INTRODUCTION:

The oral route of drug administration is the most common route of delivering active pharmaceutical ingredients (APIs) to the human body. In comparison with other routes of drug administration, the oral route is considered the safest, simplest and most convenient ⁽¹⁾. Tablet is still most popular conventional dosage forms existing today because of ease in self-administration, compact size, easy of manufacturing and it can be delivered in accurate dose. Following administration of a tablet it disintegrates into aggregates/granules, followed by disaggregation to form a suspension of fine API particles. Only then can the API dissolve before being absorbed systemically. One important drawback of solid dosage forms is the difficulty in swallowing (dysphagia) it has been reported that around one third of patients in long term care facilities experience difficulties with swallowing solid oral dosage forms^(2,3,4).

Fast dissolving tablets (FDTs) which are also referred to as orodispersible and fast disintegrating tablets, are tablets which when placed in the mouth; disperse/disintegrate rapidly before being swallowed, due to the action of saliva. Many methods are used to prepare FDTs like tablet molding, tablet loading, direct compression, lyophilization etc. Most FDT products available in the market are manufactured by lyophilization due to fast disintegration time and excellent surface characteristics they provide. The distinct advantage of FDTs is that they are products which are designed to disintegrate or dissolve rapidly on contact with saliva, thus eliminating the need to chew the tablet, swallow an intact tablet, or take the tablet with liquids. FDTs have some downsides as well, they are mostly porous in nature and therefore, have high friability values, hence they require care while handling and sometimes special packaging is also required. Formulation of FDTs must be done carefully because unless properly formulated they leave unpleasant taste or grittiness in mouth⁽⁵⁾. Lyopan® is a patented FDT technology designed by a Swiss company called ‘Pantek’. It involves filling of the component mixture in the empty blisters and freeze drying them to yield FDTs.

Amlodipine besilate (AB), (RS)-3-ethyl 5-Methyl2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-1, 4-dihydropyridine-3,5-dicarboxylate benzene sulphonate, is a potent calcium channel blocker belonging to BCS II drugs. Its free base form as well as besilate salt form is used as APIs in antihypertensive formulations. Amlodipine besilate is inherently permeable through biological membranes and has oral bioavailability of 60-70%⁽⁶⁾.

Present study is aimed at using ‘Lyopan®’ FDT technology to increase the bioavailability of a model antihypertensive drug mentioned above. FDT technology increases the solubility of the drug thereby increasing its bioavailability in turn. In the present study gelatine and mannitol are used in formulation other than API. Gelatin was used as binder while mannitol was used as bulking agent. Prepared tablets were subjected to physical testing and their physicochemical properties were evaluated using DSC, XRD, SEM, etc.

MATERIALS AND METHODS:

Materials

Amlodipine besilate was provided by JB chemicals, Mumbai. Gelatin was obtained from Loba Chemie Pvt. Ltd. Mumbai. Mannitol was obtained from Central Drug House, Mumbai. Water used was double distilled and all the other chemicals used were of analytical grade.

Estimation by RP-HPLC Method

The RP-HPLC method was selected for quantitative estimation of AB from the prepared FDT formulation. The chromatographic variables such as mobile phase, flow rate and solvent ratio were studied and the resulting chromatograms were recorded. The chromatographic system used was Shimadzu Corporation, Tokyo, Japan, UV/VIS detector (UV 2075 plus, Jasco UV-1575). The stationary phase was KYA TECH HIQ Sil C18 column (250 nm x 4.6 mm, 5 μm). The mobile phase used was acetonitrile: methanol: buffer (15:35:50). The standard solution was 10 μg/mL in strength. The sample size and flow rate were 20 μL and 1 mL/min, respectively. The samples were analysed at 237 nm.The mobile phase containing acetonitrile: methanol: buffer (15:35:50) was optimised, (where buffer is a solution prepared by dissolving 7.0 ml of triethylamine in 1000 ml of water and pH adjusted to 3 with phosphoric acid) for proper elution of Amlodipine besylate on the system as specified above. The mobile phase was filtered through 100μ nylon filter and degassed ultrasonically for 20 min⁽⁷⁾.

Into series of 10 mL volumetric flasks, 0, 2, 4, 6, 8, 10 mL (10μg/mL) solutions of AB was added and the final volume was made up to 10 mL by addition of mobile phase. A 20 μL of sample solution was injected into the injection port of chromatographic system having fixed volume loop injector. Chromatograms were noted and area under curve was plotted against concentration to get calibration curve.

Investigation of Formulation Variables

FDTs were prepared using mannitol as sugar alcohol, gelatin as matrix former and AB as active pharmaceutical ingredient. The aqueous systems consist of gelatin and mannitol dissolved in water. The batches were optimized by optimising the concentration of gelatin and mannitol in the formulation.

Optimization of matrix former

The formulation mixture for preparation of FDTs was prepared in a series of mixtures containing 5mg AB + gelatin + 5% w/v mannitol. Aliquots of the aqueous solution of gelatin were combined with 1%w/v, 1.1%w/v, 1.2%w/v, 1.3%w/v, 1.35%w/v and 1.4%w/v gelatin to produce solutions as given in Table 2. Accurately measured 2 mL aliquots of the solutions were filled into clean and labelled blisters. The blisters were kept for deep freezing at -40°C and lyophilization was carried out (Temp. -40°C and pressure 0.016 mBar).

Optimization of Mannitol Concentration

The formulation mixtures for preparation of FDTs were prepared in a series of mixtures containing 5mg AB + 1.3 % gelatin + mannitol. Aliquots of the aqueous solution of gelatin were combined with 4.5%w/v, 5%w/v and 5.5%w/v gelatin to produce solutions as given in Table 1.Accurately measured 2 mL aliquots of the solutions were filled into clean and labelled blisters. The blisters were kept for deep freezing at -40°C and Lyophilization was carried out. (Temp. -40°C and pressure 0.016 mBar).

Table 1: Optimization of gelatine and mannitol concentration

Formula	Gelatine (%w/v)	Mannitol (%w/v)	Amlodipine besylate (mg)
F1	1	5	5
F2	1.1	5	5
F3	1.2	5	5
F4	1.3	5	5
F5	1.35	5	5
F6	1.4	5	5
F7	1.3	4	5
F8	1.3	4.5	5
F9	1.3	5.5	5

Evaluation of FDTs

For FDTs, evaluation parameters of tablets mentioned in the Pharmacopoeias need to be assessed, along with some special tests.

Weight variation

A total 20 tablets were randomly selected from each formula and weighed individually to calculate weight variation.

Tablet hardness

The breaking strength of tablet was determined by using conventional hardness testers (Monsanto tablet hardness tester). The limit of hardness for the FDT is usually kept in a lower range to facilitate early disintegration in the mouth. The mean hardness in Kg (±SD) for 10 tablets was determined.

Mechanical Strength

The FDTs should possess adequate mechanical strength to bear shocks during handling in manufacturing, packaging and shipping. Thus, two important parameters like crushing strength and friability is used to determine mechanical strength of tablet⁽⁹⁾.

Crushing strength or tablet tensile strength

The force required to break a tablet by compression in the radial direction is called crushing strength. Excessive crushing strength significantly reduces the disintegration time of tablets. The crushing strength of the tablet was measured by using Pfizer hardness testers. Tensile strength for crushing (T) is calculated using equation

T= 2F / π × d × t ...... (1)

Where F is the crushing load, and d and t denote the diameter and thickness of the tablet respectively⁽¹⁰⁾.

Tablet friability

To achieve % friability within limits (0.1-0.9℅) for an FDT is a challenge for a formulator since all methods of manufacturing of FDT are responsible for increasing the % friability values. Friability of each batch was measure in “Electro lab friabilator”. Ten pre-weighed tablets were rotated at 25 rpm for 4 min or total 100 revolutions, the tablets were then reweighed and the percentage of weight loss was calculated.

Modified Disintegration Test

A fast dissolving tablet has disintegration time of about 30 secs, therefore it becomes rather tedious to evaluate by conventional methods. Hence modified disintegration test for FDTs is performed. A petridish (10cm diameter) was filled with 10 ml of water. The tablet was carefully put in the center of petridish and the time required for the tablet to completely disintegrate into fine particles with no palpable mass in petri dish was measured in seconds.

Residual Moisture

Fast dissolving tablets contain sugars and other natural ingredients which degrade in presence of moisture. It also affects the mechanical strength of FDTs. Therefore, it becomes necessary to calculate the moisture content and also assure that it should be below the limits. Moisture analysis of dried products was performed using a Karl Fischer Titrator (Vigo – Matic M.D.). Accurately 20mL of anhydrous methanol was transferred to the titration vessel and titrated to the end point. A sample of 10μl of water, accurately measured, was used to standardize the Karl Fischer reagent. Accurately weighed samples were suspended in anhydrous methanol and titration was carried out to the electromagnetic end point⁽¹⁰⁾.

Content Uniformity

AB content in the tablets under study was determined using reverse phase high performance liquid chromatogram (RP-HPLC) method. The AB content in freshly prepared known concentration solution was determined. The AB content in the tablets was measured relative to amount of AB added to solutions and each sample was assayed in triplicate. The tablet samples were reconstituted with distilled water to obtain AB concentrations of 50µg/mL and analyzed by HPLC for AB concentration. The HPLC system (Shimadzu Corp., Tokyo, Japan) consisted of UV/VIS detector (UV 2075 plus) covering the range of 200-400nm and interfaced to a computer for data acquisition. A 20μL of sample was injected at a flow rate of 1 ml/min and UV detection was carried out at λmax237nm.

Differential Scanning Calorimetry

DSC studies were performed to determine the glass transition temperature (T_g) of the formulation to allow proper design of the process and to investigate their stability by using a Model 821 DSC (Mettler Toledo). Samples of 1.5–7mg were analyzed in crimped, vented aluminum pans under a dry nitrogen purge with an automated liquid nitrogen-cooling accessory. Samples were heated from 25 to 400°C with a scanning rate of 10°C/min.

FTIR Analysis

FTIR analysis involves detection of vibration frequencies of bonds in the compound after bombarding them with IR rays. The specific vibration frequencies denote specific functional groups, and thus the FTIR spectrum represents the structure of the compound. The pellets for FTIR analysis were prepared by mixing tablet powder with potassium bromide. The spectra were recorded immediately. Care was taken to minimize sample exposure to moisture. The FTIR absorption measurements were recorded (2000–400cm-1) at room temperature using a Jasco V530 spectrometer, and analyzed using Origin8^® software (Origin Lab Corporation, USA).

X-ray Diffraction Analysis

XRPD analysis involves bombardment of x-rays on the sample and from the pattern in which they interact with it i.e. either refraction or reflection a spectrum is obtained. From the nature of the spectrum it can be predicted whether the sample is crystalline or amorphous. The XRPD analysis of the tablet was performed using X-Ray Diffractometer (PW 3710, Philips, Netherlands). Powdered product samples were scanned between 2°–100°2θ at a scan speed of 0.1°2θ/sec using 1.524Å radiations. The XRPD patterns were recorded and analyzed.

Scanning Electron Microscopy

Surface topography and amorphous state structure of product was observed by using scanning electron microscopy (SEM) JSM-6360 (JEOL Inc., Japan). The prepared tablet was fixed on SEM stub using double-sided adhesive tape and coated with Au at 20 mA for 6 min through a sputter-coater (Ion sputter JFC 1100). A scanning electron microscope with a secondary electron detector was used to obtain digital images of the samples at an accelerating voltage of 15 kV. ⁽¹¹⁾.

Short term storage stability testing

Short-term stability testing involves storing the product under various temperature and pressure conditions for a short period of time. The condition of products is evaluated after the period is over and from the results obtained stability of the sample at those conditions is determined. The optimized tablets were subjected to short-term stability studies as per ICH stability testing guidelines for biologicals. A set of 10 tablets was vacuum-sealed and stored at 2–8°Cand 40°C/75% RH for 3 months. Products under stability testing were analyzed at specific time interval. The samples were evaluated for its appearance, residual moisture content and drug content.

RESULT AND DISCUSSION:

Estimation of AB

The AB showed retention time of 2.12 min with good resolution and good peak shape. The HPLC chromatogram of pure AB (10μg/mL) and tablet formulation (10μg/mL; drug with formulation components) in acetonitrile: water: buffer (15:35:50) with retention time is shown in Fig. 1. The chromatographic conditions were optimized to provide a good performance of the assay. A five-point calibration curve was constructed with working standards and was found linear for AB over its calibration range. At wavelength 237nm none of the component of the proposed formulation interferes with the analysis of AB. Content of pure AB was found to be 98.5% with ±0.15 of SD.

Figure 1. HPLC chromatogram of 10μg/mL AB (physical mixture of drug with formulation components) in mobile phase acetonitrile: water: buffer (15:35:50)

Investigation of Formulation Variables

Optimization of matrix former

Gelatin is the main excipient used as a binder or matrix former in the formulation of lyophilized orally disintegrating tablets. Gelatin is an excellent example of solution binders i.e. they are dissolved in solvents before being used. At 40^°C it dissolves and forms a sticky solution, which on cooling to room temperature sets into jelly.

Optimization of sugar mannitol

In freeze dried formulations, excipients are mainly included to improve the functional properties and stability of the lyophilized product. These excipients are required to have regulatory acceptance as these formulations are usually meant to be taken parentally.

Research efforts in the last two decades have shown that sugars can provide significant protection to ensure storage. In addition, sugars are proven to be excellent bulking agents in case the dose of selected drug is very low. The main purpose of the bulking agent is to provide a dried matrix in which active pharmaceutical ingredient can dispersed and it offers support to prevent cake collapse even if the amorphous phase is being dried above its collapse temperature. In the present study Mannitol has been used as a bulking agent mainly due to its high water-soluble property. It also co acts as cryoprotectant i.e. it prevents damage to drug molecule from ice crystals.

Table 2:Formula of Amlodipine ODTs by lyophilization process, using a combination of optimized gelatin and mannitol as matrix with various concentrations.

Formulation code	Gelatin (mg)	Mannitol (mg)	Amlodipine besylate (mg)
F1	20	100	5
F2	21	100	5
F3	22	100	5
F4	23	100	5
F5	24	100	5
F6	25	100	5
F7	26	100	5
F8	27	100	5
F9	27	80	5
F10	27	90	5
F11	27	110	5

EVALUATION OF TABLETS

Weight variation

Weight variation test would be a satisfactory method to judge the drug content uniformity of the formulation. The test was carried out by weighing 20 tablets individually, and then taking the average weight, and comparing the individual weight to the average. The weight variation data of all the batches is graphically represented in Fig.2. Average weight of tablets in batch F8 is 39.89 mg and if we calculate according to IP limits, 19 out of 20 tablets fit between the limit of ± 10% of average weight. This proves that the batch passes the weight variation test. Thus, Batch F8 was selected as the best batch and evaluated further.

Figure 2: Graphical representation of weight variation of F8 FDT.

Mechanical Strength

Two important parameters i.e. crushing strength and friability is used to determine mechanical strength of tablet.

Tablet hardness

The resistance of tablets for capping, abrasion or breakage under conditions of storage, transportation and handling before usage depends on its hardness. Tablet hardness is defined as the load required for crushing or fracture of tablet when placed on its edge. Sometimes it is also termed as tablet crushing strength.

The hardness of tablets of all the batches are from 2 -4. Formulation with higher concentration of gelatin leads to increase the hardness due to formation of rigid three-dimensional networks after freeze drying as presence of high number of gelatin fibers leading to form cross links and inter chain H-bonds, which increase overall hardness of the tablets. The crushing strength of all tablets formulation is shown in Fig. 3.

Figure 3: Hardness of all amlodipine FDTs.

Friability test

The friability of the tablets exceeds the limit of 1% given for tablet dosage form, but as FDTs are highly porous in nature and their mechanical strength also tends to be on the lower side there is need to define special limits for FDTs and therefore special research should be done in this area. The batch F8 showed lowest friability of 4.8% shown in Fig. 4. Due to presence of higher concentration of gelatin in batch F8, tablets were free from cracks and were not broken after tumbling. As tablets exceed the limit, it indicates that they are fragile and could not be easily handled.

Figure 4: Percentage friability of all amlodipine FDTs.

Modified Disintegration Test

Freeze dried tablets are known for their shortest disintegration time (as quick as 10 seconds). This rapid disintegration assists swallowing and plays a vital role in drug absorption in buccal cavity, which helps in promoting bioavailability. Short disintegration time of these formulations is indicative of the highly porous nature of the tablet matrix. This may be due to faster uptake of water owing to the porous structure formed by gelatin, thus facilitates faster disintegration with improving dissolution. Batch with the lowest disintegration time was selected as best, i.e. batches F4 and F7 shown in Fig. 5.

Figure 5: Disintegration time of all amlodipine FDTs.

Residual Moisture

For a product prepared by lyophilization, residual moisture is a crucial component of the evaluation parameters. Presence of moisture in the Lyophilized product indicates inappropriate storage conditions, and often coincides with low quality product. Moisture also promotes bacterial growth which makes it undesirable in a pharmaceutical product. Usually Moisture content is measured with the help of Karl- Fischer titration. In the present study, the moisture contents of the optimized batches were calculated. According to USFDA limits moisture content in a lyophilized product must be below 5%, while ICH guidelines dictate the same to be below 3%. The average moisture content of the optimized batches by Karl- Fischer titration method was found to be 2.9%.

DSC Analysis

Differential scanning calorimetry (DSC) is frequently used thermal analysis technique that provides detailed information about the physical and energetic properties of substance and mixtures⁽¹²⁾. Understanding the response of drugs and their additives to thermal stresses is an integral part of the development of stable formulations. Single components can exhibit crystallization, solid-solid transitions, glass transitions and polymorphic transitions. These transitions may be endothermic or exothermic. As the temperature increases, an amorphous solid will become less viscous. At some point the molecules may obtain enough freedom of motion to spontaneously arrange themselves into a crystalline form known as the crystallization temperature (Tc). This transition from amorphous solid to crystalline solid is an exothermic process, and results in a peak in the DSC signal. These transitions appear as a step in the baseline of the recorded DSC signal ⁽¹³⁾. This is due to the sample undergoing a change in heat capacity; no formal phase change occurs. When no phase changes occur then no response is reported. As the temperature further increases the sample eventually reaches its melting temperature (Tm). The relaxation step (melting process) in the form of enthalpy relaxation is indicated by endothermic peak. The glass transition temperature (T_g) of a non-crystalline material is the critical temperature at which the material changes its behavior from being 'glassy' to being ’rubbery’. 'Glassy' in this context means hard and brittle, while 'rubbery' means elastic and flexible. The concept of Tg only applies to non-crystalline solids, which are mostly either glasses or rubbers. A glass is defined as a material that has no long-range atomic or molecular order and is below the temperature at which a rearrangement of its atoms or molecules can occur. On the other hand, a rubber is a non-crystalline solid whose atoms or molecules can undergo rearrangement. The Tg is used to characterize amorphous materials as determines the propensity of amorphous mixtures to crystallize at certain temperature and moisture defining the Tg is important in the development of dehydrated formulations and in determining the relative chemical and physical stability of amorphous compositions. Glass solution is formed when drug and polymer are entirely miscible in molten state and remain as an amorphous one-phase system when cooled. Formation of glass solution can be assured by the absence of crystallinity detected by DSC and the presence of single T_g.

Pharmaceutical materials that are processed by “high energy processes” like freeze drying are often rendered at least partially amorphous. This occurs by the virtue of the fact that such processes create conditions that can prevent crystallization while the solid structure is being formed, or by disrupting the crystalline material already present. In the region known as the glass transition temperature (T_g), the average molecular mobility is sufficiently slow that the system falls out of energetic equilibrium with its surroundings, and the material forms a so-called glassy phase. Such glasses share many of the properties of crystalline materials such as solid macroscopic appearance, low specific heat capacity, and low thermal expansivity, which makes them attractive for use in pharmaceutical dosage forms. Also amorphous forms have better aqueous solubility than crystalline forms because the energy required to transfer the molecule into solution from crystal lattice is much greater than that from a non-crystalline solid. The reported melting endothermic peak of pure AB is at 198.56 °C. The DSC thermogram of finished fast dissolving tablets shows endothermic peak at about the same temperature as that of pure drug, hence we can safely assume that there is minimal interaction between the excipients. Also, the distorted nature of the products peak shows that it is present in the amorphous state, and thus has higher solubility than pure drug, Fig. 6.

Figure 6: A DSC thermogram of F8 FDT (A), and pure Amlodipine (B).

FTIR spectroscopy

FTIR analysis is mainly used to identify the functional groups in the structure of the compound, based on vibrational frequencies detected when bombarded with IR rays. In the present FTIR studies, the region from 2000 to 200cm⁻¹ was evaluated to gain an understanding of the physicochemical state of the dried samples. FTIR spectroscopy was used to investigate the interaction between the excipients and API (if any). IR spectroscopy is very sensitive to the presence of water in any sample. Fig. 7 shows FTIR spectra of pure AB and formulation mixture. Common peaks found in the graphs are as follows. Specific functional groups found in the structure of AB were detected in both spectra. Those include but are not limited to C-Cl stretching at 753.48 cm^-1, C=O stretching at1672.98 cm^-1, C-N stretching at 1301.42 cm^-1, and C-S stretching at 631.39 cm^-1. In the graphs obtained there are no visible interactions observed.

Figure 7: A FTIR spectrogram of F8 FDT (A), and pure Amlodipine (B).

X-ray Diffraction Analysis

Biomolecules are stabilized in sugars through glass formation. The molecular mobility of biomolecules can be further enhanced by arresting its structure in glassy state. Numbers of sugars and polymers have been reported to sustain this glassy state. The excipients achieve this by increasing the Tg of sugars. The moisture increases sugar mobility and reduce the Tg. Thus, glasses mostly obtained show devitrification on storage. In processes like lyophilization the crystalline ingredient after processing turns into amorphous state product. This change is caused by physical conditions used by the process. The amorphous phase formed by the process is detected by XRPD analysis.

The XRPD pattern of AB confirmed the structure and absolute configuration, which had been previously determined by chemical studies. The XRPD pattern of pure AB is shown in Fig. 7.20. The XRPD pattern of AB used in this study showed sharp peaks at 5°, 20°, 23°, 26°, 28°2θ that were characteristic of crystalline AB. This finding was confirmed by the presence of a melting point peak observed in DSC studies at 198°C.The x-ray diffraction patterns of optimized product are shown in Fig. 8.The physical state of any mixture can be analyzed by X-ray diffraction. The XRPD gives idea about how much crystallinity is present in each mixture. For keeping formulation stability for long time, the crystallinity index should be lowest. Studies have been reported to quantify the effect of additives on physical state of sugars during lyophilization and spray drying ⁽¹⁴⁾. Crystallization of excipients during dehydration is an important factor in the stability of product. The changes in amorphous or crystalline nature of formulations significantly affect performance and stability of protein pharmaceuticals.

Figure 8: A XRD spectrogram of F8 FDT (A), and pure Amlodipine (B).

Scanning Electron Microscopy

A Scanning Electron Microscope is a type of electron microscope that produces images of a sample by scanning it with a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that contain information about the samples surface topography and composition. A high-resolution SEM can show detail down to 25 Angstroms, or better. A normal scanning electron microscope operates at high vacuum. SEM has variety of applications in number of scientific and industry related fields, especially where characterization of solid material is concerned. In addition to topographical, morphological and compositional information, SEM can detect and analyse surface fractures, provide information in micro-structures, examine surface contaminations and identify crystalline structures. The Fig. 9 is the SEM images of the finished fast dissolving tablets prepared by Lyopan® technology. The SEM shows the glassy state solid formed due to lyophilization process. The figure shows the enhanced version of the same image which clearly shows the amorphous structure formed in the tablet body. The glassy structure formed is mainly composed of Mannitol, as it was used as bulking agent.

Figure 9: A SEM image of optimized amlodipine FDTs

Short Term Storage Stability Testing

Stability testing is a routine procedure performed on drug substances and products and is employed at various stages of the product development. The major aim of pharmaceutical stability testing is to provide reasonable assurance that the products will remain at an acceptable level of fitness/quality throughout the period during which they are in market place available for supply to the patients and will be fit for their consumption until the patient uses the last unit of the product. Once the formulation has been selected, stability studies of the drug substance and drug product are required to support expiration dating by the FDA and other regulatory agencies in submissions for product approval. Such stability data submitted are the representatives of manufacturing conditions and capabilities of manufacturer. In addition, a simple visual inspection of the formulation is conducted to determine if there are changes to the formulation. The human eye is extremely sensitive to many changes observed in drug formulations, and for this reason visual inspection is a valuable tool in the bag of techniques used to assess stability. For liquid formulations, the appearance of precipitates or a change in colour of the formulation signifies trouble. For lyophilized formulations, the visual appearance of the lyophilized cake is an important characteristic of the formulation. Collapse or discoloration of the cake could indicate a compromised formulation. Moisture content is one of the essential tests for evaluating the storage stability of freeze dried dosage forms ^(15). Tablet dosage forms are usually subjected to hardness, friability and disintegration testing for stability analysis. After the optimization of the batches was done, the one giving the best results among them (batch F8)was selected and its short-term stability testing was done. The samples were stored at 25ºC/60%RH and 40ºC/75%RH up to three months. After three months, the batches were analyzed again for Hardness, Friability, drug content uniformity and disintegration time. No significant changes were observed after three months in hardness and disintegration time, although friability was found to be slightly decreased at70% humidity. Drug content remained the same after the said period (Table 3).

Batch code	Month	Hardness (kg/cm²)	Friability (%)	Disintegration Time (sec.)	Moisture content (%)
25ºC/ 60% RH
1F8	1	2.9	4.1	8	3.1
	2	2.9	4.34	8	3.3
	3	3.0	4.9	12	3.7
40ºC/ 75% RH
2F8	1	2.9	4.1	7	3.5
	2	2.8	5.3	9	4
	3	3.1	4.8	6	5.2