as a bacteriostatic agent. The following three bacteriostatic agents were evaluated: 5

- 0.9% benzyl alcohol

- 0.3% m-Cresol

- 0.5% phenol

TWEEN 20 and Pluronic® F68 were used in the following concentration range:

- TWEEN 20: range from 10 to 100 g / g 10

- Pluronic® F68: range from 10 to 100 g / g

The prepared solutions are listed in Table 1.

 Table 1: Comparative formulations

 ID 

 Na2HPO4 2H2O (mg / g) NaH2PO4 2H2O (mg / g) r-hFSH * Pluronic® F68 (µg / g) TWEEN 20 (µg / g) Bacteriostatic Agent Excipient (mg / g)

 1 P

 1.11 0.45 600 IU / g 10 - Phenol 0.5% Sucrose 70.6

 2 P

 1.11 0.45 600 IU / g 10 - Phenol 0.5% Mannitol 38.7

 3P

 1.11 0.45 600 IU / g 100 - Phenol 0.5% Sucrose 70.6

 4P

 1.11 0.45 600 IU / g 100 - Phenol 0.5% Mannitol 38.7

 5 P

 1.11 0.45 600 IU / g - 10 Phenol 0.5% Sucrose 70.6

 6P

 1.11 0.45 600 IU / g - 10 Phenol 0.5% Mannitol 38.7

 7

 1.11 0.45 600 IU / g - 100 Alc. Benzyl 0.9% NaCl 6.0

 8

 1.11 0.45 600 IU / g - 100 Alc. Benzyl 0.9% Sucrose 62.3

 9

 1.11 0.45 600 IU / g - 100 Alc. Benzyl 0.9% Mannitol 34.1

 10

 1.11 0.45 600 IU / g - 100 m-Cresol 0.3% NaCl 7.6

 eleven

 1.11 0.45 600 IU / g - 100 m-Cresol 0.3% Sucrose 78.0

 12

 1.11 0.45 600 IU / g - 100 m-Cresol 0.3% Mannitol 42.7

 13

 1.11 0.45 600 IU / g - 10 Alc. Benzyl 0.9% NaCl 6.0

 14

 1.11 0.45 600 IU / g - 10 Alc. Benzyl 0.9% Sucrose 62.3

 fifteen

 1.11 0.45 600 IU / g - 10 Alc. Benzyl 0.9% Mannitol 34.1

 16

 1.11 0.45 600 IU / g - 10 m-Cresol 0.3% NaCl 7.6

 17

 1.11 0.45 600 IU / g - 10 m-Cresol 0.3% Sucrose 78.0

 18

 1.11 0.45 600 IU / g - 10 m-Cresol 0.3% Mannitol 42.7

 19

 1.11 0.45 600 IU / g 100 - Alc. Benzyl 0.9% NaCl 6.0

 twenty

 1.11 0.45 600 IU / g 100 - Alc. Benzyl 0.9% Sucrose 62.3

 twenty-one

 1.11 0.45 600 IU / g 100 - Alc. Benzyl 0.9% Mannitol 34.1

 22

 1.11 0.45 600 IU / g 100 - m-Cresol 0.3% NaCl 7.6

 2. 3

 1.11 0.45 600 IU / g 100 - m-Cresol 0.3% Sucrose 78.0

 24

 1.11 0.45 600 IU / g 100 - m-Cresol 0.3% Mannitol 42.7

 25

 1.11 0.45 600 IU / g 10 - Alc. Benzyl 0.9% NaCl 6.0

 26

 1.11 0.45 600 IU / g 10 - Alc. Benzyl 0.9% Sucrose 62.3

 27

 1.11 0.45 600 IU / g 10 - Alc. Benzyl 0.9% Mannitol 34.1

 28

 1.11 0.45 600 IU / g 10 - m-Cresol 0.3% NaCl 7.6

 29

 1.11 0.45 600 IU / g 10 - m-Cresol 0.3% Sucrose 78.0

 30

 1.11 0.45 600 IU / g 10 - m-Cresol 0.3% Mannitol 42.7

* FSH was added to the formulations based on their biological potency instead of the protein content.

From the visual examination of the formulations it was determined that TWEEN 20 cannot be used with m-cresol and phenol because FSH formulations containing TWEEN 20 and m-cresol or TWEEN 20 and phenol had an opalescent white suspension. In contrast, FSH formulations containing Pluronic® F68 did not present this problem with m-cresol and phenol. The use of Pluronic® F68 allows the use of phenol and m-cresol.

Combination of FSH and Pluronic® F68 with antioxidants 5

The ability of the following antioxidants to inhibit oxidation of the α subunit in the presence of Pluronic® F68 was evaluated:

- Methionine: range from 10 to 100 µg / g

- Ascorbic acid: range from 10 to 100 µg / g

Sucrose and mannitol were used as tonicity agents and TWEEN 20 or Pluronic® were added at a concentration of 10 100 µg / g.

The prepared formulations are listed in Table 2. ??????????? ’

 Table 2: Comparative formulations with and without methionine

 ID 

 Na2HPO4 2H2O (mg / g) NaH2PO4 H2O (mg / g) r-hFSH * Pluronic® F68 (g / g) TWEEN (g / g) Ac. Ascorbic (g / g) Methionine (g / g) Excipient Bacteriostatic Agent

 31

 1.11 0.45 600 IU / g 100 - - - m-Cresol 0.3% Sucrose

 32

 1.11 0.45 600 IU / g 100 - - - m-Cresol 0.3% Mannitol

 33

 1.11 0.45 600 IU / g - 100 - - Alc. 0.9% benzyl sucrose

 3. 4

 1.11 0.45 600 IU / g - 100 - - Alc. Benzyl 0.9% Mannitol

 35

 1.11 0.45 600 IU / g 100 - - - Alc. 0.9% benzyl sucrose

 36

 1.11 0.45 600 IU / g 100 - - - Alc. Benzyl 0.9% Mannitol

 37

 1.11 0.45 600 IU / g 100 - - 10 m-Cresol 0.3% Sucrose

 38

 1.11 0.45 600 IU / g 100 - - 10 m-Cresol 0.3% Mannitol

 39

 1.11 0.45 600 IU / g 100 - - 100 m-Cresol 0.3% Sucrose

 40

 1.11 0.45 600 IU / g 100 - - 100 m-Cresol 0.3% Mannitol

 41

 1.11 0.45 600 IU / g 100 - 10 - m-Cresol 0.3% Sucrose

 42

 1.11 0.45 600 IU / g 100 - 10 - m-Cresol 0.3% Mannitol

 43

 1.11 0.45 600 IU / g 100 - 100 - m-Cresol 0.3% Sucrose

 44

 1.11 0.45 600 IU / g 100 - 100 - m-Cresol 0.3% Mannitol

 Four. Five

 1.11 0.45 600 IU / g - 100 - 10 Alc. 0.9% benzyl sucrose

 46

 1.11 0.45 600 IU / g - 100 - 10 Alc. Benzyl 0.9% Mannitol

 47

 1.11 0.45 600 IU / g - 100 - 100 Alc. 0.9% benzyl sucrose

 48

 1.11 0.45 600 IU / g - 100 - 100 Alc. Benzyl 0.9% Mannitol

 49

 1.11 0.45 600 IU / g - 100 10 - Alc. 0.9% benzyl sucrose

 fifty

 1.11 0.45 600 IU / g - 100 10 - Alc. Benzyl 0.9% Mannitol

 51

 1.11 0.45 600 IU / g - 100 100 - Alc. 0.9% benzyl sucrose

 52

 1.11 0.45 600 IU / g - 100 100 - Alc. Benzyl 0.9% Mannitol

 53

 1.11 0.45 600 IU / g 100 - - 10 Alc. 0.9% benzyl sucrose

 54

 1.11 0.45 600 IU / g 100 - - 10 Alc. Benzyl 0.9% Mannitol

 55

 1.11 0.45 600 IU / g 100 - - 100 Alc. 0.9% benzyl sucrose

 56

 1.11 0.45 600 IU / g 100 - - 100 Alc. Benzyl 0.9% Mannitol

 57

 1.11 0.45 600 IU / g 100-10 - Alc. 0.9% benzyl sucrose

 58

 1.11 0.45 600 IU / g 100-10 - Alc. Benzyl 0.9% Mannitol

 59

 1.11 0.45 600 IU / g 100 - 100 - Alc. 0.9% benzyl sucrose

 60

 1.11 0.45 600 IU / g 100 - 100 - Alc. Benzyl 0.9% Mannitol

 61

 1.11 0.45 600 IU / g 100 - - - Phenol Sucrose

 62

 1.11 0.45 600 IU / g 100 - - - Phenol Mannitol

 63

 1.11 0.45 600 IU / g Sucrose Phenol

 64

 1.11 0.45 600 IU / g 100 - - 10 Phenol Mannitol

 65

 1.11 0.45 600 IU / g 100 - - 100 Phenol Sucrose

 66

 1.11 0.45 600 IU / g 100 - - 100 Phenol Mannitol

 67

 1.11 0.45 600 IU / g 100-10 - Phenol Sucrose

 68

 1.11 0.45 600 IU / g 100-10 - Phenol Mannitol

 69

 1.11 0.45 600 IU / g 100 - 100 - Phenol Sucrose

 70

 1.11 0.45 600 IU / g 100 - 100 - Phenol Mannitol

* FSH was added to the formulations based on their biological potency instead of the protein content.

20 g of each formulation were prepared in Falcon polypropylene tubes and filtered through a 0.25 µm, 3 cm2 Millex-GV syringe filter unit, then analyzed according to the following scheme:

 Analytical test

 t = 0 1 week 2 weeks 3 weeks 4 weeks

 Reverse phase HPLC for the oxidized alpha subunit (%)

 X X X X X

 HPLC size exclusion for protein quantification (g / g)

 X X X X X

 HPLC for qualitative size exclusion of free subunits

 X X X X X

(X): Test performed

Inverse phase HPLC shows that in the formulations containing FSH, Pluronic® F68, m-cresol and methionine (at 5 10 and 100 g / ml), the oxidation of the un subunit of the FSH when the formulation is stored at 40 ° C was considerably reduced against the formulation that did not contain methionine as can be seen in Figure 1. Depending on the average of two experiments, in the formulation that does not contain methionine, the percentage of the oxidized subunit es is 2.3 at = 0, 4.0 at = 1 week and 7.1 at = 2 weeks. In the formulation containing 10 /g / ml of methionine, the percentage of the oxidized un subunit is 2.0 at t = 0, 3.2 at t = 1 week and 3.8 at t = 2 weeks. In the formulation containing 10 g / ml of methionine, the percentage of the oxidized un subunit is 1.8 at t = 0, 1.7 at t = 1 week and 1.3 at t = 2 weeks.

Example 2

Single dose formulation of recombinant FSH for subcutaneous or intramuscular injection.

Based on the results of Example 1, the following formulation was prepared. fifteen

Components 1 to 7 shown in Table 3 were prepared as volumetric solutions in API. Aliquots of each solution were added to a mixing vessel to form a "stock solution." The stock solution was supplied in bottles containing 10.9 micrograms (150 IU) or 5.45 micrograms (75 IU) of FSH. With recombinant FSH, bioactivity and specific activity are consistent, allowing FSH to be dosed by mass rather than by bioassay. twenty

 Table 3: Components of FSH formulations for single dose

 Component 

 Description 150 IU of FSH 75 IU of FSH

 one

 rhFSH (g / bottle) 10.9 (150 IU) 5.45 (75 IU)

 2

 Sucrose (mg / bottle) 15.00 7.50

 3

 NaH2PO4 H2O (mg / bottle) 0.111 0.0555

 4

 Na2HPO4 2H2O (mg / bottle) 0.273 0.1265

 5

 Pluronic® F68 (mg / bottle) 0.025 0.0125

 6

 Methionine (mg / bottle) 0.025 0.0125

 7

 m-cresol (mg / bottle) 0.75 0.375

 8

 pH 7.0 7.0

 9

 API c.s.p. 1 ml c.s.p. 0.5 ml

The jars were refilled and sealed under sterile conditions. The formulation has a durability of up to two years at room temperature.

Example 3

Liquid formulation for multiple doses of recombinant FSH for subcutaneous or intramuscular injection 25

Based on the results of Example 1, the following formulation for multiple doses was prepared.

Components 1 to 7 shown in Table 4 were prepared as volumetric solutions in API. Aliquots of each solution were added to a mixing vessel to form a "stock solution." The stock solution was supplied in bottles containing 22.2 micrograms (305 IU) or 33.3 micrograms (458 IU) and 66.7 micrograms (916 IU) of FSH. The resulting formulations allowed to administer a total of 300, 450 and 900 IU of 30 FSH.

The cartridges were filled and sealed under sterile conditions. The multi-dose formulation can be stored at or about 2-8 ° C, more preferably at or about 4-5 ° C, until first use for up to two years. After first use, the cartridge should be stored at or approximately 2-8 ° C, plus

preferably at or about 4-5 ° C during the multiple dose period, which may be 24 hours, 2 days or up to 12 or 14 days.

 Table 4: Components of FSH formulations for multiple doses

 Comp. 

 Description 300 IU of FSH 450 IU of FSH 900 IU of FSH

 one

 rhFSH (g / cartridge) 22.2 (305 IU) 33.3 (458 IU) 66.7 (916 IU)

 2

 Sucrose (mg / cartridge) 30.0 45.00 90.0

 3

 NaH2PO4 H2O (mg / cartridge) 0.225 0.337 0.675

 4

 Na2HPO4 2H2O (mg / cartridge) 0.555 0.832 1.665

 5

 Pluronic® F68 (mg / cartridge) 0.050 0.075 0.150

 6

 Methionine (mg / cartridge) 0.050 0.075 0.150

 7

 m-cresol (mg / cartridge) 1.50 2.25 4.50

 8

 pH 7.0 7.0 7.0

 9

 API c.s.p. 0.5 ml c.s.p. 0.75 ml c.s.p. 1.5 ml

Example 4

Liquid formulation for single dose of recombinant LH for subcutaneous or intramuscular injection 5

The following formulation was prepared.

Components 1 to 7 shown in Table 5 were prepared as volumetric solutions in API. Aliquots of each solution were added to a mixing vessel to form a "stock solution." The stock solution was supplied in bottles so that they contained 3 micrograms (75 IU) of LH. The resulting formulation provides a single dose of 75 IU of LH. 10

With recombinant LH, bioactivity and specific activity are consistent, allowing LH to be dosed by mass rather than by bioassay.

 Table 5: Components of the LH formulation for single dose

 Component 

 Description 75 IU of LH

 one

 rhLH (g / bottle) 3.0

 2

 Sucrose (mg / bottle) 52.5

 3

 NaH2PO4 H2O (mg / bottle) 0.052

 4

 Na2HPO4 2H2O (mg / bottle) 0.825

 5

 Pluronic® F68 (mg / bottle) 0.0125

 6

 Methionine (mg / bottle) 0.125

 7

 m-cresol (mg / bottle) 0.375

 9

 API c.s.p. 0.5 ml

The jars were refilled and sealed under sterile conditions. The formulation has a durability of up to two years.

Example 5 15

Formulations for multiple doses of recombinant FSH and LH (2: 1) for subcutaneous or intramuscular injection.

The following formulations were prepared for multiple doses of FSH and LH with an FSH: LH ratio of 2: 1.

Components 1 to 8 shown in Table 6 were prepared as volumetric solutions in API. Aliquots of each solution were added to a mixing vessel to form a "stock solution." The pH of the stock solution was adjusted to 8.0, if necessary, by the addition of NaOH or HCl. The stock solution was supplied in cartridges so that they contained 18.3 micrograms (457 IU) of LH with 66.7 micrograms (916 IU) of FSH, intended for 6 doses of 150 IU of FSH each; 9.2 micrograms (230 IU) of LH with 33.3 micrograms (458 IU) of FSH, intended for 3 doses of 150 IU of FSH each of FSH; and 6.1 micrograms (152.5 IU) of LH with 22.23 micrograms (305 IU) of FSH, intended for 2 doses of 150 IU of FSH each. 25

The cartridges were filled and sealed under sterile conditions. The multi-dose formulation can be stored at or about 2-8 ° C, more preferably at or about 4-5 ° C, until first use for up to two years. After the first use, the cartridge should be stored at or about 2-8 ° C, more preferably at or about 4-5 ° C during the multiple dose period, which may be 24 hours, 2 days or up to 12 or 14 days. 30

 Table 6: Components of the liquid formulations of FSH and LH (2: 1) for multiple doses

 Comp. 

 Description 6 doses 3 doses 2 doses

 one

 rhLH (g / cartridge) 18.3 (457 IU) 9.2 (230 IU) 6.1 (152.5 IU)

 2

 rhFSH (g / cartridge) 66.7 (916 IU) 33.3 (458 IU) 22.23 (305 IU)

 3

 Sucrose (mg / cartridge) 115.5 57.75 38.5

 4

 H3PO4 (mg / cartridge) 1.35 0.735 0.49

 5

 NaOH (mg / cartridge) c.s.p. pH 8.0 c.s.p. pH 8.0 c.s.p. pH 8.0

 6

 Pluronic® F68 (mg / cartridge) 375.0 187.5 125.0

 7

 Methionine (mg / cartridge) 225 112.5 75.0

 8

 m-cresol (mg / cartridge) 4.5 2.25 1.5

 9

 pH 8.0 8.0 8.0

 10

 API c.s.p. 1.5 ml c.s.p. 0.75 ml c.s.p. 0.5 ml

Example 6

Formulations for multiple doses of recombinant FSH and LH (1: 1) for subcutaneous or intramuscular injection.

The following formulations were prepared for multiple doses of FSH and LH with an FSH: LH ratio of 1: 1. 5

Components 1 to 8 shown in Table 7 were prepared as volumetric solutions in API. Aliquots of each solution were added to a mixing vessel to form a "stock solution." The pH of the stock solution was adjusted to 8.0, if necessary, by the addition of NaOH or HCl. The stock solution was supplied in cartridges so that they contained 36.6 micrograms (914 IU) of LH with 66.7 micrograms (916 IU) of FSH, intended for 6 doses of 150 IU of FSH each; 18.4 micrograms (460 IU) of LH with 33.3 micrograms (458 IU) of FSH, intended for 3 10 doses of 150 IU of FSH each; and 12.2 micrograms (305 IU) of LH with 22.23 micrograms (305 IU) of FSH, intended for 2 doses of 150 IU of FSH each.

The cartridges were filled and sealed under sterile conditions. The multi-dose formulation can be stored at or about 2-8 ° C, more preferably at or about 4-5 ° C, until first use for up to two years. After the first use, the cartridge should be stored at or about 2-8 ° C, more preferably at or about 4-5 ° C during the multiple dose period which may be 24 hours, 2 days or up to 12 or 14 days.

 Table 7: Components of the liquid formulations of FSH and LH (1: 1) for multiple doses

 Comp. 

 Description 6 doses 3 doses 2 doses

 one

 rhLH (g / cartridge) 36.6 (914 IU) 18.4 (460 IU) 12.2 (305 IU)

 2

 rhFSH (g / cartridge) 66.7 (916 IU) 33.3 (458 IU) 22.23 (305 IU)

 3

 Sucrose (mg / cartridge) 115.5 57.75 38.5

 4

 H3PO4 (mg / cartridge) 1.35 0.735 0.49

 5

 NaOH (mg / cartridge) c.s.p. pH 8.0 c.s.p. pH 8.0 c.s.p. pH 8.0

 5

 Pluronic® F68 (mg / bottle) 375.0 187.5 125.0

 6

 Methionine (g / cartridge) 225 112.5 75.0

 7

 m-cresol (mg / cartridge) 4.5 2.25 1.5

 8

 pH 8.0 8.0 8.0

 9

 API c.s.p. 1.5 ml c.s.p. 0.75 ml c.s.p. 0.5 ml

Example 7

Stability experiments for liquid formulations for multiple doses of FSH mixed with LH. twenty

7.1.- HPLC analysis in reverse phase of protein content

 The protein content in the formulation of Example 5 (6 doses), for both FSH and LH, was evaluated using the reverse phase HPLC method.

Protein content (FSH and LH) was measured at zero time and after 1, 2, 3 and 6 months of storage of the formulation at 4 ° C. The results are shown in Table 8 in micrograms of FSH or LH per gram of solvent.

7.2.- Test of the oxidized alpha subunit

The percentage of oxidized alpha subunit in a formulation of Example 5 was measured by a reverse phase HPLC method (RP-HPLC).

The percentage of oxidized alpha subunit was measured at zero time and after 1, 2, 3 and 6 months of storage at 4 ° C. The results are shown in table 8.

7.3.- In vivo test for FSH

The activity of FSH in the formulation of Example 5 (6 doses) was analyzed using the Steelman-Pohley ovarian weight gain bioassay at zero time and after 1, 2, 3 and 6 months of storage at 4 ° C. Results 5 are shown in Table 8 in international units (IU) per gram of solvent.

7.4.- In vivo test for LH

The activity of LH in the formulation of Example 5 (6 doses) was analyzed using the seminal vesicle weight gain bioassay in rats at zero time and after 1, 2, 3 and 6 months of storage at 4 ° C. The results are shown in table 8 in international units (IU) per gram of solvent. 10

7.5.- Evaluation of the free subunit (rFSH + rLH)

The percentage of free subunit was evaluated in a formulation of Example 5 by SDS-PAGE.

The measurements were made at time zero and after 1, 2, 3 and 6 months of storage at 4 ° C. The results are given as a percentage of the total protein (rFSH + rLH) and are shown in table 8.

7.5.- Evaluation of aggregates 15

The percentage of aggregates in a formulation of Example 5 was evaluated by SDS-PAGE as described above for the evaluation of the free subunit at 7.5, except that the higher molecular weight aggregates were determined as the percentage of the total protein (rFSH + rLH). The measurements were made at time zero and after 1, 2, 3 and 6 months of storage at 4 ° C. The results are shown in table 8.

7.6.- Visible particles 20

The particles were evaluated visually in the formulation of Example 5 at zero time and after 3 and 6 months of storage at 4 ° C. The results are shown in table 8.

7.7.- pH

The pH of a formulation of Example 5 was measured at zero time and after 1, 2, 3 and 6 months of storage at 4 ° C. The results are shown in table 8. 25

 Table 8. Analytical parameters for a liquid formulation of FSH and LH (2: 1) at zero time and after storage at 4 ° C for 1, 2, 3 and 6 months

 Analysis

 Time 0 1 month 2 months 3 months 6 months

 RFSH content by RP-HPLC (micrograms / g)

 46.50 46.98 46.71 46.31 44.98

 RLH content by RP-HPLC (micrograms / g)

 11.74 11.81 12.68 12.67 13.21

 % oxidized alpha subunit

 2.29 2.17 2.08 2.48 2.95

 In vivo analysis for FSH 553 (IU / g)

 566 Not analyzed Not analyzed Not analyzed 578 (23 weeks)

 In vivo analysis for LH (IU / g)

 331 Not analyzed Not analyzed 311 286

 Free subunit by SDS-PAGE (rFSH + rLH;%)

 ≤ 5 Not analyzed Not analyzed ≤ 5 ≤ 5 (23 weeks)

 Added by SDS-PAGE (rFSH + rLH;%)

 ≤ 2 Not analyzed Not analyzed> 3 4

 Visible particles

 No Not analyzed Not analyzed No No

 pH

 8,262 8,125 8,216 8,188 8,283

Example 8

Lyophilized formulation of FSH and LH for multiple doses

Two lyophilized formulations A and B were prepared having the following compositions:

Formulation A

  FSH 32g 32.75 (450 IU)

  LH g 9.0 (225 IU)

  Sucrose mg 15.0

  NaH2PO4 H2O mg 0.052 5

  Na2HPO4 2H2O mg 0.825

Pluronic® F68 mg 0.05

L-Methionine mg 0.05

Formulation B

  FSH g 65.5 (900 IU) 10

  LH g 18.0 (450 IU)

  Sucrose mg 30.0

  NaH2PO4 H2O mg 0.104

  Na2HPO4 2H2O mg 1.65

Pluronic® F68 mg 0.10 15

L-Methionine mg 0.10

The manufacturing process consisted of mixing the drug substance directly with the ingredients, filtering the solution obtained and lyophilizing the filtrate.

A description of each stage of the procedure is given below:

- Add in an API graduated container, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate monohydrate, sucrose, 5% Pluronic® F68 and L-methionine and stir for 10 minutes until the solution is complete.

- Control the pH and optionally correct it at pH 7.00 ± 0.2 with 10% NaOH or diluted H3PO4.

- Add FSH and LH to the mixture prepared above and gently stir the solution obtained for 10 minutes. 25

- Check the pH again and optionally adjust it to pH 7.0 ± 0.1 with 10% NaOH or diluted H3PO4.

- Filter the solution with a 0.22 µm Durapore membrane with a filtration ratio of not less than 15 g / cm2, with a stream of gaseous nitrogen with a pressure not exceeding 1.5 atm.

- Collect the solution in a previously sterilized bottle.

- Fill the glass container with the filtered solution, put the cap and place the filled jars in a stainless aluminum tray.

- Load the trays in the freeze dryer and freeze dry the product using the following freeze drying cycle:

 Balance at + 4 ° C for approximately 20 minutes.

 Bring the shelves to a temperature of -25ºC and keep it for 2 hours.

 Bring the shelves to a temperature of -15ºC and keep it for 1 hour. 35

 Bring the shelves to a temperature of -45ºC and keep it for 3 hours.

 Bring the condenser to a temperature of -65ºC.

 Apply vacuum to the chamber.

 When the vacuum reaches a value of 7x10-2 mbar, raise the shelf temperature to -10ºC and keep it for 14 hours.

 Increase the shelf temperature to + 35ºC in 8 hours and keep it until the end of the cycle (14 hours).

 Break the vacuum allowing nitrogen to enter the chamber.

 Make the capping with the automatic lyophilizer system. 5

 Seal the covered jars with suitable folding caps.

Formulations A and B have been stored at 25 ± 2 ° C and their stability and biological activity have been evaluated as indicated below. Before analyzing the compositions, they have been reconstituted using water for injection comprising 0.3% m-cresol as a bacteriostatic agent.

The stability and biological activity values were determined as follows:

- In vivo analysis for FSH: the FSH activity of the formulation was analyzed using the Steelman-Pohley ovarian weight gain bioassay.

- In vivo analysis for LH: LH activity of the formulation was analyzed using the seminal vesicle weight gain bioassay in rats.

- Analysis of the oxidized alpha subunit: the percentage of oxidized alpha subunit was measured by a reverse phase HPLC (RP-HPLC) method.

- Evaluation of the free subunit (rFSH + rLH): the percentage of free subunit was evaluated by SDS-PAGE.

- Evaluation of aggregates: the percentage of aggregates was evaluated by SDS-PAGE as described above for the evaluation of the free subunit.

Biological analyzes have been carried out in compliance with the regulations of the European Pharmacopoeia. In particular, the 20 analyzes have been published in the monograph "Menotropin".

Table 9 summarizes the results of the analytical tests related to the stability and biological activity of formulation A. The values were determined at 4 control points: at zero time, after 1 month, 3 months and 6 months of storage, at a storage temperature of 25  2 ° C.

TABLE 9 25

 Test

 Zero time 1 month 3 months 6 months

 Biological activity FSH U.I.

 416 420 415 417

 Biological activity LH U.I.

 276 250 259 270

 % of oxidized product

 1.95 1.81 1.95 1.57

 % of dimers / aggregates

 <2 <2 <2 <2

 % of free subunits

 <5 <5 <5 <5

Table 10 summarizes the results of the analytical tests related to the stability and biological activity of formulation B. The values were determined at 4 control points: at zero time, after 3 months, 6 months and 9 months of storage, at a storage temperature of 25  2 ° C.

TABLE 10 30

 Test

 Zero time 3 months 3 months 6 months

 Biological activity FSH U.I.

 821 850 830 838

 Biological activity LH U.I.

 570 564 580 622

 % of oxidized product

 1.0 0.9 1.0 1.0

 % of dimers / aggregates

 <2 <2 <2 <2

 % of free subunits

 <5 <5 <5 <5

From tables 9 and 10 it can be concluded that the biological activity of formulations A and B is well preserved after 9 months of storage. The formulations have high stability.

The high stability is not affected by large amounts of recombinant FSH and recombinant LH.

Sequences:

I KNOW THAT. ID. No. 1:  subunit of human glycoprotein;

I KNOW THAT. ID. No. 2: subunit  of the hFSH;

I KNOW THAT. ID: No. 3: variant 1 of the  subunit of the hFSH;

I KNOW THAT. ID: No. 4: variant 2 of the  subunit of the hFSH; 5

I KNOW THAT. ID: No. 5: variant 3 of the  subunit of the hFSH;

I KNOW THAT. ID: No. 6: subunit  of the hLH.

References

1 Burgues et al .; Subcutaneous self-administration of highly purified follicle stimulating hormone and human chorionic 10 gonadotrophin for the treatment of male hypogonadotrophic hypogonadism. Spanish Collaborative Group on Male Hypogonadotrophic Hypogonadism; Hum. Play; 1997, 12, 980-6.

2 Shome et al .; J. Clin. Endocrinol Metab 39: 187-205 (1974); Shome et al., J. Prot. Chem., 7: 325-339, 1988.

3 Keutmann et al., Structure of human luteinizing hormone beta subunit: evidence for related carboxyl-terminal sequence among certain peptide hormones; Biochem Byophys Res. Commun .; 1979, 90, 842-848; Talmadge et al .; Evolution of 15 the genes for the beta subunits of human chorionic gonadotropin and luteinizing hormone; Nature; 1984, 307, 37-40; Fiddes &Talmadge; Structure, expression and evolution of the gene for the human glycoprotein hormones; Recent Prog. Horm. Beef.; 1984, 40, 43-78.

4 Reichert, L. E., Ramsey, R. B .; Dissociation of human follicle-stimulating hormone; J. Biol. Chem; 1975, 250, 3034-3040. twenty

5 Klein et al .; Pharmacokinetics and pharmacodynamics of single-chain recombinant human follicle-stimulating hormone containing the human chorionic gonadotrophin carboxyterminal peptide in the rhesus monkey; Fertility &Sterility; 2002, 77, 1248-1255.

6 a) Fiddes, J. C. et al., J. of Mol. and Applied Genetics, 1: 3-18 (1981); b) Esch F. S. et al .; DNA 5: 363-369 (1986); c) Watkings P. C. et al., DNA 6: 205-212 (1987); d) Hirai, T. et al., J. Mol. Endocrinol 5: 147-158 (1990); e) Maurer, R. A. 25 et al., Mol. Endocrinol 1: 717-723 (1987); f) Guzman, K. et al., DNA Cell Biol. 10: 593-601 (1991); g) Kumar T. R. et al., Gene, 1995 December 12; 166 (2): 335-6; h) Kumar, T. R. et al., Gene 1995 December 12; 166 (2): 333-4.

7 Biochem. Biophys Res. Commun .; 1979, 90, 842-848.

8 Steelman et al .; Assay of the follicle stimulating hormone based on the augmentation with human chorionic gonadotrophin; Endocrinology, 1953, 53, 604-616. 30

9 Van Hell et al., Effects of human menopausal gonadotrophin preparations in different bioassay methods; Endocrinology Act, 1984, 47, 409-418.

10 Van Hell et al .; Effects of human menopausal gonadotrophin preparations in different bioassay methods; Endocrinology Act, 1964, 47, 409-418.

SEQUENCE LIST

Claims (36)

1. A liquid pharmaceutical composition comprising follicle stimulating hormone (FSH) or one of its variants, as well as a surfactant that is poloxamer 188 and also comprises methionine and a bacteriostatic agent chosen from phenol and m-cresol. 2.- A liquid pharmaceutical composition comprising follicle stimulating hormone (FSH) or a variant and luteinizing hormone (LH) or one of its variants, as well as a surfactant that is poloxamer 188 and also comprises methionine and an agent Bacteriostatic chosen between phenol and m-cresol. 3.- A liquid pharmaceutical composition comprising luteinizing hormone (LH) or one of its variants, as well as a surfactant that is poloxamer 188 and also comprises methionine and a bacteriostatic agent chosen from phenol and m-cresol. 10 4. A liquid pharmaceutical composition according to any of the preceding claims, wherein the follicle stimulating hormone (FSH) is present in a concentration of or from about 150 IU / ml to or up to about 1,200 IU / ml. 5. A liquid pharmaceutical composition according to claim 4, wherein the follicle stimulating hormone (FSH) is present in a concentration of or from about 300 IU / ml to or up to about 900 IU / ml. fifteen 6. A liquid pharmaceutical composition according to claim 5, wherein the follicle stimulating hormone (FSH) is present in a concentration of or about 600 IU / ml. 7. A liquid pharmaceutical composition according to any of claims 2 or 3, wherein the luteinizing hormone (LH) is present in a concentration of or from about 150 IU / ml to or up to about 1,200 IU / ml. twenty 8. A liquid pharmaceutical composition according to claim 7, wherein the luteinizing hormone (LH) is present in a concentration of or from about 300 IU / ml to or up to about 750 IU / ml. 9. An elaborated article comprising a lyophilized formulation comprising follicle stimulating hormone (FSH) or one of its variants, a surfactant that is poloxamer 188 as well as methionine, the article made up also comprising a solvent for reconstitution containing an agent Bacteriostatic chosen between phenol and m-25 cresol. 10. An elaborated article comprising a lyophilized formulation comprising the luteinizing hormone (LH) or one of its variants, a surfactant that is poloxamer 188 as well as methionine, the article made up also comprising a solvent for reconstitution containing a chosen bacteriostatic agent between phenol and m-cresol. 11.- An elaborated article comprising a lyophilized formulation comprising follicle stimulating hormone 30 (FSH) or one of its variants as well as luteinizing hormone (LH) or one of its variants, a surfactant that is poloxamer 188 as well as methionine , the article also comprising a solvent for reconstitution containing a bacteriostatic agent chosen from phenol and m-cresol. 12. The article made according to any of claims 9 to 11, wherein the follicle stimulating hormone (FSH) is present in a concentration (weight / weight) of or about 0.1 to 10 10g / mg of the total formulation 35. 13. The article made according to claim 12, wherein the follicle stimulating hormone (FSH) is present in a concentration of or about 0.3 to 5 µg / mg of the total formulation. 14. The article made according to claim 13, wherein the follicle stimulating hormone (FSH) is present in a concentration of or about 0.37 to 2 µg / mg of the total formulation. 40 15. The article made according to any of claims 9 to 11, wherein the luteinizing hormone (LH) is present in a concentration of or about 0.1 to 3 µg / mg of the total formulation. 16. The article made according to claim 15, wherein the luteinizing hormone (LH) is present in a concentration of or about 0.1 to 1 µg / mg of the total formulation. 17. The article made according to claim 16, wherein the luteinizing hormone (LH) is present in a concentration of or from about 0.1 to 0.6 µg / mg of the total formulation. 18. A pharmaceutical composition or article made according to any of the preceding claims, wherein the follicle stimulating hormone is a human follicle stimulating hormone and / or luteinizing hormone (LH) is a human luteinizing hormone (LH). 19. A pharmaceutical composition or article made according to claim 18, wherein the follicle stimulating hormone is a urinary human follicle stimulating hormone and / or the luteinizing hormone (LH) is a urinary human luteinizing hormone (LH). 20. A pharmaceutical composition or article made according to claim 18, wherein the follicle stimulating hormone is a recombinant human follicle stimulating hormone and / or the luteinizing hormone (LH) is a recombinant human luteinizing hormone (LH). 21. A pharmaceutical composition or article made according to any of the preceding claims, wherein the ratio between FSH and LH is in the range of or from about 6: 1 to or up to about 1: 6. 22. A pharmaceutical composition or article made according to claim 21, wherein the ratio between FSH and LH is in the range of or from about 4: 1 to or up to about 1: 2. 10 23. A pharmaceutical composition or article made according to claim 22, wherein the ratio between FSH and LH is in the range of or from about 3: 1 to or up to about 1: 1. 24. A pharmaceutical composition or article made according to claim 23, wherein the ratio between FSH and LH is in the range of or about 2: 1 and 1: 1. 25. A pharmaceutical composition or article made according to any of the preceding claims, wherein the bacteriostatic agent is m-cresol. 26.- A pharmaceutical composition or article made according to claim 25, comprising m-cresol at a concentration of or about 0.3% (mass / mass of solvent). 27.- A pharmaceutical composition or article made according to any of the preceding claims which further comprises sucrose. twenty 28. A pharmaceutical composition or article made according to any of the preceding claims further comprising a phosphate buffer at a pH of from or about 6.0 to or up to about 8.0. 29. A pharmaceutical composition or article of manufacture according to claim 28, further comprising a phosphate buffer at a pH of or about 7.0. 30. A pharmaceutical composition or article of manufacture according to claim 29, comprising the following ingredients: rFSH, poloxamer 188, sucrose, methionine, m-cresol and an aqueous phosphate buffer solution of pH at or about 7.0. 31. A pharmaceutical composition or article made according to claim 30, wherein rFSH is present at a concentration of or about 600 IU / ml, poloxamer 188 is present at a concentration of or about 0.1 mg / ml, sucrose is present at a concentration of or about 60 mg / ml, methionine is present at a concentration of or about 0.1 mg / ml, m-cresol is present at a concentration of or about 3 mg / ml and the phosphate buffer is about 10mM phosphate. 32.- An article prepared according to claim 11, comprising 32.75 µg of recombinant FSH, 9.0 g of recombinant LH, 15.0 mg of sucrose, 0.052 mg of NaH2PO4 H2O, 0.825 mg of Na2HPO4 2H2O, 0.05 mg of poloxamer 188 and 0.05 mg of L-methionine. 35 33.- An article prepared according to claim 11, comprising 65.5 g of recombinant FSH, 18.0 g of recombinant LH, 30.0 mg of sucrose, 0.104 mg of NaH2PO4 H2O, 1.65 mg of Na2HPO4 2H2O, 0.10 mg of poloxamer 188 and 0.10 mg of L-methionine. 34.- A method for preparing a pharmaceutical composition comprising the step of forming a solution of FSH, a surfactant that is poloxamer 188 and a liquid diluent and also adding methionine and a bacteriostatic agent 40 selected from phenol and m-cresol. 35.- A method for preparing a packaged pharmaceutical composition comprising placing a solution comprising FSH, a surfactant that is poloxamer 188 and also placing methionine and a bacteriostatic agent chosen between phenol and m-cresol, in a vial, ampoule or cartridge. 36.- A method for preparing an article made according to any of claims 9 to 11, comprising the step of forming a mixture of FSH with or without LH, or LH alone, as well as a surfactant that is poloxamer 188, add methionine and subjecting the mixture to lyophilization, and providing a reconstitution solvent containing a bacteriostatic agent chosen from phenol and m-cresol.