FIELD OF THE INVENTION

The present invention relates to 25-hydroxy vitamin D3, in particular to a 25- hydroxy vitamin D3 dosage regime.

STATE OF THE ART

25-hydroxy vitamin D3, known as calcifediol or (6i?)-6-[(li?,3ai?,4£ ^' ,7aR)-4-[(2Z) -2-[(5,S)-5-hydroxy-2-methylidene-cyclohexylidene]ethylidene ]-7a-methyl-2,3,3a,5,6,7- hexahydro- l//-inden- 1 -yl]-2-methyl-heptan-2-ol, has the chemical structure:

25-hydroxy vitamin D3, a metabolite determined in serum as a biomarker of vitamin D status, can be used for the treatment of diseases related to vitamin D deficiency.

Vitamin D hormones have essential roles in human health which are mediated by intracellular Vitamin D receptors (VDRs). The Vitamin D hormones participate in the regulation of cellular differentiation and growth, parathyroid hormone (PTH) secretion by the parathyroid glands, and normal bone formation and metabolism. In particular, Vitamin D hormones regulate blood calcium levels by controlling the absorption of dietary calcium and phosphorus by the small intestine and the reabsorption of calcium by the kidneys. Under normal conditions, actions of Vitamin D on stimulating intestinal calcium absorption predominate, such that dietary calcium is the main source of serum calcium. However, if dietary calcium or Vitamin D is insufficient, the parathyroid gland increases secretion of PTH to enhance calcium mobilization from bone to maintain serum calcium levels. At present, vitamin D deficiency, in some cases even severe, is significantly prevalent worldwide. It is estimated that 88.1% of the world’s population have levels of 25-hydroxy vitamin D3 below 30 ng/mL (Hilger I, Friedel A., Herr R., Rausch T., Roos F., Wahl D. A., Pierroz D.D., Weber P., Hoffmann K. A systematic review of vitamin D status in populations worldwide. Br. J. Nutr. 2014; 111:23-45). The minimum level of 25-hydroxy-vitamin D considered optimal with a certain degree of consensus is 30 ng/mL (Holick M.F., Vitamin D deficiency. N. Engl. J. Med. 2007;357:266-281). Importantly, it is estimated that 37% of the world’s population have levels of 25-hydroxy vitamin D3 below 20 ng/mL (cut-off vitamin D level for considering Vitamin D deficiency, according to Holick M.F.), and even up to 6.7% of individuals is estimated to have 25-hydroxy vitamin D3 levels below 10 ng/mL (Hilger J. et ak), a severe vitamin D deficiency that clearly puts the individual’s health at risk, both at the level of alterations in the musculoskeletal metabolism, and also in relation with the increasingly numerous extra- skeletal benefits that are being discovered in relation to vitamin D and from which patients with such low vitamin D levels would be deprived.

It is accepted that severe vitamin D deficiency occurs when serum levels of 25- hydroxy vitamin D3 are below 10 ng/mL, moderate deficiency when they are between 10 and 20 ng/mL, insufficiency between 20 and 30 ng/mL and adequate values if they are above 30 ng/mL. The US Institute of Medicine considers that healthy levels are those above 20 ng/mL.

Excessive Vitamin D hormone levels, such as those higher than 90 ng/mL, whether transient or prolonged, can lead to side effects including abnormally elevated urine calcium (hypercalciuria), blood calcium (hypercalcemia), blood phosphorus (hyperphosphatemia), and adynamic bone disease. Vitamin D hormones are also required for the normal functioning of the musculoskeletal, immune and renin-angiotensin systems.

Research suggests that vitamin D could play a role in the prevention and treatment of a number of different conditions such as serious bone disorders, including rickets and osteomalacia, type 1 and type 2 diabetes, hypertension, glucose intolerance, multiple sclerosis, osteoporosis, non-traumatic fractures of the spine and hip, obesity, muscle weakness, immune deficiencies, psoriasis, and various cancers.

Recently, it has been postulated that Vitamin D deficiency may have an effect in a person’s ability to respond to a COVID-19 infection. A principal defense against uncontrolled inflammation, and against viral infection in general, is provided by T regulatory lymphocytes (Tregs). Treg levels have been reported to be low in many COVID-19 patients and can be increased by vitamin D supplementation. Low vitamin D levels have been associated with an increase in inflammatory cytokines and a significantly increased risk of pneumonia and viral upper respiratory tract infections. Vitamin D deficiency is associated with an increase in thrombotic episodes, which are frequently observed in COVID-19. Vitamin D deficiency has been found to occur more frequently in patients with obesity and diabetes. These conditions are reported to carry a higher mortality in COVID-19.

WO 2017/182237 A1 discloses the administration of 25-hydroxy vitamin D in an amount effective to raise a patient's serum 25-hydroxy vitamin D level to greater than 90 ng/mL.

Conventional oral Vitamin D supplements are far from ideal for achieving and maintaining optimal blood 25-hydroxyvitamin D levels. These preparations typically contain 400 IU to 5,000 IU of Vitamin D3 and are formulated for quick or immediate release in the gastrointestinal tract. When administered at chronically high doses these products have significant, and often severe, limitations.

SUMMARY OF THE INVENTION

Against what was known in the prior art as administering 25-hydroxy vitamin D3 at higher doses and for relatively short periods of time, the inventors have surprisingly found that by providing a weekly dosage regime of a composition comprising an amount equal to, or less than 175 micrograms of 25-hydroxy vitamin D3, the serum levels of 25- hydroxy vitamin D can be increased and maintained at a healthy and desirable level, allowing for a consistent dosage and therefore eliminating the need for patient monitoring. In addition, the inventors were surprised to find that the increase and maintenance of the serum levels of 25-hydroxy vitamin D are independent of the subject, i.e., independent of the subject’s body mass index, age, genotype, geographic location and/or condition. This has the enormous advantage that a general and universal dosage form of 25-hydroxy vitamin D3 can now be prescribed which facilitates both practitioner’s decisions and patient’s compliance.

The present invention relates to an immediate-release oral pharmaceutical composition for use in the prevention and/or treatment of a disease or condition caused by vitamin D deficiency. The invention also relates to a method for increasing and/or maintaining the serum levels of 25-hydroxy vitamin D in a subject to a value equal to or above 20 ng/mL which comprises the administration to said subject of an immediate- release oral composition. The composition of the invention comprises from 0.050 to 0.175 mg of 25-hydroxy vitamin D3 per dosage unit and each dosage unit is for administration following a dosage regime of at least once per week wherein the dosage regime is maintained for a period of at least 1 month. The invention further relates to a method of adapting the dosage regime.

Therefore, according to a first aspect, the invention is directed at an immediate- release oral pharmaceutical composition for use in the prevention and/or treatment of a disease or condition caused by vitamin D deficiency, wherein the composition comprises from 0.050 to 0.175 mg of 25-hydroxy vitamin D3 per dosage unit and wherein each dosage unit is for administration following a dosage regime of at least once per week and wherein the dosage regime is maintained for a period of at least 1 month.

A further aspect is directed at a method for the prevention and/or treatment of a disease or condition caused by vitamin D deficiency, said method comprising administering an immediate-release oral pharmaceutical composition to a patient in need of such prevention and/or treatment, wherein the composition comprises from 0.050 to 0.175 mg of 25-hydroxy vitamin D3 per dosage unit and wherein each dosage unit is for administration following a dosage regime of at least once per week and wherein the dosage regime is maintained for a period of at least 1 month.

A further aspect of the invention is directed to the use of an immediate-release oral pharmaceutical composition as defined above, in the manufacture of a medicament for the prevention and/or treatment of a disease or condition caused by vitamin D deficiency, wherein the composition comprises from 0.050 to 0.175 mg of 25-hydroxy vitamin D3 per dosage unit and wherein each dosage unit is for administration following a dosage regime of at least once per week and wherein the dosage regime is maintained for a period of at least 1 month.

According to a further aspect, the present invention is directed to a method of adapting the dosage regime of the pharmaceutical composition as defined above, comprising: a) a non-invasive method for determining the level of 25-hydroxy vitamin D in a biological fluid sample from a subject; b) adapting said dosage regime of said pharmaceutical composition to a weekly dosage regime of at least one month period, according to the following condition:

- if the level of 25-hydroxy vitamin D in the sample determined in step (a) is equal to or lower than 30 ng/mL, then the pharmaceutical composition comprises from 0.075 to 0.150 mg of 25-hydroxy vitamin D3 per dosage unit.

A further aspect relates to a method for increasing and/or maintaining the serum levels of 25-hydroxy vitamin D in a subject to a value equal to or above 20 ng/mL which comprises the administration to said subject of an immediate-release oral composition, wherein the composition comprises from 0.050 to 0.175 mg of 25-hydroxy vitamin D3 per dosage unit and wherein each dosage unit is for administration following a dosage regime of at least once per week, and wherein the dosage regime is maintained for a period of at least one month.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the invention is directed at an immediate-release oral pharmaceutical composition for use in the prevention and/or treatment of a disease or condition caused by vitamin D deficiency, wherein the composition comprises from 0.050 to 0.175 mg of 25-hydroxy vitamin D3 per dosage unit and wherein each dosage unit is for administration following a dosage regime of at least once per week and wherein the dosage regime is maintained for a period of at least 1 month.

This aspect can be redrafted as a method for the prevention and/or treatment of a disease or condition caused by vitamin D deficiency which comprises the administration to a subject in need of said prevention and/or treatment, of an immediate-release oral pharmaceutical composition, wherein the composition comprises from 0.050 to 0.175 mg of 25-hydroxy vitamin D3 per dosage unit and wherein each dosage unit is for administration following a dosage regime of at least once per week, and wherein the dosage regime is maintained for a period of at least one month.

A further aspect is directed at a method for increasing and/or maintaining the serum levels of 25-hydroxy vitamin D in a subject to a value equal to or above 20 ng/mL which comprises the administration to said subject of an immediate-release oral composition, wherein the composition comprises from 0.050 to 0.175 mg of 25-hydroxy vitamin D3 per dosage unit and wherein each dosage unit is for administration following a dosage regime of at least once per week, and wherein the dosage regime is maintained for a period of at least one month.

The term “subject” refers to a human or an animal, preferably to a human. In a preferred embodiment, it is to be understood as synonym to the term “individual” or “person”. If said subject has a vitamin D related disease or condition, then the term “subject” can also be understood as “patient”.

Dosage regime

In the present invention, the dosage regime is maintained for a period of at least one month. In a particular embodiment, it is maintained for a period of at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months and at least 12 months. Preferably, it is maintained for a period of at least 4 months, more preferably of at least 6 months. Even more preferably, it is maintained for a period of at least 12 months.

A further aspect of the invention is related to a method of adapting the dosage regime of the pharmaceutical composition of the invention, comprising: a) a non-invasive method for determining the level of 25-hydroxy vitamin D in a biological fluid sample from a subject; b) adapting said dosage regime of said pharmaceutical composition to a weekly dosage regime of at least one month period, according to the following condition:

- if the level of 25-hydroxy vitamin D in the sample determined in step (a) is equal to or lower than 30 ng/mL, then the pharmaceutical composition comprises from 0.075 to 0.150 mg of 25-hydroxy vitamin D3 per dosage unit.

A “non-invasive method for determining the level of 25-hydroxy vitamin D in a biological fluid sample from a subject” refers to a method which is conducted outside the body, wherein said biological fluid sample has been previously removed from the body and is not returned to the same body.

In a particular embodiment, said biological fluid includes blood, particularly serum or plasma, but also breast milk, urine, semen, saliva, cerebrospinal and synovial fluid. Preferably, said biological fluid is serum or plasma, most preferably serum.

In a preferred embodiment, the level of 25-hydroxy vitamin D in a biological fluid sample from a subject is the baseline level. In a particular embodiment, the level of 25-hydroxy vitamin D in the sample determined in step (a) is equal to or less than 30 ng/mL. Preferably, it is equal to or less than 20 ng/mL.

In the present invention, the composition of the invention, when administered according to the weekly dosage regime described above, provides at least 80% of the treated subjects with a serum 25-hydroxy vitamin D3 level above 20 ng/mL after 1 month of administration, regardless of their baseline level, preferably after 2 months, more preferably after 4 months. In a preferred embodiment, said obtained serum level after 1 month of administration is above 30 ng/mL, more preferably after 2 months, even more preferably after 4 months.

In a particular embodiment, the composition of the invention, when administered according to the weekly dosage regime described above, provides at least 90% of the treated subjects with a serum 25-hydroxy vitamin D3 level above 20 ng/mL after 1 month of administration, regardless of their baseline level, preferably after 2 months, more preferably after 4 months. In a preferred embodiment, said obtained serum level after 1 month of administration is above 30 ng/mL, more preferably after 2 months, even more preferably after 4 months.

In a preferred embodiment, the composition of the invention, when administered according to the weekly dosage regime described above, provides at least 95% of the treated subjects with a serum 25-hydroxy vitamin D3 level above 20 ng/mL after 1 month of administration, regardless of their baseline level, preferably after 2 months, more preferably after 4 months. In a preferred embodiment, said obtained serum level after 1 month of administration is above 30 ng/mL, more preferably after 2 months, even more preferably after 4 months.

In a particular embodiment of the method of adapting the dosage regime, step (b) is as follows: - if the level of 25-hydroxy vitamin D in the sample determined in step (a) is equal to or lower than 30 ng/mL, then the pharmaceutical composition comprises from 0.085 to 0.135 mg of 25-hydroxy vitamin D3 per dosage unit, preferably from 0.090 to 0.130 mg, more preferably from 0.095 to 0.130 mg, even more preferably from 0.100 to 0.125 mg and most preferably 0.100 or 0.125 mg of 25-hydroxy vitamin D3 per dosage unit. In a particular embodiment of the method of adapting the dosage regime, step (b) is as follows: - if the level of 25-hydroxy vitamin D in the sample determined in step (a) is equal to or lower than 20 ng/mL, then the pharmaceutical composition comprises from 0.085 to 0.135 mg of 25-hydroxy vitamin D3 per dosage unit, preferably from 0.090 to 0.130 mg, more preferably from 0.095 to 0.130 mg, even more preferably from 0.100 to 0.125 mg and most preferably 0.100 mg or 0.125 mg of 25-hydroxy vitamin D3 per dosage unit.

The inventors have found that the dosage regime of the present invention can be further adapted to optimize results. Therefore, in a particular embodiment, the method of adapting the dosage regime of the pharmaceutical composition of the invention comprises step (a) as defined above and step (b) is as follows: b) adapting said dosage regime of said pharmaceutical composition to a weekly dosage regime of at least one month period, according to the following conditions:

- if the level of 25-hydroxy vitamin D in the sample determined in step (a) is comprised between 10 and 20 ng/mL, then the pharmaceutical composition comprises from 0.075 to 0.125 mg of 25-hydroxy vitamin D3 per dosage unit, preferably from 0.090 to 0.110 mg per dosage unit, even more preferably 0.100 mg per dosage unit; or

- if the level of 25-hydroxy vitamin D in the sample determined in step (a) is lower than 10 ng/mL, then the pharmaceutical composition comprises from 0.100 to 0.150 mg of 25-hydroxy vitamin D3 per dosage unit, preferably from 0.115 to 0.135 mg per dosage unit, even more preferably 0.125 mg per dosage unit.

In another particular embodiment, the method of adapting the dosage regime of the pharmaceutical composition of the invention comprises step (a) as defined above and step (b) is as follows: - if the level of 25-hydroxy vitamin D in the sample determined in step (a) is comprised between 10 and 20 ng/mL, then the pharmaceutical composition comprises from 0.075 to 0.125 mg of 25-hydroxy vitamin D3 per dosage unit, preferably from 0.090 to 0.110 mg and most preferably 0.100 mg of 25-hydroxy vitamin D3 per dosage unit.

The inventors have surprisingly found that the administration of the composition described in the previous paragraph, following the weekly dosage regime of the invention, provides at least 80%, at least 85%, at least 90% or at least 95% of the treated subjects with a serum 25-hydroxy vitamin D3 level above 20 ng/mL. Preferably, the serum level is above 30 ng/mL. In a preferred embodiment, these levels are obtained after 1 month of administration, preferably after 2 months, more preferably after 4 months of administration.

In yet another particular embodiment, compatible with the former, the method of adapting the dosage regime of the pharmaceutical composition of the invention comprises step (a) as defined above and step (b) is as follows: - if the level of 25-hydroxy vitamin D in the sample determined in step (a) is lower than 10 ng/mL, then the pharmaceutical composition comprises from 0.100 to 0.150 mg of 25-hydroxy vitamin D3 per dosage unit, preferably from 0.115 to 0.135 mg and most preferably 0.125 mg of 25-hydroxy vitamin D3 per dosage unit.

The inventors have surprisingly found that the administration of the composition described in the previous paragraph, following the dosage regime described therein, provides at least 80%, at least 85%, at least 90% or at least 95% of the treated subjects with a serum 25-hydroxy vitamin D3 level above 20 ng/mL. Preferably, the serum level is above 30 ng/mL. In a preferred embodiment, these levels are obtained after 1 month of administration, preferably after 2 months, more preferably after 4 months of administration.

In a particular embodiment of the method of adapting the dosage regime, described above, the weekly dosage regime is of a period of at least 4 months, preferably at least 6 months. Even more preferably, it is maintained for a period of at least 12 months.

In a particular embodiment of the method of adapting the dosage regime, described above, the weekly dosage regime achieves a level of serum 25-hydroxy vitamin D equal to or greater than 20 ng/mL.

The inventors have found that if the 25-hydroxy vitamin D3 administration is interrupted, the serum levels of 25-hydroxy vitamin D drop to the originally determined basal levels within a few weeks. Therefore, once the subject is treated with the above amounts of 25-hydroxy vitamin D3 at the above-defined dosage regime, the dosage regime can optionally be modified according to a particular embodiment of the method of adapting the dosage regime, said dosage regime as described above, wherein said weekly dosage regime is terminated and followed by the administration of a once per month regime of a pharmaceutical composition comprising at least 0.2 mg of 25-hydroxy vitamin D3 per dosage unit.

In a particular embodiment, the method of adapting the dosage regime described above, further comprises a step (c) wherein the weekly dosage regime of step (b) is maintained for a period of at least 2 months, at least 4 months, preferably at least 6 months. Even more preferably, it is maintained for a period of at least 12 months.

In another particular embodiment of the method of adapting the dosage regime described above, said subject has a disease or condition caused by vitamin D deficiency. Preferably, the disease or condition caused by vitamin D deficiency is selected from the group consisting of rickets, osteomalacia, osteopenia, osteoarthritis, osteoarthrosis, hypocalcemia, post-thyroidectomy hypocalcemia, hypophosphatemia, osteoporosis, fragility fractures, including spine, hip and distal radius fractures, hyperparathyroidism, inflammatory bowel disease, chronic kidney disease - mineral bone disorder, type 1 and type 2 diabetes, glucose intolerance, multiple sclerosis, muscle weakness, hypertension, cardiovascular complications in unstable angina, immune deficiencies, psoriasis, pneumonia, viral respiratory tract infections, severe respiratory syndrome-related coronavirus including SARS-CoV-2 and SARS-CoV, Middle East respiratory syndrome- related coronavirus including MERS-CoV, vaccine-induced immune thrombotic thrombocytopenia and cancer, including diffuse large B-cell lymphoma.

Composition

In the present invention, the composition comprises from 0.050 to 0.175 mg of 25- hydroxy vitamin D3 per dosage unit. In a particular embodiment, it comprises at least 0.050, at least 0.055, at least 0.060, at least 0.065, at least 0.070, at least 0.075, at least 0.080, at least 0.085, at least 0.090, at least 0.095, or preferably at least 0.100 mg of 25- hydroxy vitamin D3 per dosage unit. In a preferred embodiment of this particular embodiment, the composition comprises no more than 0.175 or no more than 0.125 mg of 25-hydroxy vitamin D3 per dosage unit.

In another particular embodiment, it comprises no more than 0.175, no more than 0.170, no more than 0.165, no more than 0.160, no more than 0.155, no more than 0.150, no more than 0.145, no more than 0.140, no more than 0.135, no more than 0.130, preferably no more than 0.125 mg of 25-hydroxy vitamin D3 per dosage unit. In a preferred embodiment of this particular embodiment, the composition comprises at least 0.050 or at least 0.100 mg of 25-hydroxy vitamin D3 per dosage unit.

In a more preferred embodiment, the composition comprises from 0.075 to 0.175 mg, from 0.085 to 0.155 mg, from 0.085 to 0.145 mg, from 0.090 to 0.140 mg, from 0.090 to 0.135 mg, from 0.090 to 0.130 mg, from 0.095 to 0.135 mg, from 0.095 to 0.130 mg or from 0.100 to 0.125 mg of 25-hydroxy vitamin D3. In an even more preferred embodiment, it comprises from 0.075 to 0.175 mg of 25-hydroxy vitamin D3, from 0.085 to 0.155 mg of 25-hydroxy vitamin D3, from 0.095 to 0.135 mg of 25-hydroxy vitamin D3, preferably from 0.100 to 0.125 mg of 25-hydroxy vitamin D3.

The composition of the invention is preferably a pharmaceutical composition but the skilled person will readily understand that if it is used in the context of a non- therapeutic use/method, then it can also be a nutraceutical composition.

The term “pharmaceutical composition” refers to compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a subject, preferably a human or an animal, more preferably a human. Preferably, as used herein, the term “pharmaceutic composition” means a composition that was approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The pharmaceutical composition is an immediate-release oral pharmaceutical composition. In a preferred embodiment, it is a soft gelatin capsule. In a preferred embodiment, each dosage unit is a single soft gelatin capsule comprising 25-hydroxy vitamin D3.

The term “soft capsule” is well known in the art and refers to a capsule having a soft capsule shell, as opposed to hard capsules that are made up of a rigid shell. A soft capsule shell is generally made of gelatin, water and plasticizer in various mixtures which gives elasticity and softness to the walls (shell). Soft capsules are usually formed in a single piece, as opposed to hard capsules that are made up of a shell in two pieces that fit together.

Preferred soft gelatin capsules are those described in WO 2016/124724 A1 (PCT/EP2016/052458), of Faes Farma, wherein 25-hydroxy vitamin D3 is formulated in combination with an oily component and an organic solvent and the composition is encapsulated in a soft gelatin capsule. Of the capsules described in WO 2016/124724 Al, preferred soft gelatin capsules are those wherein the soft capsule shell comprises:

- 40 to 80 wt% of gelatin,

- 10 to 30 wt% of glycerol, and

- 5 to 15 wt % of sorbitol, more preferably,

- 60 to 70 wt% of gelatin,

- 15 to 25 wt% of glycerol, and

- 5 to 15 wt % of sorbitol, the amounts by weight being expressed with respect to the total weight of the shell. The skilled person will readily understand that an amount of water is present in the shell, in an amount usually comprised between 2 and 15% with respect to the total weight of the shell, preferably between 6 and 10%.

In a particular embodiment, the pharmaceutical composition is a soft gelatin capsule as described above, which is devoid of waxes. In another particular embodiment, compatible with the former, the pharmaceutical composition is a soft gelatin capsule as described above, which is devoid of surfactants.

Serum 25-hydroxy vitamin D level

The inventors were surprised to find that the increase and maintenance of the serum levels of 25-hydroxy vitamin D is independent of the subject, i.e., independent of the subject’s body mass index, age, genotype, geographic location and/or condition.

Thus, an aspect of the invention, as already mentioned above, is directed at a method for increasing and/or maintaining the serum levels of 25-hydroxy vitamin D in a subject to a value equal to or above 20 ng/mL which comprises the administration to said subject of an immediate-release oral composition, wherein the composition comprises from 0.050 to 0.175 mg of 25-hydroxy vitamin D3 per dosage unit and wherein each dosage unit is for administration following a dosage regime of at least once per week, and wherein the dosage regime is maintained for a period of at least one month.

In the present invention, the serum 25-hydroxy vitamin D is raised to, or maintained at, a level of equal to or more than 20 ng/mL after an administration period of the composition as described herein of at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 6 months or at least 12 months, regardless of the level at the beginning of the treatment with the dosage regime of the invention. Preferably, it is raised to, or maintained at, a level of equal to or more than 30 ng/mL.

In a particular embodiment, the serum 25-hydroxy vitamin D is raised to, or maintained at, a level of equal to or more than 20 ng/mL after an administration period of the composition as described herein of 1 month, 2 months, 3 months, 4 months, 6 months or 12 months, regardless of the level at the beginning of the treatment with the dosage regime of the invention. Preferably, it is raised to, or maintained at, a level of equal to or more than 30 ng/mL.

In a particular embodiment, the serum 25-hydroxy vitamin D level is raised to, or maintained at, a value not higher than 90 ng/mL, not higher than 80 ng/mL, not higher than 70 ng/mL and preferably not higher than 60 ng/mL after an administration period of the composition as described herein of at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 6 months or at least 12 months, regardless of the level at the beginning of the treatment with the dosage regime of the invention.

In a particular embodiment, the serum 25-hydroxy vitamin D level is raised to, or maintained at, a value not higher than 90 ng/mL, not higher than 80 ng/mL, not higher than 70 ng/mL and preferably not higher than 60 ng/mL after an administration period of the composition as described herein of 1 month, 2 months, 3 months, 4 months, 6 months or 12 months, regardless of the level at the beginning of the treatment with the dosage regime of the invention.

In a preferred embodiment, the serum 25-hydroxy vitamin D is raised to, or maintained at, a level comprised between 20 and 90 ng/mL, preferably between 20 and 80 ng/mL, preferably between 20 and 70 ng/mL, preferably between 25 and 60 ng/mL, and even more preferably between 30 and 60 ng/mL after an administration period of the composition as described herein of at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 6 months or at least 12 months, regardless of the level at the beginning of the treatment with the dosage regime of the invention.

In a preferred embodiment, the serum 25-hydroxy vitamin D is raised to, or maintained at, a level comprised between 20 and 90 ng/mL, preferably between 20 and 80 ng/mL, preferably between 20 and 70 ng/mL, preferably between 25 and 60 ng/mL, and even more preferably between 30 and 60 ng/mL after an administration period of the composition as described herein of 1 month, 2 months, 3 months, 4 months, 6 months or 12 months, regardless of the level at the beginning of the treatment with the dosage regime of the invention

In the context of the present invention, the term “increase” is synonym to “raise”. The increase or raise of the serum 25-hydroxy vitamin D level is to be understood as a variation from a lower to a higher level of 25-hydroxy vitamin D in the serum. The term “maintain” is meant to describe that a subject’s serum 25-hydroxy vitamin D level throughout the administration period is kept above a given threshold value or within a given range.

For example, a method for increasing the serum levels above a certain threshold value expressed in ng/mL is a method which, upon administration of a composition comprising from 0.050 to 0.175 mg of 25-hydroxy vitamin D3 per dosage unit at least once per week and for a period of at least one month, provides an increase from a baseline serum level in a subject (for example, from a baseline value below 20 ng/mL) to a level which is above said threshold (for example, above 20 ng/mL). Similarly, a method for maintaining the serum levels above a certain threshold value expressed in ng/mL is a method which, upon administration of a composition comprising from 0.050 to 0.175 mg of 25-hydroxy vitamin D3 per dosage unit at least once per week and for a period of at least one month, is able to keep a baseline serum level in a subject above said threshold (for example, above 20 ng/mL).

In a particular embodiment of the above described method for increasing and/or maintaining the serum levels of 25-hydroxy vitamin D, the method is a non-therapeutic method.

In the present invention, serum 25-hydroxy vitamin D levels are determined by the technique:

Automated electrochemiluminescence immunoassay (ECLIA, Roche Diagnostics Cobas e 601, detection limit <3.00 ng/mL, intra-assay coefficient of variation <4.69% and inter-assay coefficient of variation 5.42%). Unless stated otherwise, the serum 25- hydroxy vitamin D levels determined by electrochemiluminescence immunoassay (ECLIA, Roche Diagnostics cobas ^® e 601) were performed following the standard procedure recommended by the manufacturer. Also, since in the present invention the increase and maintenance of the serum levels of 25-hydroxy vitamin D is independent of the subject, a particular embodiment is that wherein the subject is characterized by a body mass index of from 15 to 50, from 18 to 40, preferably from 18 to 25.

Vitamin D deficiency

The invention is directed at an immediate-release oral pharmaceutical composition for use in the prevention and/or treatment of a disease or condition caused by vitamin D deficiency or, drafted in an alternative manner, to a method for the prevention and/or treatment of a disease or condition caused by vitamin D deficiency which comprises the administration to a subject in need of said prevention and/or treatment of an immediate- release oral pharmaceutical composition. The invention is also directed at a method for increasing and/or maintaining the serum levels of 25-hydroxy vitamin D in a subject to a value equal to or above 20 ng/mL and in a preferred embodiment of this method, said subject has a disease or condition caused by vitamin D deficiency.

The term “treatment” or “to treat” in the context of this specification means administration of a composition according to the invention to ameliorate or eliminate the disease or one or more symptoms associated with said disease. “Treatment” also encompasses ameliorating or eliminating the physiological sequelae of the disease.

The term “ameliorate” in the context of this invention is understood as meaning any improvement on the situation of the patient treated.

The term “prevention” or “to prevent” refers to the reduction in the risk of acquiring or developing a given disease or disorder, or the reduction or inhibition of the recurrence or a disease or disorder.

In the present invention, vitamin D deficiency corresponds to when the serum 25- hydroxy vitamin D level of a subject is equal to or lower than 30 ng/mL, equal to or lower than 20 ng/mL, equal to or lower than 15 ng/mL or equal to or lower than 10 ng/mL. Preferably, vitamin D deficiency is a serum 25-hydroxy vitamin D level comprised between 4 and 30 ng/mL.

However, vitamin D deficiency does not necessarily express itself as a disease and a subject may otherwise be considered healthy even if the serum 25-hydroxy vitamin D level of said subject is equal to or lower than 30 ng/mL, preferably 20 ng /mL. As disclosed above, it is estimated that 88.1% of the world’s population have levels of 25- hydroxy vitamin D below 30 ng/mL and 37% below 20 ng/mL. In this way, a particular embodiment of the invention is that wherein the use, or the method of administration, as described above, correspond to a use, or method of administration, for the prevention of a disease or condition. Another particular embodiment of the invention is that wherein the aspects related to the composition for use, the method of administration or the method for increasing and/or maintaining, as described above, relates to a non-therapeutic use, or to non-therapeutic methods, respectively.

In a particular embodiment, vitamin D deficiency is responsible for causing a disease, condition or predisposition for a certain disease or condition. In the present invention, said disease or condition caused by vitamin D deficiency is selected from the group consisting of rickets, osteomalacia, osteopenia, osteoarthritis, osteoarthrosis, hypocalcemia, post-thyroidectomy hypocalcemia, hypophosphatemia, osteoporosis, fragility fractures, including spine, hip and distal radius fractures, hyperparathyroidism, inflammatory bowel disease, chronic kidney disease - mineral bone disorder, type 1 and type 2 diabetes, glucose intolerance, multiple sclerosis, muscle weakness, hypertension, cardiovascular complications in unstable angina, immune deficiencies, psoriasis, pneumonia, viral respiratory tract infections, severe acute respiratory syndrome-related coronavirus infections including SARS-CoV-2 and SARS-CoV, Middle East respiratory syndrome-related coronavirus including MERS-CoV, vaccine-induced immune thrombotic thrombocytopenia and cancer, including diffuse large B-cell lymphoma.

In the present invention, “cancer” includes breast, lung, skin, melanoma, colon, colorectal, rectal, prostate, bone cancer. It also includes lymphoma, in particular diffuse large B cell lymphoma. Diffuse large B cell lymphoma (DLBCL) is the most common lymphoma, accounting for about 25% to 30% of all the non-Hodgkin lymphoma. This aggressive disease can affect many organ systems and responds well to chemotherapy. In a preferred embodiment, cancer is DLBCL. Without wishing to be bound by a particular theory, the inventors postulate that Vitamin D supplementation can improve the prognosis of survival in patients with DLBCL treated with chemotherapy. Therefore, the composition of the invention can be used in the treatment and/or prevention of worsening of this condition. Severe acute respiratory syndrome-related coronavirus infections, include SARS- CoV-2, the virus infection commonly known as COVID-19. Vitamin D receptor stimulation is postulated to reduce acute respiratory distress syndrome (ARDS) in patients with coronavirus SARS-CoV-2 infections and there is evidence that vitamin D supplementation could reduce risk of COVID-19 infections and deaths (Grant WB, et al., Nutrients 2020; 12(4): 988 and Quesada-Gomez JM, et al, The Journal of steroid biochemistry and molecular biology 2020; 202:1057). In a particular embodiment, when the disease or condition is selected from a SARS-CoV-2 infection, the subject is at least 12 years old, preferably 18 years old.

In a particular embodiment, the composition of the invention can produce stimulation of the active immunization. Preferably, said stimulation of the active immunization occurs in a SARS-CoV-2 infection.

Without wishing to be bound by a particular theory, the inventors postulate that Vitamin D supplementation, from the administration of the composition of the invention, can reduce the number of hospital admissions in SARS-CoV-2 positive patients and can reduce the frequency of intensive care units (ICUs) admissions in hospitalized patients with SARS-CoV-2 infection and deaths. Therefore, the composition of the invention can be used in the treatment and/or prevention of SARS-CoV-2 infection, can reduce the severity of this condition and its use can be beneficial from early stages of the disease. In a particular embodiment, the SARS-CoV-2 infection is in a patient characterized by at least one of the following: 60 or more years old; body mass index equal to or higher than 30; cardiovascular chronic diseases; chronic respiratory diseases (CRDs); or chronic kidney disease (CKD).

The AZD1222 vaccine (brand names Covishield and Vaxzevria) developed by Oxford/AstraZeneca has been recently associated to rare cases of vaccine-induced immune thrombotic thrombocytopenia, a rare vaccine-related variant of spontaneous heparin-induced thrombocytopenia (A. Greinacher et al, A prothrombotic thrombocytopenic disorder resembling heparin-induced thrombocytopenia following coronavirus- 19 vaccination ; Research Square. Posted March 29, 2021). Even more recently, reports have been made available that similar rare thrombotic events occur upon the administration of COVID-19 Ad26.COV2-S Vaccine, developed by Johnson & Johnson/Janssen. Without wishing to be bound by a particular theory, the inventors postulate that the Vitamin D endocrine system can modulate the thrombocytopenic autoimmune response upon vaccination. Therefore, a particularly preferred condition of the ones listed above is vaccine-induced immune thrombotic thrombocytopenia. In this particular case, the composition of the invention can be either administered as a prevention or immediately after the appearance of the vaccine-induced thrombotic symptoms.

In a particular embodiment of said vaccine-induced immune thrombotic thrombocytopenia, the vaccine is directed at a coronavirus infection, such as the one responsible for the COVID-19.

In a particular embodiment of said vaccine-induced immune thrombotic thrombocytopenia, the vaccine is selected from vaccines comprising viral vector-based vaccines. Preferably, said viral vector-based vaccines are adenovirus/adenoviral vector vaccines.

In a preferred embodiment of said vaccine-induced immune thrombotic thrombocytopenia, the vaccine is selected from the adenovirus viral vector vaccines AZD1222 (brand names Covishield and Vaxzevria, developed by Oxford/AstraZeneca), Ad26.COV2-S (developed by Janssen Vaccines, Janssen Pharmaceuticals, subsidiary of Johnson & Johnson), Sputnik V (Gam-COVID-Vac, developed by Russia’s Gamaleya National Centre of Epidemiology and Microbiology) or AD5-nCOV (trade-named Convidecia, developed by CanSino Biologies). One of the above-listed diseases/conditions is post-thyroidectomy hypocalcemia. After total thyroidectomy, postoperative hypocalcemia is the most frequent complication (in up to 50% of cases). It is usually mild and transient and is rarely permanent (1-3%), but it is always a concern and a challenge for the surgeon and, for the patient, it entails additional treatment. Vitamin D plays a central role in calcium homeostasis, promotes intestinal absorption of calcium and phosphate, and stimulates osteoclast differentiation. Without wishing to be bound by a particular theory, the inventors have postulated that the vitamin D level could be a good indicator of the risk of postoperative hypocalcemia in patients undergoing thyroidectomy and therefore the composition of the invention can be used in the treatment and/or prevention of this condition. The present invention will be more specifically illustrated by the following examples. However, it should be understood that the present invention is not limited by these examples in any manner.

Examples

Example 1. Phase TT/TTT Efficacy Clinical Trial

ECLIA method General description:

The method involved an automated electrochemiluminescence immunoassay analysis (ECLIA).

Instrumental System:

The automated electrochemiluminescence immunoassay (ECLIA) was made up of a cobas ^® 6000 analyzer series composed of a core unit and a cobas ^® e 601 module for immunoassay tests (Roche Diagnostics, https://diagnostics.roche. com/global/en/products/instruments/cobas-e-601.html).

The detection limit of 25-hydroxy vitamin D, as determined by the manufacturer of the assay, was <3.00 ng/mL. The intra-assay coefficient of variation was <4.69% and the inter-assay coefficient of variation was 5.42%.

Serum 25-hydroxy vitamin D levels were determined following the standard procedure recommended by the manufacturer.

Phase ////// Efficacy Clinical Trial:

The efficacy of weekly dosages according to the invention versus placebo in more than 100 subjects with either vitamin D deficiency, or insufficiency, was tested by conducting a randomized, double-blind, double-dummy, multicenter clinical trial.

Subjects having a serum 25-hydroxy vitamin D baseline level above 10 ng/mL and below 20 ng/mL were allocated to Cohort 1. Cohort 1 subjects were assigned to one of the following three groups according to the amount of 25-hydroxy vitamin D3 administered: 0 mg (placebo), 0.075 mg and 0.100 mg.

Subjects having a serum 25-hydroxy vitamin D baseline level equal to or lower than 10 ng/mL were allocated to Cohort 2. Cohort 2 subjects were assigned to one of the following three groups according to the amount of 25-hydroxy vitamin D3 administered: 0 mg (placebo), 0.100 mg and 0.125 mg.

Administration was made by ingestion of soft gelatin capsules.

Eligibility criteria were as follows:

Inclusion Criteria:

• Male or female subjects > 18 years of age.

• Evidence of serum 25(OH)D levels < 20 ng/mL or < 10 ng/mL, for each cohort.

• Written informed consent.

• Females of childbearing potential who were willing to perform pregnancy tests, and who agreed to use highly effective methods of birth control throughout the study.

Exclusion Criteria:

• Subjects that were receiving any treatment with 25 -hydroxy vitamin D3, vitamin D analogues, vitamin complexes or vitamin D supplements.

• Subjects that were taking drugs that could modify vitamin D levels.

• Subjects that were taking calcium supplements.

• Uncorrected hypercalcaemia, known hypercalciuria or nephrolithiasis.

• Severe renal impairment.

• Subjects that had been diagnosed with liver or biliary failure, congestive heart failure, malabsorption, primary hyperparathyroidism, hypothyroidism, prolonged immobilisation, sarcoidosis, tuberculosis or other granulomatous diseases or hyperthyroidism.

• Any present or previous malignancy.

• Known contraindications or sensitivities to the use of the composition or any of its components.

• Pregnant women, breastfeeding women or women planning a pregnancy.

• Subjects that had received an investigational product within 30 days before the start of the screening or who were enrolled in an investigational interventional study.

• Any condition that could jeopardise the clinical trial conduct according to the protocol.

• Employees of the investigator or clinical trial site, as well as family members of the employees or the principal investigator. • Person committed to an institution by virtue of an order issued either by judicial or other authorities.

Example 2. Results

The inventors have surprisingly observed that, after 1 month of administration of the dosage regime according to the invention (in particular according to Example 1), more than 90% of all subjects of both Cohorts had their baseline 25-hydroxy vitamin D level increased to a value equal to or greater than 20 ng/mL.

After 4 months of administration of the dosage regime according to the invention (in particular according to Example 1), all subjects of both Cohorts had their baseline 25- hydroxy vitamin D level increased to a value equal to or greater than 30 ng/mL.

In particular:

All subjects of Cohort 1 (serum 25-hydroxy vitamin D baseline level above 10 ng/mL and below 20 ng/mL) had their baseline 25-hydroxy vitamin D level increased to a value equal to or greater than 20 ng/mL upon 4 months of administration of 25-hydroxy vitamin D3 at weekly dosages of 0.075 and 0.100 mg, and all had their baseline 25-hydroxy vitamin D level increased to a value equal to or greater than 30 ng/mL upon 4 months of administration of 25-hydroxy vitamin D3 at weekly dosages of 0.100 mg; and

All subjects of Cohort 2 (serum 25-hydroxy vitamin D baseline level equal to or lower than 10 ng/mL) had their baseline 25-hydroxy vitamin D level increased to a value equal to or greater than 30 ng/mL upon 4 months of administration of 25- hydroxy vitamin D3 at weekly dosages of 0.125 mg.

After 6 months of administration of the dosage regime according to the invention (in particular according to Example 1), all subjects of both Cohorts had their baseline 25- hydroxy vitamin D level increased to a value equal to or greater than 30 ng/mL. In particular:

All subjects of Cohort 1 (serum 25-hydroxy vitamin D baseline level above 10 ng/mL and below 20 ng/mL) had their baseline 25-hydroxy vitamin D level increased to a value equal to or greater than 20 ng/mL upon 6 months of administration of 25-hydroxy vitamin D3 at weekly dosages of 0.075 and 0.100 mg, and all had their baseline 25-hydroxy vitamin D level increased to a value equal to or greater than 30 ng/mL upon 6 months of administration of 25-hydroxy vitamin D3 at weekly dosages of 0.100 mg; and

All subjects of Cohort 2 (serum 25-hydroxy vitamin D baseline level equal to or lower than 10 ng/mL) had their baseline 25-hydroxy vitamin D level increased to a value equal to or greater than 20 ng/mL upon 6 months of administration of 25- hydroxy vitamin D3 at weekly dosages of 0.100 and 0.125 mg, and all had their baseline 25-hydroxy vitamin D level increased to a value equal to or greater than 30 ng/mL upon 6 months of administration of 25-hydroxy vitamin D3 at weekly dosages of 0.125 mg.

During the experiment, none of the human subjects had their baseline 25-hydroxy vitamin D level increased to a value greater than 75 ng/mL. Thus, the experiment shows that the serum 25-hydroxy vitamin D is raised to, or maintained at, a level comprised 20 and 80 ng/mL, after an administration period of the composition of the invention of 4 months, regardless of the level at the beginning of the treatment with the dosage regime of the invention. This level is maintained after 6 months. The results also show that the present invention achieves an adequate concentration of 25-hydroxy vitamin D in the body regardless of a person’s body mass index, age, genotype, geographic location and/or condition.