The present invention relates to a stable liquid oily ready-to-use formulation comprising an active pharmaceutical ingredient, which has hydrophobic and/or lipophilic properties and/or which exhibits stability problems in aqueous environments. The present invention further relates to a method for preparing said formulation, and said formulation for use in the medical treatment of particular patient groups.
Many pharmaceutical ingredients (drugs) currently exist only in a solid dosage form such as tablets or granules. It would, however, often be advantageous to have a liquid dosage form of a drug at hand. A major problem concerning liquid formulations of a drug is the instability of many drugs in liquid media so that such formulations only have a very short shelf-life and not the desired shelf-life of 2 to 3 years.
Thus, suitable liquid dosage forms do not exist for all drugs, in particular because it is often difficult to provide a stable liquid dosage form of a drug.
A lack of commercially available oral liquid dosage forms is an ongoing problem in many practice settings. A pharmacist is often challenged to provide an extemporaneous oral liquid, for example for paediatric patients, patients who are unable to swallow solid dosage forms such as tablets or capsules, patients who must receive medications via nasogastric or gastrostomy tubes or patients who require non-standard doses that are more easily and accurately measured using a liquid formulation. It is common practice for this liquid dosage form to be prepared from a commercially available oral solid dosage form by simply crushing tablets or opening a capsule and the subsequent addition of water or juice. However, these dosage forms can become complex due to the addition of excipients and while these measures are taken to improve compliance and stability of the extemporaneously prepared product, there are often limited data to support the stability or bioavailability of the final liquid dosage form, where potential interactions between the vehicle, preservative, buffering agent, flavouring agent, levigating agent, suspending agent, viscosity enhancer, storage container and the modified commercial product have yet to be established.
The pharmacist, both in community and hospital pharmacy, is often challenged with the preparation of a liquid dosage form not available commercially for paediatric patients, and those adults unable to swallow tablets or capsules. Appropriate formulations for administration to children exist for only a minority of commercially available drugs and the need for extemporaneously compounded formulations is escalating due to the release of many new drugs formulated for adults but with expected use in children. Children require titratable individualised doses in milligrams per kilogram of body weight and most children under six years of age cannot swallow tablets.
There are many reasons for the lack of commercially available paediatric formulations. The overall size of the paediatric market is much smaller than for adults, especially for common diseases such as hypertension. The industry is thus reluctant to commit resources to seek labelling for infants and children (unless a disease occurs exclusively or frequently in the paediatric population), since the formulation has to have been adequately studied in paediatric patients. It has been estimated that more than 40% of doses given in paediatric hospitals require compounding to prepare a suitable dosage form since crushing a tablet and/or sprinkling the contents of a capsule over food or mixing in a drink may lead to errors in preparation or delivery of doses.
Another practice seen in paediatric care is to use injectable solutions for oral administration. This is generally cost prohibitive and presents with many problems including the following:
(i) drugs and/or vehicles may be mucosal irritants, vesicants, nauseants, or cauterants;
(ii) drugs may undergo extensive first-pass metabolism or may have poor bioavailability after oral administration (e.g. cefuroxime and enalapril);
(iii) drugs and/or vehicles suitable for injection may be unpalatable;
(iv) excipients included in the formulation may have toxic effects when cumulative oral ingestion is considered; and
(v) co-solvents used in the commercial formulation may be diluted when mixed with syrup or water, thus allowing the drug to precipitate.
In most cases the pharmacist will therefore prepare an oral liquid dosage form with the active ingredient dissolved or suspended in a simple syrup or sorbitol mixture. Since pure crystalline powders of drugs are not usually accessible to pharmacies, the active pharmaceutical ingredient (API) is often obtained by modifying a commercially available adult solid dosage form by crushing a tablet or opening a capsule. When a drug is formulated for paediatrics use, several factors unique to paediatrics must be considered such as the immaturity of the intestinal tract and the subsequent influence on gastrointestinal absorption. Additives, including preservatives and sugar must be chosen carefully.
Formulations may also contain preservatives; an excipient considered to be largely inert in adults, however, may lead to life threatening toxicity in paediatrics when multiple doses of medications with the same preservative are employed. This is particularly the case with benzyl alcohol and benzoic acid. The physical, chemical, microbial and therapeutic stability of the above paediatric extemporaneous preparations may not have been checked at all. In addition, the increased potential for calculation or dispensing errors may prove the practice of modifying commercially available products to be extremely unsafe.
The stability of a pharmaceutical formulation is a major factor in ensuring the quality of the drug product and consequently, the efficacy of the treatment. Several parameters can influence the stability of a pharmaceutical formulation. These include the exposure of the product to a number of environmental conditions such as temperature, humidity and light. The chemical composition, the physical-chemical properties, the quantity of ingredients (both drug and excipients) in the formulation and the manufacturing process including storage and conditions during transportation may also affect the stability.
As an example, angiotensin-converting enzyme (ACE) inhibitors are widely used in the treatment of paediatric hypertension and have been found to be particularly effective treatments for hypertension in infants. ACE inhibitors are currently the principal agents for antihypertensive therapy in children both because of their effectiveness and their beneficial influence on cardiac and renal function and peripheral vasculature. However, drug administration to paediatric patients presents a number of challenges, since the pharmacist has to prepare an extemporaneous suspension by dispersing marketed tablets following their disintegration within liquids.
An enalapril suspension as a liquid dosage form was designed to address specific objectives such as ease and reproducibility of preparation for the pharmacy, ease of dosing, protection from microbial contamination, stability to support the suspension shelf-life, and acceptable taste for the patient (Rippley, R. K., Connor, J., Boyle, J., Bradstreet, T. E., Hand, E., Lo, M-W., Murphy, M. G., Biopharm. Drug Dispos. 2000, 21: 339-344). In this study, there were no adverse experiences related to formulation taste. However, this formulation is not a ready-to-use preparation, does not possess long term stability under standard storage conditions and has to be extemporaneously prepared by a pharmacist. Hence, there still is a need for a stable liquid ready-to-use dosage form of enalapril.
Despite many attempts to design a liquid dosage form of enalapril, up to now, there is no commercial ready-to-use liquid formulation of enalapril meeting health regulatory authorities requirements. This is probably due to the drug sensitivity in the liquid medium and lack of data on prolonged stability of the active compound in such medium over sufficient time to allow the design and the marketing of a viable commercial product.
In addition to enalapril, it would be beneficial to develop liquid dosage formulations for other active pharmaceutical ingredients that have hydrophobic and/or lipophilic properties and/or which exhibit stability problems in aqueous environments.
It is therefore an object of the present invention to provide a pharmaceutical formulation that is suitable for the preparation of a ready-to-use liquid formulation. The ready-to-use formulation shall prevent the potential side effects that can be elicited by extemporaneous formulations which are not well characterised. The liquid formulation shall be suitable for the formulation of a wide variety of drugs and, in particular, for the formulation of drugs which have hydrophobic and/or lipophilic properties and/or exhibits stability problems in aqueous environment. Said formulation shall allow an accurate and precise dosing of the drug contained therein and shall be particularly suitable in the medical treatment of patient groups with swallowing problems such as paediatric or elderly patients. The drug shall be stable within said preparation for a long shelf-life.
The present invention is based on the unexpected and surprising finding that stable liquid formulations of drugs which have hydrophobic and/or lipophilic properties and/or exhibit stability problems in aqueous environments can be prepared using specific oily vehicles as a basis, in which the active pharmaceutical ingredient is dissolved or dispersed.
Thus, the present invention relates to a stable liquid oily ready-to-use formulation, comprising:
(i) an active pharmaceutical ingredient, which has hydrophobic and/or lipophilic properties and/or which exhibits stability problems in aqueous environments,
(ii) an oily vehicle, in which the active pharmaceutical ingredient is dissolved or dispersed, and which is selected from vegetable oils, synthetic oils, fatty acids or combinations thereof; and optionally one or more of a thickening/suspending agent, an antioxidant, a preservative, a flocculating agent, an entero-coated polymer, a dipolar solvent (such as alcohol, glycerine etc.), a surface stabilising agent, a sweetener, a flavouring agent, an emulsifier, and a colouring agent, or combinations thereof.
The invention further relates to the stable liquid oily ready-to-use formulation for use in the medical treatment of a patient group selected from (i) paediatric patients, (ii) elderly patients, (iii) patients suffering from dysphagia, or (iv) patients requiring medication via nasogastric or gastrostromy tubes.
The invention further relates to a method for preparing said stable liquid oily ready-to-use formulations comprising the steps of
The active pharmaceutical ingredient comprised in the formulation according to the invention is an active pharmaceutical ingredient which has hydrophobic and/or lipophilic properties and/or which exhibits stability problems in aqueous environments. Pharmaceutical ingredients which have hydrophobic and/or lipophilic properties are classified as class 2 according to the FDA BCS classification. A drug has hydrophobic properties if the Log P of the drug product is >5 and has hydrophilic properties if the Log P is <5. Log P measurements are common technology in the field of pharmaceutical ingredients. A person skilled in the art is well in a position to carry out a corresponding test on the basis of common textbooks. According to a preferred embodiment of the invention, the active pharmaceutical ingredient has a Log P>5. Pharmaceutical ingredients which exhibit stability problems in aqueous environments are sensitive principles which can be degraded as a result of hydrolysis, pH or oxidation. Examples thereof are omeprazole or aspirin.
The formulation according to the present invention is particularly suitable for the following active ingredients:
Domperidone, enalapril maleate, omeprazole, bisacodyl, lamotrigine, atorvastatin, famotidine, gabapentin, granisetron HCl, itraconazole, ketoconazole, lansoprazole, levodopa/carbidopa, losartan potassium, ondansetron HCl, amiodarone HCl, captopril, rifampicin, sildenafil citrate, dexamethasone, verapamil, spironolactone, tacrolimus, tramadol HCl, tramadol with paracetamol, baclofene, terbinafine HCl, spironolactone with hydrochlorothiazide.
The formulation according to the present invention is also suitable for the following active ingredients: Acyclovir, Valacyclovir, Divalproex, Simvastatin, Atomoxetine, Amphetamine salts, Dilitazem, Candesartan, Amlodipine, Valsartan, Atenolol, Amitriptyline, Clopidogrel, Fenofibrate, Glipizide, Allopurinol, Warfarin, folic acid.
Preferably, the active pharmaceutical ingredient is enalapril maleate, omeprazole, atorvastatin or lamotrigine. Optionally, the active pharmaceutical ingredient is used together with an emulsifier (emulgator). Preferably, enalapril maleate or omeprazole are used together with an emulsifier. In other embodiments two or more of the active pharmaceutical ingredients may be combined in a formulation of the invention.
According to the invention, the oily vehicle is selected from vegetable oils, synthetic oils, fatty acids or combinations thereof.
According to a preferred embodiment of the invention, the oily vehicle is a vegetable oil selected from wheat germ oil, soybean oil, olive oil, arachis oil, corn oil, cottonseed oil, linseed oil, coconut oil, rapeseed oil, borage seed oil, apricot kernel oil, peanut oil, sunflower oil, sesame oil, safflower oil or sweet almond oil or combinations thereof.
According to a preferred embodiment of the invention, the oily vehicle is a synthetic selected from medium chain triglycerides (MCTs), propylene glycol dicaprylocaprate, glyceryl caprylate/caprate, glyceryl cocoate, butylene glycol dicaprylate/caprate, coco caprylate/caprate, glyceryl behenate, glyceril monolinoleate, glyceryl oleate, hydrogenated vegetable oils, refined vegetable oils, glyceryl laurate, glyceryl myristate or combinations thereof.
According to a preferred embodiment, the oily vehicle is a fatty acid selected from C8 to C22 fatty acids saturated and unsaturated, omega-3, omega-6 or omega-9 fatty acids. For example caprilic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, margaric acid, heptadecic acid, stearic acid, oleic acid, linoleic acid, alpha and gamma linolenic acid, arachidic acid, eicosenoic acid and its' derivates (EPA, PGs, etc.), arachidonic acid, behenic acid, decosaenoic acid or a derivates of thereof, or a combination thereof. Preferably, the oily vehicle is soybean oil or a MCT. Examples of MCTs are caproic acid (C6), caprylic acid (C8), capric acid (C10) and lauric acid (C12). MCTs are composed of a glycerol backbone and three of these fatty acids. The approximate ratios of these fatty acids in commercial MCT products derived from coconut oil is 2 (C6):55 (C8):42 (C10):1 (C12).
According to a preferred embodiment of the invention, a thickening/suspending agent is selected from polysaccharides such as alginates, carageenan, xantangum, acacia, tragacanth, pectin, locust bean gum, guar gum; clays, such as magnesium aluminium silicates (veegum), kaolin, bentonite, hectorite; aliphatic acids and stearic salts, such as aluminium monostearates; colloidal silicon dioxide; long chain alcohols such as cetostearyl alcohol, cetyl alcohol; waxes such as beeswax; long-chain diacylglycerols such as glyceryl distearate, or combinations thereof. More preferably, the thickening/suspending agent is beeswax, cetyl alcohol, glyceryl distearate or a combination thereof.
A thickening agent is an agent which increases the viscosity of the solution and subsequently reduce the sedimentation rate of the dispersed particles. A suspending agent promotes dispersion of the particles in the liquid medium without necessarily increasing the viscosity by preventing aggregation or close contact between the disperse particles. The precipitate forms loose flocks that can be easily re-dispersed and return to the initial homogeneity and particle size distribution. The suspending agent can be a structured vehicle that prevents close contact between the dispersed particle upon sedimentation by forming a sieve structure network able to entrap the particles and avoid direct contact between them. It should be kept in mind that the shorter the distance between particles, the stronger the attraction forces between them resulting in the formation of aggregates that cannot be anymore separated into individual particles.
According to a preferred embodiment of the invention, the antioxidant may be selected from propyl gallate, octyl gallate, dodecyl gallate, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), tocopherol, D-alpha-tocopherol, DL-alpha-tocopherol, tocopheryl acetate, D-alpha-tocopheryl acetate, DL-alpha-tocopheryl acetate, diferoxamine mesylate, dilauryl thiodipropionate, or combinations thereof. The preservative may be selected from propyl gallate, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), D-alpha-tocopherol, dilauryl thiodipropionate or a combination of thereof.
An antioxidant is a substance capable of inhibiting oxidation, which may be added for this purpose to pharmaceutical products subject to deterioration by oxidative processes, as for example the development of rancidity in oils and fats or the inactivation of some medicinal in the environment of their dosage forms (Remington 21th ed.). A preservative is, in common pharmaceutical sense, a substance that prevents or inhibits microbial growth, which may be added to pharmaceutical preparations for this purpose to avoid consequent spoilage of the preparations by microorganisms (Remington 21th ed.). Substances of both categories may be added to the formulations of the present invention.
Preferably, the antioxidant/preservative is BHT and/or propyl gallate.
Suitable surface stabilising agents may be selected from kaolin, magnesium aluminium silicates, glycerine, bentonite, dimethicone and pectin or combinations thereof.
The formulation may optionally further comprise one or more emulsifier(s). Examples thereof are polysorbate 20, polysorbate 80, linoleoyl macrogolglycerides, lauroyl macrogolglycerides, oleoyl macrogolglycerides (polyoxylglycerides).
The ratio of the active pharmaceutical ingredients and the oily vehicle ranges from 0.1% to 20% preferably from 0.5% to 10%, more preferably from 1% to 10%. The further ingredients of the claimed ready-to-use formulation may be present in an amount from 0.1% to 99.9%.
A preferred formulation of the invention comprises an active pharmaceutical ingredient selected from enalapril maleate (1%) or omeprazole (10%) or atorvastatin (2%) or lamotrigine (0.4-8%), an oily vehicle (90-99%) selected from soybean oil or medium chain triglyceride, thickening/suspending agent selected from beeswax (5%) or glyceride palmitostearate (3%), a preservative being propyl gallate (0.01%) and a surface stabilising agent being bentonite (6.5%).
The formulations according to the present invention will be used for the medical indication as given by the respective active pharmaceutical ingredient. A dosage will be determined by a physician in relation to the active ingredient and in relation to the kind and severity of the condition to be treated, the age and weight of the patient.
The formulation according to the present invention is particularly suitable for the medical treatment of patient groups having problems in swallowing a drug. Patients who are unable to swallow a drug are paediatric patients, elderly patients, patients suffering from dysphagia or patients requiring medication via nasogastric or gastrostromy tubes. The invention thus also relates to a method of treatment of said patients with the formulation according to the present invention.
According to a preferred embodiment of the invention, the formulation may be in the form of an oral, topical or parenteral formulation.
According to a preferred embodiment of the invention, the stable liquid oily ready-to-use formulations are used for the manufacture of a medicament for the medical treatment of a patient group selected from (i) paediatric patients, (ii) elderly patients, (iii) patients suffering from dysphagia, or (iv) patients requiring medication via nasogastric or gastrostomy tubes.
The formulation according to the present invention may basically be prepared according to methods generally known in the art. According to a preferred embodiment, said method comprises:
The method of preparing the formulation is according to general methods of the art (Remington 21th ed; Drugs and the pharmaceutical sciences textbook, Vol. 105 Pharmaceutical emulsions and suspensions, ed. by James Swarbrick 2000; The pharmaceutical codex 12th ed. Walter Lund 1994).
In a preferred embodiment, the active pharmaceutical ingredient may be granulated or encapsulated into microparticles prior to adding/dispersing the active pharmaceutical ingredient in the vehicle. In the case of pH sensitive molecules in the acidic range, then it will be worthy to protect the particles from degradation by coating them with an entero-coated polymer.
The liquid oily ready-to-use formulations according to the present invention have the advantage that they are ready-to-use formulations, i.e. they may easily be dosed for application to people having a disease which makes swallowing difficult or to children and elderly who cannot easily swallow solid dosage forms. Thus, an extemporaneous compounding is not required, and calculation or dispensing errors, which involve risks in the practice of modifying commercially available products are eliminated.
The enclosed Figures illustrate the subject-matter of the invention:
The following examples illustrate further embodiments of the invention:
In the following a general flowchart of the manufacturing process according to the present invention is given.
The following compositions were prepared according to the following method:
The optional emulsifying agent may be polysorbate 20, polysorbate 80, linoleoyl macrogolglycerides, lauroyl macrogolglycerides.
The stability of the formulations was checked according to USP/EP pharmacopeial specifications. Assay and Related substances, and Dissolution were measured by HPLC validated method based on EP and USP.
Any of the following compositions may contain flavouring and/or sweetening and/or colouring agents.
All amounts given in the examples are % w/w.
The following method example details an exemplary method of preparing the formulation of the invention further.
The oily vehicle is heated to 65-70° C. in water bath under mild stirring. The antioxidant/preservative (e.g. propyl gallate) is added. Then, a thickening/suspending agent, such as glyceryl palmitostearate or beeswax is dissolved or melted until a clear solution is obtained. The mixture is cooled down to the room temperature while stirring. Around 35° C., silica dioxide and bentonite (if used) are added, followed by the active pharmaceutical ingredient. At this point the sweetener, flavour and/or colour may be added. The volume is then completed with the oily base (MCT or soybean oil). Optionally, homogenisation is performed at the end.
During the entire procedure, it is important to maintain constant stirring in order to get the homogenous thickening effect and homogenous distribution of the active pharmaceutical ingredient.
The formulations of Examples 1 to 13 were stable formulations, whereas the formulations according to Comparative Examples 1 to 5 were not physically stable according to visual evidence of sedimentation volume (example of preliminary results for 3 months stability in room temperature, 4° C. and 30° C./65% (relative humidity=RH). The results are presented in the following Table 1:
This method is intended for estimation of degree of Flocculation when two layers are observed. The Sedimentation Volume, F, is the ratio of the equilibrium volume of the sediment, Vu, to the total volume of the suspension, V0. When F=1, no sediment is apparent even though the system is flocculated. This is the ideal suspension for, under these conditions, no sedimentation will occur. Caking will also be absent.
The test was performed with two separate bottles. The bottles should stand upright and should not be disturbed before the examination. The total height (V0) of the suspension in each bottle was measured with the caliper. The sedimentation height (Vu) was measured with the caliper. The sedimentation volume (F) is calculated according to the following formula:
This test is intended to determine, under defined conditions, the suspendability of suspensions, i.e., the ability of a suspension to be re-dispersed by an appropriate procedure such as shaking.
Three separate containers were tested. The sample was brought to the temperature of 20-25° C. The container to be tested was carefully held. The container was shaken vigorously many times. The container was opened and its content was poured into a beaker. It was noted whether there is any precipitate left on the bottom of the container.
The results were determined as follows:
Easy re-dispersion of the settled particles that form flock and not aggregates. Chemical stability of the active ingredient was evidenced by validated HPLC techniques based on USP/EP monographs.
Further, the method for preparing the stable liquid oily ready-to-use formulation according to the present invention is described in the following exemplary method.
Assay on impurities and degradation products and dissolution test
The following formulations were tested:
Omeprazole magnesium—5%
Silica dioxide—6%
Propyl gallate—0.01%
Mixing MCT oil with propyl gallate, adding bentonite and silica dioxide with stirring, when homogeneity is reached, adding the omeprazole and mixing until final homogeneity
For Assay and Impurities and Degradation Products Determination test 1 g of sample are weighted into 200 ml volumetric flask, then 20 ml of methanol are added and the sample is sonicated for 10 minutes, then it is diluted to volume with methanol, further on the above solution is diluted 5.0:50 with the same solvent and is filtered through 0.45μ PTFE filter prior to injection. Standard solution is 0.05 mg/ml of Omeprazole Mg.
The test performs with Betasil C8, 5μ, 4.6*150 mm column, at 30° C.; eluent is 0.01M phosphate buffer pH 7.6/acetonitrile (65:35 v/v).
Enalapril maleate—1%
Glyceryl distearate—3%
Propyl gallate—0.02%
Soybean oil—q.s.
Mixing MCT oil with propyl gallate, sucralose and flavour, adding pre-melted glyceryl distearate and mixing until homogeneity is reached, adding enalapril maleate and mixing until homogeneity.
For Assay and Impurities and Degradation Products Determination test 4 g of sample are weighted into 100 ml volumetric flask, then 20 ml of methanol are added and the sample is shaken for 30 minutes at ambient temperature, then it is diluted to volume with 0.05M phosphate buffer pH
6.8 and is filtered through 0.45μ GHP filter prior to injection. Standard solution for assay level is 0.4 mg/ml and for IDD level is 0.002 mg/ml of Enalapril Maleate in 0.01M phosphate buffer solution pH 2.2.
The test performs with Acclaim C8, 5μ, 4.6*250 mm column, at 50° C.; eluent is 0.01M phosphate buffer pH 2.2/acetonitrile (75:25 v/v).
The dissolution test is performed at followed conditions: 0.25% Sodium lauryl sulphate (so called “SLS”), 500 ml, 75 rpm (Apparatus 2—paddle). The results are obtained by HPLC method. The dissolution sample is filtered prior to HPLC analysis.
The dissolution test has been carried out according to European pharmacopeia 6th edition 2010, Chapter 2.9.3.
The following results were obtained:
The following enalapril formulations were prepared:
The above formulations were prepared according to the general description of the above method example. The formulations were tested for the following parameters:
The physical stability of the following formulation has been tested.
The formulations were prepared as follows:
The formulation was tested as follows:
Dissolution Adjustment:
Preliminary stability for 6 m at 40° C./75%RH, 30° C./65%RH and RT
Viscosity Adjustment:
The results are summarized in the following Tables 5 to 7.
The above test demonstrated that Amiodarone is stable at accelerated conditions (40° C./75% RH) for three months. Due to the lipophilic nature of the active material there is a need for emulsifiers to ensure dissolution. It has further been found this formulation has a thixotropy characteristic, i.e. it is thick and getting flow after shaking. Heat, even for a short period of time, destroys the thixotropy characteristics of the formulation. The formulation looses its viscosity permanently after heating even for a short term. Moreover, the formulation's odor is changing.
Monolayers of differentiated Caco-2 (epithelial colorectal adenocarcinoma cells) cells show morphological and biochemical similarity to normal intestinal enterocytes, and they develop tight effective junctions. Thus, Caco-2 cell monolayer is considered an established model to investigate the mechanisms involved in oral absorption including the effect of the P-gp pump. The P-gp expression of the actual Caco-2 monolayer was evidenced (data not shown) using the validated monoclonal antibody C219 technique. The following oily enalapril formulations of the invention were tested:
Caco-2 monolayer were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% nonessential amino acids, and 5% antibiotic-antimycotic solution at 37° C. in humidified air, 5% CO2 atmosphere. The culture medium was replaced every 96 h. The uptake studies were conducted with monolayers (passages 73 to 79) in wells of 2 cm2 which reach confluency following 4 days of incubation in culture. Prior to the experiments, the culture medium was replaced with transport medium and cell monolayers were subsequently equilibrated for 30 min at 37° C. before the uptake study. The transport medium was Hanks buffer composed of 136.89 mM NaCl, 5.36 mM KCl, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, 0.41 mM MgSO4 7H2O, 19.45 mM glucose, 1.26 mM CaCl2, 0.49 mM MgCl2 6H2O, 4.17 mM NaHCO3, 10 mM HEPES, and the pH was adjusted to 7.4. The cell monolayers were washed twice with 1 ml Hanks solution and then combined with the transport medium (1 ml) which contained 25 μg/ml of enalapril maleate (provided by CTS, RLB: 802871, E65 mean diameter smaller that 100 μm) dissolved in an appropriate solvent preferably in water, or aliquot of enalapril tablet CTS (Xanef® 20 mg tablets. Batch number, 30056.03.00 Expiry date 0.1-2011) or as enalapril CTS oil formulation (ENL drops 10 mg/ml lot. 030301). In addition, the influence of increasing concentrations of intestinal juice (IJ) was also tested. Xanef® comprises the following excipients: sodium hydrogen carbonate, lactose monohydrate, corn starch, pregelatinized corn starch, magnesium stearate, iron (III) oxide E172 (colorant), iron (II) hydroxide-oxide.
The following formulation was tested: CTS-COCO2-no 1
A tablet of enalapril maleate was grounded and sieved through a sieve of 366 μm aperture. The powder (10 mg equivalent to 1 mg enalapril maleate) was weighted dispersed and fully dissolved in 1 ml Hanks buffer. 25 μl from this solution were placed in Caco-2 wells with 975 μl of Hanks buffer resulting in a final concentration of 25 μg/ml of enalapril maleate.
A 100 μl of parent CTS oil formulation was withdrawn and diluted with soybean oil USP to give 1 mg/ml of enalapril maleate.
A 25 μl of the diluted oil solution was placed into the transport medium to give a 25 μg/ml of enalapril maleate in each well (dilution 1:40).
These experiments was conducted under similar experimental conditions as described in section b. except; the dilution medium containing 0.1%; 0.01% of intestinal juice.
The final objective of this study was to design and partially validate a Caco-2 cells method by which usually water soluble formulations are tested, to be able to teach on the potential absorption of CTS oil formulation following oral administration to humans as compared to standard tablets of enalapril.
All the uptake experiments were performed over 3 hours at 37° C. Following incubation the transport medium was collected in clean tubes stored at −80° C. up to the quantitative determination of the enalapril levels by HPLC (The HPLC method was provided by CTS). Regarding the cell monolayer in the wells, 1 ml of 0.2% SDS was added to each well to elicit lysis of the cell monolayer. The lysate were collected in a clean tube and enalapril was determined by CTS HPLC technique.
Analysis was determined with the Tukey-Kramer multiple comparisons test calculated by InStat software (version 3.01). The level of significance was corrected using a post test analysis. Statistical significance was set with one * for p<0.05 and with *** for p<0.001 while values are presented as mean±S.D.
In preliminary studies, the incubation time of the various CTS formulations with the Caco-2 cells was investigated and the data indicated that no difference could be detected when the formulations were incubated over 1 h only and 3 hours were needed to elicit a significant enalapril uptake by the Caco-2 cells. Indeed, enalapril residual concentration in the wells diminished by more than 50% in the presence of Caco-2 cells from the apical side when dissolved in Hanks buffer or released from the tablet whereas about 80% of the initial enalapril concentration remained in the well and only 20% of the enalapril was up taken by the Caco-2 cells following incubation over 3 h. This probably was due to lack of partition of the drug from the oil towards the Hank buffer solution because the oil solution did not disperse well into the aqueous phase (
This was confirmed by the data presented in
The apparent permeability (Papp, cm/s) was calculated according to the following equation described by Schrickx and Fink-Gremmels (Schrickx, J. and Fink-Gremmels, J. P-glycoprotein-mediated transport of oxytetracycline in the Caco-2 cell model. J. Vet Pharmacol. Therap. 30: 25-31 (2007)).
Where Q is the total amount of permeated drug throughout the incubation time period (μg t−1), A is the diffusion area (2 cm2), Co is the initial concentration (25 μg/ml) and t is the incubation time (3 hours=10800 seconds) in seconds.
Statistical analysis of the apparent permeability (Papp) values calculated from equation 1 (Table 1), clearly indicated that there was no significant difference between the enalapril aqueous formulation (25.09±0.50 10−6, cm/s), CTS oil formulation+0.1% intestinal juice (21.85±2.44 10−6 cm/s) and the Tablet formulation (20.89±2.93 10− cm/s).
It can be concluded from the overall results that the CTS oil formulation elicits a similar enalapril uptake compared to the tablet and aqueous solution of enalapril maleate under experimental conditions mimicking the normal physiological GI conditions in Caco-2 monolayer cells.
Number | Date | Country | Kind |
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102010015143.2 | Apr 2010 | DE | national |
Number | Date | Country | |
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Parent | PCT/IB2011/001351 | Apr 2011 | US |
Child | 13649744 | US |