METHODOLOGY - FORMULATION AND EVALUATION OF SUSTAINED RELEASE MATRIX TABLETS OF STAVUDINE

 

METHODOLOGY

 

Drug name	Source
Stavudine	Alchem laboratories, Mumbai , India

 

Excipients and chemicals used in the study

4.1 List of Equipments used in the study

Equipment Name	Manufacturer
16-station tablet	Cadmach Co, Ahmedabad, India
compression machine
Electronic balance	ER182A, Mettler Toledo
UV visible	Schimadzu, UV-1700 E 23
spectrophotometer
Hardness tester	MHT-20, Campbell Electronics, Mumbai, India
Friability tester	FTA-20, Campbell Electronics, Mumbai, India
FT-IR	Thermo Nicolet 670 spectrometer
DSC	Mettler Star SW 8.10, USA
Sonicator	Power sonic 505, HWASHIN Technology Co,
	USA
Dissolution apparatus	LABINDIA, DISSO-2000, Mumbai, India
Scanning electron	JEOL JSM -5200, USA
microscope
Overhead 3-blade	Model RO 123, RemiÒ, Mumbai
medium duty stirrer
Stability chambers	Thermolab Scientific Equipments Pvt Ltd, India
HPLC	Shimadzu Scientific Instruments , Japan
Vortex mixer	Remi Equipment, Mumbai, India

 

DOSAGE FORMS SELECTED IN THE PRESENT STUDY

Single unit system (matrix tablets)

          Matrix drug delivery systems consist of a polymer, drug, and other excipients distributed throughout the matrix. This system is dependent on polymer wetting, polymer hydration, and polymer dissolution for the controlled release of drug. At the same time, other soluble excipients or drug substances comprising the tablet will also become wet, dissolve, and diffuse out of the matrix, while insoluble excipients or drug substances will be held in place until the surrounding polymer/excipient/drug complex erodes or dissolves away.

Microparticles

          These are particles with size more than „1mm, containing the polymer. At present, there is no universally accepted size range that particles must have in order to be classified as microparticles.

          However, many workers classify the particles smaller than 1000mm, as nanoparticles and those more than 1000 mm, as macroparticles.

Classification:

        

Microcapsules:

          Microcapsules are small particles that contain an active agent or core material surrounded by a coating or shell. (Commercial microcapsules typically have a diameter between 3 & 800 micrometer and 10-90% core).

Microspheres:

          Microspheres are solid, spherical particles containing dispersed drug molecules, either in solution or crystalline form, among the polymer molecules.

General Methods in the Preparation And Characterization Of Matrix Tablets

 

Preparation  of  Matrix  Tablets  using  direct  compression method

          The drug, polymer(s) and all other excipients sifted through 425 μm sieve (ASTM mesh no 40) and mixed uniformly. The dry mix blend was then pre lubricated with respective excipients and lubricated with magnesium stearate. The lubricated granules were directly compressed on 16-station tablet compression machine using respective punches. (Cadmach Co, Ahmedabad, India).

Preparation of Matrix Tablets using Wet granulation method

          The drug, polymer and other excipients were sifted through 425 μm sieve (ASTM mesh no 40) and mixed uniformly. The dry mix blend was then granulated with respective granulation fluid. The wet granules were dried at 60 °C until the complete evaporation of granulation fluid from the granules. The dried granules were again sifted through ASTM mesh no 30. The dried and sifted granules were then pre lubricated with respective excipients and then lubricated with magnesium stearate. The lubricated granules were compressed on 16-station tablet compression machine using respective punches. (Cadmach Co, Ahmedabad, India).

 

Solubility determination of drugs

          Solubility study of the active drug was investigated in four different media as follows:

1. Purified water
2. 0.1 N hydrochloric Acid (HCl),  USP
3. Acetate buffer pH 4.5, USP
4. Phosphate buffer pH 6.8 USP

 

          Required quantity of above media was transferred in to a volumetric flask and heated up to 37 ±0.5 oC using magnetic stirrer provided with heat. Previously weighed quantity of active drug was added to the above volumetric flask until the saturation point occurs. The total quantity of drug added was recorded. Stirring was continued up to 5 hours at 37 ±0.5 oC. The sample was filtered through 0.45 µm filter. A measured quantity of filtered sample was transferred in to another volumetric flask and further dilutions made. The absorbance was measured using UV visible spectrophotometer (Schimadzu, UV-1700 E 23).

Construction of standard calibration curves

          Accurately weighed quantity of active drug was transferred in to the volumetric flask. Required quantity of media was added to the above volumetric flask. Shake the volumetric flask until the complete solubility of the drug and make up the volume with remaining quantity of media. Similarly stock solutions were prepared in all the media. Standard calibration curves in different media were constructed using the above stock solutions. The samples were scanned for l_max  at the UV range of 200-400 nm. After 1 day again the samples were scanned for l_max. The l_max at initial and 1 day were compared for the stability of pure drug in the respective media. From the above stock solutions different concentrations of the solutions were prepared and standard calibration curves were prepared by plotting the absorbance values vs concentration.

Differential scanning calorimetry (DSC)

          Thermal properties of pure drug, initial formulation and stability samples of both matrix tablets and microcapsules were evaluated by Differential scanning calorimetry (DSC) using a Diamond DSC (Mettler Star SW 8.10). The analysis was performed at a rate 5⁰ C min-1 from 50⁰ C to 200⁰ C temperature range under nitrogen flow of 25 ml min-1.

Drug content estimation

          The drug content of the prepared matrix was determined in triplicate. From each batch, 20 tablets were taken, weighed, crushed and finely powdered. An accurately weighed quantity of this powder was taken and suitably dissolved under sonication (Power sonic 505, HWASHIN Technology Co.) in pH 6.8 phosphate buffer and filtered through 0.45µ (Millipore) filters. The          sample was analyzed after making appropriate dilutions using the developed analytical method.

Hardness, weight variation and friability determination

          The weight variation was determined by taking 20 tablets using an electronic balance (type ER182A, Mettler Toledo). Tablet hardness was determined for 10 tablets using a Monsanto tablet hardness tester (MHT-20, Campbell Electronics, Mumbai, India). Friability was determined by testing 10 tablets in a friability tester (FTA-20, Campbell Electronics) for 300 revolutions at 25 rpm.

In vitro drug release studies of prepared matrix tablets  

          The in vitro dissolution studies were performed up to 14 hours and more using dissolution apparatus (LABINDIA, DISSO-2000, Mumbai, India). The dissolution medium consisted of phosphate buffer pH 6.8 (900 mL), maintained at 37 ±0.5⁰C. An aliquot (5 mL) was withdrawn at specific time intervals and filtered through 0.45 µ (Millipore) filter. After appropriate dilution the samples were analyzed and cumulative percentage of the drug released was calculated. 6 tablets from 3 different batches were used in analysis.

Accelerated stability studies on the prepared formations

          Selected formulations from prepared formulation were filled in HDPE containers and stored at the following conditions like 40°C/75% RH for about 3 months as per ICH guidelines. The samples were characterized for percent drug content, FTIR and DSC study.

 

Kinetic analysis of dissolution data

          The release rate and mechanism of drug release from the prepared formulations were analyzed by fitting the dissolution data into the zero-order equation

Q = k0t

 

where Q is the amount of drug released at time t, and k0 is the release rate constant,

The dissolution data was fitted to the first order equation

ln (100–Q) = ln 100 – k1t.

where k1 is the release rate constant.

The dissolution data was fitted to the Higuchi‟s equation

Q = k2 t1/2

Statistical Comparison of Dissolution Profiles

          Dissolution studies of the prepared matrix tablets and microcapsules for all the formulations were determined. A statistical comparison such as similarity factor (f2 factor) among some formulations was used. This statistical model is suitable only when three or more dissolution time points are available. The similarity factor (f2) is a logarithmic reciprocal square root transformation of the sum of squared error and is a measurement of the similarity between two curves in the dissolution. The following equation represents a similarity factor (f2):

where 1) f2  similarity factor, log is logarithm to base 10,

2) P is number of sampling time points

3) Σ is the summation of over all time points,

4) μti    is  the  dissolution  measurement  (in  mean  percent  labeled amount) at time point “t” of the first batch (test batch) profile,

5) μri is the dissolution measurement (in mean percent labeled amount) at time point t of the second batch (reference batch) profile.

Encapsulation efficiency (EE)

          Drug loaded microcapsules (100 mg) were powdered and suspended in water. Then the contents suspended in the water were kept for sonication (Power sonic 505, HWASHIN Technology Co) for about 20 mins and shaked using mechanical shaker (ORBITEX, Scigenics Biotech) for about 20 mins for the complete extraction of drug from the microcapsules. The resultant solution was filtered through 0.45 µm filter. Drug content was determined by UV- visible spectrophotometer (Schimadzu, UV-1700 E 23).

          The percent entrapment was calculated by using the following formula.

Scanning electron microscopy (SEM)

          Morphological characterization of the microcapsules was done by using Scanning electron microscope (JEOL JSM -5200). The samples were coated to 200 A° thickness with gold-palladium prior to microscopy.

 

Pre formulation Studies for Stavudine (STAV)

Multimedia    dissolution    of    conventional    Stavudine formulation

          The drug release study from conventional stavudine capsules (Zerit-30 mg, Batch No-4B76334 manufactured by Bristol Mayer Squibb) was done using USP type 1 at 75 rpm, 900 ml of dissolution medium at 37 ±0.5 °C. The dissolution media used were water, 0.1 N HCl, acetate buffer pH 4.5 and phosphate buffer pH 6.8 of USP standard. A sample of 5 ml was withdrawn from the dissolution medium and replaced with 5 ml of blank medium. The samples were withdrawn at 5, 10, 15, 30 and 45 minutes and analyzed for drug content using UV visible spectrophotometer after suitable dilution.

Fourier Transform Infrared spectroscopy (FT-IR)

          The FT-IR spectrum was taken for pure STAV powder, initial formulation and stability samples were determined by the method.

Analytical Method

Ultraviolet Spectroscopy

          The UV spectroscopic method for STAV was developed in the four different pH media to study the solubility, dissolution and drug content estimation using the method. Finally, the quantity of STAV was calculated from the regression equation of the calibration curve.

 

 

DRUG PROFILE

Stavudine (D4T)87

US FDA approval date: June 1994

Structural formula of stavudine

PHYSICOCHEMICAL  PROPERTIES OF STAVUDINE

i.       Description           A white to yellowish, odorless, crystalline solide.

ii.     CAS No                  3056-17-05

iii.  Molecular formula   c12H10N2O4

iv.   Molecular weight     224.2.

v.      Chemical name       2,3'-didehydro-3'-deoxythymidine

vi.   Melting Range         About 165-166°C

vii. Solubility                Soluble in water and propylene glycol

Description: Stavudine is an antiretroviral medication used to prevent and treat HIV-AIDS. Stavudine is a nucleoside analogue of thymidine. It is phosphorylated by cellular kinases into an active typhosphate. Stavudine tryphosphate inhibits HIV’s reverse transcriptase by competing with the natural substrate, thymidine tryphosphate. Stavudine is rapidly absorbed after oral Administration. The elimination half-life of Stavudine is 1.6 hours.

Mechanism of Action

Stavudine, a nucleoside analogue of thymidine, is phosphorylated by cellular kinases to the active metabolite stavudine triphosphate. Stavudine triphosphate inhibits the activity of HIV-1 reverse transcriptase (RT) by competing with the natural substrate thymidine triphosphate (Ki = 0.0083 to 0.032 μM) and by causing DNA chain termination following its incorporation into viral DNA. The action of Stavudine triphosphate is mainly by inhibiting the cellular DNA polymerases β and γ and reduces the mitochondrial DNA synthesis.

Antiviral Activity

            The in vitro antiviral activity of stavudine was measured in peripheral blood mononuclear cells, monocytic cells, and lymphoblastoid cell lines. The concentration of drug necessary to inhibit HIV-1 replication by 50% ranged from 0.009 to 4 mm against laboratory and clinical isolates of HIV-1.

Pharmacokinetics of Stavudine

Absorption	After oral administration Stavudine is rapidly absorbed
	with peak plasma concentration occurred in 1 hour after dosing
Distribution	Binding of Stavudine to serum proteins is negligible over
	the concentration range of 0.01 to 11.4 μg/ml. Stavudine
	distributed equally between red blood cells and plasma.
Metabolism	The metabolic fate of Stavudine has not been elucidated
	in humans
Excretion	In humans, renal elimination accounts for about 40% of
	the overall clearance of stavudine (Table 2).
	. The elimination half-life of stavudine is 1.6 hours.

 

Pharmacokinetic	Parameter	Value
	Availability (Oral)	86.4
Parameters	T max	About 3hours
	Elimination Half-life	1.5  hours
	Volume of distribution	58 liters

 

 

Administration: It can be administered orally with food or with out food. It can be used to prevent Human immunodeficiency virus from multiplying in the body. Less than 60 kg patients must take 30 mg orally every 12hours. The patients is near about 60 kg, 40 mg doses should be taken orally every 12 hours.

 

Table 4.2: Reference marketed products of Stavudine.

	Brand name	Company
	Stadin	Sain medicaments Pvt.Ltd.
	Stadine	Emcure pharaceuticals
	Stag	Genix pharma Pvt.Ltd.
	Stavir	Cipla

 

Drug interactions

          The regimen of stavudine, nevirapine and lamivudine is widely used as first- line therapy for the treatment of HIV & AIDS in the Adults. When Stavudine is taken with Didanisine a dangerous inflammation of the pancreas and lactic acidosis as a side effect may occur.

Adverse effects:

1. Common side effects:

Nausea

Vomiting

Diarrhea

Headche

Upset stomach

2. Severe side effects:

Peripheral neuropathy

Lactic acidosis

Pancreatitis

Hepatotoxicity

Lipoatropy

Hydroxy propyl methyl cellulose (HPMC)

Structural formula

R is H, CH₃, or CH₃CH(OH)CH₂

 

Functional Category: Coating agent; extended release agent

Applications: Tablet binder, in film-coating, and as a matrix for use in extended-release tablet formulations.

Description: Hypromellose is an odorless and tasteless, white or creamy-white fibrous or granular powder.

Glass transition temperature: 170–180°C.

Melting point: 190–200°C.

 

Solubility: Soluble in cold water, forming a viscous colloidal solution; practically insoluble in chloroform, ethanol (95%), and ether, but soluble in mixtures of ethanol and dichloromethane, mixtures of methanol and dichloromethane, and mixtures of water and alcohol. Few grades of HPMC are soluble in acetone, mixtures of dichloromethane and propanol, and other solvents.

Viscosity: Wide range viscosity grades are available in the market.

Stability and Storage Conditions: Hypromellose powder is a stable material, although it is hygroscopic after drying.

Polyethylene oxide (PEO)

Structural formula

 

Functional Category: Polyethylene oxide (PEO) is used as a matrix binder for tablets and capsules.

Applications: Extended release.

Description: It is a white , tasteless , free flowing powder.

Melting point: 68°C

 

Solubility: Polyethylene oxide (PEO) is soluble in water.

Molecular Weight : 100000-700000.

Stability and Storage Conditions : Polyethylene oxide (PEO) is stored in a well-closed air tight light resistance container in a cool, dry place.

 

Ethyl cellulose (EC)

 

 

Functional Category: Ethyl cellulose (EC) is used as an enteric film coating material, or as a matrix binder for tablets and capsules and also as tablet diluent.

 

Applications: binders, fillers, granulation aids, protective and controlled release coatings, taste masks and flavor fixatives.

 

Description: It is a white, tasteless, free flowing powder.

 

Glass transition temperature: 129-133°C

 

Melting point: 165-173°C.

 

Solubility: Practically insoluble in water, freely soluble in chloroform, soluble in dichloromethane.

 

Viscosity: Various grades of ethyl cellulose are commercially available having viscosities ranging from 3-385 mPa s.

 

Stability and Storage Conditions: cellulose acetate butyrate is stable if stored in a well-closed container in a cool, dry place

Magnesium stearate

Non-proprietary names	:	Magnesium stearate   (BP, USP)
		Magnesii stearas (PhEur)
Synonym	:	Dibasic magnesium stearate, Magnesium distearate
Chemical Name	:	Octadecanoic acid magnesium salt
Empirical Formula	:	C36H70MgO4
Molecular Weight	:	591.34 g/mol
Functional Category	:	Tablet and capsule lubricant

Description : It is a very fine, light white, precipitated or milled powder of low bulk density, having a faint odor of stearic acid and a characteristic taste. The powder is greasy to the touch and readily adheres to the skin.

Physicochemical Properties

Density (bulk)	:	0.159 g/cm3
(tapped)	:	0.286 g/cm3
(true)	:	1.092 g/cm3
Flowability	:	poorly flowing, cohesive powder.
Melting range	:	117–150°C (commercial samples)
		126–130°C (high purity magnesium stearate)
Solubility	:	Practically insoluble in ethanol (95%), ether and
		water; slightly soluble in warm benzene and
		warm ethanol (95%).

 

Stability and Storage : Magnesium stearate is stable and should be stored in a well closed container in a cool, dry place.

 

Standards : Magnesium stearate contains not less than 3.8% and not more than 5.0% of magnesium, calculated on the dried basis.

 

Identification : To 5gm add 50 ml of ether, 20 ml of 2 M nitric acid and 20 ml of distilled water and heat under a reflux condenser until fully dissolved. Allow to cool. Separate the aqueous layer and shake the ether layer with two quantities (each 4 ml) of distilled water. Combine the aqueous layers, wash with 15 ml of ether and dilute to 50 ml with distilled water. Evaporate the ether layer and dry the residue at 105˚ C. The freezing point of the residue is not lower than 53˚ C.

Assay : Weigh accurately about 0.75 gm, add 50 ml of a mixture of 1-butanol and ethanol, 5 ml of strong ammonia solution, 3 ml of ammonia buffer pH 10.0, 30 ml of 0.1M disodium edetate and 15 mg of mordant black mixtures. Heat to 45˚ to 50˚ C and titrate with 0.1 M zinc sulphate until the colour changes from blue to violet. Repeat the operation. The difference between the titrations represent the amount of disodium edentate. Each ml of disodium edetate is equivalent to 0.002431 gm of Magnesium.

Applications: It is widely used in cosmetics, foods, and pharmaceutical formulations. It is primarily used as a lubricant in capsule and tablet manufacture at concentrations between 0.25% and 5.0% w/w. It is also used in barrier creams.

 

Carboxy methylcellulose(CMC)

Synonyms:                    Carmellose, E466, cellulose gum

CAS No.:                        9004-32-4

Description:                  White or light yellow powder, granule or fiber                                             solid, odorless, tasteless and non-toxic.

Chemical Name:            Cellulose

Density (Bulk):              0.52 g/cm³

Density (tapped):           0.78 g/cm³

Dissociation constant: pKa= 4.30

Functional category:    Coating agent, stabilizing agent, suspending                                              agent, tablet and capsule dis-integrant, tablet                                            binder, viscosity increasing agent and water

                                      absorbing agent.

Melting point:               Browns at approximately 227⁰c, and chars at                                             approximately 252⁰c

 

Solubility: Practically insoluble in acetone, ethanol (95%), ether and touelene, easily dispersed in water at all temperature, forming clear, colloidal solutions. The aqueous solubility varies with the degree of substitution (DS).

 

Viscosity: Aqueous 1% w/v solutions with viscosities of 5-1 000m pa s (5-13000 cp) may be obtained. An increase in concentration results in an increase in aqueous viscosity. The viscosity of carboxy methylcellulose solutions is fairly stable over a pH range of 4-10. The optimum pH range is neutral.

 

Stability and storage conditions: Carboxy methylcellulose is a stable, through hygroscopic material. Under high humidity conditions, carboxy methylcellulose can absorb a large quantity (˃ 50%) of water. In tablets, this has been associated with decrease in tablet hardness and an increase in disintegration time. The bulk material should be stored in a well- closed container in a cool place.

 

Incompatibilities: Carboxy methylcellulose sodium is incompatible with strongly acidic solutions and with the soluble salts of iron and some other metals, such as aluminium, mercury and zinc. Precipitation may occur at pH ˂ 2, and also when it is mixed with ethanol (95%).

 

Polyethylene glycol (PEG)

 

Description:                  It is clear, colourless liquid.

Molecular formula:        H(OCH₂CH₂)_nOH,

Molar mass:         18.02 + 44.05n g/mol

Flash point:                   182–287 °C; 360–549 °F; 455–560 K

Solubility:                     Soluble in water and ethanol

Packaging and storage:  Preserve in tight containers.

DILUENTS

Micro crystalline cellulose

Nonproprietary Names	:	Microcrystalline cellulose (BP),
		Cellulosum microcristallinum (PhEur)
Synonyms	:	Avicel pH, cellulose gel, crystalline cellulose
Chemical name	:	Cellulose
Empirical Formula	:(C6H10O5)n, where n = 220.
Molecular Weight	:	370.351 g/mol

 

 

Description : It is a purified, partially depolymerized cellulose that occurs as a white, odorless, tasteless, crystalline powder composed of porous particles. It is commercially available in different particle sizes and moisture grades that have different properties and applications.

Structural formula        :

Chemical structure of microcrystalline cellulose

Density (Bulk)	:	0.337 g/cm3
Density (Tapped)	:	0.478 g/cm3
Density (True)	:	1.512-1.668 g/cm3
Loss on drying	:	≤ 7.0%
Melting Point	:	chars at 260-270˚C
Ash value	:	0.1%
Moisture content	:	less than 5% w/w

 

Functional category : Adsorbent, suspending agent, tablet and capsule diluent and tablet disintegrant.

 

Solubility : Slightly soluble in 5% w/v sodium hydroxide solution. Practically insoluble in water, dilute acids and most organic solvents.

 

Stability : Though it is a hygroscopic material, it is stable.

 

Storage : To be stored in a well closed container in a cool, dry place.

 

Lactose

Synonyms              : Aero flow , fast flow, flowlac, milk sugar,

Funtional category:   Tablet and capsule diluents and channeling agrnt.

Description : White to off- white crystalline particles or powder.Lactose is odourless and slightly sweet taste.

 

Physical properties

Solubility : Highly soluble in water practically insoluble in chloroform , ethanol and ether

Bulk density                 : 0.62 g/cm³        

Tapped density             : 0.94 g/cm³

True density                  :1.522 g/cm³

Specific rotation           :+52° to +52.6°

 

 Stability and storage Conditions:

          Lactose may develop a brown coloration on storage, the reaction being accelerated by warm damp conditions. Lactose should be storage in a well-closed container in a cool, dry place.

 

Incompatibilities: A Maillard-type condensation reaction is likely to occur between lactose and compounds with a primary amine group to form brown, or yellow-brown-colored products.