Technical field

The present invention relates to the treatment of patients suffering from major depressive disorder (MDD). It also relates to personalized medicine.

Background of the invention

Trintellix® is an FDA approved medical drug comprising vortioxetine hydrobromide. Commercially available Trintellix® tablets comprise 5 mg, 10 mg, 15 mg, or 20 mg vortioxetine (as hydrobromide). Trintellix® is mostly used for treatment of depression. In some European countries, a corresponding product is or was available as Brintellix®. Similar to Trintellix®, Brintellix® can only be obtained with a prescription and is available as tablets (5, 10, 15 and 20 mg) and oral drops (20 mg/ml). The most common side effects reported with vortioxetine are nausea, diarrhea, dry mouth, constipation, vomiting, flatulence, dizziness and sexual dysfunction.

To study mood, depression and anxiety in rats or mice, several behavioral assays have been developed. The forced swim test (FST) measures the presence of or reduction in positive coping skills in rats and mice. When animals are treated with an effective anti-depressant prior to the test, they show reduced immobility and more climbing, suggesting that the animals do not give up, while untreated animals show increased immobility and will float more in the water. Therefore, reduced immobility and more climbing is thought to be an anti-depressive, positive coping phenotype in the FST. Guilloux et al. (2013) have found that vortioxetine (2.5-5 mg/kg) increased mobility in the FST [see Guilloux, J. P. et al., “Antidepressant and anxiolytic potential of the multimodal antidepressant vortioxetine (Lu AA21004) assessed by behavioural and neurogenesis outcomes in mice”, Neuropharmacology, 73, 147-159, 2013],

The German G-BA (“Gemeinsamer Bundesauschuss”) evaluates the additional benefit of drugs with new active ingredients compared to a comparative therapy. Such benefit assessment is done in accordance with Section 35a of the German Social Code, Book V (known as the AMNOG procedure). The result of this benefit assessment is the starting point for price negotiations between the National Association of Statutory Health Insurance Funds (“GKV- Spitzenverband”) and the pharmaceutical manufacturer. At the end of the negotiations, it is determined how much the statutory health insurance will pay for the drug in the future. In Germany, the benefit assessment of vortioxetine has been rather negative. As a result, the originator company appears to have withdrawn Brintellix® from the German market.

Whereas vortioxetine surely is an effective drug, there is a need for an improved vortioxetine therapy. Summary of the invention

According to the present invention, vortioxetine therapy is improved by taking into consideration the vitamin D level of the patient to be treated.

Hitherto, every vortioxetine patient has been treated in the same manner - regardless of his or her vitamin D status. However, to be effective, vortioxetine requires a certain level of vitamin D in the body of the patient. The inventors have unveiled that patients who lack vitamin D are more likely to not sufficiently respond to a vortioxetine treatment.

The present invention relates to a fixed-dose combination (FDC) that is suitable to improve the vortioxetine treatment of patients who lack vitamin D. The preferred fixed-dose combination of the invention is a solid oral dosage form which comprises - in addition to vortioxetine - also vitamin D.

Preferably, the solid oral dosage form of the invention is similar to the commercially available vortioxetine tablet (Brintellix®). Thus, the solid oral dosage form of the invention is preferably a compressed tablet that comprises 5 mg, 10 mg, or 20 mg vortioxetine hydrobromide.

In deionized water at 37°C, a 10 mg Brintellix® tablet has an in vitro disintegration time of less than 1 minute. As shown in the examples herein, initially, disintegration time of a Brintellix® tablet was significantly increased when adding vitamin D. This is disadvantageous, as there is a linear relationship between disintegration of the tablet and dissolution of the drug; disintegration testing is an appropriate drug product quality control method for evaluating drug release from this dosage form (Nickerson et al., “Correlation of dissolution and disintegration results for an immediate-release tablet”, Journal of Pharmaceutical and Biomedical Analysis 150 (2018), page 339, left column, section “4. Conclusions”).

It would be desirable to find a way of adding vitamin D to Brintellix® without increasing disintegration time of the obtained tablet in an unacceptable manner.

One embodiment of the invention relates to a vortioxetine tablet comprising calcifediol instead of vitamin D3. When replacing vitamin D3 with the corresponding amount of calcifediol, the increase of the tablet's disintegration time is less pronounced. For human consumption, calcifediol is commercially available under the tradename ampli-D® at DSM® Nutritional Products (Switzerland). Calcifediol is also available as Rayladee® (OPKO Health, Miami, FL) and as Hidroferol® oral solution (Faes Farma, S.A., Spain).

One embodiment of the invention relates to a method of manufacturing a solid oral dosage form, wherein vitamin D3 and/or calcifediol are added to vortioxetine granules before compressing the obtained blend into tablets. Surprisingly, such extra-granular addition also reduces the unwanted increase of the tablet's disintegration time. Thus, the use of granules comprising both, vortioxetine and vitamin D, for manufacturing tablets is not preferred. Figures

Figures 1 and 2 relate to Example 1 (vide infra).

Figure 1 shows a lack of effects of acute vortioxetine treatment in the forced swim test (FST) in male Balb/cJ mice that were vitamin D deficient. Vortioxetine was dosed at 5 mg/kg bw at 24, 6 and 1 hours before the FST. The immobility time in seconds [s] is shown and is expressed as means ± SE (standard error). The experiment duration was 6 minutes. In the control group, a vehicle was applied. Surprisingly, vortioxetine had no positive effect in vitamin D deficient mice. Despite of vortioxetine treatment, immobility time was about as long as in the control group.

Figure 2 shows the effects of vitamin D supplementation in the forced swim test (FST) in male Balb/cJ mice that were treated with vortioxetine. Vortioxetine (5 mg/kg bw) alone or in combination with vitamin D at 0.05 (low) or 0.10 (high) mg/kg bw was given at 24, 6 and 1 hours prior to the FST. The immobility time was recorded and the data in seconds [s] is expressed as means ± SE (standard error). The experiment duration was 6 minutes. Significance (*) of p< 0.05 was tested using one-way RM ANOVA with Tukey’s post-hoc analysis. Figure 2 shows that in vitamin D deficient mice, vortioxetine shows positive effect only if vitamin D is coadministered.

Figures 3 and 4 relate to Example 2 (vide infra).

Figure 3 shows a lack of effects of acute vortioxetine treatment on cerebral blood flow (CBF) in male Balb/cJ mice that were vitamin D deficient. Vortioxetine was dosed at 5 mg/kg bw at 24, 6 and 1 hours before the CBF. In the control group, a vehicle was applied. The mean percentage above baseline ± SE (standard error) is shown. Trial time post dipyridamole injection lasted for 10 minutes. It is surprising that vortioxetine has no effect on CBF in vitamin D deficient mice.

Figure 4 shows effects of acute vortioxetine treatment in combination with different doses of vitamin D on cerebral blood flow (CBF) in male Balb/cJ mice that were vitamin D deficient. Vortioxetine (5 mg/kg bw) alone or in combination with vitamin D at 0.05 (low) or 0.10 (high) mg/kg bw was given at 24, 6 and 1 hours priorto the CBF measurement. The mean percentage above baseline ± SE (standard error) is shown. Trial time post dipyridamole injection lasted for 10 minutes. Significance (*) of p<0.05 was tested using one-way RM ANOVA with Tukey’s post- hoc analysis. Figure 4 shows that in vitamin D deficient mice, vortioxetine has an effect on CBF if vitamin D is co-administered.

Figure 5 relates to Example 3 (vide infra) and shows effects of acute vortioxetine treatment in the forced swim test (FST) in male Balb/cJ mice that were vitamin D deficient. Vortioxetine (5 mg/kg bw) alone or in combination with calcifediol at 0.025 mg/kg bw was given at 24, 6 and 1 hours prior to the FST. The immobility time was recorded and the data in seconds [s] is expressed as means ± SE (standard error). The experiment duration was 6 minutes. Significance (*) of p< 0.05 was tested using one-way RM ANOVA with Tukey’s post-hoc analysis. A comparison of Figure 5 with Figure 2 shows that a low dosage of the selected vitamin D metabolite (calcifediol) is as effective as a high dosage of vitamin D3.

Figure 6 relates to Example 3 (vide infra) and shows effects of acute vortioxetine treatment in combination with different doses of vitamin D on cerebral blood flow (CBF) in male Balb/cJ mice that were vitamin D deficient. Vortioxetine (5 mg/kg bw) alone or in combination with vitamin D at 0.05 or 0.10 mg/kg bw, or in combination with calcifediol at 0.025 or 0.050 mg/kg bw, was given at 24, 6 and 1 hours prior to the CBF measurement. The mean percentage above baseline ± SE (standard error) is shown. Trial time post dipyridamole injection lasted for 10 minutes. Significance (*) of p<0.05 was tested using one-way RM ANOVA with Tukey’s post-hoc analysis.

Detailed description of the invention

The inventors have unveiled a reason for poor response to vortioxetine treatment: lack of vitamin D. The surprising interaction of vitamin D and vortioxetine allows to improve various aspects of the current vortioxetine treatment.

Definitions

Direct compression is the shortest, most effective and least complex way to produce tablets. The manufacturer can compress a blend of the drug with excipients. No additional processing steps are required. Unfortunately, some powders are difficult to compress even if a readily compactable binder/adhesive is included in the blend. However, granules of the same powders are often more easily compressed.

Although inactive in a therapeutic sense, pharmaceutical excipients are critical and often essential components of a modern drug product. In many products, excipients make up the bulk of the total dosage form. In the context of the present invention, a “tableting excipient” is a pharmaceutical excipient that is useful when compressing a tablet. Exemplary tableting excipients are, amongst others, diluents, fillers, binders, disintegrants, lubricants, coloring agents and preservatives.

“Granules” are formed by granulation. Granulation is the process of forming granules and involves agglomeration of fine particles into larger granules. Wet granulation and dry granulation are two types of granulation technologies. The granules of the present invention are preferably formed by wet granulation. Wet granulation requires a processing solvent. In the context of the present invention, water is the preferred processing solvent. For agglomeration of fine particles into larger granules, a binder might be necessary. When doing wet granulation, the binder must be adapted to the chosen processing solvent. The granules of the present invention are preferably formed by wet granulation, wherein water is used as processing solvent and wherein a binder suitable for aqueous wet granulation is used. Typically, such binders are soluble in water. Preferred binders are PVP (polyvinylpyrrolidone, also commonly called polyvidone or povidone) and HPMC (hydroxypropyl methylcellulose; short: hydromellose).

When manufacturing a solid pharmaceutical dosage form, a particular compound may be blended with other ingredients prior to granulation and is thus incorporated within the obtained granules. This is referred to as “intra-granular addition”. Alternatively, a particular compound (or a mixture of compounds) may be mixed with already existing, prefabricated granules. This is referred to “extra-granular addition”. After extra-granular addition, the obtained blend can then be further processed (e.g. can be compressed into tablets or can be filled into sachets). Thus, an “extra-granular” compound is not part of a granule (i.e. is outside of a granule) whereas an “intra-granular” compound is part of a granule.

An “intra-granular composition” is a composition obtained by a granulation process. An example of an intra-granular composition are the granules described herein.

In the context of the present invention, the term “vortioxetine” refers to vortioxetine or a pharmaceutically acceptable salt thereof. Thus, the term “vortioxetine” is to be understood in a broad manner. Preferably, the term “vortioxetine” refers to a pharmaceutically acceptable salt of vortioxetine. Most preferably, the term “vortioxetine” refers to vortioxetine hydrobromide.

In the context of the present invention, the term “vitamin D” refers to any kind of vitamin D, including all vitamers of vitamin D and also including metabolites of all vitamers of vitamin D. A vitamin D metabolite is thereby a molecule that is either a precursor, an intermediate or the final, physiologically modified product of the vitamin D pathway in the human body. Thus, in the context of the present invention, the term “vitamin D” is to be understood in a very broad manner. Preferably, the term “vitamin D” refers to D3 (cholecalciferol), vitamin D2 (ergocalciferol), to a vitamin D metabolite or a mixture thereof. More preferably, the term “vitamin D” refers to vitamin D3 (cholecalciferol), vitamin D2 (ergocalciferol) or a mixture thereof. The most preferred vitamin D metabolite is calcifediol (25-hydroxyvitamin D3, also referred to as "25-OH D3"). Thus, in an also preferred embodiment, the term “vitamin D” refers to calcifediol. Alternative vitamin D metabolites are 1 -alpha, 25-vitamin D3 and 1 -alpha, 25-vitamin D2.

Patent claims relating to a “pharmaceutical combination” are product claims. A “pharmaceutical combination for simultaneous administration” is a combination that is suitable for simultaneous administration. By "simultaneous administration", it is meant that vitamin D and vortioxetine are administered on the same day. Said two active ingredients can be administered at the same time (for fixed combinations) or one at a time (for free combinations).

A “fixed combination” is a pharmaceutical combination that delivers both actives (i.e. vitamin D and vortioxetine) at the same time to a patient. A solid oral dosage form (e.g. a tablet) comprising both, vitamin D and vortioxetine, is an example of a fixed combination. A liquid oral dosage form (e.g. oral drops) comprising both, vitamin D and vortioxetine, is another example of a fixed combination. A “free combination” is a pharmaceutical combination that allows to administer both actives (i.e. vitamin D and vortioxetine) separately, i.e. one at a time. Treatment regimens in which vortioxetine and vitamin D are not administered by the same route of administration and/or are not administered at the same time require free combinations.

A “kit” is a set of articles or equipment needed for a specific purpose. The preferred kit of the invention comprises the herein described free combination and optionally means for measuring the vitamin D level of a patient. An alternative kit of the invention comprises the herein described fixed combination and means for measuring the vitamin D level of a patient.

A “pharmaceutical combination for sequential administration” is a combination that is suitable for sequential administration. By "sequential administration", it is meant that during a period of two or more days of continuous treatment, only one of vortioxetine and vitamin D is administered on any given day. By way of example, a patient's vitamin D deficiency may be treated first (i.e. before initiating the vortioxetine treatment). Vortioxetine treatment then starts afterwards. Sequential administration requires a free combination.

Fixed combinations are not suitable for sequential administration. In comparison, free combinations are more versatile: they are suitable for sequential administration and - if both actives are administered on the same day - also for simultaneous administration.

Fixed combinations are suitable for simultaneous administration if both actives (i.e. vortioxetine and vitamin D) are to be administered at the same time of the same day. However, if vortioxetine and vitamin D are to be administered on the same day but one at a time, fixed combinations are not suitable.

By "separate administration", it is meant that vitamin D and vortioxetine are administered one at a time. Thus, separate administration can referto both, sequential administration and - when referring to the administration of both actives on the same day but one at a time - also to simultaneous administration.

A “patient” is a human subject who is in need of medical advice and/or is suffering from a disease. In the context of the present invention, a “patient” is preferably a human subject that is in need of pharmaceutical drug. More preferably, it is a human subject that is in need of vortioxetine or a pharmaceutically acceptable salt thereof.

For human beings, vitamin D is essential. In that sense, everybody needs vitamin D. However, this is not what is meant by the expression “in need of vitamin D”. A patient who is “in need of vitamin D” is a patient whose vitamin D level is below the recommendation limit of a governmental or non-governmental medical organization. The recommendation limit of the Institute of Medicine (IOM) is 50 nmol calcifediol per liter blood. The recommendation limit the medical ‘Endocrine Society’ association is 75 nmol calcifediol per liter blood. Thus, a patient who is “in need of vitamin D” is preferably a patient whose calcifediol blood concentration is lower than 75 nmol/L or is lower than 50 nmol/L. A patient who is in need of vitamin D benefits from vitamin D supplementation (e.g. oral vitamin D supplementation). A patient whose vitamin D level is in a healthy range is not in need of vitamin D supplementation. A patient whose calcifediol blood concentration is 75 nmol/L or more is not in need of vitamin D supplementation.

A patient who is “vitamin D insufficient” has a calcifediol blood concentration preferably lower than 50 nmol/L. A patient who is “vitamin D deficient” has a calcifediol blood concentration preferably lower than 30 nmol/L. In case of a calcifediol blood concentration lower than 10 nmol/L, the patient is severely vitamin D deficient.

A “vortioxetine non-responder” is a patient who is in need of antidepressant treatment but shows no positive response to the vortioxetine treatment. Preferably, “no positive response to vortioxetine treatment” means that a daily dosage of 20 mg vortioxetine (as hydrobromide) does not relieve the patient's symptoms.

A “vortioxetine low-responder” is a patient who is in need of antidepressant treatment but shows poor response to the vortioxetine treatment. Preferably, “poor response to vortioxetine treatment” means that a daily dosage of 10 mg or 15 mg vortioxetine (as hydrobromide) does not relieve the patient's symptoms. Vortioxetine non-responders may also be considered as vortioxetine low-responders. However, not every vortioxetine low-responder is necessarily also a vortioxetine non-responder. A not sufficient vitamin D level is believed to be a reason for being a vortioxetine non-responder or a vortioxetine low-responder.

Vitamin D

The herein disclosed pharmaceutical combination comprises vitamin D. Several kinds (vitamers) of vitamin D exist. The two major vitamers are vitamin D3 and vitamin D2. Thus, a preferred pharmaceutical combination of the present invention comprises vitamin D3, vitamin D2 or a mixture thereof. In the context of the present invention, there is no reason for mixing vitamin D3 with vitamin D2. Thus, more preferably, the pharmaceutical combination of the present invention comprises either vitamin D3 or vitamin D2, but not a mixture thereof.

Calcifediol is a vitamin D metabolite, more specifically a vitamin D3 metabolite. In the human body, calcifediol is the most abundant metabolite of vitamin D. When determining a patient's vitamin D status (i.e. vitamin D level), it is common to directly or indirectly measure the concentration of calcifediol in a sample of the patient's blood. When orally administered, calcifediol has excellent bioavailability and hence, is very well absorbed by the body. This helps reaching optimal vitamin D levels faster. Thus, in an also preferred embodiment, the pharmaceutical combination of the present invention comprises at least one vitamin D metabolite, preferably at least one vitamin D3 metabolite and most preferably calcifediol.

Calcifediol for human consumption is commercially available as ampli-D® (DSM® Nutritional Products, Switzerland). Without wishing to be bound by theory, it is believed that calcifediol is improving vortioxetine treatment more effectively because a metabolic step can be skipped in the patient's body: whereas most kinds of vitamin D first need to be hydroxylated in the liver at position 25, this is not needed if calcifediol is administered. Although there is no need to do so, a mixture comprising vitamin D and a metabolite thereof could be used. Thus, an alternative embodiment of the present invention relates to a pharmaceutical combination comprising vitamin D3 and at least one vitamin D metabolite, or vitamin D2 and at least one vitamin D metabolite, or vitamin D3, vitamin D2 and at least one vitamin D metabolite. Thereby, calcifediol is the preferred vitamin D metabolite.

Vortioxetine

The herein disclosed pharmaceutical combination comprises vortioxetine. Several kinds of vortioxetine exist, including pharmaceutically acceptable salts of vortioxetine. Most if not all commercially available products comprise a pharmaceutically acceptable salt of vortioxetine. Commercially available, solid mono-preparations comprise vortioxetine hydrobromide.

The purpose of the present invention is to improve existing vortioxetine treatments. In consideration thereof, pharmaceutically acceptable salts of vortioxetine are preferred. Vortioxetine hydrobromide is the most preferred pharmaceutically acceptable salt of vortioxetine. However, in case of liquid oral dosage forms, vortioxetine (D.L)-lactate is the preferred pharmaceutically acceptable salt of vortioxetine.

Pharmaceutical combination comprising vortioxetine and vitamin D

The pharmaceutical combination of the present invention comprises vortioxetine and vitamin D. Regarding vortioxetine and vitamin D, the above-mentioned preferences apply. Thus, the herein disclosed pharmaceutical combination comprises preferably a pharmaceutically acceptable salt of vortioxetine, wherein said pharmaceutically acceptable salt of vortioxetine is more preferably vortioxetine hydrobromide (with the proviso that it is not a liquid oral dosage from).

A preferred pharmaceutical combination of the present invention comprises a pharmaceutically acceptable salt of vortioxetine and vitamin D3, wherein said pharmaceutically acceptable salt of vortioxetine is preferably vortioxetine hydrobromide. An also preferred pharmaceutical combination of the present invention comprises a pharmaceutically acceptable salt of vortioxetine and calcifediol, wherein said pharmaceutically acceptable salt of vortioxetine is preferably vortioxetine hydrobromide. An alternative pharmaceutical combination of the present invention comprises a pharmaceutically acceptable salt of vortioxetine and vitamin D2, wherein said pharmaceutically acceptable salt of vortioxetine is preferably vortioxetine hydrobromide. In a less preferred embodiment, the alternative pharmaceutical combination of the present invention comprises vortioxetine and a mixture of various kinds of vitamin D such as a mixture comprising vitamin D2 and D3, or a mixture comprising vitamin D2 and calcifediol, or a mixture comprising vitamin D3 and calcifediol. Also not preferred are pharmaceutical combinations comprising vortioxetine base instead of a pharmaceutically acceptable salt of vortioxetine. These preferences apply regardless of whether pharmaceutical combination is for simultaneous administration or for sequential administration. Most often, intramuscular (i.m.) or intravenous (i.v.) injections are more effective and efficient than orally consumed supplements (e.g. in pill form). Vitamin D i.m. or i.v. injections are therefore sometimes recommended if someone's vitamin D level is low (e.g. a calcifediol blood concentration of less than 30 nmol/L) or even very low (e.g. a calcifediol blood concentration of less than 20 nmol/L). Vortioxetine, on the other hand, is almost never injected. In situations like this, relating to different routes of administration, free combinations are preferred or even needed. A preferred kit of the invention comprises means for vitamin D injection (e.g. a prefilled syringe or a vial) and at least one oral dosage form, said at least one oral dosage form comprising a pharmaceutically acceptable salt of vortioxetine being preferably vortioxetine hydrobromide. Optionally, such kit may further comprise means facilitating the ex vivo measurement of calcifediol in a blood sample.

Free combinations comprising means for a vitamin D injection and at least one oral dosage form that comprises vortioxetine (or a pharmaceutically acceptable salt thereof) can be administered simultaneously or sequentially. If it is recommended to administer the injection and the oral dosage form on the same day, said free combination is for simultaneous administration. If it is recommended to administer the injection and the oral dosage form on different days, said free combination is for sequential administration. Such recommendations can be found e.g. in a package leaflet, in a Summary of Product Characteristics (SmPC) or in guidelines for practitioners/ pharmacists. It is also possible that said free combination is either for simultaneous or for sequential administration (i.e. the practitioner and/or pharmacist can deicide).

Whereas the above example (relating to vitamin D injections) is suitable to illustrate the concept of a free combination, the free combination of the present invention is not limited to this example. A more general embodiment of the present invention relates to a free combination for simultaneous administration or preferably for sequential administration. A preferred embodiment of the present invention relates to a free combination comprising vitamin D3 and vortioxetine hydrobromide and to kit comprising such free combination. An also preferred embodiment of the present invention relates to a free combination comprising calcifediol and vortioxetine hydrobromide and to kit comprising such free combination. A preferred kit comprises at least one oral dosage form, wherein said at least one oral dosage form is a solid or liquid oral dosage form that comprises vortioxetine hydrobromide. An alternative embodiment of the present invention relates to a free combination comprising vitamin D3 and vortioxetine hydrobromide and to kit comprising such free combination.

Oral dosage forms comprising vitamin D may be liquid or solid. Oral dosage forms comprising vortioxetine may also be liquid or solid. Whereas it is possible to combine two liquid oral dosage forms into one combined liquid oral dosage form, it is difficult or even impossible to combine a liquid oral dosage form with a solid oral dosage form. In this situation, a free combination is preferred. One embodiment of the present invention relates to the herein described pharmaceutical combination, wherein said pharmaceutical combination is a free combination that comprises at least one solid oral dosage form and at least one liquid oral dosage form. A preferred embodiment of the present invention relates to a pharmaceutical combination comprising at least one solid oral dosage form and at least one liquid oral dosage form, wherein said at least one solid oral dosage form comprises vitamin D3 and wherein said at least one liquid oral dosage form comprises a pharmaceutically acceptable salt of vortioxetine (or vice versa). An also preferred embodiment of the present invention relates to a pharmaceutical combination comprising at least one solid oral dosage form and at least one liquid oral dosage form, wherein said at least one solid oral dosage form comprises calcifediol and wherein said at least one liquid oral dosage form comprises a pharmaceutically acceptable salt of vortioxetine (or vice versa). An alternative embodiment of the present invention relates to a pharmaceutical combination comprising at least one solid oral dosage form and at least one liquid oral dosage form, wherein said at least one solid oral dosage form comprises vitamin D2 and wherein said at least one liquid oral dosage form comprises a pharmaceutically acceptable salt of vortioxetine (or vice versa). Such pharmaceutical combinations are free combinations for simultaneous and/or sequential administration.

Brintellix® solution (oral drops, 20 mg/ml) is a commercially available liquid oral dosage form of vortioxetine. Each drop contains vortioxetine (D.L)-lactate equivalent to 1 mg vortioxetine. Thus, in case of a liquid oral dosage form of vortioxetine, vortioxetine (D.L)-lactate is the preferred pharmaceutically acceptable salt of vortioxetine.

The present invention also relates to kits comprising such free combinations for simultaneous and/or sequential administration. Optionally, such kits may further comprise means facilitating the ex vivo measurement of calcifediol in a blood sample.

In terms of administration, free combinations are more versatile than fixed combinations. However, free combinations also have some downsides such as higher manufacturing cost, increased complexity of supply chain and/or reduced patient compliance. Therefore, there are situations where fixed combinations are the better option. For simultaneous administration, fixed combinations often increase patient compliance.

One embodiment of the present invention relates to the herein described pharmaceutical combination, wherein said pharmaceutical combination is for simultaneous administration, and wherein said pharmaceutical combination is preferably a fixed combination, and wherein said pharmaceutical combination is more preferably a solid or liquid oral dosage form, and wherein said pharmaceutical combination is most preferably a solid oral dosage form. A preferred embodiment of the present invention relates to a solid or liquid oral dosage form comprising vitamin D3, a pharmaceutically acceptable salt of vortioxetine and at least one pharmaceutically acceptable excipient, wherein said at least one pharmaceutically acceptable excipient is preferably a pharmaceutically acceptable carrier. An also preferred embodiment of the present invention relates to a solid or liquid oral dosage form comprising calcifediol, a pharmaceutically acceptable salt of vortioxetine and at least one pharmaceutically acceptable excipient, wherein said at least one pharmaceutically acceptable excipient is preferably a pharmaceutically acceptable carrier. An alternative embodiment of the present invention relates to a solid or liquid oral dosage form comprising vitamin D2, a pharmaceutically acceptable salt of vortioxetine and at least one pharmaceutically acceptable excipient, wherein said at least one pharmaceutically acceptable excipient is preferably a pharmaceutically acceptable carrier. A preferred kit of the invention comprises such solid or liquid oral dosage form and means facilitating the ex vivo measurement of calcifediol in a blood sample. Some examples of materials which can serve as pharmaceutically acceptable excipient and/or carrier include: liquid carriers (such as water, ethanol, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol and phosphate buffer solutions), sugars (such as lactose, glucose and sucrose), starches (such as corn starch and potato starch), cellulose and its derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate), powdered tragacanth, malt, gelatin, talc, fat and oils (such as cocoa butter, waxes, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil), glycols (such as propylene glycol), polyols (such as glycerin, sorbitol, mannitol and polyethylene glycol), esters (such as ethyl oleate and ethyl laurate), agar, buffering agents (such as magnesium hydroxide and aluminum hydroxide), alginic acid and other non-toxic materials employed in pharmaceutical formulations.

Regardless of whether it is a free or fixed combination, there is usually a reason why two active ingredients are combined. In some cases, the dosage of one active ingredient depends on the dosage of the other active ingredient. In the context of the present invention, this is typically not the case: the amount of vitamin D that needs to be administered to a given patient does usually not depend on the amount of vortioxetine that is administered to the patient. The required vitamin D dosage rather depends on the vitamin D level of the patient and on the chosen vitamin D vitamer. If the vitamin D status (i.e. vitamin D level) of a patient is very low, often a relatively high amount of vitamin D needs to be administered. Similarly, if the vitamin D level of a patient is almost sufficient, often a relatively small amount of vitamin D will suffice. In addition, the inventors have found that calcifediol is more effective than other vitamin D vitamers such that a relatively low amount of calcifediol may be sufficient to bring the vitamin D level of the patient back to normal.

The herein described pharmaceutical combination is preferably a fixed combination that comprises preferably from 10 pg to 2000 pg cholecalciferol or from 5 pg to 100 pg calcifediol, and that comprises more preferably from 10 pg to 1000 pg cholecalciferol or from 5 pg to 80 pg calcifediol, and that comprises most preferably from 15 pg to 100 pg cholecalciferol or from 5 pg to 35 pg calcifediol. A further embodiment of the invention relates to a solid oral dosage form, wherein said solid oral dosage form comprises (in addition to vortioxetine hydrobromide) preferably from 10 pg to 2000 pg cholecalciferol or from 5 pg to 100 pg calcifediol, more preferably from 10 pg to 1000 pg cholecalciferol or from 5 pg to 80 pg calcifediol, and most preferably from 15 pg to 100 pg cholecalciferol or from 5 pg to 35 pg calcifediol. Another embodiment of the invention relates to a kit comprising a free combination as herein described, wherein said kit comprises at least one solid oral dosage form, and wherein said at least one solid oral dosage form comprises preferably from 10 pg to 2000 pg cholecalciferol or from 5 pg to 100 pg calcifediol, and that comprises more preferably from 10 pg to 1000 pg cholecalciferol or from 5 pg to 80 pg calcifediol, and that comprises most preferably from 15 pg to 100 pg cholecalciferol or from 5 pg to 35 pg calcifediol.

The purpose of the present invention is to improve existing vortioxetine treatments. Commercially available solid mono-preparations comprise 5 mg vortioxetine hydrobromide or 10 mg vortioxetine hydrobromide or 15 mg vortioxetine hydrobromide or 20 mg vortioxetine hydrobromide. Therefore, the herein described solid fixed combination preferably also comprises 5 mg vortioxetine hydrobromide or 10 mg vortioxetine hydrobromide or 15 mg vortioxetine hydrobromide or 20 mg vortioxetine hydrobromide.

A preferred embodiment of the invention relates to the herein described pharmaceutical combination, wherein said pharmaceutical combination is a solid oral dosage form comprising: 5 mg vortioxetine hydrobromide or 10 mg vortioxetine hydrobromide or 15 mg vortioxetine hydrobromide or 20 mg vortioxetine hydrobromide; and from 10 pg to 2000 pg cholecalciferol or from 5 pg to 100 pg calcifediol; and at least one pharmaceutically acceptable excipient.

Such solid oral dosage form may be part of a kit and is not suitable for separate administration.

An alternative embodiment of the invention relates to the herein described pharmaceutical combination, wherein said pharmaceutical combination is a free combination comprising at least one solid oral dosage form, said solid oral dosage form comprising 5 mg vortioxetine hydrobromide or 10 mg vortioxetine hydrobromide or 15 mg vortioxetine hydrobromide; or said solid oral dosage form comprising 20 mg vortioxetine hydrobromide, or from 10 pg to 2000 pg cholecalciferol or from 5 pg to 100 pg calcifediol, and said solid oral dosage form further comprising at least one pharmaceutically acceptable excipient.

Solid oral dosage forms of this alternative embodiment comprise vitamin D or vortioxetine, but not both. They are therefore suitable for sequential administration. In both embodiments, the above-mentioned materials can serve as the at least one pharmaceutically acceptable excipient.

Novel use of a vitamin D measurement device

In some cases, there is no need to improve the current vortioxetine treatment. In other cases, however, a patient turns out to be a vortioxetine low-responder or (even worse) a vortioxetine non-responder. A reason for a sub-optimal response maybe a lack of vitamin D. If this is the case, vortioxetine treatment can be relatively easy improved by raising the patient's vitamin D level. It is therefore meaningful to measure the vitamin D status of patients that are in need of vortioxetine. In one embodiment of the invention, the vitamin D level of a vortioxetine low-responder or of a vortioxetine non-responder is measured. If the patient is in need of vitamin D supplementation, the patient's current vortioxetine medication is preferably switched to the herein described pharmaceutical combination. In another embodiment of the present invention, the vitamin D level of a patient is measured before initiating a vortioxetine treatment. If the patient is in need of vitamin D supplementation, the herein described free combination may be used (e.g. to raise the patient's vitamin D level before initiating the vortioxetine treatment). In either case, it is meaningful to measure the vitamin D level of a patient that is in need of vortioxetine.

Measurement devices suitable for measuring the vitamin D levels are well known and commercially available. In the context of the present invention, such measurement devices are used for assessing the benefit of the herein described pharmaceutical combination for a predetermined patient. Such approach is particularly meaningful if vortioxetine treatment of said predetermined patient has not yet been initiated or if said predetermined patient is a vortioxetine low-responder or a vortioxetine non-responder.

To determine the vitamin D level of a patient, it is preferred to measure (directly or indirectly) the patient's calcifediol blood concentration. Preferably, said measurement is done ex vivo in a blood sample of the patient. One embodiment of the present invention relates to the use of a measurement device for assessing the benefit of the herein described pharmaceutical combination, wherein the benefit for a predetermined patient is assessed and wherein said measurement device is suitable for measuring ex vivo the concentration of calcifediol in a blood sample of said predetermined patient, and wherein said predetermined patient is preferably a vortioxetine low-responder or a vortioxetine non-responder. A preferred embodiment of the present invention relates to the use of a measurement device for assessing the benefit of the herein described pharmaceutical combination, wherein the benefit for a predetermined patient is assessed and wherein said measurement device is suitable for measuring ex vivo the concentration of calcifediol in a blood sample of said predetermined patient, and wherein vortioxetine treatment of said predetermined patient preferably has not yet been initiated. Also disclosed is a method of identifying patients that are likely to benefit from the pharmaceutical combination of the invention, characterized in that a device is used which is suitable for measuring ex vivo the concentration of calcifediol in a blood sample.

Some vitamin D measurement devices are relatively small and inexpensive. They may therefore be included in the kit of the invention. One embodiment of the present invention relates to a kit comprising means facilitating the ex vivo measurement of calcifediol in a blood sample, wherein said means is preferably a device for measuring ex vivo the concentration of calcifediol in a blood sample. Some parts of said device may be reusable, whereas other parts may be expendable material that is consumed when running the device. Parts that are reusable do not need to be included in each and every kit. Therefore, a preferred kit of the invention comprises means facilitating the ex vivo measurement of calcifediol in a blood sample, wherein said means are preferably expendable materials that are consumed when running a device for measuring ex vivo the concentration of calcifediol in a blood sample.

Medical use

The present invention also relates to a pharmaceutical combination for use as a medicament, wherein said pharmaceutical combination comprises i) vitamin D, and ii) vortioxetine or a pharmaceutically acceptable salt thereof, and wherein i) vitamin D and ii) vortioxetine or a pharmaceutically acceptable salt thereof are administered simultaneously or sequentially.

Thereby, the herein described preferences apply. Accordingly, vortioxetine is preferably vortioxetine hydrobromide, whereas vitamin D is preferably vitamin D3, vitamin D2, at least one vitamin D metabolite or a mixture thereof.

The inventors have found that the presence of vitamin D is necessary for vortioxetine to exert its effects. This surprising finding suggests that many vortioxetine non-responders and low- responders have a suboptimal level of vitamin D in their blood (i.e. are in need of vitamin D supplementation). Therefore, one embodiment of the present invention relates to the herein described pharmaceutical combination for use in the treatment of a vortioxetine non-responder and/or in the treatment of a vortioxetine low-responder.

In case of a vortioxetine low-responder, the positive effects of vortioxetine may be enhanced by replacing a low-responder's daily oral dosage of e.g. 10 mg vortioxetine hydrobromide with a daily oral dosage of 10 mg vortioxetine hydrobromide plus a suitable amount of vitamin D. One embodiment of the invention relates to the herein described pharmaceutical combination for use in the treatment of a patient that is suffering from depression, anxiety and/or chronic pain, wherein the patient's medical symptoms remain at least partially despite that said patient is being treated with vortioxetine medication, characterized in that the patient's vortioxetine medication

• comprises not more than 0.001 pg vitamin D and not more than 0.001 pg of a vitamin D metabolite, and

• is preferably free of vitamin D and metabolites thereof.

Alternatively, a low-responder's daily oral dosage of e.g. 10 mg vortioxetine hydrobromide may be replaced by a daily dosage of 5 mg vortioxetine hydrobromide plus a suitable amount of vitamin D. Replacement of a high dosage with a lower dosage will reduce many, most or even all dose-dependent side-effects of vortioxetine. One embodiment of the invention relates to the herein described pharmaceutical combination for use in the treatment of a patient who’s previous vortioxetine treatment has been stopped due to vortioxetine induced side effects, wherein the patient's previous vortioxetine medication

• was free of vitamin D and metabolites thereof, or • comprised not more than 0.001 pg vitamin D and not more than 0.001 pg of a vitamin D metabolite.

In another embodiment of the invention, the patient to be treated is a vortioxetine non-responder. In this embodiment, a patient who has so far not benefited from the positive effects of vortioxetine is now treated by use of the pharmaceutical combination of the present invention.

The pharmaceutical combination of the present invention is also useful if vortioxetine treatment has not yet been initiated (i.e. before a patient's response to the vortioxetine treatment is known). One embodiment of the present invention relates to the herein disclosed pharmaceutical combination for use in the treatment of a patient who is in need of vortioxetine or a pharmaceutically acceptable salt thereof. Such treatment is of particular benefit if the patient to be treated is also in need of vitamin D. A preferred embodiment of the present invention relates to the use of the herein disclosed pharmaceutical combination for use in the treatment of a patient who is in need of vortioxetine or a pharmaceutically acceptable salt thereof, wherein said patient is vitamin D deficient or insufficient, and/or wherein said patient has a calcifediol blood concentration that is lower than 75 nmol/L, preferably lower than 50 nmol/L, more preferably lower than 30 nmol/L, yet more preferably lower than 25 nmol/L, even more preferably lower than 20 nmol/L and most preferably lower than 12 nmol/L. These calcifediol blood concentrations are preferably concentrations that have been measured before initiating oral administration of vitamin D, or that have been measured less than 3 days or preferably less than 2 days after initiating oral administration of vitamin D. Thus, the present invention also relates to a pharmaceutical combination for use in the treatment of a patient is suffering from depression, anxiety and/or chronic pain, wherein said pharmaceutical combination comprises i) vitamin D, and ii) vortioxetine or a pharmaceutically acceptable salt thereof, and wherein i) vitamin D and ii) vortioxetine or a pharmaceutically acceptable salt thereof are administered simultaneously or sequentially, and wherein said patient is vitamin D deficient and/or has a calcifediol blood concentration that is lower than 75 nmol/L, preferably lower than 50 nmol/L, more preferably lower than 30 nmol/L, yet more preferably lower than 25 nmol/L, even more preferably lower than 20 nmol/L and most preferably lower than 12 nmol/L.

Thereby, the pharmaceutical combination is preferably a fixed combination, is more preferably a solid or liquid oral dosage form, and is most preferably a solid oral dosage form. Thus, a particularly preferred embodiment of the invention relates to the herein described a solid or liquid oral dosage form for use in the treatment of a patient who

(a) is suffering from depression, anxiety and/or chronic pain; and

(b) has a calcifediol blood concentration that is lower than 75 nmol/L, preferably lower than 50 nmol/L, more preferably lower than 30 nmol/L, yet more preferably lower than 25 nmol/L, even more preferably lower than 20 nmol/L and most preferably lower than 12 nmol/L, and wherein said solid or liquid oral dosage form is preferably a solid oral dosage form, and wherein said solid oral dosage form preferably comprises:

5 mg vortioxetine hydrobromide or 10 mg vortioxetine hydrobromide or 15 mg vortioxetine hydrobromide or 20 mg vortioxetine hydrobromide, from 10 pg to 2000 pg cholecalciferol or from 5 pg to 100 pg calcifediol, and at least one pharmaceutically acceptable excipient, and wherein said at least one pharmaceutically acceptable excipient is preferably a starch, a cellulose, sodium carboxymethyl cellulose, ethyl cellulose cellulose acetate, powdered tragacanth, malt, a gelatin, talc, a fat, an oil, a glycol, a polyol, an ester, agar, a buffering agent and/or alginic acid.

An also preferred embodiment of the invention relates to a liquid oral dosage form for use in the treatment of a patient who

(a) is suffering from depression, anxiety and/or chronic pain and

(b) has a calcifediol blood concentration that is lower than 75 nmol/L, preferably lower than 50 nmol/L, more preferably lower than 30 nmol/L, yet more preferably lower than 25 nmol/L, even more preferably lower than 20 nmol/L and most preferably lower than 12 nmol/L, and wherein said liquid oral dosage form comprises vortioxetine (D,L)-lactate, vitamin D and at least one pharmaceutically acceptable excipient, wherein said at least one pharmaceutically acceptable excipient is preferably a pharmaceutically acceptable liquid carrier, and wherein said vitamin D is preferably vitamin D3, vitamin D2, at least one vitamin D metabolite or a mixture thereof, and is more preferably vitamin D3 or calcifediol.

In case of sequential administration, the pharmaceutical combination is preferably a kit comprising the herein described free combination. Thus, one embodiment of the present invention relates to a free combination for use as a medicament or for use in the treatment of a patient who is suffering from depression, anxiety and/or chronic pain, and wherein said free combination comprises i) vitamin D, and ii) vortioxetine or a pharmaceutically acceptable salt thereof, and wherein said patient is vitamin D deficient and/or has a calcifediol blood concentration that lower than 75 nmol/L, preferably lower than 50 nmol/L, more preferably lower than 30 nmol/L, yet more preferably lower than 25 nmol/L even more preferably lower than 20 nmol/L and most preferably lower than 12 nmol/L.

Cerebral blood flow (CBF) is the blood supply to the brain in a given period of time. In a human adult, CBF is typically 750 millilitres per minute or 15% of the cardiac output. Cerebral blood flow is determined by a number of factors, such as viscosity of blood, how dilated blood vessels are, and the net pressure of the flow of blood into the brain, known as cerebral perfusion pressure, which is determined by the body's blood pressure. When treating a patient with the pharmaceutical combination of the present invention, the patient's cerebral blood flow (CBF) is increased. This suggests that abnormal cerebral blood flow is associated with a patient's lower or poor responsiveness to a vortioxetine treatment. Therefore, one embodiment of the present invention relates to the pharmaceutical combination of the present invention for use in the treatment of a patient suffering from major depressive disorder (MDD), wherein said patient has a suboptimal cerebral blood flow.

A suboptimal cerebral blood flow is associated with several cerebrovascular diseases such as cerebrovascular atherosclerosis. In fact, depression and cerebrovascular atherosclerosis often occur in comorbidity showing neuropsychological impairment and poor response to antidepressant treatment. Therefore, one embodiment of the present invention relates to the pharmaceutical combination of the present invention for use in the treatment of depression that is associated with a cerebrovascular disease. A further embodiment of the present invention relates to the herein described pharmaceutical combination for use in the treatment of a patient who is suffering from depression and from at least one cerebrovascular disease, and is preferably for use in the treatment of a patient who is suffering from depression that is associated with cerebrovascular atherosclerosis.

Examples

In below Examples 1 to 4, the following materials and methods were used:

Test Article Identification and Preparation. Pre-formulated diets lacking vitamin D (Modified AIN-93G Rodent Diet with no added vitamin D3: Research Diets, New Brunswick, NJ; catalogue No. D10073001 , lot No. 20090401) was shipped ambient and stored at -20°C prior to use. ROVIMIX® Hy-D® 1.25% (Hy-D® is calcifediol, DSM Nutritional Products, Lot No. WB01990008) and dry Vitamin D3 100 CWS/AM (DSM Nutritional Products, lot No. UT19010007) were prepared for PO dosing at 10 mL/kg in vehicle (1 % fish gelatin in PBS). ROVIMIX® Hy-D® 1.25% is a formulation comprising 1.25 weight-% 25-Hydroxy-vitamin D3, based on the total weight of the formulation. Vortioxetine (Advanced Chemblock Inc., Cat. P50895, CAS 960203-27-4, Batch 19394) was prepared in for PO (per os) dosing at 10 mL/kg in vehicle. The purchased vortioxetine is vortioxetine hydrobromide. Thus, in the context of the examples, “vortioxetine” refers to vortioxetine hydrobromide. The vehicle for mock PO dosing of the test articles was sterile saline. Dipyridamole was used for induction of vasodilation during the assays of cerebral blood flow. Dipyridamole (Millipore-Sigma, catalog No. D9766; Lot No. BCCB0805) was prepared fresh in a 20% b-cyclodextrin solution in sterile water (VetOne, lot No. A1806022) on study days 64 through 67 for intraperitoneal (IP) injection at 1 mL/kg.

Test System Identification. Male Balb/cJ mice (N = 96; stock number 000651) were obtained from The Jackson Laboratory (Bar Harbor, ME). The mice were 4 weeks old and weighed approximately 17 to 22 grams (mean of 20.2 g) at enrollment on study day 0. The animals were identified by marks at the base of tail delineating animal number. After randomization, all cages were labeled with protocol number, group number, and animal numbers. Environment and Husbandry. Upon arrival, the animals were housed 3 per cage in polycarbonate cages with wire tops, wood chip bedding, and suspended food and water bottles. The mice were housed either in shoebox cages (static airflow, approximately 0.045 m2 floor space) with filter tops or in individually ventilated pie cages (passive airflow, approximately 0.045-0.048 m2 floor space). An attending veterinarian was on site or on call during the live phase of the study. No concurrent medications were given.

The animals were acclimated for 7 days prior to being placed in the study. During the acclimation and study periods, the animals were housed in a laboratory environment with temperatures ranging 19°C to 25°C and relative humidity of 30% to 70%. Automatic timers provided 12 hours of light and 12 hours of dark. Animals were allowed access ad libitum to Envigo Teklad 8640 diet or vitamin D-deficient diet and fresh municipal tap water.

Animal care including room, cage, and equipment sanitation conformed to the guidelines cited in the Guide for the Care and Use of Laboratory Animals and the applicable SOPs.

Experimental Design. The animals were given immediate ad libitum access to water and standard rodent chow and acclimated to the facility for 7 days prior to any further intervention. After 7 days of acclimatization, the animals were randomized by weight, placed on chow deficient in vitamin D, and subjected to mock oral gavage on a twice-weekly basis for 6 weeks. For behavioral testing the study was divided into 4 cohorts of 24 animals each (3 mice/group per cohort) with dosing and testing of each cohort proceeding on successive days. The mice were dosed by oral gavage 24, 6, and 1 hour prior to behavioral testing.

Example 1. Vortioxetine alone is not effective in vitamin D deficient animals. A combination of vitamin D and vortioxetine is needed for anti-depressive effects in the forced swim test.

The forced swim test (FST) is a test centered on a rodent’s response to unescapable stress. Results of this test have been interpreted as a measure of susceptibility to negative mood. It is used to measure the effectiveness of antidepressants. Forced swim tests were performed in groups of 12 mice, per treatment. We provided the substances to the animals per gavage at 24, 6 and 1 hours before the FST. The animals were acclimated to the testing room for 1 hour prior to testing. Four animals were tested simultaneously. The animals were each placed in a plexiglass cylindrical container (45 cm x 20 cm; Stoelting Co., Wood Dale, IL) filled with fresh water (25- 27°C) to 30 cm above the bottom. The animals remained in the containers for 6 minutes and were monitored by a computer program with tracking software (Ethovision, Noldus). The mice were observed constantly to mitigate the outside chance that an animal drowned or began to drown (i.e., spent more than 5 seconds completely submerged). After 6 minutes, the animals were removed, towel-dried, and returned to their holding cages. The chambers were drained and cleaned with 70% ethanol then refilled between tests. The animals were returned to their home cage and room after all of the animals in the cohort were tested. All videos were checked and threshold-ed post-hoc by an experimenter blind to the group assignments. Total time swimming or immobile was then acquired via computer-driven video analysis. Swimming was defined as movement of the forelimbs and hind limbs that did not break the surface of the water. Immobility was defined as absence of any movement except for slight movements necessary for the animal to keep its head above water. Data was statistically analyzed using the GraphPad Prism software (San Diego, CA, USA).

Surprisingly, it was observed that in the forced swim test (FST) vitamin D deficient mice treated with vortioxetine did not differ from the vehicle control group as shown in Figure 1 . This result of Example 1 differs from those of Guilloux et al. (2013) in which vortioxetine (5 mg/kg) increased the time in mobility in the FST.

In contrast to the animals of Example 1 , the animals of Guilloux et al. (2013) were not vitamin D deficient. This suggests that the presence of vitamin D might be necessary for vortioxetine to exert the effects. We tested this hypothesis by combining vortioxetine with vitamin D at two different doses. Interestingly, in the acute FST depression model we could show that the combination of vortioxetine together with a sufficient dose of vitamin D improved the depression scores. In Figure 2, mice having sufficient vitamin D in the vortioxetine combination (vortioxetine_D2_high) showed reduced immobility scores.

Example 1 shows the benefit of administering vortioxetine together with vitamin D when treating vitamin D deficient subjects.

Example 2. In vitamin D deficient animals, vortioxetine showed an improvement in cerebral blood flow only in combination with vitamin D.

For the cerebral blood flow (CBF) measurement, the animals were anesthetized one hour after the last test article administration using 2% isoflurane (VetOne, catalogue No. 502017), and each animal's skull was exposed by a midline scalp incision. The scalp was removed to visualize the brain without disturbing the overlying skull. The animals were then injected (IP) with dipyridamole (DP; 10 mg/kg) and placed in the prone position under the laser Doppler device (MoorLDI2; Moor Instruments, Wilmington, DE). Body temperature was maintained at approximately 30°C with a heating pad, and anesthesia and immobility were maintained with 1 .5% isoflurane. The surface of the brain was diffusely illuminated by a 785-nm laser light. The scattered light was filtered and detected by a digital camera positioned above the animal's head. Raw speckle images were used to compute the speckle contrast, which is a measure related to the number and velocity of moving particles (i.e., CBF). Thirteen, color-coded, blood-flow images were compiled for each animal at 45-second intervals, resulting in CBF monitoring for 10 minutes post-DP injection. The first 5 scans were averaged as baseline CBF, and all subsequent scans were normalized to the baseline as the CBF response to DP.

Interestingly, vortioxetine did not show any significant change in CBF when compared to the control group in vitamin D deficient animals (Figure 3). It can be assumed that vitamin D deficiency is the explanation why vortioxetine did not exert its effects in the forced swim test (cf. Example 1). Therefore, we were interested to see additional hint in the brain when adding vitamin D to the vortioxetine treatment. And surprisingly, the combination of vortioxetine and vitamin D was effective in the CBF experiment. Interestingly enough, using the CBF methodology, the combined effect of vortioxetine can already be seen at the lower dose of vitamin D (0.05 mg/kg bw) (Figure 4). The observation that cerebral blood flow is improved in the brain of vitamin D deficient subjects by administering vortioxetine in combination with vitamin D is consistent with the results shown in Figure 2.

Example 3. The combination of vortioxetine and calcifediol, a metabolite of vitamin D3, relieved the depressive mood in mice in the forced swim test (FST).

Vortioxetine alone was not effective in ameliorating depressive mood in vitamin D deficient Balb/cJ mice in the forced swim test (cf. Example 1). After having observed the vitamin D effect in vortioxetine treatment (cf. Example 2), we were interested whether other vitamin D metabolites could have a similar action. Therefore, calcifediol (25-hydroxy vitamin D3, 25D3) co-administration was tested in the FST. Intriguingly, a low dose of calcifediol (0.025 mg/kg bw) had the same effect like the high dose of vitamin D3 (0.10 mg/kg bw), when together with vortioxetine to vitamin D deficient mice. Figure 5 shows that the combination of calcifediol with vortioxetine effectively improves the anti-depressive behavior of vitamin D deficient mice in the FST as seen by reducing the immobility per minute.

Example 4. Both combinations, ‘vortioxetine and vitamin D’ and ‘vortioxetine and calcifediol’, improved cerebral blood flow in a dose-dependent manner.

Similar to Example 2, calcifediol was tested in two different concentrations (0.025 mg/kg bw; 0.050 mg/kg bw) in combination with vortioxetine in the CBF experiment vitamin D deficient mice. A dose dependent effect was observed. A two times higher oral dose of vitamin D resulted in less improved cerebral blood flow than calcifediol (Figure 6).

The result of Example 4 shows a dose-effect relationship for both, vitamin D and calcifediol.

Example 5a. Mono-therapy; comparison with comparator therapy.

In Example 5a, vortioxetine is compared with another therapy (“comparator therapy”) to assess its added benefit. Both drugs are approved for patient treatment by the drug authorities. They have a positive benefit-risk ratio, i.e. both are effective and safe.

To assess the added benefit, 100 patients are split into two groups. A first group is treated with a vortioxetine, whereas a second group is treated with a comparator therapy. Patient-relevant endpoints (“positive effects") from which the additional benefit is to be derived are defined. An additional benefit is attested if, for example, a significant improvement of disease symptoms or an avoidance of side effects can be shown compared to the comparative therapy.

Example 5a shows that vortioxetine has little added benefit. Example 5b. Mono-therapy; subjects with low vitamin D level are excluded.

In Example 5b, the assessment of Example 5a is repeated. However, to participate in the assessment, patients must have a healthy vitamin D level (i.e. low vitamin D level is an exclusion criterion).

100 patients that have a calcifediol blood concentration of more than 50 nmol/L are split into two groups. A first group is treated with vortioxetine, whereas a second group is treated with a comparator therapy. The same patient-relevant endpoints (“positive effects") as in Example 5a are applied. The duration of the assessment is as long as in Example 5a, but at least 6 months.

In Example 5b, vortioxetine has a significant added benefit.

A comparison of Examples 5a and 5b shows the relevance of a healthy vitamin D level when treating a patient with vortioxetine: to unfold the full potential of vortioxetine, the patient to be treated should have a healthy vitamin D level.

Example 5c. Combination therapy; low vitamin D subjects receive vitamin D supplementation.

In Example 5c, the assessment of Example 5a is repeated. However, to participate in the assessment, patients must have a low vitamin D level (i.e. normal vitamin D level is an exclusion criterion).

100 patients that have a calcifediol blood concentration of less than 30 nmol/L are split into two groups. A first group is treated with a fixed combination (namely with a solid oral dosage form comprising vortioxetine and calcifediol), whereas a second group is treated with a comparator therapy. The same patient-relevant endpoints (“positive effects") as in Example 5a are applied. The duration of the assessment is as long as in Example 5a, but at least 6 months.

In Example 5c, the combination therapy (vortioxetine + calcifediol) has a significant added benefit.

Examples 5c shows that a low vitamin D level of a patient does not need to be a show-stopper. Oral supplementation of vitamin D (via fixed combination) brings the patient's vitamin D level back to normal and thus, makes vortioxetine treatment successful. Example 6a. In the presence of vortioxetine, adding Vitamin D3 increases disintegration time of the tablet.

In Example 6a, vitamin D3 has been added intra-granularly.

The materials were first blended and then granulated in a high shear granulation device (Diosna P 1/6 mixer). An aqueous suspension of Klucel (6 w/w %) and water were sprayed on the blend. The speed of impeller: 900rpm; the speed of chopper: l OOOrpm. The suspension was manually added at a constant speed through the syringe for binding with the powder blend. Then, the water was added for wetting. The granulates were dried overnight at around 22-25°C, RH 24- 27%.

The resulting granules were sieved manually with the fraction range of 600 pm - 150 pm. LOD of granules determined with HR73 Halogen Moisture Analyzer (Mettler). Moisture content in the trial was between 0.6 - 1 %. After blending with the rest of the excipients for 7minutes at 23 rotations/minute in a Turbula mixer, the mixture was used for tableting. A Korsch XP-1 single punch press equipped with a 7mm round curved punch was used for tableting. The blend was compressed into tablets at 8kN compression force.

Disintegration time of the obtained tablets was measured using a disintegration apparatus (Sotax DT50, Sotax AG, Switzerland). According to the European Pharmacopeia guidelines, the disintegration time of 6 tablets in one batch was measured simultaneously in distilled water (800 mL) at 37 °C. The mean value of the 6 tablets was considered the disintegration time of one sample (European Directorate for the Quality of Medicines. "Healthcare. Council of Europe Disintegration of tablets and capsules (monograph 2.9. 1)." European Pharmacopeia; Council of Europe: Strasbourg, France (2019): 323-325).

Disintegration time can be adjusted by adding a disintegrant such as sodium starch glycolate (Desai et al., “Review of Disintegrants and the disintegration Phenomena”, Journal of Pharmaceutical Sciences 105 (2016) 2545-2555).

In the following examples, the same type and the same amount of sodium starch glycolate was used in all trials.

Table 1 : Comparison of disintegration times for a tablet comprising vortioxetine alone (VXT), or vortioxetine in combination with vitamin D3 (VXT + Vit.D3)

Example 6a shows that disintegration time increases massively (from 30 seconds to more than 7 minutes) when adding vitamin D3 intra-granularly.

Example 6b. In the absence of vortioxetine, the effect of vitamin D3 on the tablet's disintegration time is less severe.

In Example 6b, vitamin D3 has also been added intra-granularly. However, to better understand the issue unveiled in Example 6a, further tablets with vitamin D3 but without vortioxetine were produced.

Table 2: Comparison of disintegration times for a tablet comprising vortioxetine alone (VXT), vortioxetine in combination with vitamin D3 (VXT + Vit.D3), or vitamin D3 alone (Vit.D3)

Example 6b shows that the negative effect of vitamin D3 addition on disintegration time of the obtained tablet is less pronounced in the absence of vortioxetine (increase to only 4'47" instead of increase to more than 7 minutes). Thus, there is an unforeseable, surprising interaction of vortioxetine with vitamin D3. In the context of the present invention, this interaction is unwanted (i.e. unwanted synergistic effect).

Example 7. Increase of disintegration time of the tablet can be reduced by adding vitamin D3 extra-granularly instead of intra-granularly.

In Example 7, the trial of Example 6a is repeated. However, in contrast to the method of Example 6a, vitamin D3 is added extra-granularly. Table 3: Comparison of disintegration times for a tablet comprising vortioxetine alone (VXT), or vortioxetine in combination with vitamin D3 (VXT + Vit.D3) which was added intra- or extra- granularly

Example 7 shows that the negative effect of vitamin D3 addition on disintegration time of the obtained tablet is less pronounced if vitamin D3 is added extra granularly instead of intra granularly. In case of extra granular addition, the increase of the disintergration time is approx. 3 minutes instead of more than 7 minutes (intra granular addition). Considering the resting time of a Brintellix® tablet in the patient's stomach and also considering that Brintellix® tablet is a immediate release formulation, a compressed tablet having an approximately 3-time longer disinteration time may be acceptable.

Example 8a. Increase of disintegration time of the tablet can be reduced by adding calcifediol instead of vitamin D3

In Example 8a, the trial of Example 6a is repeated. However, in contrast to Example 6a, calcifediol was added instead of vitamin D3. Both kinds of vitamin D were added intra-granularly. Calcifediol increases the vitamin D blood level of a patients much more efficient. In terms of patient benefit, 8.67 mg Vitamin D3 correspond to 2.89 mg Calcifediol. Table 4: Comparison of disintegration times for a tablet comprising vortioxetine alone (VXT), or vortioxetine in combination with vitamin D3 (VXT + Vit.D3) or calcifediol (VXT + AmpliD) which was added intra-granularly

Example 8a shows that the negative effect of vitamin D3 addition on disintegration time of the obtained tablet is less pronounced if calcifediol instead of vitamin D3 is added. In case of calcifediol, the increase of the disintergration time is approx. 2 minutes instead of more than 7 minutes (intra granular addition).

Example 8b. the increase of disintegration time due to the addition of calcifediol can be reduced by extra-granular addition instead of intra-granular addition

In Example 8b, the trial of Example 8a was repeated. However, in contrast to the method of Example 8a, calcifediol has been added extra-granularly. Table 5: Comparison of disintegration times for a tablet comprising vortioxetine alone (VXT), or vortioxetine in combination with calcifediol (VXT + AmpliD) which was added intra- or extra- granularly

Example 8b shows that the negative effect of calcifediol addition on disintegration time of the obtained tablet is less pronounced if calcifediol is added extra granularly instead of intra granularly. In case of intra granular addition, disintergration time is approx. 2 minutes. In case of extra granular addition, disintergration time is approx. 1 minutes. This is roughly the same disintegration time as Brintellix® tablet, considering the resting time of a Brintellix® tablet in the patient's stomach and also considering that Brintellix® tablet is a immediate release formulation.

Example 9. In the absence of vortioxetine, calcifediol increases of the tablet's disintegration time is less than vitamin D3.

In Example 9, the effect of calcifediol vs. vitamin D3 on disintegration time are compared. Table 6: Comparison of disintegration times for a tablet comprising vortioxetine alone (VXT), vitamin D3 alone (Vit.D3), or calcifediol alone (AmpliD), the vitamin being added intra- or extra- granularly

Example 9 shows that intra granular addition of vitamin D increases disintegration time of the tablet more than extra granular addition of vitamin D, regardless whether vitamin D3 or calcifediol is added. However, if vitamin D3 is replaced by a corresponding amount of calcifediol, the increase in disintegration time is 3-4 times smaller.

The examples also show that adding vitamin D increases disintegration time of the tablet, regardless whether vortioxetine is present or not. However, if vitamin D3 is replaced by a corresponding amount of calcifediol, the increase in disintegration time is 3-4 times smaller.