GELS:

Gels are transparent or translucent semisolid formulations containing a high ratio of solvent / gelling agent.¹

CLASSIFICATION OF GELS⁶: Gels are classified by two methods based on:

a) Nature of colloid phase:

i) Inorganic gels.

ii) Organic gels.

b) Based on nature of solvent:

i) Aqueous gels.

ii) Non-aqueous gels.

MECHANISM OF DRUG ABSORPTION^5,6:

The rate of permeation across various layers of skin tissues in the course of topical application can be expressed mathematically as:

dQ/dt = P_s(C_d- C_r)

Where dQ/dt = rate of permeation across various layers.

C_d = Concentration of drug in the donar phase e.g. on the surface of stratum corneum of vagina.

C_r = Concentration of drug in the receptor phase e.g. body.

P_s = Permeability co-efficient of the skin tissues.

The concentration in the systemic circulation which is penetrating in the form of pharmacological activity, the permeability co-efficient is given by the relationship:

P_s = K_s.D_ss/ h_s.

Where K_s= Partition coefficient of the penetrant molecules.

h_s = Overall thickness of the skin tissues.

D_ss = Apparent diffusivity for the steady state diffusion of penetrate moles.

If C_d >> C_r than the equation is written as:

dQ/ dt = P_s.C_d.

PREPARATION OF INTRA – VAGINAL GEL:

For the preparation of gel the polymer and purified water I.P. should take in a glass beaker and allowed to soak for 24 hrs and to this required amount of drug and other additives should added with continuous stirring to form a semisolid mass.^7,8 The prepared gel should packed in laminated aluminum collapsible tubes or sterile lacquered collapsible aluminum tubes or in glass containers.^8,9,10 It should be kept in cool place for further study.

METHODS USED FOR THE EVALUATION OF INTRA – VAGINAL GEL:

For the evaluation of gel different parameters should be covered:

1) Percent Yield of gel:

The percent yield of gel should calculate by using the following relation¹¹:

Percent Yield = [Weight of formulated gel (W_g) / total weight of material taken for formulation (W_m)] × 100.

2) Estimation of Drug in Vaginal Gel:

Accurately weighed 1.0 gm of vaginal gel suspend into 50 ml of volumetric flask to which 20 ml of suitable stimulated vaginal fluid (SVF) add with continuous shaking to dissolve the gel and then make up the volume up to mark with the same fluid. Plain bases should also treat in similar manner for blank determination. Then filter these solutions using filter paper and analyzed by using UV-Visible spectrophotometer after suitable dilution at scanned wavelength.¹²

3) Homogeneity of gel:

Initially the formulations should test for homogeneity by visual inspection. To further ensure homogeneity of drug content in formulation of gel, six tubes should take randomly from different locations in mixer and assayed for the drug content using UV-Visible spectroscopy at scanned wavelength. Studies should perform in triplicate and mean values should used for the analysis of data.¹²

4) Determination of pH:

Digital pH meter should used for the determination of pH of gel. For the determination of pH of gel 2.5 gram of vaginal gel should dissolve in 25 ml of distilled water and then dip the electrode into this gel formulation for some time and note the constant reading.^7,12,13

5) Colour of gel:

Initially the gel should test visually for colour inspection. Then should test at UV – visible by dissolving 1 gm of gel in SVF and analyze at UV - Visible spectrophotometer.^12,14

6) Strength of gel:

A sample of 50 gm of gel place in a 100 ml graduated cylinder and gelled in a thermostat at 37°C. The apparatus for measuring gel strength (shown in fig. 1) should allow penetrating in the gel. The gel strength which means the viscosity of the gels at physiological temperature was determined by the time (s), the apparatus should be to sink 5cm down through the prepared gel.^15,16

FIG.1. MEASUREMENT OF STRENGTH OF INTRA – VAGINAL GEL

7) Determination of Spreadability:

Spreadability was determined by apparatus suggested by Mutimer et al (1956)¹⁷ which was suitably modified in the laboratory and used for the study. It consists of a wooden block, which was provided by a pulley at one end. By this method, spreadability was measured on the basis of ‘Slip’ and ‘Drag’ characteristics of gels.¹² A ground glass slide should fix on this block. An excess of gel (about 2.0 gm) under study should place on this ground slide. The gel should then sandwiched between this slide and another glass slide having the dimension of fix ground slide and provide with the hook. A 1.0 Kg weight should place on the top of the two slides for 5 minutes to expel air and to provide a uniform film of the gel between the slides. Excess of the gel should scrap off from the edges. The top plate should then subject to pull of 80 gms. With the help of string attach to the hook and the time (in seconds) require by the top slide to cover a distance of 7.5 cm be note. A shorter interval indicates better Spreadability.^7,8,12,17,18 The Fig. 2 shows the determination of spreadability of intra – vaginal gel.

FIG.2. DETERMINATION OF SPREADABILITY OF INTRA – VAGINAL GEL

Spreadability should then calculate using the following formula:

S = M × L/ T

Where, S = is the spreadability,

M = is the weight in the pan (tied to the upper slide),

L = is the length moved by the glass slide and

T = represents the time taken to separate the slide completely from each other.

8) Extrudability Study:

It is a usual empirical test to measure the force required to extrude the material from tube. The method applied for determination of applied shear in the region of the rheogram corresponding to a shear rate exceeding the yield value and exhibiting consequent plug flow one such apparatus is described by wood et al.¹⁸

In the present study, the method adopted for evaluating gel formulation for extrudability should based upon the quantity in percentage of gel and gel extrude from lacquered aluminum collapsible tube on application of weight in grams require to extrude at least 0.5 cm ribbon of gel in 10 seconds. More quantity extrudes means better will be extrudability. The measurement of extrudability of each formulation should in triplicate and the average value should present.¹⁹ The extrudability should than calculate by using the following formula¹⁹:

Extrudability = Applied weight to extrude gel from tube (in gm) / Area (in cm²)

9) Rheological study of gel:

Katarina Edsman has studied the dynamic rheological measurement on gels containing four different Carbopol polymers and the corresponding mixtures with porcine gastric mucin and bovine sub - maxillary mucin. The method does not give the same ranking order when two different comparison strategies were used. The results were contrast to the results obtained with the tensile strength measurements.²⁰

Hassan developed a simple viscometric method to quantify mucin – polymer bioadhesive bond strength. Viscosities of 15 % w/w porcine gastric mucin dispersion were measured with Brookfield’s viscometer. In absence or presence of selected neutral, anionic and cationic polymer, viscosity components and the forces of bioadhesion were calculated. He observed a positive rheological synergism when chitosan solutions prepared in pH 4.7 acetate buffers and in 0.1 M Hcl, were mixed with a fixed amount of porcine gastric mucin. The mixture with mucin showed a viscosity greater than the sum of polymer and mucin viscosities.²⁰

Mortazavi and Smart investigated the effect of Carbopol 934 P on rheological behaviour of mucus gel and role of mucus and effect of various factors such as ionic concentration, polymer molecular weight, its concentration and the introduction of anionic, cationic and neutral polymers on mucoadhesive mucus interface.²¹

Carla Caramella et al investigated the influence of polymer concentration and polymer: mucin weight ratio on chitosan – mucin interaction, assessed by means of viscosimetric measurements. Two hydration media, distilled water and 0.1 M Hcl were used. Chitosan solutions were prepared at concentrations greater than the characteristics entanglement concentration and mixed with increasing amounts of porcine gastric mucin. Viscosity measurements were performed on the polymer – mucin mixtures and on polymer and mucin solutions having the same concentrations as in the mixtures. The formation of chitosan – mucin interaction products was determined on the basis of the changes in low shear viscosity and high shear viscosity of the mixtures as a function of polymer: mucin weight ratio. Rheological synergism parameter was also calculated. The results obtained suggest that two different types of rheological interaction occur between chitosan and mucin in both media, depending on polymer concentration and polymer: mucin weight ratio.²²

The rheological studies should carry out using Brookfield viscometer (or Brookfield programmes DV II + model). The gel under study should place in a small sample holder with a facility of water circulation. Water in the jacket circulated with the help of water immersion pump. Initially ice – cold water should circulate and then hot – water to raise the temperature gradually. The temperature – sensing probe should lowered in the gel and the temperature of gel should be recorded. Spindle should lower vertically in it. The spindle should be rotate at varying speed. Temperature should increased in the range of 10°C up to 40°C and viscosity should be recorded.¹⁵

10) Swelling Index:

Swelling of the polymer depends on the concentration of the polymer, ionic strength and presence of water. To determine the swelling index of prepare intra-vaginal gel, 1.0 gm of gel should take on porous aluminum foil, and then place separately in a 50 ml beaker containing 10 ml acetate buffer pH 4.7. Then remove the aluminum foil at predetermine period of time from beaker then put it on dry place for some time and reweigh. Swelling index should calculate by following^23,24:

Swelling Index (S_W) % = [(W_t – W_o) / W_o] × 100.

Where, (S_W) % = Equilibrium percent swelling,

W_t = Weight of swollen gel after time t,

W_o = Original weight of gel at zero time.

Test for swelling index of all formulations should repeat for three times.

11) Partition Coefficient Determination:

An equal volume from n-octanol and acetate buffer pH 4.7 should saturate with each other for 24 hours; the two phases should separate. Certain weight of either the drug alone or an equivalent weight of the gel should dissolve in 10 ml of the aqueous phase to give the concentration of 0.5 mg/ml. The final solution should transfer to a stoppered glass bottle containing 10 ml of n-octanol. The systems should agitate in a thermostated water bath at 37 ± 1ºC for 24 hours, the phases should then separate, the aqueous phase should filter and the concentration of the drug should determine spectrophotometrically at λ_max 316 nm against a blank solution prepare in an analogous manner. The concentration of the drug in octanol should calculate from the difference between the initial and final concentrations of the drug in the buffer phase. The partition coefficient should calculate according to Nernst equation.^25,26

C_organic

K = ----------------

C_aqueous

Where, K = partition coefficient

C_organic = concentration of the drug in organic phase (octanol)

C_aqueous = concentration of the drug in aqueous phase (buffer)

12) Drug interaction study of gel:

Dried gel/hydrogels (oven at 40°C) should powder in a mortar. IR spectra should record over the range 400 – 4000 cm^-1 (150 scans, resolution 1 cm^-1) in a Bruker IFS 66V FTIR spectrophotometer (Germany) or Shimadzu 8400S FTIR spectrophotometer (Japan), using the potassium bromide pellet technique.^27,28

13) Vaginal Irritation Test:

For vaginal irritation study rabbits (like New-Zealand white female rabbits) are suitable animal having large size of vagina and easily available. All vaginal gel samples should test for vaginal irritancy for atleast 48 hrs. But vaginal irritation study for 10 days or more than 10 days is the best for proper vaginal irritation study.²⁶

For the vaginal irritation study, total 18 female rabbits should use for proper study of one batch formulation of gel to avoid variations. From that total animal, 6 female rabbits should keep without any formulation (as blank), 6 female rabbits should treat intra – vaginally with standard irritant like benzalkonium chloride (BZK) for positive irritation effect and 6 female rabbits should treat intra-vaginally with 500 mg of intra-vaginal formulated gel, once per day for 14 consecutive days. Animal should kill on day 15 and parts of the cervico – vagina (upper), mid – vagina (middle) and uro – vagina (lower) of each animal should fix in 10% neutral – buffered formalin. Fix vaginal tissues should embedded in paraffin, sectioned and stained with hematoxylin and eosin (H & E). Stained sections should examine by light microscopy. Each of the 3 regions of vagina should score blindly for epithelial ulceration, leukocyte infiltration, edema and vascular congestion. The irritation scores should assign on the bases of semi – quantitative score system of Eckstein et al³⁵, which should as follows: individual score, 0 = none; 1 = minimal; 2 = mild; 3 = moderate; and 4 = intense irritation. This scoring system correlates to human irritation potential as follows: scores of 0 to 8 are acceptable; scores of 9 to 10 indicate borderline irritation potential; and scores of 11 and above are indicative of significant irritation potential.^26,30,31

14) In-vitro drug diffusion studies of intra – vaginal gels:

Kiescary Chien (KC) diffusion cell or other should use for in-vitro drug release study of intra-vaginal gel by using suitable stimulated vaginal fluid (SVF) as diffusion medium. The processed cellophane membrane should use for stimulating the vaginal in-vivo condition like vaginal epithelial barrier.³²

For this study, 1.0 gm of gel should keep in donor compartment. The entire surface of membrane should in contact with the receptor compartment containing 85 ml of acetate buffer pH 4.7 (or SVF). The receptor compartment should continuously stir (100 rpm) using a magnetic stirrer. The temperature in whole study should be 37 ± 1°C. The sample should withdraw at predetermined period of time and same volume should replace with fresh acetate buffer (or SVF). The absorbance of withdraw sample should measure at a particular λ_max of drug e.g. 267nm to estimate Zidovudine in using UV – visible spectrophotometer. But for analysis other instruments also used sometimes like HPLC, etc. The experiment should carry out in triplicate and average values should report.^32,33

15) In-vitro Drug Release Kinetic Study:

In order to determine the release kinetic model and mechanisms, the diffusion data should fit in the zero – order, first – order, Higuchi diffusion, Korsmeyer – Peppas, etc.³²

The zero – order, first – order, Higuchi diffusion / plot are the models which give information about the drug release kinetic, but Korsmeyer – Peppas is the model which gives the information about the drug release mechanism.

To analyze the in vitro release data various kinetic models should use to describe the release kinetics. The zero order rate Eq. (1) describes the systems where the drug release rate is independent of its concentration (Hadjiioannou et al., 1993). The first order Eq. (2) describes the release from system where release rate is concentration dependent (Bourne, 2002). Higuchi (1963) describe the release of drugs from insoluble matrix as a square root of time dependent process based on Fickian diffusion Eq. (3).

C = k_ot -------------------------------- (1)

Where, K_o is zero-order rate constant expressed in units of concentration/time and t is the time.

LogC = LogC_o - kt / 2.303 -------- (2)

Where, C_o is the initial concentration of drug and K is first order constant.

Q = Kt^{1/ 2} ----------------------------- (3)

Where, K is the constant reflecting the design variables of the system.

The following plots should made: cumulative % drug release vs. time (zero - order kinetic models); log cumulative of % drug remaining vs. time (first - order kinetic model); cumulative % drug release vs. square root of time (Higuchi model) and log cumulative % drug release vs. log time (Korsmeyer – Peppas model).³³

Korsmeyer et al (1983) derived a simple relationship which described drug release from a polymeric system Eq. (5). To find out the mechanism of drug release, first 60% drug release data was fitted in Korsmeyer–Peppas model:

M_t/M_∞ = Ktⁿ ---------------- (4)

Where M_t/M_∞is fraction of drug released at time t, K is the rate constant and n is the release exponent. The n value is used to characterize different release mechanisms as given in table 1 for cylindrical shaped matrices.^33,34

TABLE 1: DIFFUSION EXPONENT AND SOLUTE RELEASE MECHANISM FOR CYLINDRICAL SHAPE^35-37:

DIFFUSION EXPONENT (n)	OVERALL SOLUTE DIFFUSION MECHANISM
0.43	Fickian diffusion
0.43 < n < 0.85	Anomalous (non - Fickian) diffusion
0.85	Case – II transport
n > 0.85	Super case – II transport

16) Ex-Vivo Permeation Studies of intra-vaginal gel:

Kiescary Chien (KC) diffusion cell mounted with hairless goat vaginal membrane or other animal or human cadaver skin³⁸ should use for the permeation study of intra – vaginal gel. 1.0 gm of intra – vaginal gel should take into the donor compartment with 1.0 ml of acetate buffer pH 4.7 (or SVF) and phosphate buffer pH 7.4 (or stimulated blood fluid or SBF) should take into receptor compartment which should agitate using magnetic stirrer (100 rpm) and temperature maintain to 37 ± 1°C. The sample should withdraw at predetermine intervals of time and same volume should replace with fresh SVF. Absorbance should measure at particular λ_max of drug if using UV – Visible spectrophotometer as instrument for analysis or otherwise use any other instrument for analysis.⁷

17) Determination of the Permeability Parameters:

The amount of the drug permeates per unit surface area (µg/cm²) should plot versus time (minutes) and the flux (µg/cm² min^-1) should calculate from slope of line. The method³⁹ reported by Yoneto et al.,⁴⁰ should use for the analysis of the permeation data.

a) Flux determination:

The flux values sometimes also are calculated by using the following relations^41,42:

J_ss = ΔM / A. Δt

Where

ΔM is the amount of drug transported across the membrane during time Δt and

A is the diffusional area.

b) Permeability Coefficient determination:

The permeability coefficient should then calculate according to the following equation.^25,42

P_m = J_ss / C_d

Where

P_m = permeability coefficient

J_ss = flux

C_d = concentration of the drug in the donor side.

The partition coefficient, which is an indication of the distribution of drug between the gel bases and the receiving medium, should calculate by using the following equation:

P_m = K_p D / h

Where

P_m = permeability coefficient

K_p = partition coefficient

D = Diffusion coefficient

h = thickness of the membrane (cm)

In order to normalize the permeability data for each enhancer treat formulations in respect to its own control, enhancement factors as percent should calculate as reported by Shojaei et al.^25,44

P_enh

Enhancement factor (%) = --------------- × 100

P_control

Where

P_enh = permeability coefficient obtained for gels containing enhancer.

P_control = permeability coefficient obtained for gels without enhancer.

18) Ex-vivo bioadhesive strength measurement of gel:

A modified balance method should use for determining the ex-vivo mucoadhesive strength.⁴⁵ Fresh goat vaginal membrane should use for this study and you should use it within 2 hours of slaughter. The vaginal membrane should separate by removing the underlying fat and loose tissues. The membrane should wash with distilled water and then with acetate buffer pH 4.7 (or SVF).^46,47

The modified Patel et al (2007)⁴⁶ method should use for the measurement of bioadhesive strength. The fresh vaginal membrane should cut into pieces and wash with acetate buffer pH 4.7 (SVF). Than these two pieces of vaginal membrane tie to the two glass slide separately from that one glass slide fix on the wooden piece and other piece should tie with the balance on right hand side. The right and left pans should balance by adding extra weight on the left – hand pan. 1 gm of intra-vaginal gel was placed between these two slides containing vaginal membrane pieces, and extra weight from the left pan should remove to sandwich the two pieces of vaginal membrane and some pressure should apply to remove the presence of air. The balance should keep in this position for 5 minutes. Weight should add slowly at 200 mg/ min to the left – hand pan until the patch detach from the vaginal membrane surface. The weight (gram force) requires detaching the gel from the vaginal membrane surface give the measure of bioadhesive strength. The experiments should perform in triplicate and average values with standard error of mean were report.^46-48 The bioadhesive strength should calculate by using following:

Bioadhesive Strength = Weight required (in gms) / Area (cm²)

19) Retention time of intra – vaginal gel in Vagina:

Retention time of intra-vaginal gel in excised vaginal tube and environment should study by an in – vitro method based on the principle of measuring weight of formulation/dispersion falling drawn (or retained) as a function of time, from an isolated intact tubular portion of vagina (suspended in vertical position). Dispersion of vaginal formulation (one gram of gel dispersed in 2 ml of SVF) placed inside a vertically suspended excised goat vaginal tube, should allow to fall under the influence of gravity. The weight of formulation falling down should record as percentage leak out and percentage retain should plot against time. Also this test should repeat into inclined position at angle of 45⁰.⁴⁸

The percent gel remains in vagina should calculate by using:

Percent intra-vaginal gel remained in vagina = {(Initial weight – Final weight)/ Initial weight} × 100.

20) In-vivo drug release study of intra-vaginal gel:

For in-vivo drug diffusion studies rabbits (like New-Zealand white female rabbits) should use, because the size of rabbit vagina is large and easily available. For this study apply intra – vaginal gel on rabbit vagina with the help of applicator. Withdraw blood samples in predetermined interval of time and then estimate the concentration of drug in blood plasma by using C₁₈ column (ODS) in HPLC or by using a particular drug λ_max for absorbance analysis in case of UV – Visible spectrophotometer.^49-52

Some of the important parameters which should be study for in – vivo evaluation of intra – vaginal gel. The highest observe concentration during the study period; C_max, and time, at which C_max should observe, T_max, should obtain directly from the plasma concentration – time profiles. The area under the plasma concentration – time curve (AUC_0-t, and AUC_t_-α, µg.h/ml) should calculate based on the trapezoidal rule. The volume of distribution (V_d), total body clearance (Cl_T), elimination rate constant (K_E) and half – life (t_1/2) should also calculate.⁵³

21) Stability Studies of Intra – vaginal gel:

The stability of intra – vaginal gel should perform as per the International Conference on Harmonization (ICH) guidelines of stability testing of new drug substances and drug product, October 27, 1993 or otherwise others.^21,54

The formulated gel should fill in the sterile lacquered collapsible aluminum tubes^21,55,56,57 and store at different temperature condition viz. 25 ± 2°C (refrigerator temperature) and 40 ± 2°C (condition of accelerated stability testing)^21,54,58 for a period of three months and study for colour, pH, extrudability, spreadability, drug content, etc.^21,54,59

CONCLUSION:

The present study gives the information to prepare an ideal vaginal gel. These types of gels today are useful for scientists and researchers in major types of investigation. Intra – vaginal gel is more useful today, because these prepare a transparent protective membrane in vagina and does not produces any type of irritation etc. This type of therapy may be useful in the treatment of HIV infection in future. So, this article provides full information about the preparation and evaluation of most effective Intra – vaginal gel. Almost all the parameters used for evaluation of intra-vaginal gel are discussed here. In conclusion, intra-vaginal gel drug delivery is the best for deliver drug into vagina, method of preparation and evaluation of intra-vaginal gel is easier. This intra-vaginal gel drug delivery has more advantage over other routes, it avoids hepatic first-pass metabolism of drug, and it also avoids the hepato-gastrointestinal side effects and improves the user compliance.

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Received on 08.12.2009

Accepted on 13.01.2010

Research Journal of Pharmaceutical Dosage Forms and Technology. 2(1): Jan. –Feb. 2010, 07-13