TECHNICAL FIELD

The invention is related to therapeutic compositions comprising SMEDDS formulation (OM-SMEDDS) and SMEDDS pre-formulation comprising Olmesartan medoximil. Moreover the invention is related to usage of the formulation for a test model and applications in determining oral bioavailability and biodistribution by marking the pre-formulation with NIR dyes.

PRIOR ART Olmesartan Medoximil (OM) is an active agent developed by Daiichi Sankyo. Initially it has been marketed as a mono product with the brand name Olmetec® in the year 2002. It is being marketed in USA with the names Benicar®, Benicar® HCT, Azor®, Tribenzor®. In Japan it is available in the market as Olmetec®, Rezaltas®. In Europe there are several drugs that comprise olmesartan from single therapy to ternary combination forms. Since then dual combinations with Hydrochlorothiazide and Amlodipin and ternary combinations with Hydrochlorothiazide + Amlodipin have been developed.

Presently, olmesartan is found in 10, 20, 40 mg tablets in the market and while its usage in children with the ages of 6-16 is 2.5-20mg for children weighing between 20-35kg, the dose in children weighing 35kg and more is 5-40 mg. The dose of olmesartan for geriatric patients (between the age of 65-74 and >75) and adults is 20-40mg. In the package leaflet of the original drug (Benicar® tablet) in USA, the base suspension, is obtained by mixing two standard carriers (Ora Sweet®, Ora Plus®) and it is recommended that twenty tablets within the box are powdered and suspended by the pharmacist and given to the elderly and children in a measured amounts. This process is a dosing process that can only be carried out by the pharmacist. As such carrier products are not available in the market in our country, the tablet containing Olmesartan Medoxomil, is powdered with a difficult process as it is hydrophobic and is diluted and given to children and elderly patients. Due to this reason a new formulation is required for the drug to be given with low and correct doses, suitable per kilogram.

It has been reported by FDA that Olmesartan had celiac alike side effects in the intestines following usage for 3-6 months in humans and that it caused diarrhea and weight loss and this information has been added into the prospectus of the drug. Rather than eliminating the side effect caused by olmesartan with an additional agent, a brand new formulation is required to prevent the side effects by reducing the side effects of the drug caused by intestinal exposure. The developed OM-SMEDDS shall prevent the side effects of olmesartan that has been approved by the FDA and included in the prospectus of the drug. As a result of our studies, it has been histologically proved by us that OM-SMEDDS did not cause these side effects in the intestines.

OM-SMEDDS shall increase bioavailability 10 times (according to (IVIS) test the optical imaging of oral biodistribution was increased 24 times, antihypertensive effects was increased at least 3.1 times with pharmacodynamic (NIBP) test and it is 100 times more permeable according to the PAMPA test results) and which was 26% for the olmesartan tablet that is the only preparation available in the market and it shall provide better efficiency with lower amounts of active agent thereby providing pharmaeconomic advantage, it shall increase patient tolerability and it shall make dosing feasible.

With the sweetened SMEDDS to be developed, different alternative formulations will be offered to elderly and pediatric patients such as SMEDDS liquid loaded tablets and to adults such as SMEDDS hard or soft gelatin capsule formulations.

In addition to these findings, during the evaluation of the bioavailability of OM-SMEDDS, a new method has been discovered in which the real-time oral biodistribution of drugs can be monitored in the body by marking OM-SMEDDS with near infra red fluorescent dyes. At the same time, as a result of the 1 month pharmacodynamic test, the test for comparing the effect potential of pure olmesartan suspension and OM-SMEDDS it has been discovered that after the application of olmesartan suspension at a treatment dose for one month the exact celiac disease model could be created in rats. In the present situation, the celiac animal model is an expensive model that necessitates a transgenic animal. By means of this animal model, as the celiac disease can be created exactly in healthy rats, numerous repeats of the disease is possible and a sustainable and economic model can be provided in the celiac diagnosis and treatment field. These methods that have been discovered are techniques that have not been tried before and these methods are unique in the world.

BRIEF DESCRIPTION OF THE INVENTION

Our invention is a SMEDDS formulation that has been developed according to the physical-chemical properties of Olmesartan medoxomil which is an antihypertensive active agent that is widely used in treating hypertension which is a diseases that has been defined as the most frequent cause of death by WHO.

The present invention comprises the following features:

The Olmesartan SMEDDS formulation subject of our invention is a pharmaceutical pre-mixture that comprises oleic acid as a fat, at least two surface active agents, an inactive surface active agent, an active agent and water. It comprises approximately 10-20% (14.72%) oleic acid as a fat, approximately 10-20% (16.218%) Tween 80 and approximately %10-20% (16.218%) Span 80 as surface active agents, approximately 25-35% (32.435%) Transcutol as inactive surface active agent, and approximately 15-25% (20.41%) water, and approximately 10, 20, 40 mg Olmesartan medoximil as an active agent. The surface active agent/inactive surface active agent ratio is approximately 1:1. The ratio of the surface active agents relative to each other, Tween 80/Span 80 is approximately 1:1. It is possible for the developed OM-SMEDDS pre-formulation or intermediate product to be used in obtaining pharmaceutical compositions that provide an economic advantage as a result of increased patient tolerability, eliminated celiac side effects and increased bioavailability. OM-SMEDDS can be sweetened and flavorized and can be placed in the market in liquid form, tablet loaded with liquid and or in hard or soft gelatin capsules. The side effects of drug molecules that have other bioavailability problems, that cause celiac alike side effects or gastrointestinal side effects can be reduced by being formulated with SMEDDS. Moreover, after the oral administration of SMEDDS other lipid based micro/nano particle drug carriers that have been marked with Vivotag 680 XL, Xenolight DiR or other fluorophores, the real time biodistribution of these drugs in the body can be monitored, the bioavailability rate can be measured, the organ location of the drug can be monitored, organ targeting can be performed with the drug, it could be checked if the blood brain barrier has been crossed and all of these parameters can be illustrated mathematically. Our invention includes a method in which different SMEDDS and other lipid-based micro/nanoparticle formulations prepared specifically for the physicochemical properties of the active substances to be monitored in the body, can be marked with different NIRs and monitored in the body.

In order to compare the side effects of our invention, celiac disease can be formed in rats by means of the suspension we have been using in the Celiac rat model that has been established during the experiment, and this model can be used for the diagnosis and treatment of the disease. The ARB’s that cause celiac side effects or the gastrointestinal side effects of other problematic drug molecules can be compared with the prepared SMEDDS and suspension.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises the following features: a) The Olmesartan SMEDDS formulation subject of our invention is a pharmaceutical pre-mixture that comprises oleic acid as a fat, at least two surface active agents, an inactive surface active agent, an active agent and water. It comprises approximately 10-20% (14.72%) oleic acid as a fat, approximately 10-20% (16.218%) Tween 80 and approximately %10-20% (16.218%) Span 80 as surface active agents, approximately 25-35% (32.435%) Transcutol as inactive surface active agent, and approximately 15-25% (20.41%) water by weight to the total weight of the composition, and approximately 10, 20, 40 mg Olmesartan medoximil as an active agent. The surface active agent/inactive surface active agent ratio is approximately 1:1. The ratio of the surface active agents relative to each other, Tween 80/Span 80 is approximately 1:1. b) Olmesartan SMEDDS is prepared after mixing homogenously oleic acid, surface active agents (Tween 80, Span 80) and the inactive surface active agent (Transcutol) at the determined amounts for 5-20 minutes at approximately 300 rpm (25 ± 0.5°C) and water is added titration is continued until a turbid mixture is obtained. OM addition to the Formula is carried out with the prior dissolving of the active agent in transcutol. c)It is possible to treat hypertension with OM-SMEDDS as it provides an economic advantage as a result of increased patient tolerability, eliminated celiac side effects and increased bioavailability. d) Olmesartan SMEDDS can be sweetened and flavorized and can be placed in the market in liquid form, tablet loaded with liquid and or in hard or soft gelatin capsules. e)By means of the method of our invention, after the oral administration of SMEDDS other lipid based micro/nano particle drug carriers that have been marked with Vivotag 680 XL, Xenolight DiR or other fluorophores, the real time biodistribution of these drugs in the body can be monitored, the bioavailability rate can be measured, the organ location of the drug can be monitored, organ targeting can be performed with the drug, it could be checked if the blood brain barrier has been crossed and all of these parameters can be illustrated mathematically. f) Our invention includes a method in which different SMEDDS and other lipid-based micro/nanoparticle formulations prepared specifically for the physicochemical properties of the active substances to be monitored in the body, can be marked with different NIRs and monitored in the body. g) In the method of our invention, the control dye solution marked with Vivotag® 680 XL, comprises 0.45 mg VivoTag® 680 XL per 150 mΐ per mouse (such that it comprises 3 mg/ml Vivotag 680 XL per one mouse). The solution is prepared with 270 mΐ stock dye, 480 mΐ water and 150 mΐ buffer (50 mM NaHCOs) as 900 mΐ by volume for 6 mice. The OM-SMEDDS solution marked Vivotag 680 XL is prepared with 270 mΐ stock dye, 480 mΐ OM-SMEDDS and 150 mΐ buffer (50 mM NaHCOs) as 900 mΐ by volume for 6 mice. During the washing process carried out in order to remove excess dye, 900 mΐ PBS (pH 7) is added to this mixture, it is vortexed and is centrifuged for 10 minutes at 15300 rpm. The washing stage is repeated twice. Every time, 900 mΐ supernatant is removed. The OM-SMEDDS that is precipitated is transferred into another eppendorf with care with a micropipette. The remaining washed section is given with a 150m1 oral gavage to mice. The control dye solution marked Xenolight DiR comprises 0.3 mg Xenolight® DiR in 150m1 liquid to be given per mouse. The solution is prepared as 900m1 with 720m1 PBS, 180m1 stock dye for 6 mice (such that it comprises 2 mg/ml 1 Xenolight® DiR per 2 mice). The OM-SMEDDS solution marked with Xenolight® DiR is prepared as 900m1 with 720m1 OM-SMEDDS and 180m1 stock dye according to the same calculation. The excess dye is removed with the same method mentioned above. h) By means of our invention a rat test model can be developed in order to carry out assays for the treatment of celiac disease. Our invention comprises a rat animal model for establishing enteropathy similar to celiac with histological imaging following the oral application of the suspension to rats at a dose of 1.3 mg/kg for 1 month comprising 1 mg/ml OM and 0.25% w/v CMC (carboxymethylcellulose). The OM suspension has been prepared by topping up 0.25 g CMC and 100 mg OM with 100 ml distilled water and suspending the mixture. The amount of dispersant in the suspension and/or type of dispersant and the carrier body solvent can change. If needed, a model study can be carried out with other experimental animals instead of the rat animal model or cell cultures. g) Our invention can comprise OM or other drug molecules that have other bioavailability problems that cause celiac alike side effects or gastrointestinal side effects as the active agent. i) The animal model our invention comprises can be used at a number or amount that can be repeated or that is sustainable in the diagnosis and treatment of celiac disease. It can be beneficial in scientific studies by allowing endless numbers of simulation of the disease. It can also explain the mechanisms of action of other drugs that lead to gastrointestinal side effects. This data, can be supported, confirmed by immuno histochemical studies, gene expression and ELISA based antibody analysis, and thereby the severity and progress of the disease can be measured. These models can serve humanity in the pharmaceutical sector and the celiac diagnosis and treatment field, by being widening the scope and by variations using different animal species and cell cultures.

A) Premix

The premix of the invention is a premix that comprises, a fat, at least two surface active agents, an inactive surface active agent, an active agent and water and it is prepared by titrating in order to create microemulsion (SMEDDS). The premix comprises by weight 10-20% (14.72%) fat, approximately 10-20% (%16,218) two surface active agents, approximately 25-35% (32,435%) an inactive surface active agent and approximately 15-25% (20.41%) water and 10-40 mg olmesartan medoxomil.

In one embodiment of the present invention, the premix comprises at least two surface active agents selected from nonionic, anionic, cationic and zwitterionic surface active agents. For this purpose, non-ionic surfactans such as Sorbitan esters (Span), Polysorbates (Tween), Ethylene glycol distearate Glyceril monostearate Propylene glycol monostearate Glyceryl monostearate Diethylene glycol monolaurate, poloxamer 188, anionic surfactants such as acacia, sodium lauryl sulfate, cationic surfactants such as cetrimonium bromide, cetylpridine chloride can be used.

Products sold under the commercial name Span® in the market, are non-ionic surface active agents containing Sorbitan Monolaurate, Monopalmitate, Monostearate, Tristearate, Monooleate or Trioleate esters. The product sold in the market with the commercial name Tween® (polysorbates) are nonionic surface active agents that comprise polyoxyethylene sorbitan esters.

In an embodiment of the invention the pre-mix comprises approximately 10-20% oleic acid as fat, 10-20% Tween and approximately 10-20% Span mixture as surface active agent, and approximately 25-35% Transcutol as the inactive surface active agent and distilled water.

In another embodiment of the invention the premix comprises 10, 11, 12, 13, 14,

15, 16, 17, 18, 19 or 20% oleic acid as fat, approximately 10, 11, 12, 13, 14, 15,

16, 17, 18, 19 or 20% Tween 80 as surface active agent, approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% Span 80 inactive surface active agent, approximately 25-35% or approximately 30% Transcutol as the active agent and distilled water.

The formulation of the invention comprises Tween 80/Span 80 surface active agent mixture, approximately at a rate of 25-35% (32.435%) Tween or Span 80. Preferably the ratio of the Tween 80 or Span 80 in the mixture is approximately 10-20% (16,218%).

The compositions of the present invention comprise at least one inactive surface active agent. As used in this description document, the inactive surface active agent is added into the premix in order to form an emulsion, stability and/or emulsification together with at least a surface active agent. As the inactive surface active agent Transcutol with 6 carbons (2-(2-ethoxyethoxy) ethanol), short chain alcohols with 1 to 6 carbons (for example ethanol), benzyl alcohol, alkane diols and triols (for example Propylene Glycol, glycerol, polyethylene glycols such as PEG and PEG 400), glycol ethers such as tetraglycol and glycorol (for example tetrahydrofurfuryl PEG ether), pyrrolidone derivatives such as N-methyl pyrrolidone (for example Pharmasolve®)and 2-pyrrolidone (for example Soluphor® P) and bile salts, for example sodium deoxycholate, ethyl oleate can be used. Transcutol has been selected in the formulation of the invention. Transcutol can be used in the formulation at the ratio of 20-40%, 25-35%, 30-35%, approximately 30, 31, 32, 33, 34, 35% approximately 32,435%.

In the formulation of the present invention the oleic acid that forms the fat portion is located in the formula as 10-20% (14,72%) by weight relative to the total weight of the pre-mix.

In the formulation of the invention the ratio of the surface active agent mixture (Tween 80 - Span 80) to the inactive surface active agent (transcutol) is between 0.8: 1.2 and 1.2:0.8, approximately 1/1. The preferred surface active agent - inactive surface active agent ratio (Tween 80 /Span 80 mixture transcutol ratio) is approximately 50% (1:1).

The mixture ratio of the surface active agents Tween80/Span80 in the present invention among themselves is 1/1 and the ratio by weight of these surface active agents within the total surface active agent in the formulation according to the weight of the premix is 25-35% (32,435%).

The mixture ratio of the surface active agent/ inactive surface active agent in the formulation of the present invention (Ratio of Transcutol to 1/1 Tween80-Span80 mixture) is approximately 1:1 and the inactive surface active agent according to the weight of the premix is approximately 25-35% (32,435%) by weight.

The formulation of the present invention is an oil/water type formulation and this type of formation is provided by HLB calculations carried out. In the formulation the water part, is approximately 15-25% (20.41%) by weight relative to the total weight of the pre-mix. The formulation that provides the highest microemulsion area has been determined with a triangular phase diagram method (Figure 1). The formulation comprises approximately 10 mg, 20mg or 40mg Olmesartan medoximil as active agent.

The pre-mix of the present invention can be in the form of self nano emulsifying drug release systems (SNEDDS), self micro emulsifying drug release systems (SMEDDS) or self emulsifying drug delivery systems (SEDDS). The pre-mix forms an emulsion in the body comprising micelle particles inside the aqueous solution when it comes into contact with the gastric and/or intestinal medium. SNEDDS / SMEDDS / SEDDS, can contain a particle size (particle diameter) that varies between approximately 20 nm to approximately 200 nm. For example in some formulation arrangements particle size varies between approximately 5nm to 50 nm or approximately 100 nm to approximately 150 nm.

In the SMEDDS form in order to prepare the pre-mix fat, oleic acid, surface active agents (Tween 80, Span 80) and the inactive surface active agent (Transcutol) at determined amounts is mixed at a suitable rate for example 300 rpm at a suitable temperature for example (25 ± 0.5°C) for a suitable period of time, for example 5-20 minutes and the mixture is titrated with a measured amount of water until a turbid product is obtained. The active agent addition to the Formula is carried out with the prior dissolving of the active agent in transcutol. The pre-mix comprising olmesartan medoximil has been prepared with the mentioned method.

The pre-mix and/or SNEDDS/SMEDDS/SEDDS may also contain at least an antioxidant. Suitable antioxidants can be a-tocopherol (vitamin E), calcium disodium EDTA, a-tocopherol acetates, butylhyroxytoluenes (BHT) and butyl hydroxyanisoles (BHA).

During in vitro solubility, diffusion and permeability studies, OM determinations have been carried out with an HPLC method that has been developed and validated beforehand (Figure 2 A). The droplet size, zeta potential, and polydispersity index of the SMEDDS formulas have been directly measured using a ZetaSizer device and the Laser Ray Scattering Size Analysis technique.

5

The refraction index of the SMEDDS formulas has been measured with a refractometer by adding a drop of diluted liquid SMEDDS.

The self emulsion time of SMEDDS on its own, has been evaluated in a pH 1.2 gastric fluid medium with a USP Type II dissolution apparatus at 50 rpm. The 10 dispersion time of SMEDDS has been recorded as the microemulsification time. SMEDDS pH measurements have been conducted using pH meter.

SMEDDS electrical conductivity has been evaluated using a conductivity meter in order to determine the oil/water microemulsion type.

SMEDDS viscosity has been determined using Brookfield viscometer.

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The stability studies of SMEDDS has been carried out on three series for 3 months at 25±2°C/75%±5% RH and the physical appearance, droplet size and drug content of SMEDDS has been monitored. The results of these examinations have been given in Table 1.

Table 1. Characterization of SMEDDS

25 The in vitro Solubility Studies of SMEDDS; has been carried out in 900 ml gastric fluid (0.1 N HC1 medium , pH 1,2) at 37 ± 0.5 ° C that has been simulated at 50 rpm using a USP Type II dissolution apparatus in order to compare the release of a large gelatin capsule with a size of 000 comprising 10 mg/ml OM with the commercial tablet comprising the same amount of OM (n=3). Drug release percentages have been calculated with ANOVA and they have been evaluated statistically (Figure 2B).

The in vitro Diffusion Studies of SMEDDS has been carried out by monitoring the diffusion of 10 mg/ml OM-SMEDDS from a diffusion floating tube (Sigma Aldrich) of 1 kDa MWCO (cut in molecular weight) in order to determine the release profile of SMEDDS from the low porous membrane in gastric fluid. The diffusion tubes loaded with SMEDDS have been monitored for 6 hours in a beaker with 50 rpm magnetic mixing and in a medium of 100 ml pH 1.2 (0.1 N HC1) at 37 ± 0.5 ° C (n = 3).

Permeability speed studies; have been carried out with PAMPA (parallel artificial membrane assay) assay systems and OM-SMEDDS has been found to be at least 100 times more permeable in comparison to the suspension of the pure drug (Figure 2C).

B) Development of the Pharmaceutical Dosage Forms

Olmesartan SMEDDS can be sweetened and flavorized and can be placed in the market in liquid form, tablet loaded with liquid and or in hard or soft gelatin capsules.

Accordingly at least an excipient (inactive agent) is added into the formulation in order to stabilize the form efficiently, such that is suitable and/or acceptable for usage, and to conserve, colour, add flavor to said formulation. Colloidal silicon dioxide, crospovidone, lactose monohydrate, lecithin, microcrystalline cellulose, polyvinyl alcohol, povidone, sodium lauryl sulfate, sodium stearyl fumarate, talc, titanium dioxide and xanthan gum can be selected as at least one inactive agent.

The tablet formulation that can be loaded with liquid, can contain magnesium aluminometasilicate, crospovidone XL 10 and magnesium stearate talk.

SGcaps® Capsugel, Posilok®, Qualicaps, Licaps Qualicaps can be used in the hard gelatin capsule SMEDDS formulation. The flavored and aromatized SMEDDS liquid formulation can comprise Kollidon® 25, Kollidon® 30, glycerol, sodium cyclamate, orange or raspberry aroma.

The pharmaceutical compositions can also comprise at least a superdisintegrant. The pharmaceutical composition can comprise approximately 1% to approximately 25% of at least a super disintegrant by weight of the composition, it can contain, for example approximately 1% to approximately 20% or approximately 1% to approximately 15% super disintegrant by weight of the invention. The compositions that comprise at least a super disintegrant can be in the form of a tablet. In addition to these but not limited thereto, croscarmellose (a cross-linked cellulose), crospovidone (a cross-linked polymer), sodium starch glycolate (cross-linked starch) and soy polysaccharides are available as super disintegrants. Commercial examples are Kollidon® (BASF), Polyplasdone® XL (ISP) and Ac-Di-Sol (FMC BioPolimer).

The pharmaceutical composition can be in the form of syrup, tablet, a liquid loaded tablet or any other drug delivery form. For example, the pre-mix can be encapsulated within a hard or soft gelatin capsule. In some of the embodiments of the invention the capsule filling content can be approximately between 0.400 g to 1.600 g.

Pharmaceutical compositions can be used for any kind of therapeutic treatment and/or program related to at least a health issue such as treatment of hypertension or cardiovascular diseases that result in heart attacks, heart or kidney failure, strokes and blindness. It is possible to treat hypertension with Olmesartan SMEDDS that provides economical advantage as it has increased patient tolerability, the celiac side effects are eliminated and it has increased bioavailability.

By using the developed OM-SMEDDS composition, dose adjusted base SMEDDS can be prepared for children, geriatric patients and adults. SMEDDS syrup formulations for children and geriatric patients by selecting suitable sweeteners and flavors and SMEDDS hard or soft gelatin capsules and liquid loaded tablets for adults can be developed.

C) Application for determining bioavailability, biodistribution and Other biological parameters with OM-SMEDDS.

With the present invention, in vivo and ex vivo imaging following oral application has been provided after marking fluorescently marked SMEDDS and other micro / nanoparticle drug systems with an infrared fluorescent dye and obtaining marked drug systems.

In the prior art, it is not possible to monitor the real time oral biodistribution of drugs in vivo. By means of our invention OM-SMEDDS has been able to be fluorescently marked with Vivotag 680 XL and Xenolight DiR and the real time oral bioavailability of the drug has been determined. OM-SMEDDS subject to our invention, that has been fluorescently marked with Vivotag and Xenolight has included the hydrophobic active agent into an oil phase and it has improved solubility and adsorption of the drug and increased its bioavailability and thereby a different distribution pattern has been observed in comparison to the control dye and it has been enabled for the drug to be monitored real-time in the body.

Determining the in vivo and in vitro activity of a drug, understanding the molecular and visible release levels, determining the biodistribution, determining the location in the body in real time, targeting, sequential and dynamic monitoring are problems and they require appropriate analysis. Following the oral application of the fluorescent marked SMEDDS, the optical imaging method enables to see the real-time biodistribution of the drug, the metabolism, elimination, and efficiency against the disease and the tumor in real time.

Although several studies have been conducted using the in vivo imaging technique to determine the biological properties, biological distribution, efficiency, and toxicity of the drug carriers after intravenous administration, there are no oral biodistribution studies related to optical imaging of the biodistribution following the oral application of the fluorescent marked SMEDDS drug system.

Imaging studies are generally techniques involving radiation, and this condition poses a health problem for both those exposed and those around them. The optical imaging system with fluorescent marking allows imaging without radiation.

Creating formulations with drugs that are poorly soluble and poorly permeable, that have high molecular weight, that are chemically unstable and that have a short half-life is a tremendous challenge. Therefore the importance of in vivo monitoring of drug carriers has gained importance. SMEDDS subject to our invention that is marked with Vivotag 680 XL and Xenolight DiR has ground breaking properties in determining oral bioavailability. Our invention provides a solution as it can be prepared according to the physical-chemical properties of these types of problematic active agents.

Another problem is the visualization of deep tissues, this is possible with SMEDDS that is marked with Vivotag 680 XL and Xenolight DiR that provides high penetration between infrared light (NIR) 700 and 900 nm.

The present invention may comprise any kind of active agent that allows drug targeting with bioavailability and biodistribution assay. By means of the present invention the real-time biological distribution of the self microemulsifying drug release system (SMEDDS) comprising an active agent that is marked (tagged) with fluorescent dyes can be determined by the real-time optical imaging method. SMEDDS containing the fluorescently marked active ingredient will enable to determine increased bioavailability as it will exhibit stronger fluorescent emission in predetermined minutes, in vivo, in comparison to the control dye solution.

For marking VivoTag® 680 XL (N-hydroxysuccinimide(NHS)- ester) and Xenolight DiR (DMC18 (7) or l,l’-dioctadecyltetramethyl indotricarbocyanine iodine) fluorescent dyes have been used. By means of the method, after the oral administration of SMEDDS other lipid based micro/nano particle drug carriers that have been marked with Vivotag 680 XL or Xenolight DiR or other fluorophores, the real time biodistribution of these drugs in the body can be monitored, the bioavailability rate can be measured, the organ location of the drug can be monitored, organ targeting can be performed with the drug, it could be checked if the blood brain barrier has been crossed and all of these parameters can be illustrated mathematically.

Our invention includes a method in which different SMEDDS and other lipid-based micro/nanoparticle formulations prepared specifically for the physicochemical properties of the active substances to be monitored in the body, can be marked with different NIRs and monitored in the body following oral application.

Therefore a control dye solution marked with Vivotag 680 XL has been prepared and 150plcontrol dye solution has been applied per mouse. This amount of solution comprises 0.45 mg VivoTag® 680 XL (3 mg/ml Vivitag 680 XL). The solution is prepared with 270 mΐ stock dye, (dissolved in 5 mg VivoTag® 680 XL, 500 mg dimethylsulfoxide), 480 mΐ water and 150 mΐ buffer (50 mM NaHC03) as 900 mΐ by volume for 6 mice. OM-SMEDDS solution marked Vivotag 680 XL is prepared with 270 mΐ stock dye, 480 mΐ OM-SMEDDS and 150 mΐ buffer (50 mM NaHCOs) as 900 mΐ by volume according to the same calculation. During the washing process carried out in order to remove excess dye, 900 mΐ PBS (pH 7) is added to this mixture, it is vortexed and is centrifuged for 10 minutes at 15300 rpm. The washing stage is repeated twice. Every time, 900 mΐ supernatant is removed. The OM-SMEDDS that is precipitated is transferred into another eppendorf with care with a micropipette. The remaining washed section is given with a 150m1 oral gavage to mice.

The control dye solution marked Xenolight DiR however comprises 0.3 mg Xenolight® DiR in 150m1 liquid to be given per mouse. The solution is prepared as 900m1 for 6 mice (such that it comprises Xenolight® DiR 2 mg/ml per mouse) with 180m1 stock dye, (25 mg dye is dissolved in 2.5 ml ethanol) 720m1 PBS. The OM-SMEDDS solution marked with Xenolight® DiR is prepared as 900m1 with 720m1 OM-SMEDDS and 180m1 stock dye according to the same calculation. During the washing process carried out in order to remove excess dye, 900 mΐ PBS (pH 7) is added to this mixture, it is vortexed and is centrifuged for 10 minutes at 5 15300 rpm. The washing stage is repeated twice. Every time, 900 mΐ supernatant is removed. The OM-SMEDDS that is precipitated is transferred into another eppendorf with care with a micropipette. The remaining washed section is given with a 150m1 oral gavage to mice.

10 In this study, Vivotag 680 XL, which is strongly bound to small molecules and proteins, and Xenolight® DiR fluorescent dyes with lipophilic properties were used to display the lipid characteristic of OM-SMEDDS (Table 2). The detailed labeling procedures of each dye have been described below.

Table 2. Fluorescent dye properties*

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* Perkin el er protocol

Following oral administration, the biological distribution and absorption of fluorescently labeled OM-SMEDDS was monitored using IVIS® technique in a 20 total of 12 male Swiss mice at different predetermined time points compared to the mice that were applied the control dye.

As a result of the study, a real-time biological distribution of fluorescently labeled OM-SMEDDS has been observed with optical marking, and an optical 25 imaging technique has been developed that can visualize the biodistribution of the bioavailable drug that cross the gastrointestinal system and blood brain barrier. The oral biodistribution of OM-SMEDDS that has been fluorescently labeled with NIR fluorescent dye VivoTag® 680 XL and Xenolight DiR, has been able to be successfully monitored using 2-D fluorescent imaging tomography (IVIS) on mice. OM-SMEDDS, containing nano-sized droplets, is a novel technique developed for the first time to display low resolution class drug biodistribution, and it has given promising results. Vivotag 680 XL marked OM-SMEDDS emits 4.2 times stronger in the body than the control dye group at the end of the 5th hour and this rate increased 24 times at the end of the 6th hour. The real-time biodistribution of Xenolight DiR marked OM-SMEDDS has shown 2 times stronger emission in the body. Ex vivo organ findings (heart, liver, lung, kidney, spleen, colon) were evaluated 7 hours after the application of VivoTag® 680 XL and Xenolight DiR-labeled OM-SMEDDS to confirm the in vivo imaging results of real-time biodistribution. The mice group that were administered OM-SMEDDS labeled with Vivotag® 680 XL and Xenolight® DiR, have emitted 3.96 and 1.7 times stronger fluorescence respectively in comparison to mice that were administered with the control dye. According to ex vivo and in vivo findings it has been determined that OM-SMEDDS showed strong localization in the stomach and intestines while the control dye was leaving the body at the same time points (Figure 3, 4), (Table 3, 4). Table 3. Comparison of the Ex vivo IVIS® emission findings in organs (liver, lung, stomach, colon and ileum) following the application of the control dye for 6 hours and the Vivotag 680 XL fluorescent marked SMEDDS. The ROI signal intensity has been expressed as (photons / s / cm ² / sr). The SMEDDS formulation provided 3.96 times more emission in comparison to the control group and the findings have shown that the absorption in tissue and organs of the group that had taken SMEDDS was higher.

Table 4. Comparison of the Ex vivo IVIS® emission findings in organs (liver, lung, stomach, colon and ileum) following the application of the control dye for 7 hours and the Xenolight DiR fluorescent marked SMEDDS. The ROI signal intensity has been expressed as (photons / s / cm ² / sr). The OM-SMEDDS formulation provided 2 times more emission in comparison to the control group and the findings have shown that the absorption of OM-SMEDDS in tissue and organs of the group that had taken OM-SMEDDS was higher.

D) Creating a Celiac Model in Hypertensive Test Animals induced with L-Name for tracking pharmacodynamics and side effects

In this study, in order to compare the pharmacodynamic efficiency of OM and the side effect of Celiac-like enteropathy effect, rats that were given a suspension comprising 1.3 mg / kg dose of OM and the rats that were given the same dose of OM-SMEDDS formulation were compared with the control group that were not given any medication. During the pharmacodynamic experiment, anti-hypertensive response created by OM-SMEDDS and OM-suspension against the artificial hypertensive response (systolic blood pressure) caused by L-NAME (N-nitro-l-arginininmethylester) in rats, was determined. As a result of the experiment, the antihypertensive treatment effect of OM-SMEDDS was 3.1 times higher in hypertensive rats where said hypertension was created with L-Name compared to the effect of suspension, and it was observed that OM-SMEDDS did not cause celiac-like enteropathy and diarrhea (Figure 2D, Figure 5).

The developed OM-SMEDDS formulation has the advantage that it does not cause celiac side effects in comparison to OM suspension.

An aspect of the invention is that it provides an animal model for celiac disease to be established following the oral application of the olmesartan suspension in a test subject. The model comprises the below mentioned steps:

(a) giving a suspension comprising 1 mg /ml OM to the test subject at a treatment dose of 1.3 mg/kg for one month.

(b) continuing the application for one month, in order for the symptoms of this application to be created,

(c) imaging of the histology of the created celiac table with suitable techniques following the dying process and thereby determining the enteropathy degree that has been created in the test subject.

From another point of view, the creating the celiac disease artificially in animals, clinically is difficult and it is an expensive process. Nowadays highly expensive animals models whose numbers have been increasing day by day are used in order to test the targeted new treatments.

Although attempts to create celiac disease artificially, in transgenic animals have been carried out before, no animal model and intestinal histology studies are available regarding the formation of this disease with olmesartan exposure.

The experimental model obtained with the present invention can be used for the diagnosis, treatment and follow-up of celiac disease, and the modeling process can be repeated and olmesartan in treatment doses, can be given for prolonged periods of time. There is no cure for celiac disease other than the gluten diet today and it is important to try alternative treatment methods by creating an artificial animal model. Due to this reason, animal models that can be easily created are needed; therefore the usage of our model during drug analysis, to monitor the side effects of such drugs that cause these types of intestinal inflammation shall make it easier to compare the advantages of the formulations.

Other active agents that cause enteropathy similar to celiac or gastrointestinal problems due to intestinal exposure, which can be hydrolized (hydrolized in paraoxanase and carboxymethylenebutelonides that take part in the disintegration of olmesartan and gluten) with the same steps and that have the same mechanism of action, other ARB’s can create enteropathy similar to celiac with cell lines and different animal models instead of the active agent olmesartan used in the present invention.

In order to create celiac disease in test animals according to the present invention, the OM suspension obtained by the addition of 1 mg / ml active agent and suspension thereof, to the 0.25% w/v CMC suspension (0.25g CMC per 100 ml) has been given to the rats by oral route at a dose of 1.3 mg/kg for 1 month.

OM-Suspension and OM-SMEDDS has been given to two different groups of rats 1 time a day by means of oral gavage, for 1 month, and in comparison to the control group that was not given any medication and the group given OM-SMEDDS, the rat intestinal images of the group that was given OM suspension showed intensive leucocyte infiltration, histologically.

The present invention comprises a rat animal model for establishing enteropathy similar to celiac with histological imaging following the oral application of the suspension to rats at a dose of 1.3 mg/kg for 1 month comprising 1 mg/ml OM and 0.25% w/v CMC (carboxymethylcellulose) to prove that the SMEDDS formulation comprising Olmesartan medoximil does not lead to celiac side effects. A celiac disease model can be created in test animals by giving the suspension loaded with the pure drug that creates enteropathy similar to celiac for 1 month at the treatment dose. This model simulates celiac disease and it can be beneficial to scientific studies as it can be used any number of times or scale, that can be repeated or that is sustainable in the diagnosis and treatment of celiac disease. It can also explain the mechanisms of action of other drugs that lead to gastrointestinal side effects. These models can serve humanity in the pharmaceutical sector and the celiac diagnosis and treatment field, by being widening the scope and by variations using different animal species and cell cultures.

The present invention provides an economic and practical model in the related experiments with the diagnosis and treatment of celiac disease by means of the “celiac rat model induced with olmesartan”. In addition, with this model, the mechanisms of action of other drugs that cause intestinal inflammation can be investigated in detail by immunohistochemical, genetic, histological and ELISA-based antibody analysis.

The present invention is related to being used in both bioavailability and efficacy studies in the pharmaceutical sector and in Celiac research in clinic.

As a result our invention OM-SMEDDS shall prevent the side effects of olmesartan that has been approved by the FDA and included in the prospectus of the drug. It has been reported by FDA that Olmesartan had celiac alike side effects in the intestines following usage for 3-6 months in humans and that it caused diarrhea and weight loss and this information has been added into the prospectus of the drug. (FDA Safety Announcement 7-3-2013). In patients with this phenomenon, complaints of chronic diarrhea, vomiting, severe abdominal pain, bloating and weight loss intestinal enteropathy are observed and this phenomenon has been the subject of many lawsuits. The originating company (Daichii Sankyo) has spared a very high budget in order to pay the compensations of the lawsuits related to these side effects.

It is quite clear that a novel formulation is required to prevent the side effects by reducing the side effects of the drug caused by intestinal exposure. As a result of our studies, it has been histologically proved by us that OM-SMEDDS did not cause these side effects in the intestines. Our invention, OM-SMEDDS, can be presented for use as dosage forms that can be easily given in measured doses to children and elderly hypertensive patients by sweetening and aromatizing them, or as hard gelatin capsules or in tablet forms that can be loaded with liquid to adults as dosage forms that reduce the side effects of the drug. This is a solution to the problems caused by the tablet form available in the market. Moreover, our invention, OM-SMEDDS, has increased its bioavailability by including hydrophobic OM active ingredient in the oil phase and improving the solubility and absorption of the drug as a solution to the solubility and bioavailability problem of olmesartan.

DESCRIPTION OF THE FIGURES

Figure 1. Triangular phase diagram that provides the ideal micro-emulsion area (Formula 2) and HLB calculations.

HLB calculations (YEM and YYEM ratios)

YEM/YY Fat YEM/YEM YYEM YEM/YYE Fat YEM/YEM YYEM

EM _ Ratio M Ratio Ratio

Ratio ( oleic (tween80/ (transcu (oleic (tween80/ ( transcu acid ) span80 ) tol) acid) span80) tol)

Formulal 2/3 Formula 3 2/3

1:1 1 1.8/2.7 4.5 2:1 1 2.4/3.6 3

1:1 2 1.6/2.4 4 2:1 2 2.13/3.2 2.67

1:1 3 1.4/2.1 3.5 2:1 3 1.86/2.81 2.33

1:1 4 1.2/1.8 3 2:1 4 1.6/2.4 2

1:1 5 1/1.5 2.5 2:1 5 1.33/2 1.67

Formula2 1/1 Formula 4 1/1 1:1 1 2.25/2.25 4 2:1 1 3/3 3

1:1 2 2/2 4 2:1 2 2.66/2.67 2.67

1:1 3 1.75/1.75 3.5 2:1 3 2.33/2.33 2.33

1:1 4 1.5/1.5 3 2:1 4 2/2 2

1:1 5 1.25/1.25 2.5 2:1 5 1.66/1.67 1.67

Triangular phase diagram findings Formula A(Fat) B( YEM/YYEM ) C(Water) X Y Microemulsion

(1/1) Area

~1 13.75 66.09 20.16 0.76 0.17 15839

2 14.72 64.87 20.41 0.75 0.18 215.27

3 11.94 6692 21 15 0 77 0 18 12451

13.9 65.88 20123 0 76 0 18 25944 Figure 2. Solubility and permeability studies

A. Chromatogram injected with Olmesartan medoximil.

B. Release graphic of olmesartan from SMEDDS in the hard gelatin capsule compared to olmesartan tablet form in pH 1.2 medium. The solubility values of SMEDDS is higher than the market formulation.

C. Calculated permeability rates. The calculated permeability rate of the SMEDDS formulation according to the formula is -4,014 (Pe = 9,895 x 10-5). Since the CMC suspension permeability rate can only be calculated when there is a transition in detectable concentration, the OM-suspension transition rate has been calculated using the detection limit of the OM-suspension which is 0.05 pg value, and the OM-suspension permeability rate was calculated as -6.395 (Pe = 4.030). x 10-7). When the antilogarithm is taken it has been noted that the permeability of SMEDDS was at least 100 times more than the suspension.

D. Graph Pad statistical image of the blood pressure graphs following application in test animals whose hypertension was lowered. The difference between Olmesartan medoximil OM-SMEDDS, control Group and the olmesartan medoksomil suspension was found to be statistically significant (p <0.0001).

Figure 3. IVIS® images showing in vivo biodistribution by hours of mice that have been given Vivotag 680 XL OM-SMEDDS formulation and control dye at lst(A), 2nd(B), 3rd(C), 4th(D), 5th(E) and 6th(F) hours (n = 3). When compared with the control group (left) mice that were given OM-SMEDDS with fluorescent Xenolight® DiR dye label (right) gave a stronger signal. (G), (H) Following the application of the control dye and the Xenolight DiR fluorescent marked SMEDDS for 6 hours, ex vivo IVIS® findings in organs (liver, lung, stomach, colon and ileum). SMEDDS group had stronger emission in comparison to the control group. (I) the OM-SMEDDS group organs (top) emitted higher emission signals in comparison to the control group (bottom).

Figure 4. real time biodistribution images of rats that were given Xenolight® DiR marked OM-SMEDDS formulation and the Control dye at the 20th(A), 40th(B), 90th(C), 120th (D), 165th(E) minute and 5th(F), 6th(G), 7th(H) hours (n = 3). Compared to the control group (right), mice given OM-SMEDDS with fluorescent Xenolight® DiR dye label (left) gave a stronger signal. (I, J) Ex vivo IVIS® findings of the organs (liver, lung, stomach, colon and ileum) following the 7 hour application of Xenolight DiR. (K) The OM-SMEDDS group organs (top) emitted higher emission in comparison to the control group (bottom).

Figure 5. Images of histopathologic examinations carried out in rat duodenum following a 1 month treatment with Olmesartan SMEDDS or suspension. Enteropathic findings similar to celiac have not been observed in the instestinal images of the control group (Al, A2, A3) that have been given olmesartan SMEDDS (Cl, C2, C3) contrary to the rats that were given the suspension. The biopsy of the duodenum did show enteropathic findings similar to celiac related to olmesartan in rats that were given the suspension (Bl, B2, B3). The arrows show increased mononuclear cell infiltration. Scaling has been carried out as 20 pm in A2, 50 pm in A3, B3, C3, 100 pm in Al, B2, C2 and 500 pm in Bl, Cl.