Site-specific oral controlled release metformin tablets - development, in vitro, ex vivo and in vivo evaluation
V Prakya, K Vemula, K Devi, S Sonti
bioadhesion test, controlled release, in vivo studies, metformin hydrochloride, site-specific, stability studies.
V Prakya, K Vemula, K Devi, S Sonti. Site-specific oral controlled release metformin tablets - development, in vitro, ex vivo and in vivo evaluation. The Internet Journal of Pharmacology. 2009 Volume 8 Number 1.
Oral absorption of metformin is confined to the upper part of intestine posing problems in the formulation of extended release tablets. Therefore, the objective of the present study was to develop controlled-release mucoadhesive core tablets and confine the tablets to the specific site in the gastrointestinal tract. A projective coat protects the core tablets from mucoadhesion till the targeted site is reached. Once the tablet reaches the specific site, the coat dissolves exposing the core tablet for mucoadhesion. In vitro coat intactness test and ex vivo tablet bioadhesion test confirmed that the tablets were targeted and contained in the upper intestine. Further, the pharmacokinetic parameters obtained for metformin from the site-specific coated formulation were better (P<0.05) than that of the non-site-specific uncoated formulation in the in vivo studies. Standardized formulation was stable during the stability studies conducted as per ICH Q1C guidelines.
Metformin is a widely used biguanide anti-diabetic drug for the management of non-insulin dependent diabetes mellitus. Oral absorption of Metformin is confined to the upper part of the intestine, i.e., the duodenum, jejunum and to a lesser extent ileum1. Therefore, the bioavailability of this drug even from aqueous solution or rapidly dissolving tablets is relatively low2. A pharmacokinetic-pharmacodynamic rationale for development of metformin controlled release formulations was established and concluded that clinical advantage could be obtained from gastro-retentive systems3. However, only a 15% increase in bioavailability was produced by gastro-retentive swelling tablets based on high molecular weight polyethylene oxide compared to the currently marketed immediate release products4. In yet another study, compressed matrix tablets were prepared using pH-sensitive polyethylene oxide-eudragit-L100 compounds by a co-evaporation process, which gave a gradual and complete release of metformin from stomach to jejunum without getting affected by gastric pH fluctuations5. But the drug release was limited to only 4 hours with such a formulation. Hence to optimize oral metformin therapy, there is a need for development of metformin tablets, which confine to specific site in the upper intestine. Therefore, in the present study, an attempt was made to develop oral mucoadhesive controlled release metformin hydrochloride (MH) tablets using hydroxypropylmethyl cellulose (HPMC). These mucoadhesive tablets were intended to target the upper part of the intestine by pH sensitive polymer coating with eudragit-L100. As eudragit-L100 is soluble only above pH 6, coat dissolves upon reaching upper intestine and releases the core tablet to adhere at the specific site thus facilitating drug release for an extended duration of time.
Materials And Methods
MH was supplied as a gift sample by Micro Labs Ltd., Hosur, India and eudragit-L100 by Degussa, Goa, India. All other materials used were of analytical grade and were obtained from s.d.fine-chemicals limited, Mumbai-25, India. Animal studies were conducted in accordance to the Institutional Animal Ethics Committee of Al-Ameen College of Pharmacy (Ref. No: AACP/P-05; Date: 07/07/2002), Bangalore, India.
Method of preparation of the mucoadhesive core tablets
Metformin controlled release mucoadhesive core tablets each containing 500mg dose of drug were prepared by conventional wet granulation as well as by direct compression methods. Polyvinylpyrrolidone–K30D (PVP-K30D) was used as a binding agent and microcrystalline cellulose (MCC) was included as filler, both of which improve the compressibility of MH. Colloidal silicon dioxide and magnesium stearate were used as lubricants.
In case of wet granulation method, the drug MH, 30% of total quantity of the HPMCs and other ingredients except colloidal silicon dioxide and talc were blended together thoroughly after passing through 60 mesh. The powder blend was wetted with 95% alcohol solution and granulated using 16 mesh. The granules were then dried at 500C ± 5°C in an oven till the moisture content fell to 2% level (moisture content was determined by using HR73 halogen moisture analyzer, Mettler, Toledo). After drying, the dried granules were regranulated using 22 mesh and the granules were mixed with the remaining quantity of the HPMC mixture along with the lubricants. In case of direct compression method, all the ingredients were blended thoroughly after passing each ingredient through 60 mesh and compressed into tablets to maximum hardness. The hardness of the tablets formulated by both the methods ranged from 2 to 6 Kg/cm2. The tablets were compressed using 13mm normal concave punches to a constant pressure of 6 tons for 10 seconds in a single punch tablet compression machine (Cadmach, Ahmedabad, India).
Evaluation of the mucoadhesive core tablets
The properties of the core matrix tablets, such as hardness, friability and weight variation were determined as per USP 24 & NF 19. Briefly, for each batch, hardness was determined by using Pfizer hardness tester (12061 [USP], Secor, India). Friability was determined using friability testing apparatus (EF-2 friabilator [USP], Electrolab, India). Weight variation of tablets was determined as per official procedure on randomly selected 20 tablets.
Uniformity of drug content in the tablets was determined on 10 randomly selected samples, which were crushed independently in a mortar. The drug equivalent to 400 mg was weighed into 100mL volumetric flasks and the volume was made up with water. After filtration through 0.45 µm membrane filter and further dilutions with water, the samples were analyzed using UV double beam spectrophotometer (Pharmaspec UV-1700, Shimadzu, Japan) at 233nm and drug content was calculated taking 806 as the value of A (1%, 1 cm) at the maximum at 233nm as per BP 2001.
Ex vivo bioadhesion strength measurement
Bioadhesive strength was assessed in terms of the weight in grams required to detach the tablet from the membrane. Bioadhesive strength of the formulations was studied using freshly excised mucosal membrane of the upper intestine of the pig. The mucosal membrane was equilibrated at 37°C ± 1°C for 30 minutes in pH 6.8 buffer solution before the bioadhesion evaluation study. A modified balance was used to study the bioadhesive strength of the tablets6, 7. The left pan of a chemical balance was removed and a string with a cylindrical shaped base was hung from the left side pan hook. A tablet was fixed to the bottom portion of the cylindrical shaped base with ‘feviquick’ glue. The string with tablet was hung in such a way that the tablet was just in contact with the surface of the mucosal side of the upper intestinal portion of the pig when the balance was in a balanced position. The intestinal mucosa was tied to the cylindrical wooden base fixed on the inside base of a glass beaker containing pH 6.8 buffer. Both sides of the balance were balanced by hanging required weight to the left side pan hook. The balance was left in a balanced position for fixed time of 5 minutes and then slowly weights were increased on the right pan till the tablet detaches from the surface of the intestinal mucosa. The weights on right side pan gave the mucoadhesive strength of the tablet in grams.
In vitro dissolution studies
The drug release profiles from the formulated core tablets was studied using dissolution apparatus I (TDT-06T, Electrolab, India) in 900mL of 0.68% w/v solution of potassium dihydrogen orthophosphate adjusted to pH 6.8 by the addition of 1M sodium hydroxide as per BP 2001 and rotating the basket at 50 rpm. The temperature of the dissolution medium was maintained at 37
Development and standardization of tablet coat formula
Various coat formulas were developed using pH sensitive eudragit-L100 polymer along with other additives. Crospovidone was used as a channeling agent, triethyl citrate (TEC) was used as plasticizer and erythrocin was used as the coloring agent. Isopropyl alcohol was the solvent whereas titanium dioxide was used as an opaquant.
Method of preparation of coat solution
Eudragit-L100 and TEC were added to 3/4th of the total volume of isopropyl alcohol and stirred at 35 rpm using Eltek motor stirrer for ½ hour till the solution was clear. Talc, titanium dioxide, crospovidone and coloring agent were triturated thoroughly in a mortar and added to the above solution and stirring continued further. Finally, the volume was made up with isopropyl alcohol.
Coating of the mucoadhesive core tablets
Coating was carried out using 1 kilogram of core tablets in Neocota 12inch (Smart Coat) at an inlet temperature of 50C and outlet temperature of 40C with a pan rotation of 15 rpm. The spray rate of coating solution was 10ml/min. Distance between the sprayer and the tablet bed was 30cm.
Evaluation of the film coated tablets
Physicochemical characterisation and disintegration test
The film coated tablets were characterized for all the physicochemical parameters in the similar way as that described under the core tablets. Disintegration test
In vitro dissolution studies
The drug release profiles from the coated tablets was studied in the similar way as that described under core tablets in 900mL of various simulated gastrointestinal fluids viz., in 0.1N HCl (pH 1.2) for the first 2 hours, pH 4.5 phosphate buffer for the next 1 hour and finally in pH 6.8 phosphate buffer till 100% of the drug was released9, 10.
In vivo studies
12 male albino Newzealand rabbits of average weight 2.5+/-0.010kg were used for the study. The rabbits were divided into 2 groups of 6 rabbits each (n=6). All the rabbits were fasted overnight with ad libitum access to water. One group of the animals received coated tablets whereas the other group received uncoated core tablets as reference. The order of administration was randomly selected. The tablets were administered through oral wooden gag with a central opening of 9mm diameter. The tablet was placed deep into the throat through the opening and immediately 20mL of water was administered by syringe to facilitate swallowing of the tablet intact and to prevent it from sticking to the animal’s throat. Aliquot blood samples were collected using 27 gauge needle from the marginal ear vein into heparinized tubes at time intervals of 0, 1, 2, 3,4, 5, 6, 8, 10, 12 and 24 hours. Xylene was applied to the shaved marginal ear vein, which causes blood vessel to dilate. The blood was immediately centrifuged at 6000 rpm for 10 minutes to separate the plasma and stored at –200C until analysis. The samples were analysed using micro titer plate reader and the drug concentration was determined from the calibration curve.
Determination of MH in the plasma by micro titer plate reader
A stock solution was prepared by dissolving 10mg of MH in water and various dilutions in the same solvent were made to get working standards of concentrations 10 to 100ng/50mcL. Plasma solutions were prepared by spiking various volumes of the working standards of the drug into the healthy plasma.
Drug extraction procedure from plasma
200mcL volumes of spiked plasma samples were transferred to effendorf tubes. Extraction was performed by adding 100mcL of 8M sodium hydroxide and 1.3mL of 1-butanol/n-hexane (50:50, v/v) to the tubes and shaking for 2 minutes. After centrifugation at 11300 rpm for 2 minutes, the whole organic layer was separated and transferred into another tube. To the organic layer, 100mcL of 1% acetic acid was added. After the mixture was vortex-mixed and centrifuged for 2 minutes, the organic phase was discarded and the aqueous phase was analysed for MH content11 at 233nm using micro titer plate reader against the blank and a calibration curve was constructed.
Stability studies were conducted on the standardized formulation. The standardized formulation was strip packed in Alupoly strip of 0.04mm thickness and was exposed to 400C ± 20C / 75% ± 5% RH and 300C±20C/65%±5% RH as per ICH guidelines12 Q1C: “Stability testing of new dosage forms.” Sampling was done at predetermined time intervals of 0, 90 and 180 days. The tablets were evaluated for various physico-chemical parameters viz., appearance, drug content, hardness, and in vitro drug release profiles. To confirm the similarity of drug release profiles before and after stability studies, a model-independent statistical tool for comparison of dissolution profile “
The results were analyzed by student’s t-test using Graph Pad Prism software (Version 3.02). A difference below the probability level of 0.05 was considered statistically significant.
Formulation development and evaluation of core tablets
Various batches of mucoadhesive core tablets from F-1 to F-4 were developed as indicated in Table 1 by both wet granulation and direct compression method.
The physico-chemical properties of all the four formulations F-1 to F-4 were evaluated as per official procedures the results of which are given in the Table 2.
Ex vivo bioadhesive strength of formulations was analysed using a modified balance. The in vitro drug release profiles of the selected 3 core formulations are shown in the Figure 1.
Coat formula development and evaluation of coated tablets
Various coat solution formulas were developed as shown in the Table 3.
Out of the various coat formulas developed, the best coat formula was chosen for coating the standardized core formulation. Coated tablet samples were collected at various weight gains of the initial total tablet weight to get formulations CF-1, CF-2 and CF-3. All the three formulations were evaluated for various physicochemical parameters including DT.
In vitro dissolution studies were carried out in various simulated ascending GI fluids to find out the drug release profiles from all the batches of the coated formulations. The drug release profiles obtained are illustrated in the Figure 2.
In vivo and stability studies
The pharmacokinetic parameters obtained with the most satisfactory coated formulation from the in vivo studies in rabbits was compared to that of the uncoated core formulation. Plasma concentration-time profiles obtained after oral administration of coated and uncoated formulations are illustrated in Figure 3.
The pharmacokinetic parameters derived from the plasma data are presented in Table 4.
Each value represents the mean ±S.D. for 6 rabbits.
Finally, stability studies were performed on the most satisfactory coated formulation.
Formulation development and evaluation of core tablets
Various batches of mucoadhesive core tablets from F-1 to F-4 were developed by both wet granulation and direct compression methods as the methods of preparation for comparison. A combination of various viscosity grades of HPMCs viz., HPMC K4M, HPMC K15M and HPMC K100M in a ratio of 1:1:1 was incorporated in the formulations to achieve formulations with optimum physicochemical properties. The average weight of the formulation F-1 was 830mg, formulations F-2 and F-3 were 840mg, whereas formulation F-4 was 850mg. All the formulations were punched using 13mm round normal concave punches to a pressure of 6 tons for 10 seconds.
The appearance of all the formulations prepared by both the methods were satisfactory whereas hardness values of the tablets prepared by direct compression method were lower with consequent higher friability values compared to the formulations prepared by wet granulation method. Therefore, it was concluded that wet granulation method improved the physico-chemical properties like increase in hardness and decrease in friability values of the tablets suitable for coating compared to direct compression method. The average percentage deviation of 20 tablets of each formula was within ±5%, which provided good weight uniformity as per USP 24 & NF 19 requirements. In all the formulations, the assay for drug content was found to be uniform among different batches of the dosage form and met official requirements. Only 30% of the total quantity of HPMC mixture was used during granulation stage of wet granulation method while the rest of the quantity was incorporated during lubrication stage to improve the physicochemical properties of the tablets.
Hardness of the formulations improved with the incorporation of binding agent as seen Formulations F-2 and F-3 prepared by both wet and direct compression methods differ only in the type of binding agent used. It was observed that with the formulation F-3, which contained PVP-K30D as the binding agent, hardness increased and friability value decreased when compared with formulation F-2 (p<0.05), which contained pregelatinised starch (PGS) as the binding agent at the same concentration. Hardness and friability values further improved with increase in the concentration of the binding agent, as is seen with formulation F-4, prepared by both the methods (p<0.05). Weight variation of all the formulations was within ±5% as per official requirements of the tablets.
Out of the various formulations of mucoadhesive core tablets developed, only formulations F-3 and F-4 prepared by wet granulation method and formulation F-4 prepared by direct compression technique were taken up for carrying out further studies viz., ex vivo bioadhesion strength measurement and in vitro dissolution studies as they possessed maximum hardness of 5.5(
The bioadhesive strength of formulations F-3 and F-4 prepared by wet granulation method and formulation F-4 prepared by direct compression technique was found to be 34.75(
During the in vitro drug release studies, formulations F-3 and F-4 prepared by wet granulation technique released drug for about 8 hours (R2=0.9531) and 9 hours (R2=0.9567) respectively whereas it was 7 hours (R2=0.9614) with formulation F-4 prepared by direct compression method. As the formulation F-4 prepared by wet granulation method possessed maximum bioadhesion strength and longer duration of drug release with required burst release, only that formulation was chosen for coating.
Coat formula development and evaluation of coated tablets
Out of the various coat solution formulas developed, as formula C-3 gave a better coat in terms of texture during trial coating of the placebo tablets, it was chosen for coating the standardized core formulation F-4 prepared by wet granulation method. Coated tablet samples of formulation F-4 were collected when the weight gain was at 3%, 4% and 5% level of the initial total tablet weight and were coded as CF-1, CF-2 and CF-3 respectively.
All the batches of the coated formulations CF-1 to CF-3 possessed satisfactory appearance along with other required physico-chemical characters. Hardness indicates strength of the tablets and the results obtained were found to be 7.23kg/sq.cm (±0.058), 7.4kg/sq.cm
The pH of the human gastrointestinal tract (GIT) increases progressively from the stomach (pH 2-3), small intestine (pH 6.5-7) to the colon. The coat was proposed to protect the core tablet from mucoadhesion till it reaches the upper part of the small intestine from where the bioavailability of MH is maximum. Therefore, DT was carried out on all the coated formulations from CF-1 to CF-3 to find out the coat intactness in various simulated ascending GI fluids of the upper GIT as the formulations were site specific dosage forms coated with a pH sensitive polymer, eudragit-L100, which is soluble only in a buffer of pH-6 and above. The coat was expected to expose the core tablet during the first hour of the studies in pH-6.8 buffer after 2 hours and 1 hour studies in pH 1.2 and pH 4.5 buffers respectively. The coat was intact during the initial 2 hours in pH-1.2 and next 1 hour in pH-4.5 buffers, whereas it dissolved completely in pH-6.8 buffer during the 4th hour of the studies with all the formulations from CF-1 to CF-3. This ensures that the tablet is available for mucoadhesion only at the specific site in the GIT. Since all the coated formulations from CF-1 to CF-3 possessed the required physico-chemical parameters and passed DT, dissolution studies were carried out on the same.
In vitro dissolution studies were carried out in various simulated ascending GI fluids as described under DT to find out the drug release profiles from the coated formulations. There was a complete and uniform drug release from formulation CF-1 (r2=0.9886) compared to formulations CF-2 (r2=0.9862) and CF-3 (r2=0.9821), which possessed minimum coat thickness. Once coating had dissolved completely in pH-6.8 buffer, there was a sharp increase in the drug release profiles from all the developed formulations. The drug release during the initial 3 hours could be due to the diffusion of the drug through the pores created in the coat by crospovidone, which swells in the presence of body fluids14. Even though the drug release was lower during the first 3 hours, which may be due to the presence of coat around the core tablet, the amount of the drug released met the required therapeutic levels15 only from the formulation CF-1.
As the formulation CF-1 possessed the required physicochemical properties, passed DT for site specificity and released therapeutic concentrations of drug from the 1st hour onwards, it was chosen as the standardized formulation, and further studies like in vivo studies in rabbits and stability studies were carried out on the same.
In vivo studies
The pharmacokinetic parameters obtained with the most satisfactory coated formulation CF-1 from the in vivo studies in rabbits was compared to that of the uncoated core formulation F-4. Since the experimental animals chosen were rabbits for in vivo studies, the dose and therefore the size of the tablets were reduced. CR mucoadhesive core tablets of about 170mg weight containing 100mg of barium sulphate instead of drug were prepared and film coated. The method of preparation of the formulations, composition of the core tablets, coat formula used, method of coating and coat weight gain of the tablets represented the actual formulations. Core tablets were punched using 7mm round normal concave punches to a constant pressure of 3 tons.
Drug in plasma was estimated using micro titer plate reader. A calibration curve was constructed from concentrations 10ng/50mcL to 100ng/50mcL (r2=0.9980) in water at 233nm as per BP 2001. The average percentage recovery of MH from plasma was found to be 94.52% (±0.9714).
Plasma concentration-time profiles of the coated and uncoated formulations were obtained to derive pharmacokinetic parameters. In case of MH, even though the plasma levels from the coated tablets was slower during the initial 3 hours, it was faster and higher there after leading to a steady state of drug release than those achieved with uncoated tablets. A sharp increase in plasma MH levels between 2nd and 3th hour (tmax was 2.45 hours) with a steady plasma levels there after for an extended duration clearly indicated that the coat had targeted and confined the CR mucoadhesive core tablets to the specific site in the GIT from where the bioavailability of metformin is maximum. The AUC0-24 and AUC0-α of the coated tablets were 1.39 and 1.44 folds greater than that of the uncoated tablets respectively.
In addition, the Cmax value [1.201mcg/mL (±0.03012)] of coated tablets was 1.32 times greater than that of the uncoated tablets [0.911mcg/mL (±0.0895)]. This enhancement in the values of AUCs and Cmax of the coated tablets compared to the uncoated tablets can be attributed to the site specific delivery of MH. This could also be the reason for the extended t1/2 of coated tablets [9.32 hours (±1.9516)] compared to uncoated tablets [6.43 hours (±1.3222)] with reduced Ke of coated tablets [0.083 hour-1 (±0.0224)] compared to uncoated tablets [0.119 hour-1 (±0.0321)]. Especially, the appearance of peak levels of the drug in plasma was rapid from coated tablets with Tmax at 2.45 hours (±0.6732) whereas it was 5.15 hours (±0.5468) with uncoated tablets. The derived pharmacokinetic parameters were further subjected to statistical analysis by unpaired two-tailed t-test, which showed that there was significant difference (p<0.05) in all the derived parameters between the coated and uncoated tablets. Thus, the pharmacokinetic parameters of coated tablets were found to have improved as they were site-specific compared to the non-site specific uncoated tablets for the absorption window limited drug MH.
Stability studies were performed under accelerated storage conditions as per ICH guidelines Q1C on the most satisfactory formulation CF-1 to find out the effect of various temperature and humidity conditions on the formulation. There was no change in the appearance of the formulation at all temperature and humidity conditions during the course of the study. There was a slight decrease in the hardness values during the stability studies, which was statistically insignificant (p>0.05) when subjected to one-way ANOVA analysis and the drug content was found to be within the official limits. The calculated f2 values obtained from in vitro drugs release profiles of the formulation CF-1 before stability studies versus after stability studies at 300C±20C/65%±5%RH and 400C±20C/75%±5%RH were 93.21 and 75.12 respectively. These findings suggested that the in vitro drug release profiles investigated were therefore similar.
It can therefore be concluded that the standardized formulation CF-1 satisfied the physico-chemical parameters, in vitro and in vivo drug release profile requirements for a site-specific oral controlled release dosage form of metformin. Thus, oral controlled release of metformin can be standardized by ensuring that optimum amount of drug releases at the specific site in GIT from where it is absorbed maximum.