Cancer is a highly invasive disease characterised The pharmaceutical industry has become more interested in creating bilayer tablets, which combine two or more API in a single dose form, in the past ten years to improve patient compliance and convenience. To prevent chemical incompatibilities between API by physical separation and to facilitate the creation of distinct drug release profiles (immediate release paired with sustain release), bilayer tablets may be the principal choice. Despite their benefits, the mechanical structures of this drug delivery system have grown quite complex due to the use of various materials and intricate geometric boundaries between the adjacent layers. This has resulted in the need for complex tablet architectures and patient-friendly administration, which presents significant challenges to pharmaceutical scientists and engineers. The main obstacles to bilayer compression are covered in depth in this oral presentation, along with a logical plan of action to achieve the intended bilayer tablet performance. Delamination, or layer separation, is one of the main problems caused by inadequate bonding and adhesion at the interface between adjacent compacted layers. This is frequently the result of an interfacial crack caused by residual stresses in the tablet that propagate a finite distance within the tablet and may not always be noticeable right away after compaction (e.g., during storage, packaging, shipping). Additionally, an inadequate link between the compacted layers might result in impaired mechanical integrity if they are too soft or too rigid. Establishing the layer sequence order, layer weight ratio, elastic misfit of the neighboring layers, initial layer tamping force, and cross contamination between layers are further development obstacles. These elements will affect the bilayer compression process itself (an ineffective or uncontrolled one) as well as the quality characteristics of the bilayer tablets (enough mechanical strength to preserve the tablet's integrity and individual layer weight management), if they are not properly managed or optimized. Consequently, in order to allow the creation of a resilient product and process, it is imperative to get insight into the underlying reasons. Cimetidine is a member of the class of guanidine that consists of guanidine carrying a methyl substituent at position 1, a cyano group at position 2 and a 2-{[(5-methyl-1H-imidazol-4-yl) methyl]sulfanyl}ethyl group at position 3. It is an H2-receptor antagonist that inhibits the production of acid in stomach. It has a role as a H2-receptor antagonist, a P450 inhibitor, an anti-ulcer drug, an analgesic and an adjuvant. It is a member of guanidine's, a member of imidazole's, an aliphatic sulfide and a nitrile. Ciprofloxacin HCl is a fluoroquinolone antibiotic used to treat a number of bacterial infections. This includes bone and joint infections, intra-abdominal infections, certain types of infectious diarrhea, respiratory tract infections, skin infections, typhoid fever, and urinary tract infections, among others. For some infections it is used in addition to other antibiotics. It can be taken by mouth, as eye drops, as ear drops, or intravenously. Materials used in the study:-Cimetidine and Ciprofloxacin HCl was obtained from Virion Enterprise. All HPMC grade were obtained from Ashland India Pvt Ltd. Sodium Starch Glycolate was purchased from DFE Pharma Excipients. Croscarmellose and Sodium & Cross povidone were obtained from Hetero Labs Ltd. All the reagents, chemical and solvent were of analytical grade.

METHODS:-

FORMULATION DEVELOPMENT:-

To formulate bilayer tablets, fast-release, and sustained-release layers were initially prepared separately to study the dissolution profile of each layer with an objective of selecting the optimized combination of excipients for the formulations. The optimized formulation of each layer was then compressed to bilayer tablet and further in vitro drug dissolution data

DEVELOPMENT OF CIMETIDINE SUSTAINED RELEASE (SR) TABLETS:-

The composition of various batches is given in Table 1. Different batches of cimetidine sustained release tablets were formulated using different polymer in varying concentration of 26,28 and 30%w/w. cimetidine and excipients were passed through ASTM (American society for testing and materials) 40 mesh sieve (425 µ) and dry blended for 10 min in motar aerosol 200P (0.5%w/w) and magnesium stearate (0.5%w/w) were sifted through ASTM 60 mesh sieve (250 µ) and added extra granularly and lubricated for 3 min. Compression was carried out using ten station single rotary tablet compression machine equipped with beveled flat-faced punches of 12.7 mm diameter at a tablet weight of 700 mg. Various excipients were screened and used in preparation of sustained release orally disintegrating tablets. The excipients chosen are as follows:

Sustained Release Polymer:-

HPMC K4M, HPMC K15M, HPMC K100M, HPMC E3LV. Diluent:- Microcrystalline cellulose. Lubricant:-Mg stearate, talc. Carbon Dioxide Generating Agent:- sodium bicarbonate and citric acid

DEVELOPMENT OF CIPROFLOXACIN HCL (IR) TABLETS:-                              

Ciprofloxacin HCL IR tablet formulations (F1-F8) were prepared by dry granulation technique as given in Table 2. All the powders were passed through ASTM 40 mesh sieve (425 µ). Required quantities of ciprofloxacin hcl and super disintegrant were mixed thoroughly in motar  Avicel PH 101 and lactose monohydrate were added to the above blend and mixed for 5 min. The powder was lubricated for 3 min with magnesium stearate (0.5 %w/w) and Aerosol 200 P (0.5%w/w) (ASTM 60 mesh sifted). The tablets were compressed at a weight of 550 mg on pilot press tablet compression machine equipped with bevelled flat-faced punches 12.7 mm in diameter at a tablet weight of 300 mg.

Super disintegrants:

Sodium starch glycolate, Cross povidone Cross carmellose sodium Diluent: Microcrystalline cellulose Lubricant: Mg stearate, Talc

COMPOSITION OF FORMULATION BATCHES:-

SUSTAIN RELEASE LAYER :-

Table No. 1: Sustain Release Layer

OPTIMIZATION OF IR TABLET:-

Table No. 3:-Optimization Of Super Disintegrants

OPTIMIZED FORMULA:-

Table No. 4:- Optimized Formula

EVALUATION:-

EVALUATION OF TABLET BLEND:

1. Bulk density

The bulk density of a powder is the ratio of the mass of an untapped powder sample and its volume including the contribution of the inter particulate void volume. Hence, the bulk density depends on both the density of powder particles and the spatial arrangement of particles in the powder bed. The equation for determining bulk density is

?b = m/ Vb

where, m= Weight of sample taken,

Vb= Bulk volume

2. Tapped density

It is the maximum packing density of a powder [or blend of powders] achieved under the influence of well defined, externally applied forces. The tapped density is a limiting density attained after “tapping down,” usually in a device that lifts and drop volumetric measuring cylinder containing the powder a fixed distance. The tapped density is calculated using the equation:

?t = m/ Vt

where, m = Weight of sample taken,

Vt = Tapped volume

3. Compressibility

Compressibility is indirectly related to the relative flow rate, cohesiveness and particlesize distribution of the powder. Powders with compressibility values lesser than 20% have been found toexhibit good flow properties. Tapped [?t] and Apparent Bulk density [?b] measurements can be used toestimate the compressibility of a material. It is often referred to as Carr’s Index.

4. Compressibility index = Tapped density – Bulk density

Tapped density X 100

5. Angle of repose

The angle of repose gives an indication of the flow ability of the substance. Funnel was adjusted such that the stem of the funnel lies 2 cm above the horizontal surface. The drug powder was allowed to flow from the funnel under the gravitational force till the apex of the pile just touched the apex of the funnel, so the height of the pile was taken as 2 cm. Drawing a boundary along the circumference of the pile and taking the average of six diameters determined the diameter of the pile. These values of height and diameter were then substituted in the following equation:

             Angle of repose [?] = tan -1[2h/d]              

6. Hausner’s ratio 51

Hausner’s ratio is an indication of the flowability and compressibility of a powder. It measures the friction condition in a moving powder mass. It is the ratio of bulk volume to tapped volume or tapped density to bulk density. Hausner’s ratio less than 1.5 indicates good flowability.

Hausner’s Ratio = Vb / Vt

 EVALUATION OF TABLET:-

1. Weight variation

Twenty tablets were randomly selected and individually weighed, the average weight and standard deviation of 20 tablets was calculated.

2. Thickness

The thickness of each tablet was measured using digital Vernier calliper.

3. Friability

It is the ability of tablets to withstand mechanical shocks during handling and transportation. Friability of the tablets was measured in Roche friabillator. Twenty pre-weighed tablets are placed in the friabillator that revolves at 25 rpm, dropping the tablets at a distance of six inches with each revolution. Normally, it is operated for 100 revolutions. The tablets are then dusted and reweighed.

4. Drug content

Three tablets were crushed and powder weight equivalent to one tablet weight was accurately weighed. The powder was dissolved in 100ml of pH 6.8 phosphate buffer and sonicated for about 20 mins. An aliquot was withdrawn and diluted with pH 6.8 phosphate buffer and measured at ? 320 nm.

5. In vitro dissolution

In vitro dissolution studies were performed on the optimized tablets. In vitro drug release was studied up to 8 hrs. using USP Type 2 [Paddle] dissolution apparatus in 900 ml of pH 1.2 phosphate buffer at 37.5°C ±0.5°C. The stirring speed was set at 50 rpm. Five milliliter sample was withdrawn at intervals of 1hr and replaced with fresh dissolution medium. After appropriate dilution, the samples were analyzed by UV-visible spectrophotometer at 245 and 276 nm.

MATHEMATICAL TREATMENT OF IN VITRO RELEASE DATA

Several mathematical equations which generally define the dissolution profile. Once an appropriate function has been selected, the evaluation of dissolution profile can be carried out and hence the drug release profile can be correlated with drug release kinetic models. Various mathematical models are employed to understand drug release kinetics which is explained below.

The model dependent approaches include:

• Zero order kinetic models
• First order kinetic model
• Higuchi model

STABILITY INDICATING HPLC METHOD DEVELOPMENT AND VALIDATION :-

High Performance Liquid Chromatography [HPLC] is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase). The main purposes for using HPLC are for identifying, quantifying and purifying the individual components of the mixture. A stability-indicating assay is a validated quantitative analytical procedure that can detect the changes with time in the pertinent properties of the drug substance and drug product. A stability-indicating assay accurately measures the active ingredients, without interference from degradation products, process impurities, excipients, or other potential impurities.

APPARATUS AND CHROMATOGRAPHIC CONDITIONS:

FOR CIMITIDINE

Equipment:

High Performance Liquid Chromatography [Isocratic]

Mobile phase :

methanol : hplc water [80:20]: 0.3% phosphoric acid

Flow rate :

1ml/min Injection volume 25?L

Detection wavelength:

218 nm Retention time: 2.88 mins

FOR CIPROFLOXACIN HCL

Equipment: High Performance Liquid Chromatography [Isocratic]

Mobile phase :

methanol : hplc water [80:20]: 0.3% phosphoric acid

Flow rate :

 1ml/min Injection volume 25?L

Detection wavelength:

276 nm Retention time: 2.37 mins

FORCED DEGRADATION:-

Forced degradation plays an important role in the development of stability indicating analytical methodology. In addition to demonstrating specificity, forced degradation studies can be used to determine the degradation pathways and degradation products of the APIs that could form during storage and facilitate formulation development, manufacturing and packaging. The drug products were subjected to forced degradation at various stressed conditions like Hydrolytic degradation under acidic condition, Hydrolytic degradation under alkaline condition, Photolytic degradation & Oxidative degradation. All the samples were placed at reflux at elevated temperature i.e 60ºC for a period of 24 hours separately. The degraded samples are then neutralized using suitable acid, base or buffer to avoid further decomposition. The samples were then analyzed with HPLC for the degradation.

HYDROLYTIC DEGRADATION UNDER ACIDIC CONDITIONS:-

The stock solution of Cimetidine and Ciprofloxacin HCL was prepared having a concentration of 1 mg/ml with water in 100 ml of volumetric flask. A 25ml of the above drug solution was placed in 50 ml of round bottom flask, to it 25ml of 1M solution of HCl was added. Then the round bottom flask was kept at the 60°C for reflux for 24 hours and further, it was neutralized with 1M NaOH. The resultant solution was filtered with 0.45 microns syringe filters and placed in the vials.

HYDROLYTIC DEGRADATION UNDER ALKALINE CONDITIONS:-

The stock solution of Cimetidine and Ciprofloxacin HCL was prepared having a concentration of 1mg/ml with water in 100ml of volumetric flask. A 25ml of the above drug solution was placed in 50ml of round bottom flask, to it 25ml of 1M solution of NaOH was added. Then the volumetric flask was kept at 60°C for reflux for 24 hours and further, it was neutralized with 1M HCl. The resultant solution was filtered with 0.45 microns syringe filters and placed in the vials.

OXIDATIVE DEGRADATION:-

The stock solution of Cimetidine and Ciprofloxacin HCL was prepared having a concentration of 1mg/ml with water in 100 ml of volumetric flask. A 25 ml of the above drug solution was placed in 50 ml of volumetric flask, to it 25ml of 1% solution of H2O2 was added. Then the volumetric flask was kept at the 60°C for reflux for 24 hours. The resultant solution was filtered with 0.45 microns syringe filters and placed in the vials.

VALIDATION OF HPLC METHOD

1. LINEARITY

100 mg of Cimetidine and Ciprofloxacin HCL was placed in 100 ml of volumetric flask and dissolved with the mobile phase to get 1000 ppm. 1 ml of the solution from the stock solution of [1000ppm] was transferred and diluted to 10ml with sufficient mobile phase so as to get 100 ppm solution. Then 0.2 ml, 0.4 ml, 0.6ml, 0.8ml, 1.0ml, 1.1 ml,1.2 ml, 1.4ml was taken in 10 ml volumetric flask and diluted with mobile phase to get 2,4,6,8,10,12,14 ppm solutions respectively. A plot of peak area against concentration was calculated and subjected to linear regression.

2. ACCURACY

Accuracy was determined by calculating recovery of Cimetidine and Ciprofloxacin HCL by the standard addition method. The standard solution was added at 80, 100 and 120% were injected into chromatographic systems and calculated the amount found and amount added for Cimetidine and Ciprofloxacin HCL and further calculated the individual recovery.

3. PRECISION

Interday and intraday precision was carried out by injecting the standard solutions of 60, 80 and 100 ?g/ml in triplicates twice a day.

Next day same concentration [60, 80 and 100 ?g/ml] were injected and evaluated to find the concentrations based on the responses [Area]. Standard deviation and % Relative standard deviation was calculated.

4. LIMIT OF DETECTION [LOD] AND LIMIT OF QUANTITTATION [LOQ] 61

Detection limit [DL] expressed as: DL = 3.3 ?/ S

Where,

? = Standard deviation of the response

S = Slope of the calibration curve Quantitation limit [DL]

STABILITY STUDIES:

• Stability testing of pharmaceutical formulations is one of the key aspects of formulation development. It is performed at various stages of formulation development on drug substances and products. In early stages, accelerated stability testing [at relatively higher temperature and/or humidities] can be used for “worst case” evaluation to determine what types of degradation products may be found after long term storage. Testing under gentle conditions [long term shelf storage] slightly elevated temperatures can be used to determine a product’s shelf life and expiration dates. During stability testing, the products are tested for content uniformity, degradation products, dissolution time, appearance.
• The optimized formulation of Cimetidine and Ciprofloxacin HCL were subjected to accelerated stability studies testing under storage condition. The formulation was placed in an Alu-Alu pack at a condition of 250C ± 2?C/60% RH ± 5% ; 300C ± 2?C/65% RH ± 5% RH & 40?C ± 2?C/75% RH ± 5% RH for a period of one month.

RESULT AND DISCUSSIONS

Different types of studies ranging from physicochemical to in vitro assay were performed in order to optimize and to assess the potential of the drug delivery system developed. Results along with detailed discussion are being given here with justification in support of the rationale for development of the delivery system.

A. PREFORMULATION STUDIES:

1. ORGANOLEPTIC EVALUATION:

Cimetidine were examined for their organoleptic properties like colour and odour. Cimetidine sample is a white, odorless amorphous powder.                                          

• The melting point determination of a drug is done as it is an important property that proves the authentication of the drug since presence of relatively small amount of impurity can lower as well as widen the melting point range. The melting point of cimetidine was found to be 142°C.
• The melting point determination of a drug is done as it is an important property that proves the authentication of the drug since presence of relatively small amount of impurity can lower as well as widen the melting point range. The melting point of ciprofloxacin HCL was found to be 290°C.

3. PH SATURATED SOLUBILITY:

Table No. 7:- Solubility Of Cimetidine In Different Media

Cimetidine shows maximum solubility in pH 1.2 Acid buffer

Table No. 8:- Solubility Of Ciprofloxacin In Different Media

4. DRUG EXCIPIENT COMPATIBILITY STUDY:-

The drug and excipients were found to be compatible with each other and did not show any change in their physical properties.

B) AUTHENTICATION OF CIMETIDINE AND CIPROFLOXACIN HCL:

1.SPECTRUM OF THE CIMETIDINE

Simple, sensitive, accurate, precise and rapid ultraviolet spectrophotometric method was developed for estimation of cimetidine. The aim was to develop accurate, precise and sensitive UV spectrophotometric method for estimation of cimetidine by UV- visible spectrophotometer.

Determination of ? max:

The absorption maxima of cimetidine was determined by scanning the drug solution using double beam ultraviolet spectrophotometer.

Procedure:

Accurately weighed 100 mg of Cimetidine was dissolved in 10 ml pH 1.2 phosphate buffer in 100 ml of volumetric flask and then made up the volume up to 100 ml with pH 1.2 phosphate buffer. 1 ml of the solution was diluted to 100 ml with pH 1.2 phosphate buffer in 100 ml volumetric flask to obtain concentration of 10 ?g/ml. The spectrum of this solution was run between 200- 400 nm range using UV- visible spectrophotometer.

The absorption maximum was found to be 245 nm.

?max = 245nm

1. UV SPECTRUM OF THE CIPROFLOXACIN HCL

Simple, sensitive, accurate, precise and rapid ultraviolet spectrophotometric method was developed for estimation of ciprofloxacin HCL The aim was to develop accurate, precise and sensitive UV spectrophotometric method for estimation of ciprofloxacin HCL by UV- visible spectrophotometer.

Determination of ? max:

The absorption maxima of ciprofloxacin HCL was determined by scanning the drug solution using double beam ultraviolet spectrophotometer.

Procedure:

Accurately weighed 100 mg of CIPROFLOACIN HCLwas dissolved in 10 ml pH 1.2 phosphate buffer in 100 ml of volumetric flask and then made up the volume up to 100 ml with pH 1.2 phosphate buffer. 1 ml of the solution was diluted to 100 ml with pH 1.2 phosphate buffer in 100 ml volumetric flask to obtain concentration of 10 ?g/ml. The spectrum of this solution was run between 200- 400 nm range using UV- visible spectrophotometer.

The absorption maximum was found to be 276 nm.

?max = 276nm

2. FOURIER-TRANSFORM INFRA RED SPECTROSCOPY [FTIR]:

The sample of Cimetidine showed a characteristic vibrational peak for N-H stretching at 3226 cm-1. IR peaks at 1622 cm-1 were assigned to C=N stretching, respectively. The C=N-C=C asymmetric stretching was assigned to peak at 1586 cm-1, N-SH bending vibration peak was attributed to peak at 1506cm-1, and C-C stretching peak was assigned to peak at 1425-1426 cm-1. The absorption peaks at 1387 and 668 cm-1 were assigned to CH3 bending and C-S asymmetric stretching, respectively.

The sample of Ciprofloxacin HCL showed a characteristic vibrational peak for O–H stretching in COOH at 3530–3373 cm?1. IR peaks N–H stretching of the ionized amine (>NH2+) at 2689–2463 cm?1, respectively. The C=O stretching in COOH and 4-quinolone ring at 1703 cm?1 and 1623 cm?1, C=C stretching in aromatic ring at 1588–1495 cm?1, and C-C stretching peak was assigned to peak at 1425-1426 cm-1. The C–H bending at 1449 cm?1, the C–N stretching at 1384 cm?1, the C–O stretching in COOH at 1267 cm?1, and the C–F stretching vibrations in the range of 1250–1100 cm?1

3. CALIBRATION CURVE OF CIMETIDINE: -

Calibration curve was plotted and the correlation coefficient ‘r?2;’values were calculated. The method for estimation of cimetidine showed linear relationship in the concentration range 20 to 180 ?g/ml as shown in figure no. 2.

Calibration data of Cimetidine

Calibration curve was plotted and the correlation coefficient ‘r?2;’values were calculated. The method for estimation of ciprofloxacin HCl showed linear relationship in the concentration range 2 to 18 ?g/ml as shown in figure no. 3.

4. EVALUATION:

EVALUATION OF TABLET BLEND:

The powder blend before tablet compression was evaluated for various parameters such as bulk density, tapped density, Carr’s compressibility index, Hausner’s ratio and angle of repose. Bulk density was found in the range of 0.64 g/ml and the tapped density between 0.69 g/ml indicating both parameters were found to be within the limits. Using the above two density data, Carr’s compressibility index was determined. The compressibility index and Hausner’s ratio were found to be 6.333 ± 0.57 and 1.068 ± 0.005 respectively indicating that the powder blend showed significantly acceptable flow properties. The flow property of all powder blends was better explained from angle of repose. The angle of repose was found to be 32.2?. The results of angle of repose showed all powder blends exhibited good to acceptable flow property.

2. EVALUATION OF TABLET:

DISSOLUTION STUDIES:

Dissolution test was carried out in accordance with USP Type 2 [Paddle type apparatus], 50 rpm speed, at temperature of 37°C, in pH 1.2 0.1N HCL at time interval for 8 hours. The USP drug release requirement is given below for Cimetidine sustained release tablet and Ciprofloxacin HCL immediate release tablets in Table No 23 and the requirement was NLT 85% drug release in 25 Minutes for ciprofloxacin HCL & NLT 95% drug release in 8 Hours for Cimetidine.

5. RELEASE KINETIC STUDIES:

FOR CIPROFLOXACIN HCL :-

The In vitro drug release data of optimized formula was analysed for determining kinetics of drug release. The obtained data was used to find whether the release obeyed zero order kinetics, first order kinetics and Higuchi model. The highest correlation coefficient [r2] obtained from them gives an idea about model best fitted to the release data. From the results of kinetic studies, the examination of correlation coefficient ‘r’ indicated that the drug release followed Higuchi model release kinetics

FOR CIMETIDINE :-

The In vitro drug release data of optimized formula was analyzed for determining kinetics of drug release. The obtained data was used to find whether the release obeyed zero order kinetics, first order kinetics and Higuchi model. The highest correlation coefficient [r2] obtained from them gives an idea about model best fitted to the release data. From the results of kinetic studies, the examination of correlation coefficient ‘r’ indicated that the drug release followed Higuchi model release kinetics.