Shenyang Pharmaceutical University LAB 5: OINTMENT PHARMACEUTICS ILABORATORY 5: Preparation and evaluation of the effectof ointment bases onin vitro drug release1. LABORATORY OBJECTIVESa) To learn the preparation methods of different types of ointment bases.b) To learn the methods for determining drug release from an ointment and compare the impact of different ointment bases on drug release.2. INTRODUCTIONOintments are semisolid preparations intended for external application to the skin or mucous membranes. They are used topically for several purposes such as protectants, antiseptics, emollients, antipruritics, kerotolytics, and astringents. Also, some drugs can be absorbed into the general circulation through the skin and produce systemic therapeutic effects.In the formulation of ointments, the base forms the main part of the product. An ointment base is of prime importance because it does not only serve as the vehicle but also functions as the carrier of the medicinal agents in the product. It affects the overall quality of an ointment, the release rate of the drug substance, as well as its absorption into the body. The commonly used ointment bases are classified into three different groups:Oleaginous bases: This group includes hydrocarbon bases, fat and fixed oil bases. A simple ointment which is prepared from the fusion of beeswax and vegetable oil or petrolatum alone can be applied as ointment bases. Other oleaginous ingredients such as liquid paraffin and lanolin are commonly used to adjust the viscosity of the bases.Emulsion bases: Emulsion bases are composed of semisolid or solid oleaginous components, water soluble ingredients, and emulsifying agents. The commonly used emulsifiers include soap, high molecular weight fatty alcohols, fatty alcohol sulfate, polybasic alcohol ester such as triethanolamine soaps, sodium lauryl sulfate, Tween-80, etc. By selecting different types of emulsifying agents, O/W and W/O emulsion based ointments can be prepared. An emulsion based ointment is also called a cream.Water-soluble bases: Water-soluble bases include natural and synthetic water soluble high molecular substances, commonly used are glycerinated gelatin, cellulose derivatives, polyethylene glycol, and polypropylene acid, etc.In addition to the solid drug substance which can dissolve in or form eutectic mixture with the ointment base, insoluble drug substances should be pulverized prior to being incorporated in the ointment base. If necessary, penetration enhancers, humectants and preservatives can be added in the ointment. Ointments can be prepared by the28Shenyang Pharmaceutical University LAB 5: OINTMENT PHARMACEUTICS Iincorporation method, fusion method or emulsification method depending on the properties of the drug substances and bases.Ointments containing semisolid and liquid ingredients can be prepared using the incorporation method which includes levigating the drug powder with a suitable amount of liquid or a portion of the base followed by incorporating the mixture into the base with geometric dilution to ensure uniform distribution of the active ingredient. When the mixing is completed, the product should not show grittiness when it was applied to the skin. For ointments containing ingredients with different melting points, which cannot be mixeduniformly at room temperature, the fusion method should be applied. In this process, the drug substance which is soluble in the base can be directly incorporated in the molten base and the insoluble drug substance can be added to the molten or softened base with constantagitation until it congeals. Emulsion based ointment can be prepared by using the emulsification method. The oil phase is first heated to 70~80 ℃ (if necessary, the oil phase can be filtered when it is hot to get rid of foreign matter) and the ingredients soluble inwater is added to the aqueous phase. The aqueous phase is subsequently heated to the same or a slightly higher temperature compared with that of oil phase. The water phase is added into the oil phase gradually with constant agitation until the final product congeals.In addition to the determination of melting range, pH, viscosity, drug stability andskin irritation, ointments are also evaluated with respect to the in vitro drug release rate.Drug release rate and percutaneous absorption mainly depend on the nature of the drug itself. However, the composition and properties of the ointment bases also influence drug release and percutaneous absorption to a certain extent. Furthermore, the different drug incorporation techniques have also been shown to affect the drug release rate. In general, the fastest drug release rate is obtained with water soluble and emulsion ointment bases and the drug release rate from hydrocarbon ointment base is usually the slowest.Several methods can be used to evaluate the influence of different types of bases ondrug release from ointments. The measurement of drug release rate from an ointment to the release medium can be performed through a semi-permeable membrane. Gel diffusion or excised animal/human skin method also can be employed to study the drug release rate from ointments. Release of the drug from ointments normally follows the Higuchi equationwhich shows that the cumulative release amounts of drug is proportion to t 1/2 as shownbelow:M = kt 1/23. METHODS3.1 Preparation of a simple ointment29Shenyang Pharmaceutical UniversityLAB 5: OINTMENTPHARMACEUTICS IBeeswax6.6gVegetable oil15mL （ 6.7g）Melt the beeswax in an evaporating dish using a water bath. When the wax has completely melted, add the vegetable oil slowly with constant stirring. When the mixture is homogeneous, remove the dish from the heat and stir the mixture until it begins to congeal.Upon the addition of the vegetable oil, stir the mixture constantly until the mixture is uniform. After removing from heat, stir the mixture continuously until it begins to congeal to prevent stratification.3.2 Preparation of O/W emulsion basesStearyl Alcohol1.8 gWhite petrolatum2.0 gLiquid paraffin1.3 mLSodium lauryl sulfate0.2 gEthylparaben0.02 gGlycerin0.1 gDistilled waterq.s.Total:20 ga) Melt the oleaginous phase (stearyl alcohol, white petrolatum and liquid paraffin) in an evaporating dish using a water bath at the temperature of 70~80℃.b) Dissolve the water-soluble ingredients (sodium lauryl sulfate, ethylparaben, glycerin ) in the calculated amount of water in an evaporating dish or a beaker and heat the solution to 70～ 80℃ .c) Add the aqueous phase to the oleaginous phase slowly and heat the mixture on the water bath for additional several minutes under constant stirring.d) Remove the dish/beaker from the heat and stir the mixture at room temperature until it congeals.3.3 Preparation of water soluble bases30Shenyang Pharmaceutical University LAB 5: OINTMENT PHARMACEUTICS IGlycerin24.4 gWater24.7 g1% (w/v) sodium benzoate1 mLCarbopol 9400.25 gTriethanolamine0.25 ga) Disperse Carbopol 940 uniformly in water under vigorous stirring.b) Add glycerin to the mixture prepared above and stir until uniform. Add triethanolamine and 1% (w/v) sodium benzoate and stir until a homogenous solution is formed.Preparation of 1% (w/v) sodium benzoate solution: Add 1 g of sodium benzoate in water with the final volume of 100mL.3.4 Preparation of 5% Diclofenac Potassium ointmentsa) Preparation of diclofenac potassium ointment with a simple oleaginous ointment base: Place the preweighed amount of diclofenac potassium powder (0.5 g) in a mortar and then triturate the drug powder with 9.5 g of the ointment base using the geometric dilution method.b) Preparation of diclofenac potassium ointment with a paraffin base: Heat and melt9.5 g of paraffin in an evaporating dish using a water bath. Add 0.5 g of diclofenac potassium powder in the liquefied base and stir constantly. Remove the dish from the water bath and stir until the mixture begins to congeal.c) Preparation of diclofenac potassium ointment with an O/W emulsion base: Place 0.5 g of diclofenac potassium powder in a mortar. Triturate the drug powder with9.5 g of emulsion base using the geometric dilution method.d) Preparation of diclofenac potassium ointment with a water soluble base: Place 0.5 g of diclofenac potassium powder in a mortar. Triturate the drug powder with 9.5 g of water soluble base using the geometric dilution method.Diclofenac potassium should be milled into a fine powder (passing through a 100-mesh sieve according to the China Pharmacopeia) before being used for preparing different ointments.31Shenyang Pharmaceutical University LAB 5: OINTMENT PHARMACEUTICS I3.5 Comparison of drug release rate from ointments prepared with different basesa) Each of the four products is filled into a short glass tube with an internal diameter of 2 cm and 2cm in length. The length of the filled ointment is about 1.5 cm. The opening is covered carefully with a piece of glassine paper and a rubber band. Make sure that there are no air bubbles underneath the surface of the glassine paper.b) Add the tube with the sample prepared in a) in a beaker containing 100 mL of37℃ distilled water, which is placed in a thermostatic water bath of 37℃±1o C. Immerse the sealed tube in the beaker and make sure that the dialysis glassine paper is approximately 1 mm below the water level. Record the time as time zero.Stir the solution gently but continuously with a stirring bar on a magnetic stirring plate. At specified time points of 15, 30, 45, 60, 90, 120, 150 min, withdraw approximately 5 mL and replace with distilled water of the same volume. Measure the content of diclofenac potassium in the release medium using the method as described in the following part c).c) Drug content measurement: Accurately weigh diclofenac potassium reference standard and prepare the stock solution in a 100 mL volumetric flask. Dilute with water to prepare solutions with different concentrations of 5, 7.5, 10, 12.5, 15 and 17.5 μg/mL. Measure the absorbance of the standard solutions at 275nm (CP 2005 version appendices IVA). Using the drug concentration as the X axis and the absorbance as the Y axis, a standard curve can be established. Dilute 5 mL of the drug release samples collected at different time points with water to 50 mL in a volumetric flask and measure the absorbance at 275 nm. Calculate the concentration of diclofenac potassium in the medium using the standard curve. "Zero" the machine before each reading using water as the blank.If the absorbance value is higher than 0.8, dilute the solution with water and take the measurement again. In this case, be sure to calculate the concentration with the correct dilution factor.4. RESULTS AND DISCUSSION4.1 Apply all four types of diclofenac potassium ointments on your skin. Check if they are smooth in texture, compare their viscosity and spreadability, and discuss the function of each ingredient in these four formulations.4.2 Record the amount of drug released at different time points and record the results in Table 1.32Shenyang Pharmaceutical University LAB 5: OINTMENT PHARMACEUTICS ITable 1. Amount of diclofenac potassium released from four different ointments as a function of timeBaseSimplePetrolatum O/W emulsion Water soluble Ointmenttime (min)1530456090120Plot the cumulative amount of diclofenac potassium released as a function of time for the four ointment preparations and discuss the difference in drug release rate for these four ointment bases.5. QUESTIONSa) How many methods can be used to incorporate drug substances during ointment preparation?b) Which are the factors influencing the drug release behavior from ointments?References[1] Leon Lachman, Herbert A. Lieberman, Joseph L. Kanig. Theory and Practice of Industrial Pharmacy, 3rd ed., Lea & Febiger, 1986.[2] H. C. Ansel, N. G . Popovich and L. V . Allen, Jr. Pharmaceutical Dosage Forms andDrug Delivery Systems, 8th ed., Williams and Wilkins, Baltimore, 2005.(Cuifang CAI)33。