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FORMULATION, DEVELOPMENT AND EVALUATION OF ESTRADIOL VAGINAL TABLETS


Kumkum Sarangdevot 1*, Bhawani Singh Sonigara2
1. Pacefic College of Pharmacy, Udaipur, Rajasthan, India
2. Bhupal Nobles’ College Of Pharmacy, Udaipur, Rajasthan, India

ABSTRACT

As the vaginal walls become thinner and drier and lose some of the folds that allow them to stretch, some women never notice any symptoms. Others may experience a burning sensation, a feeling of dryness, some itching, or a thin, watery discharge. While symptoms of vaginal dryness and atrophy can be very bothersome, the good news is that effective treatment options are available. These include different forms of low-dose estrogens applied directly to the vagina, as well as non-hormonal treatments. In the present study an attempt was made to formulate robust vaginal tablets suitable for scale up manufacturing and commercial use. Preliminary trials were planned using different grades of Hypromellose which showed good results with respect to flow property and compressibility but a variation in release profile form tablet o tablet. Seeing this later it was decided to formulate the tablet using Low viscosity polymer for better drug release and uniformity. Marketed formulation, falls within the USP specification. From the results of present investigation, it can be concluded that the tablet dosage form was successfully formulated and it has matched the target dissolution profile with specific time point with innovators patent and the final optimized formulation has shown the expected result for all the evaluation parameters.

Keywords: Vaginal walls, Estrogens, Hypromellose, Drug release, vaginal atrophy.

INTRODUCTION

INTRODUCTION OF DISEASE 

Vaginal Atrophy

Vaginal atrophy is most likely to occur after menopause, when   ovaries naturally decrease estrogen production and periods stop. It can also happen after sur­gical removal of   ovaries. Sometimes   ovaries shut down even if only   uterus is removed.   Ovaries also produce less estrogen at a time of breast-feeding, causing temporary vaginal atrophy. In this case, the vaginal walls return to their usual moist, stretchy state after   baby stops, or decreases, nursing1-6.

Medications used to treat conditions of the reproductive system, such as endometriosis or fibroids, can decrease estrogen levels and cause temporary vaginal atrophy. It can also be caused by medications used to treat some cancers. Vaginal atrophy usually occurs gradually, not immediately after estrogen levels decrease. It may take several years before notice the changes7.

Figure 1: Anatomy of vaginal wall 

As the vaginal walls become thinner and drier and lose some of the folds that allow them to stretch, some women never notice any symptoms. Others may experience a burning sensation, a feeling of dryness, some itching, or a thin, watery discharge. One of the most common symptoms is pain when inserting anything into the vagina, such as dur­ing intercourse, masturbation, or other sexual contact. After intercourse, patient may experience some light bleeding or spotting. During a pelvic examination,   health care provider may see the changes in   vagina8-10.

Menopause and aging can affect the vagina in the following ways:

  • Vaginal tissues become thin, dry, and less elastic
  • Vaginal secretions decrease with reduced lubrication
  • Vaginal infections increase (as the healthy acidic pH of the vagina increases)
  • Discomfort with urination and increased urinary tract infections can occur
  • Fragile, dry, inflamed vaginal tissues may tear and bleed
  • Women with menopause induced by cancer treatments may have additional injury to the vaginal tissues from chemotherapy or pelvic radiation
  • Aromatase inhibitors taken by many women with breast cancer result in extremely low estrogen levels, often causing severe symptoms of vaginal dryness and decreased lubrication
  • Vaginal changes often result in pain during intercourse or pelvic exams
  • Women with discomfort from vaginal atrophy often engage in less frequent intercourse, which can cause the vagina to become shorter, narrower, and less elastic
  • For some women, pain, narrowing of the vagina, and involuntary tightening of vaginal muscles (veganism’s) can intensify to the point where sexual intercourse is no longer pleasurable or even possible 6.

Figure: 2: Anatomy of vaginal wall 

Treatment options:

While symptoms of vaginal dryness and atrophy can be very bothersome, the good news is that effective treatment options are available. These include different forms of low-dose estrogen applied directly to the vagina, as well as non-hormonal treatments. You may combine non-hormonal and hormonal treatments for optimal symptom relief 9-16.

Non-hormonal remedies:

  • Vaginal lubricants reduce discomfort with sexual activity when the vagina is dry by decreasing friction during intercourse. Water-soluble products are advised because the oil in some products may cause vaginal irritation. There are many effective brands available without a prescription, including K-Y Jelly, Astroglide, K-Y Silk-E, Slippery Stuff, and Just Like Me.
  • Vaginal moisturizers line the wall of the vagina and maintain vaginal moisture. Like your face or hands, the vagina should be moisturized on a regular basis, for example, several times weekly at bedtime, including Replens and K-Y Liquibeads.
  • Regular sexual stimulation promotes blood flow and secretions to the vagina. Sexual stimulation with a partner, alone, or with a device (such as a vibrator) can improve vaginal health.
  • Expanding your views of sexual pleasure to include such “outercourse” options as extended caressing, mutual masturbation, and massage is an effective way to make painful vaginal penetration (intercourse) more comfortable, or provide a way to remain sexually intimate in place of intercourse.
  • Vaginal dilators can stretch and enlarge the vagina after many years of severe vaginal atrophy, especially if sexual activity is infrequent and the vagina has become too short and narrow for intercourse. Involuntary tightening of vaginal muscles (vaginismus), a learned response to pain, often contributes to uncomfortable intercourse. In addition to regular use of vaginal estrogens, lubricants, and moisturizers, several months of daily “exercises” with lubricated vaginal dilators can help. Dilators can be purchased from pharmacies and medical supply stores and used with the guidance of a gynecologist, physical therapist, or sex therapist. Remember, the vagina can diminish in size and its supporting muscles can weaken, so “use it or lose it”!
  • Pelvic floor exercises can both strengthen weak vaginal muscles and relax tight ones.

Vaginal estrogen therapy:

  • An effective and safe treatment available by prescription, low-dose local estrogen is applied directly to the vagina to increase the thickness and elasticity of vaginal tissues, restore a healthy vaginal pH, increase vaginal secretions, and relieve vaginal dryness and discomfort with sexual activity. Improvements usually occur within a few weeks, although complete relief may take several months.
  • Short-term treatment may even be an option for women with a history of breast or uterine cancer, but only after careful consideration of risks and benefits with a healthcare provider.
  • Government-approved low-dose vaginal estrogen products are available by prescription as vaginal creams (used 2 or 3 nights weekly), a vaginal estradiol tablet (used twice weekly), and an estradiol vaginal ring (changed every 3 months). All are highly effective. You may wish to try several different forms and choose the one you prefer.
  • Higher doses of estrogen therapy provided to treat hot flashes also treat vaginal dryness, although some women still need additional low-dose vaginal estrogen treatment. If only vaginal symptoms are present, low doses of estrogen applied to the vagina are recommended 16-28.

MATERIAL AND METHODS 

Preformulation Studies

Organoleptic Properties

  1. Colour: A small quantity of Estradiol hemihydrate powder was taken in butter paper and viewed in well-illuminated place.

Physico-mechanical Characteristics

  1. Solubility studies
  2. Loss on drying
  3. Micromeritic properties
  4. Compatibility studies
  5. Solubility studies of Estradiol Hemihydrate:
  • An accurate quantity of Estradiol Hemihydrate was taken separately and added in different solvents like methanol, water, Isopropyl alcohol and Different pH buffer solutions.
  • These solutions were shaken well for few minutes. Then the solubility was observed.

Table 1: Solubility studies

Descriptive Term Parts of Solvent Required for 1 part of Solute
Very soluble Less than 1
Freely soluble From 1 to 10
Soluble From 10 to 30
Sparingly soluble From 30 to 100
Slightly soluble From 100 to 1,000
Very slightly soluble From 1,000 to 10,000
Practically insoluble or Insoluble Greater than or equal to 10,000

  1. b) Loss on drying studies
  • Weigh accurately about 1 g of the test substance and transfer it to a weighing bottle of weight W1 with a stopper, previously dried at 105 ± 2oC for 30 minutes. Note down the weight of the weighing bottle with the sample (W2). Distribute the sample evenly in the weighing bottle by sidewise shaking to a height of 5 mm.
  • Place the weighing bottle uncovered at 105 ± 2oC in an oven for 3 hours. After 3 hours, open the oven and immediately place the stopper on the weighing bottle.

Place the weighing bottle in desiccators to reduce the temperature to room temperature. Determine the weight of weighing bottle with the contents. Note down the weight (W3). Calculate loss on drying with the following formula.

 

  • Not more than 0.5 per cent (for the anhydrous form) and 8.0 per cent to 9.5 per cent (for the hydrated form), determined on 1.0 g by drying in an oven at 1050C.
  1. c) Hygroscopicity
  • Weighed quantity of the pure drug was taken in a petridish and the initial weight of petridish with the drug was recorded.
  • Then, it was kept openly in environment chamber maintained at 75% RH.
  • The weight of the petridish was measured periodically and the weight gain or loss was calculated in reference to the initial weight of the petridish.
  1. d) Drug-Excipient Compatibility Studies

Drug and excipients different concentration mixtures were prepared. These samples were loaded for stress study i.e. 2 week, 60°C and 4 week, 40°C/75 % RH, in glass vial open condition.

Method

Samples mixtures were prepared in different ratio manually and were loaded in to chamber. Samples were analysed for Related substance and Physical appearance.

5.2. Formulation of Estradiol Vaginal Tablets:

General Manufacturing Procedure:

Sifting:

  • Sift the materials Estradiol Hemihydrate, Lactose 30 GR, Hypromellose, Pregelatinised starch, Magnesium stearate individually through SS sieve 40 # and collect the sifted materials separately and label them.

API Geometric premix preparation:

  • Mixing geometrically API: Lactose in uniform ratio.
  • Geometric mix of the above API Premix with rate controlling polymer.

Pre lubrication:

  • Charge sifted Hypromellose (Rate controlling polymer), API Premix into Double cone Blender and mix for 30 min at 20 RPM.
  • Sift the Above prelubricated blend through 40 #.

Lubrication:

  • Charge the sifted granules of above step and add Magnesium stearate (lubrication) into Double cone Blender and mix for 5 min.
  • Collect the resulted granules in polybag, label and record the weight of the granules.

Compression:

Lubricated granules were subjected to compression using B-Tooling of 6 mm standard concave punch.

 

Film Coating:

Film coating of tablets were done using PEG 6000 and Hypromellose E 3 Dissolved in purified water.

Potency Calculation for Estradiol Hemihydrate

Actual quantity of estradiol Hemihydrate required per tablet

Experimental Trials

Table 2: Formulation trials

B.No: Z1 Z2 Z3 Z4 Z5 Z6
Batch Size 161.2 g (2000 tablets) 161.2 g (2000 tablets) 161.2 g (2000 tablets) 161.2 g (2000 tablets) 161.2 g (2000 tablets) 161.2 g (2000 tablets)
Qty Per tablet (mg)
Estradiol Hemihydrate 0.01 0.01 0.01 0.01 0.01 0.01
Hypromellose (HPMC)
K 100 premium LV CR
___ ___ 45 55 55 55
Hypromellose (HPMC)
k 4 M
___ 10 ___ ___ ___ ___
Hypromellose (HPMC)
k 15M
10 ___ ___ ___ ___ ___
Lactose monohydrate
30 GR
59.60 59.60 24.60 14.60 8.60 4.60
PG starch 10 10 10 10 16 20
Magnesium Stearate 0.4 0.4 0.4 0.4 0.4 0.4
Total Core Weight 80 80 80 80 80 80
Film coating
Hypromellose E 3 LV 0.54 0.54 0.54 0.54 0.54 0.54
PEG 6000 0.06 0.06 0.06 0.06 0.06 0.06
Purified water qs. qs. qs. qs. qs. qs.
Total weight 80.60 80.60 80.60 80.60 80.60 80.60

 

Evaluation of granules

Evaluation was performed to assess the physicochemical properties and release characteristics of the developed formulations.

Precompression studies

  1. a) Bulk density (Db)

It is the ratio of total mass of powder to the bulk volume of powder. It was measured by pouring the weighed powder (passed through standard sieve # 20) into a measuring cylinder and the initial volume was noted. This initial volume is called the bulk volume. From this, the bulk density is calculated according to the formula mentioned below. It is expressed in g/cc and is given by: 

  1. b) Tapped density

It is the ratio of total mass of powder to the tapped volume of powder. The volume was measured by tapping the powder for 500 times. Then the tapping was done for 750 times and the tapped volume was noted (the difference between these two volumes should be less than 2%). If it is more than 2%, tapping is continued for 1250 times and tapped volume was noted. It is expressed in g/cc and is given by:

  1. c) Flow properties (Angle of Repose (θ))

Angle of repose is used to characterize the flow particles of particles. It is a characteristic related to interparticulate friction or resistance to movement between particles.

This is the maximum angle possible between the surface of a pile of powder or granules and the horizontal plane.

The angle of repose of granules was determined by funnel method. The funnel was fixed at a particular height (2.5 cm) on a burette stand. The powder sample was passed through the funnel until it forms a heap. Further adding of granules was stopped as soon as the heap touches the tip of the funnel. A circle was drawn across it without disturbing the pile. The radius and height of the heap was noted down. The same procedure was repeated for three times and the average value was taken. The angle of repose was calculated by using equation.                     tan θ = h/r

θ = tan –1(h/r)

where,

θ = Angle of repose

h = Height of the heap

r = Radius of the heap.

  1. d) Compressibility Index

Compressibility Index has been proposed as indirect measurement of bulk density, size, shape, surface area, moisture content and cohesiveness of material because all of these can influence the observed. The flow ability of powder can be evaluated by comparing the loose Bulk density (LBD) and Tapped bulk density (TBD) of powder and the rate at which it packed down.  Compressibility index of the granules was determined by the Carr’s compressibility index:

  1. e) Hausner’s Ratio

It is measurement of frictional resistance of the drug. The Ideal range should between 1.2 – 1.5, it was determined by the ratio of tapped density and bulk density.

In-Vitro dissolution study (USP Pharmacopoeial specification)

       Dissolution Parameters

  • Medium :    500 ml Phosphate buffer
  • pH :     75
  • Apparatus :     USP apparatus-I (Basket type)
  • RPM :     40
  • Time :     3,5,10 hours
  • Temperature             :     37 ± 0.5 0C

Medium preparation:

500 ml of dissolution medium was placed in the vessels of the apparatus, the apparatus was assembled and the dissolution medium was equilibrated at 37°C ± 0.5°C. Placed one tablet in each of the vessel and immediately operated the instrument at specified RPM. At the end of the specified time, the solution was withdrawn from the vessel. a portion of this solution was Centrifuged at 3000 rpm for 3 minutes and the clear supernatant solution was used and injected.

Weighed 6 tablets and dropped into separate dissolution bowl after run time, samples were collected from each dissolution bowl and replaced with buffer in specified intervals.

The % of Estradiol dissolved was determined by employing the following method:-

Preparation of Mobile Phase:

Organic: Acetonitrile and Methanol (50: 50, v/v).

Prepare a degassed mixture of filtered Water and Organic (55: 45, v/v)

 

Table 3: Chromatographic conditions

Column Waters X-Bridge TM, C18. 75 mm x 4.6 mm, 3.5 µm or equivalent
Column Temperature 30oC
Flow rate 1.5 mL/Min
Wavelength 285 nm
Injection volume 200 µL
Run time 10 minutes
Retention time for Estradiol 3.0 to  5.0 minutes

  

Preparation of standard solutions:

Preparation of Estradiol standard stock solution (Solution A):

About 20 mg of Estradiol hemihydrates was weighed and transferred accurately working standard into 200 ml volumetric flask. about 170 ml of alcohol was added  and sonicated to dissolve.

Allowed it to come to room temperature and diluted up to the 1nark with alcohol and mixed.

(Concentration of Estradiol is 100.0 ~g/ mL)

Preparation of standard solution:

5 ml of Solution A was pipette out into 50 ml volumetric flask, diluted up to the mark with dissolution medium and mix. 4 ml of this solution into 100 ml volumetric flask, diluted up to the mark with dissolution medium and mixed. Further 5 ml of this solution was pipetted out into 100 ml volumetric flask, diluted up to the mark with dissolution medium and mixed.

(Concentration of Estradiol is 0.02 µgl ml)

 

 

Procedure:

The equal specified volumes of dissolution medium, standard solution- I (five injections) and standard solution-II (two injections) were separately injected into the chromatograph. The chromatograms recorded and measured the peak responses due to Estradiol. The sample solution was injected and calculated the amount of Estradiol dissolved in terms of percentage by using formula given under calculations.

ASSAY

Table 4: Chromatographic conditions

Column Waters X-Bridge TM, C18. 75 mm x 4.6 mm, 3.5 µm or equivalent
Column Temperature 30oC
Flow rate 1.5 mL/Min
Wavelength 285 nm
Injection volume 200 µL
Run time 10 minutes
Retention time for Estradiol 3.0 to  5.0 minutes

 

Preparation of Mobile Phase:

Organic: Acetonitrile and Methanol (50: 50, v/v).

Prepare a degassed mixture of filtered Water and Organic (55: 45,v/v),

Preparation Diluent: Prepare mixture of Water and Acetonitrile (50: 50, v/v)

 

Preparation of Standard solutions:

About 25 mg of Estradiol hemihydrates working standard was weighed and transferred accurately into 100 ml volumetric flask. About 60 ml of diluent was added and sonicated to dissolve. Allowed it to come to room temperature and diluted up to the mark with diluent and mixed. 5 ml of this solution was added into 50 ml volumetric flask, diluted up to the mark with diluent and mixed. 4 ml of this solution was added into 100 ml volumetric flask, and 5 ml of acetone was added quantitatively with the help of pipette. Diluted up to the mark with diluent and mixed.

(Concentration of Estradiol is 1.0 µg/ mL)

 

Preparation Sample solution:

10 intact tablets were weighed and transferred into a 100 ml volumetric flask. About 5 ml of Acetone was added and shaken to disperse tablets completely. About 70 ml of diluent was added and shaken on mechanical shaker for 1 hour, then sonicated at room temperature for about 20 minutes with intermittent vigorous shaking. Allow it to come to room temperature, diluted up to the mark with diluent and mixed. the portion of this solution was Centrifuged at 3000 RPM for 10 minutes and injected.

(Concentration of Estradiol is 1.0 µg/ ml).

 

Stability Studies

Objective of the study

The purpose of stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors such as temperature, humidity and light, enabling recommended storage conditions, re-test periods and shelf-lives to be provided.

 

 

Table 5: Storage Conditions

Study Storage condition Minimum time period covered by data at submission.
Long term 25ºC ± 2 ºC/ 60% RH ± 5% RH 12 months
Intermediate 30ºC ± 2 ºC/ 65% RH ± 5% RH 6 months
Accelerated 40ºC ± 2 ºC/ 75% RH ± 5% RH 6 months

Method

The selected formulations were packed. They were then stored at 40 0C/75% RH for one month and evaluated for their physical appearance, drug content and in-vitro dissolution time at specified intervals of time.

Studies designed to increase the rate of chemical degradation or physical change of an active drug substance or drug product by using exaggerated storage conditions as a formal, definitive storage programme.

When significant change occurs at any time during testing at the accelerated storage condition, additional testing at the intermediate storage condition should be conducted and evaluated against significant change criteria.

RESULTS AND DISCUSSION  

Preformulation Studies

Preformulation investigations are designed to identify those physicochemical properties and excipients that may influence the formulation design, method of manufacture, and pharmacokinetic-biopharmaceutical properties of the resulting product. Followings studies performed for in the Preformulation study.

API characterization

  • Appearance

White or off-white crystalline powder or colorless crystals.

  • Specific Rotation

The specific rotation is +76.0° ≤ [α]20D ≤+83.0° (calculated on the anhydrous and solvent-free basis)

  • pKa values

The chemical structure of EH does not show any groups, which can be protonated or deprotonated in the biologically relevant range of pH 2 to pH 10. Therefore, EH can be considered as a neutral molecule in this pH range.

Pka = 10.3 (hydroxyl group)

  • Partition coefficient

The partition coefficient of EH is 3.30

  • Hygroscopicity

     Estradiol was kept at controlled humid condition at 75 % RH for 7 days. And continuous monitoring of weight was done. It was observed there was no change in weight. EH is not hygroscopic.

  • Polymorphism and pseudo-polymorphism

EH was examined for polymorphism and pseudo-polymorphism according to the ICH guidelines Q6A by instrumental methods of analysis, crystallization experiments from different solvents and from the melt.

Estradiol Hemihydrate (EH), contains between 2.9% and 3.5% water (Karl Fischer titration). The calculated water content of the hemihydrate is 3.2%. The hemihydrate may lose water between 80°C and 174°C depending on the particle sizes. In addition, estradiol forms two unstable anhydrous forms, polymorph I with a melting point of approx. 178°C and polymorph II with a melting point of approx. 169°C, which are readily transformed into the hemihydrate under the influence of atmospheric moisture. EH forms also unstable solvates with methanol and ethanol. The amorphous form (glass transition point between 56°C and 84°C) can exist at room temperature.

The identity of the hemihydrate is controlled by IR spectroscopy.

  • Solubility

Table 6: Solubility of drug

Solvent Solubility[mg/mL] Temp.[°C] Descriptive term
Water 0.002 20 Practically insoluble
Water 0.003 37 Practically insoluble
Buffer pH 1.0 0.003 37 Practically insoluble
Buffer pH 4.5 0.003 37 Practically insoluble
Buffer pH 6.8 0.003 37 Practically insoluble

 

Organic Solvent solubility:

Table 7: Solubility of drug

Sr. No. Media Class Description
1 Methanol Freely soluble
2 Ethanol
3 Iso propyl alcohol

 

The solubility data confirms that Estradiol Hemihydrate solubility is pH independent and is freely soluble in organic solvents.

  • UV spectrum and specific absorbance

Ultraviolet–visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region. This means it uses light in the visible and adjacent (near-UV and near-infrared (NIR)) ranges. The absorption or reflectance in the visible range directly affects the perceived color of the chemicals involved. In this region of the electromagnetic spectrum, molecules undergo electronic transitions. This technique is complementary to fluorescence spectroscopy, in that fluorescence deals with transitions from the excited state to the ground state, while absorption measures transitions from the ground state to the excited state.

The UV spectrum was recorded using EH dissolved in methanol (1.92mg/50ml)

Absorbance maximum: 281nm

Molar absorption coefficient: 2080 L/mol*cm

Figure 3: U.V Spectrum of EH

  • DSC of EH

Differential Scanning Calorimetry enables the quantitative detection of all process in which energy is required or produced (i.e. endothermic or exothermic phase transformation). samples were weighed (8-10mg) and heated at a scanning rate of 20 ºC/min under air flow (100 mL/min) between 50 ºC to 400 ºC. Aluminium pans and lids were used for all samples. From the results of DSC graph it was found that drug EH showed endothermic peak at 181.38 ºC due to melting point of EH.

Figure 4: DSC spectra of EH

STRESS TESTING OF API

  • Thermal Stress:

When stored at a 50°C to 90°C for up to 30 days EH micronized drug substance did not show any significant degradation. The same applies when the material is exposed to 50°C to 90°C at 75% relative humidity.

  • Hydrolytic Stress:

When suspended in buffer solutions at pH 1, 7 and 9 at temperatures of 50, 70 and 90°C EH shows no significant degradation.

  • Oxidative Stress:

EH is very stable when suspended in water and treated with hydrogen peroxide solution. After 24 hours at 50°C and treatment of 0.3% hydrogen peroxide solution a very slight increase in impurities is detected (0.33% of unknown impurities).

Figure 5: Chromatogram of unstressed EH

Figure 6: Chromatogram of EH suspended in 0.3% hydrogen peroxide solution heated at 50°C and stored for 24 hours.

 

  • Photo stability:

The test on photo stability was performed with the solid substance as well as with a suspension in water according to ICH Q1B. The experiment was performed in a light chamber with Hg lamp, day light type. The overall illumination was 1.28 Million. Lux hours.

Neither solid drug substance nor water suspension showed degradation or change in appearance.

Nevertheless, since the description in the respective USP monograph says “preserve in tight, light resistant container” EH micronized drug substance is stored “protected from light”.

Result Table:Table 8: Result table of stress testing of API

Sr. No. Test condition Temperature/RH (°C/%) Time (days) Total impurities (%) Assay (%)
1. Influence of light (20,000 LUX) on solid and suspension of substance
Solid substance 64hr 0.30 99.70
Suspension 64hr 0.30 99.70
2. Influence of temperature on solid substance
Solid substance 90/75 30 0.20 99.80
3. Influence of buffer solutions, acid and alkali on suspension of substance at 90°C
suspension pH 1 30 0.60 99.40
suspension pH 7 30 0.40 99.60
suspension pH 9 30 0.40 99.60
4. Influence of hydrogen peroxide on solution of substance at 50°C
suspension H2O2 0.3% 1 0.60 99.40
5. Influence of organic solvent on solution of substance at 50°C
solution Ethanol 3 0.40 99.40

 

API-EXCIPIENTS COMPATIBILITY STUDY

Study detail:

  • API: EH
  • Incubation study:

Compatibility study of

  1. Individual API
  2. Individual API with each of the excipients

Excipients and API also placed separately in closed vials as control samples.

  • Sample preparation process
  1. API + Excipients
  2. Shift separately the EH and excipients through #40 sieve.
  • Mix API and Excipients thoroughly in proposed ratios.
  1. Pass the bends of step iii) individually through #40 sieve and mix properly on butter papers. Use separate butter paper for each blend.
  2. Transfer suitable quantity of individual blend of step iv) into vials and pack as per pack T or pack H (explained below).
  3. Incubate the prepared samples in respective chambers
  • Pack storage conditions:

Keep all initial samples at 25°C/60%RH till analysis.

Table 9: Pack storage conditions

Condition Pack Period Test details* No. of vials
25°C/60%RH Initial a,b,c 1
25°C/60%RH Control 4 weeks a,b,c 1
60°C ± 2°C T 2 weeks a,b,c 1
40°C/75%RH H 4 weeks a,b,c 1

 

 

*Test details:

a = Appearance/physical characteristics

b = Related substance

c = DSC (if applicable)

Pack T and control sample packing: clear glass vials should be closed with butyl rubber caps and sealed with Alu seal

Pack H: Clear glass vials should be closed with aluminum foil with hole.

Table 10: Sample preparation plan for API and excipients alone

Sr. No. Ingredients Quantity per vial (mg)
API alone
1. EH 500
  Excipients alone
1. Hypromellose K 100M LV 1000
2. Hypromellose K 15M 1000
3. Hypromellose K 4M 1000
4. Hypromellose E 3 LV 1000
5. Lactose monohydrate 30GR 1000
6. Pregelatinised starch 1000
7. Magnesium stearate 1000
8. PEG 6000 1000

 

Table 11: Sample preparation plan for EH and excipients

Sr.No. Ingredients Ratio
1. EH + Hypromellose K 100M LV 1:25
2. EH + Hypromellose K 15M 1:700
3. EH + Hypromellose K 4M 1:400
4. EH + Hypromellose E 3 LV 1:700
5. EH + Lactose monohydrate 30GR 1:25
6. EH + Pregelatinised starch 1:50
7. EH + Magnesium stearate 1:50
8. EH + PEG 6000 1:25

Result Table: 

Table 12:  Result table of API-excipients compatibility study

Sr. No. Sample condition Total impurities (%)
1 API Initial 0.16
2 week, 60°C 0.24
4 week, 40°C 0.23
2 API + Hypromellose K 100M LV Initial 0.18
2 week, 60°C 0.23
4 week, 40°C 0.23
3 API + Hypromellose K 15M Initial 0.51
2 week, 60°C 0.69
4 week, 40°C 0.58
4 API + Hypromellose K 4M Initial 0.22
2 week, 60°C 0.26
4 week, 40°C 0.19
5 API + Hypromellose E 3 LV Initial 0.30
2 week, 60°C 0.28
4 week, 40°C 0.27
6 API + Lactose monohydrate 30GR Initial 0.27
2 week, 60°C 0.24
4 week, 40°C 0.27
7 API + Pregelatinised starch Initial 0.44
2 week, 60°C 0.49
4 week, 40°C 0.46
8 API + Magnesium stearate Initial 0.26
2 week, 60°C 0.26
4 week, 40°C 0.26
9 API + PEG 6000 Initial 0.27
2 week, 60°C 0.25
4 week, 40°C 0.25


Formulation of Estradiol Vaginal Tablets:

Introduction

Numerous oral extended DDS have been developed to prolong drug release, the real challenge in the development of an extended release DDS lies mainly in prolonging the residence of the dosage form the drug is completely released in the desired time period

A matrix device consists of drug dispersed homogenously throughout a polymer matrix. There are two major types of materials used in the preparation of matrix devices.

Matrix devices offer several advantages relative to other extended release dose forms:

  • Ease of manufacture, since the dosage form is prepared with a single manufacturing step
  • Versatility, effectiveness and low cost
  • Can be made to release high molecular weight compounds
  • As the drug is dispersed homogeneously throughout the polymeric matrix, accidental leakage of the total drug component is less likely to occur, althoug

Occasionally, cracking of the matrix material can cause unwanted release, since the drug is dispersed in the matrix system.

  • Challenges of matrix systems:
  • The remaining matrix should be removed after the drug has been completely released to avoid accumulation following multiple administrations.
  • The drug release rates vary with the square root of time. Release rate continuously diminishes due to an increase in diffusional resistance and/ or a decrease in effective area at the diffusion front
  • However, a substantial sustained effect can be produced through the use of very slow release rates, which in many applications are indistinguishable from zero-order.

Various experimental trials have been taken with varying concentrations of polymers:

Trials:

Trail Z1:

  • Batch was manufactured using Hypromellose K 15M as a rate controlling agent.
  • The manufacturing process was direct compression.

Figure 7: Dissolution release profile of Trial Z1 

Physical parameters Of Granules:

Table 13: Physical parameters of Trial Z1

Parameter Observation
BD 0.397 g/mL
TD 0.485 g/mL
HR 1.22
CI 18.14

Physical parameters Of Tablets:

Table 14: Physical parameters of Trial Z1

Uncoated tablet Coated tablet
Average weight 79.9-80.7 80.5-81.5
Hardness 30-40 N
Thickness 2.80-2.95 2.90-3.05
Friability Nil

Dissolution release profile and Assay:

Table 15: Dissolution release profile of Trial Z1

Time in Hours Z1 RLD
3 5 43
5 23 64
8 47 92
Assay 99.3 99.7

Observation:

  • The release of drug was retarding, not uniform, not within the limits required.
  • An alternate trial replacing Hypromellose K 15 M with Low viscosity Hypromellose K 4 M was planned by keeping all parameters constant.

Trail Z2:

  • Batch was manufactured using Hypromellose K 4 M as a rate controlling agent.
  • The manufacturing process was direct compression.

Figure 8: Dissolution release profile of Trial Z2

 

Physical parameters Of Granules:

Table 16: Physical parameters of Trial Z2

Parameter Observation
BD 0.416 g/mL
TD 0.505 g/mL
HR 1.21
CI 17.62

Physical parameters Of Tablets:

Table 17: Physical parameters of Trial Z2

Uncoated tablet Coated tablet
Average weight (mg) 79.5-80.3 80.0-81.2
Hardness 30-40 N
Thickness 2.85-2.98 2.95-3.10
Friability Nil

Dissolution release profile and Assay:

Table 18: Dissolution release profile of Trial Z2

Time in Hours Z2 RLD
3 18 43
5 43 64
8 74 92
Assay 98.5 99.7

Observation:

  • The release of drug was retarding, their was tablet to tablet variation in release profile & it is not within the limits required.
  • An alternate trial replacing Hypromellose K 4 M with Low viscosity Hypromellose K 100 LV CR was planned by keeping all parameters constant.

Trail Z3:

  • Batch was manufactured using Hypromellose K100 LV CR as a rate controlling agent.
  • The manufacturing process was direct compression.

 

Figure No 9: Dissolution release profile of Trial Z3  

Physical parameters Of Granules:

                     Table 19:  Physical parameters of Trial Z3

Parameter Observation
BD 0.382 g/mL
TD 0.472 g/mL
HR 1.24
CI 19.07

 Physical parameters Of Tablets:

                      Table 20: Physical parameters of Trial Z3

Uncoated tablet Coated tablet
Average weight 79.2-80.1 79.8-81.1
Hardness 30-40 N
Thickness 2.85-2.93 2.95-3.0058
Friability Nil

Dissolution release profile and Assay:

Table 21: Dissolution release profile of Trial Z3

Time in Hours Z3 RLD
3 62 43
5 85 64
8 99 92
Assay 99.1 99.7

Observation:

  • The release of drug was retarding, uniform it is not within the limits required.
  • An alternate trial changing concentration of Hypromellose K 100 LV CR was planned by keeping all parameters constant.

Trail Z4:

  • Batch was manufactured using Hypromellose K100 LV CR as a rate controlling agent.
  • The manufacturing process was direct compression.

 Figure 10: Dissolution release profile of Trial Z4

 

Physical parameters Of Granules:

Table 22: Physical parameters of Trial Z4

Parameter Observation
BD 0.370 g/ml
TD 0.493 g/ml
HR 1.33
CI 24.95

 Physical parameters Of Tablets:

Table 23: Physical parameters of Trial Z4

Uncoated tablet Coated tablet
Average weight (mg) 79.8-80.1 80.3-81.0
Hardness 30-40 N
Thickness 2.85-2.93 2.98-3.05
Friability Nil

 Dissolution release profile and Assay

Table 24: Dissolution release profile of Trial Z4

Time in Hours Z4 RLD
3 62 43
5 85 64
8 99 92
Assay 98.9 99.7

 Observation:

  • The release of drug was retarding, uniform it is near to the limits required.
  • An alternate trial changing (increasing) concentration of Hypromellose K 100 LV CR was planned by keeping all parameters constant. 

Trail Z5:

  • Batch was manufactured using Hypromellose K100 LV CR as a rate controlling agent.
  • The manufacturing process was direct compression.

Figure 11: Dissolution release profile of Trial Z5

Physical parameters Of Granules:

Table 25: Physical parameters of Trial Z5

Parameter Observation
BD 0.382 g/mL
TD 0.472 g/mL
HR 1.24
CI 19.07

Physical parameters Of Tablets:

Table 26 Physical parameters of Trial Z5

Uncoated tablet Coated tablet
Average weight (mg) 79.9-80.4 80.3-81.0
Hardness 30-40 N
Thickness 2.88-2.96 2.94-3.05
Friability Nil

 Dissolution release profile and Assay

Table 27: Dissolution release profile of Trial Z5

Time in Hours Z5 RLD
3 40 43
5 72 64
8 92 92
Assay 99.2 99.7

 Observation:

  • The release of drug was retarding, uniform it is near to the limits required.
  • An alternate trial changing concentration of Pregelatinized starch was planned by keeping excipients and parameters constant.

Trail Z6:

  • Batch was manufactured using increasing Pregelatinized starch.
  • The manufacturing process was direct compression.

Figure 12: Dissolution release profile of Trial Z6

Physical parameters Of Granules:           

Table 28:  Physical parameters of Trial Z6

Parameter Observation
BD 0.370 g/mL
TD 0.493 g/mL
HR 1.33
CI 24.95

Physical parameters Of Tablets:

Table 29: Physical parameters of Trial Z6

Uncoated tablet Coated tablet
Average weight (mg) 79.7-80.1 80.2-81.1
Hardness 30-40 N
Thickness 2.85-2.93 2.95-3.10
Friability Nil

Dissolution release profile and Assay:

Table 30: Dissolution release profile of Trial Z6

Time in Hours Z6 RLD
3 26 43
5 51 64
8 82 92
Assay 99.6 99.7

 Observation:

  • The release of drug was retarding, uniform it is near to the limits required.
  • An alternate trial changing concentration of Pregelatinized starch was planned by keeping excipients and parameters constant.

COMPARISON OF BATCHES:

Table 31: Evaluation parameters of Tablets for different trials

Evaluation Parameter Z1 Z2 Z3 Z4 Z5 Z6
Physical Attributes
Appearance White to off white, Round, biconvex film coated tablets plain on both sides.
Average Weight (mg) 80.8 80.7 80.7 80.7 80.7 80.8
Thickness (mm) 3.05 3.10 3.08 3.05 3.05 3.10
Friability (%) 0.15 0.13 0.11 0.20 0.14 0.15
Hardness (N) 30-40 N
Quality Attributes
Bulk Density (g/mL) 0.397 0.416 0.382 0.370 0.382 0.370
Physical Parameters Tapped Density (g/mL) 0.485 0.505 0.472 0.493 0.472 0.493
Car’s Index 1.22 1.21 1.24 1.33 1.24 1.33
Hausner ratio 18.14 17.62 19.07 24.95 19.07 24.95
Assay (%) 99.3 98.50 99.10 98.90 99.2 99.6
Dissolution (%) 3 h (25-50) 5 18 62 55 40 26
5 h (40-80) 23 43 85 81 72 51
10 h (NLT 75) 47 74 99 94 92 82

Figure 13: Dissolution release profile of all Trials Vs RLD

DISCUSSION

  • In trial Z5 formulation, drug content was found in between 90-110%, which was in the limits and ensured the uniformity of the drug content in the formulation.
  • Dissolution of optimize batch was close to the marketed product.
  • A value of 100% for the similarity factor (f2) suggests that the test and reference profiles are identical. Values between 50 and 100 indicate that the dissolution profiles are similar whilst smaller values imply an increase in dissimilarity between release profiles. Similarity factor f2 72 for marketed formulation and optimized formulation was calculated and that shown that similarity between drug release profile of marketed formulation and optimized formulation.

Drug Release kinetics:

Table 32: Kinetic parameters

Release Exponent (n) Drug transport mechanism Rate as a function of time
0.5 Fickian diffusion t-0.5
0.45 < n= 0.89 Non- Fickian diffusion tn-1
0.89 Case II transport Zero order release
Higher than 0.89 Super case II transport tn-1

 

Conclusion: Based on the R2 values of all models it can be confirmed that both Trial formulation and RLD are following Zero order drug release dissolution profile.

Swelling index study

To confirm release study swelling pattern of tablets marketed Vs. Lead batch Z5 was studied. Photographs of swelling study for marketed formulation and Z 5 shown in figure.

Initial in SVF (RLD, Z5, Z4)

Initial in Water (RLD, Z5, Z4)

Swelling pattern after 8 hours in SVF

Swelling pattern after 8 hours in water

Swelling pattern study clearly shows similarity in swelling pattern.

Figure 15: Swelling pattern of tablets Trial Z5, Z4 Vs RLD

Stability Testing

Table 33: Stability testing of tablets

Sr. No. Sample condition Hardness % Assay % Drug release
3 h 5 h 10 h
1 Z5 Initial 30-40N 99.20 40 72 92
40°C, 15 days 30-35N 99.20 38 70 90
40°C, 30 days 30-35N 98.90 41 72 91
40°C, 1 month 30-35N 98.98 40 68 88

 

The stability data shows that tablet remains stable at 40°C up to 1 month. Total impurities were not more than even 1%. No any significant change observed in the dissolution of the tablets and also no change in color or any other physical parameter observed in the formulation, which shows that present formulation is stable in the criteria of impurities, dissolution and physical appearance.

CONCLUSION

In the present study an attempt was made to formulate robust vaginal tablets suitable for scale up manufacturing and commercial use. Dissolution profile of the optimized formulation was matched with that of the innovator, which was challenging and fruitful for the company beneficial. According to the review of work done on Estradiol vaginal tablets it was decided for a controlled release tablet for enhanced effect/Hormone replacement therapy for the patient.

Preformulation study revealed that Estradiol hemihydrate was stable with all the excipients used within the core part with all directly compressible excipients. There was not any Physical, Chemical or therapeutic incompatibility was found between drug and excipient in present study. The innovator product was evaluated for the cumulative percentage drug release of estradiol to design formulation similar to that of innovator product.

Preliminary trials were planned using different grades of Hypromellose which showed good results with respect to flow property and compressibility but a variation in release profile form tablet o tablet. Seeing this later it was decided to formulate the tablet using Low viscosity polymer for better drug release and uniformity. Hypromellose K 100 premium LV CR directly compressible was then selected for formulation. Trial Z5 which was formulated and evaluated. It was found to give satisfactory results and was comparable to that of marketed formulation, falls within the USP specification.

From the results of present investigation, it can be concluded that the tablet dosage form was successfully formulated and it has matched the target dissolution profile with specific time point with innovators patent and the final optimized formulation has shown the expected result for all the evaluation parameters.

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