Metronidazole Retention Enema Experiment and Study

Metronidazole Retention Enema Experiment and Study1. Introduction1.1. Rectal dosage lay downsThe most leafy vegetable and usually most convenient route for administering conventional pharmaceutical dosage forms is orally, where the do drugs dissolves in the gastric and/or intestinal fluids and is absorbed to reach the localise of activity. Dissolution and absorption of the drug from the gastro-intestinal environment depends upon many factors e.g. the physico-chemical substance properties of the drug, enzymes, metabolism, pH of the fluids etc. and these are considered as major drawback when localized drug delivery in the colon is required. Colonic drug delivery system offers advantages over oral dosage forms in improving the efficacy and achieving high concentrations with minimal side-effects1.1.1.1. Advantages and limitations of rectal dosage formsAdvantages golosh and painless form of administration.Drugs liable to degradation in the gastrointestinal tract can be administration .First pass elimination (drugs liable to degrade in the beginning reaching the site of action) of high clearance drugs is partially owing to bypassing the liver.Even larger social diseases can be administered.Drugs can be administered rectally in the long full term care of geriatric and terminally ill patients.Administration of rectal suppositories or capsules is a simple procedure that can be under taken eve by unskilled healthcare personnel and patients.LimitationsPatients acceptability and compliance is poor, especially for long term therapy.Suppositories can leak.Drug absorption from suppositories is slow in resemblance to oral or intravenous administration.1.2. Different rectal drug formulationsMany formulations are developed for rectal use and these include suppositories (in the form of solid dose suspensions and emulsions), irrigations, gelatine capsules (used for non-steroidal anti-inflammatory drugs, encapsulated in a soft gelatine capsules as a end micellar solution f or rectal application) 2, and clysters.Enemas are aqueous solutions are suspensions intend for instillation into the rectal region for evacuation of bowel and to treat microbial infections. Enemas are of two types macro enemas (100ml or more) and micro enemas (1.3. Anatomy of Rectum and lower colonThe terminal 15- 19cm portion of the large intestine is rectum it has a circumference of 15-35cm. The rectal pH is around 7-8. The absorptive area of rectum is less when compared tothat of small bowel because the rectal surface area is 200-400cm2 compared to that 200m2 of small bowel, due to very much shorter surface area per unit length 4. Generally medications for rectal delivery are cave in absorbed as weakly alkaline solutions. For rapid absorption of the medication aqueous solutions are preferable rather than suppositories or suspensions. Rectal absorption takes positioning by active absorption and for maximum retention without any rectal urgency to vacate bowels small bulk of th e fluid is recommended5.The diagram with a particle of colon is shown below in fig-1.1.3.1. Physiological considerations of colon1.3.1a. Intestinal colonic micro floraThe human colonic atmosphere supports over 400 perspicuous species of bacterium with a population of 1011 to 1012 CFU/ml with mostly Eubacterium, Bacteriodes etc6. The enzymes produced by these bacterium has wide spectrum of action, that being hydrolytic and reductive in nature, these enzymes are actively involved in many processes, such as steroidal transformation, protein and carbohydrate fermentation, and destruction of mutagenic metabolism. Nitroreductase, azoreductases, and N-oxide and sulfoxide reductases are the most extensive reductive enzymes produced by the intestinal flora7.1.3.1b. Colonic gestureUnder the normal physiological conditions the colonic motility is exposit as irregular alternation of inactive, non-propagative, segmental contractions and infrequent propagative contractions that can be further class into high amplitude contractions ( 100 mmHg) and low amplitude contractions (1.3.1c. Ascending colonic volumeThe ascending colonic volume was found to be 17040 ml as per the studies conducted on healthy subjects using single photon emission computed tomography11.MetronidazoleMetronidazole is a nitro-imidazole bactericidal agent primarily used against control anaerobic bacteria including Bacteroides, Clostridium spp., and certain protozoal parasites like Trichomonas vaginalis, Entamoeba histolytica, Giardia lamblia, Blastocysistis hominis, Balantidium coli and also some of the facultative anaerobes Gardnerella vaginalis and Helicobacter pylori16.Gram negative anaerobes like bacteroides and fusobacterium species and the gram overconfident anaerobes like Peptosteptococci and clostridium species typically test sensitive to metronidazole. It is particularly used against Helicobacter pylori associated to duodenal ulcers and gastritis. Metronidazole is also used against anaerobic bowel flora for the prophylaxis and for the word of Crohns disease where patients might develop complications of infections in bowel12. Metronidazole supports the overgrowth of aerobic microbial flora of the large intestine by reducing the number of anaerobic micro-organisms with acceptable profile of adverse effects13.1.4.1. Anti microbial action of MetronidazoleMetronidazole as anti microbial agent was first introduced in 1959 for the sermon of Trichomonas vaginalis infections, and used subsequently for invasive giardiasis and amebiasis. Metronidazole is highly effective and show rapid onset of action against anaerobic infections. Antimicrobial action of Metronidazole is in the main due to the toxic intermediates which are produced during the reduction of the compound14. Interaction of these intermediates with deoxyribonucleic acid in protozoan inhibits nucleic acid synthesis and there by exerts antimicrobial effects15. Mechanism of action of Metronidazole is shown in the fig- 2.1.4.2. Physico-chemical propertiesChemically metronidazole is 2-methyl-5-nitroimidazole-1-ethanol or 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole. Its formula is C4H9N3O and its chemical formula is in the fig-3.Metronidazole is sporty to pale yellow coloured, odourless, crystalline powder. It is meagerly soluble in water, alcohol and slightly soluble in ether16.It is generally stable in aqueous solutions of pH 2.0-7.017.1.4.3. Metabolism of MetronidazoleMetronidazole is metabolised in the liver into two metabolites. These metabolites include 2-hydroxy-methyl-5-nitroimdazole-1-acetic acid (HM) and 2-methyl-1-2-nitroimidazole-1-acetic acid (MAA). Hydroxy metronidazole is pharmacologically active with antimicrobial action and its potency is 30% to that of metronidazole and the acetic acid metabolite of metronidazole is pharmacologically inactive but its activity is noticed in patients with renal dysfunction, small amount of metronidazole is oxidised to acetamide18. The metabolizati on pathway and chemical formulas can be seen in the figure 419.Study objectiveMetronidazole is commercially available in the form Tablets, Suppositories, Gels, and suspensions20. But metronidazole in the form of enema is not available commercially. Olumide F et al. 1976, described metronidazole in the form of enema (2gm. of metronidazole in 200ml of normal saline) for management of severe intestinal amoebiasis21. No extensive pharmaceutical data about the formulation and stableness of the metronidazole enema is available at this moment. Hence, the development of a metronidazole enema was undertaken.The present objective of the study is to ruminate Metronidazole retention enema in a suitable aqueous media using carbopol and tragacanth as suspending and viscosifying agents. Chemical stability of the hypothesise enema is analysed with an analytical method high doing liquid chromatography (HPLC) and viscousness of the formulation is checked by Brook field viscometer.AimTo formulate a metronidazole retention enema and analyze physico-chemically.2.0. Materials and Method2.1. ChemicalsMetronidazole (98.9% pure) used is a gift sample from Thrope Laboratories (India) Ltd, Mumbai. 2-methyl-5-nitro-imidazole is used as a well-worn impureness. Cardopol (35% w/v),tragacanth, methyl paraben, propyl paraben, tris pilot film were purchased from Aldrich. The methanol and acetonitrile used were of high performance liquid chromatography (HPLC) grade along with distilled water former(a) materials namely honey oil hydrogen phthalate (KHP), sodium hydroxide (NaOH), sodium chloride (NaCl), potassium di-hydrogen phosphate (KH2PO4), acetic acid (CH3C OOH), sodium acetate (CH3COONa), sodium citrate (Na3C6H5O7) and 0.1M hydrochloric acid (HCl) were of analytical quality.2.2. orchestrationHigh Performance Liquid Chromatography apparatus and conditionsThe liquid chromatography used was a Varian Prostar HPLC system (Model 410), equipped with an auto sampler serial mode system wit h a 20l loop. Detection is accomplished with a UV-Visible detector. Integration and the system parameters were controlled by Galaxy software running on a PC.ViscometerThe viscometer used was Brook field viscometer (Model LVDV II + PRO) displays temperature (C or F), viscosity, % scales, shear rate, shear stress, speed, % torque, and spindle. Technical specifications include 0.01 to 200/rpm, viscosity order of 1 6Million cP, with 4 supplied spindles.2.3. Preparation of buffer solutions22, 232.3.1. Preparation of Phthalate bufferPhthalate buffer was prepared by dissolving 20.42g of potassium hydrogen phthalate in 1000ml of water (0.1M). The pH was familiarised with 0.1M hydrochloric acid, 0.1M sodium hydroxide. Preparation of different pH ranges of phthalate buffer is shown in the table-12.3.4. Preparation of tris bufferTris buffer was prepared by dissolving 12.11g of tris (hydroxymethyl aminomethane) in 1000ml of water (0.1M). The pH of the buffer was adjusted with 0.1M hydrochlo ric acid. Preparation of different ph ranges of tris buffer is shown in the table-4.2.4. Pre-formulation studies2.4.1. Pre-formulation solvability studiesThe main problem associated with developing any of the solution formulation of a compound is its aqueous solubility. Metronidazole is poorly aqueous soluble drug24. For enhancing the poorly aqueous solubility of drug there are several alternatives and these include pH manipulation, co-solvency, surfactants, chelating agents and emulsion formation25.In the present experiment the solubility of metronidazole, 2-methyl-5-nitro-imidazole in water, buffered solutions of phosphate, acetate and tris in various pH ranges were determined by adding a weighed amount of drug to the solvent by stir with a chalk rod at 202oC. Excess amount of solvent was added until the drug completely dissolved in that solvent.2.5. Stability Indicating HPLC studiesThe stability studies are particularly demonstrated to analyse the quality, concentration and pur ity of the pharmaceutical dosage form. For demonstrating stability of pharmaceutical dosage forms HPLC is prominently used. Szepesi et al.26 described some special stability-indicating requirements for HPLC and these include2.5.1. Stability- indicating assayThe peaks of the drug substance and its decomposition product should not elute at same condemnation any reduce in the active drug concentration should be detected by the method.Stability-indicating purityThe resolutions between the active component peak and the adjacent peak should be higher to identify any decomposition of the active component similar in its structure formed during different storage conditions.2.5.3. The main impurity peak(s) should be separated from spendthrift product peak(s) of different chemical structure, so that evaluation of purity and assay are carried out together.2.5.4. The peaks of the degraded products or secondary degraded products formed by the decomposition of by-product can also be separated f rom other peaks.2.6. Stability-indicating purityPre-formulation stability studies were conducted to determine the stability of metronidazole in water and various buffered pH ranges using reverse phase high performance liquid chromatography (HPLC).All the solutions were stored at room temperature 202oC for approximately 20 days in glass beakers and then analysed for stability.Results and DiscussionSolubility study of MetronidazoleThe aqueous solubility and pH solubility profiles for Metronidazole and 2-methyl-5-nitro-imidazole are shown in Table-7, 8, 9. Overall solubility of Metronidazole and 2-methyl-5-nitro-imidazole is determined at all pH values. Both Metronidazole and 2-methyl-5-nitro-imidazole exhibited high solubility at a pH range 4. For example, at room temperature the aqueous solubility of Metronidazole was 100mg/50ml, respectively. Metronidazole, being a weak base, appears to dissolve maximally at a pH 4.0.High performance liquid chromatography method developmentThe new method developed in this present study was very closely related to that of British Pharmacopoeia. British pharmacopoeia specifies reverse phase chromatography carried out using stainless steel column (20cm-4.6 mm) packed with octadecylsilyl silica gel of particle size 10m (spherisorb ODS), using a mixture of 30 volumes of methanol and 70 volumes of 0.01M potassium di-hydrogen orthophosphate as the restless phase with a flow rate of 1ml per minute and a detecting wavelength of 315nm.To establish a new stability indicating chromatographic nature of the HPLC method, we have changed the column to 5m C18 (150 - 4.60 mm i.d., Phenomenex) stainless steel column, packed with Sphereclone octadecylsilane (ODS) and an (eluent) mobile phase to carry out the stability analysis in the solution at ambient room temperature with a flow rate of 1.0ml/min and at a detection wave length of 325nm.Each chromatographic run required about 10 minutes and the elution time obtained for metronidazole and the standard impurity were different for different mobile phases.For methanol KH2PO4 in the volume of 3070v/v retention times of drug and the impurity was1.89min and 2.00min, for methanol KH2PO4 in the volume of 8020v/v retention times were very narrow, like wise all the mobile phase in different proportion showed a littler retention time gap between drug and impurity. When acetonitrile and sodium citrate was used in the volumes of 1090 v/v retention time gap of 2min was achieved. Elution of Metronidazole and 2-methyl-5-nitro-imidazole was achieved with a retention time of 3.19Min and 4.99Min respectively.Stability indicating solubility studiesMetronidazole was observed to be relatively stable in water and buffer pH conditions. The results obtained in our stability indicating solubility study showed an agreement with solubility and studies conducted by Yunqi et al., 200527.Test for clarity and sedimentationSedimentation was observed when the water (400C) solubilized metronidazole is s tored for 20 days, this might be due to the super saturation of the solution during solubilization of the drug at high temperature. Rest of the solutions were free from particles and sedimentation when observed against a black and white back ground.Solubility studies of metronidazole and its standard impurity (2-methyl-5-nitro-imidazole), sharpens solubility of metronidazole was more in aqueous solvents including various buffered pH systems. These solubility profiles help to understand the chemical nature (polarity) of drug and the impurity.Solubility of metronidazole was observed to be high in acetate buffer (100mg/30ml) than any other buffer solutions. Taking the solubility profiles and colonic pH (5.5-7) into consideration, acetate buffer of pH 6 was used to formulate enema4. Formulation of enemaOur puzzle to formulate retention enemas is based on the studies conducted by Nyman-Pantelidis et al., 1994. There study proved low viscous enemas superiority over high viscous enemas i n retention and colonic spread28.4.1. Preparation of metronidazole- tragacanth enemaMetronidazole-tragacanth enema was prepared by simple titration technique using motor and pestle. Metronidazole retention enema prepared was an aqueous formulation with a viscosity of 6.00 cPas, containing metronidazole in a buffered pH. The formulation includes tragacanth as suspending and viscocifying agent. Methyl paraben as a preservative and NaOH (0.1M) was used to oppose the pH of the formulation. Metrinidazole was dissolved in buffered solution and then added to tragacanth gum, while triturating methyl paraben was added and pH of the final formulation is maintained at pH-6 using NaOH. The composition of the enema prepared in the study contains 1g of Metronidazole as shown in table 9.Preparation of metronidazole- carbopol enemaFor the treatment of anaerobic infection of colon we have formulated Metronidazole as enema using carbopol, a high molecular weight polymer of acrylic acid cross linked to allyl sucrose. Carbopol increases the viscosity of the enema and so help in retaining in the colon for maximum efficacy of metronidazole29.Metronidazole was dissolved in buffered solution 1.0g/300ml. This solution was added under constant stirring to carbopol. Methyl paraben was dissolved in water and underconstant stirring this mixture was added to the suspension. The pH of the final formulation is maintained at 6 by the addition of NaOH. The composition of the enema formulated is shown below in the table-10.Storage of enemas for stability and viscosity studiesThe formulated enemas were filled in 100 ml glass beakers and stored for 2 days at refrigerated conditions 40C, room temperature 200C and at accelerated conditions 400C and analysed for stability and viscosity.Stability studies of the formulated enemasBoth the enemas were found to be stable for 3 days at various temperatures (2-100C, 2520C, 4020C). The chromatograms show no peak of degraded metronidazole. Chromatograms of both(prenominal) the enemas is shown in the fig-9, fig-10.Viscosities of the formulated enemasThe viscosity measurements for both rectal enemas were performed by Brooks field viscometer, using spindle 62 revolving at 22 rpm. Both the rectal enemas were prepared with a viscosity of 6.0 cP using tragacanth and carbopol. The viscosities of both rectal enemas were then analysed after storage for 2 days at 2-100C, 2020C, and 4020C respectively and there results are shown in table-10.There was a little variation of viscosities in the formulated enemas after their storage at various temperatures, and this variation is seen especially in enemas stored at accelerated temperature (4020C) for 3 day. Because viscosity is inversely related to temperature, as temperature increases viscosity decreases.Test for clarity and sedimentationBoth the formulated enemas were clear without any particles when observed against a black and white background. When the enemas are further analysed, phase separati on was observed in the enema formulated using tragacanth and stored at 2-100C, 2020C, and 4020C.ConclusionThe solubility studies results indicate that metronidazole showed a good solubility at various pH levels (4.0). Maximum solubility of metronidazole was shown in acetate buffer of pH 6 (100mg/30ml). The stability studies indicate that metronidazole was stable at all the pH ranges without any degradation.The Metronidazole enema formulated using tragacanth and carbopol was chemically stable with no degradation when stored for a period of 3 days at 2100C, 2020C, 4020C respectively. And there is no remarkable effect on the viscosities and pH of the enemas when stored at these temperatures.Future workIn the present study stability studies were conducted for only 3 days, the results would be more appropriate if the stability studies of the formulated enema were carried for some more days.The new methodology developed for demonstrating High Performance Liquid Chromatography indicating s tability studies using acetinitrile and sodium citrate in the volumes of 1090v/v should be validated.