Formulation and evaluation of emulgels loaded with microspheres containing Baricitinib

The main objective of the present research work was to formulate and evaluate transdermal gel loaded with microspheres of Baricitinib to increase bioavailability and to reduce the dosing frequency and to improve patient compliance. Gel loaded with microspheres of Baricitinib was prepared by taking different ratios and grades of Carbopol polymers. The prepared formulations (F1–F9) were evaluated for pre-formulation studies, percentage yield, particle size, content Uniformity, drug content entrapment efficiency and in vitro dissolution studies. The optimized gel loaded with microspheres of F8 formulation (drug: Carbopol 974polymer in 1:1 ratio) is more effective compared to all formulations. The prepared gel showed acceptable physical properties such as particle size of (126.5 microns), percentage yield of (96.5%), content uniformity (97.8), and drug content entrapment efficiency (90.5%). In vitro dissolution studies have shown 100.1% drug release in 12 h. With the optimized formulation Baricitinib emulgels are formulated using different grades and ratios of Carbopol and optimized microsphere formulation F8. The Emulgel formulation F7 showed the acceptable results with a yield of 92.3%, Drug content (100.2%), Spread ability (13.38 g.cm/s), Viscocity (112.3 cP), pH off 6.05 and it was observed that formulation F7 showed 100.2% drug release within 12 hours. All the results show that the gel loaded with microspheres of Baricitinib can be effectively used for the treatment of viral infections like small fox.


Introduction
Microspheres are small spherical particles, with diameters in the micrometer range (typically 1 μm to 1000 μm).Microspheres are sometimes referred to as microparticles.Microspheres can be manufactured from various natural and synthetic materials.Microspheres vary widely in quality, sphericity, uniformity of particle and particle size distribution.The appropriate microsphere needs to be chosen for each unique application.The range of techniques for the preparation of microspheres offers a variety of opportunities to control aspects of drug administration.This approach facilitates the accurate delivery of small quantity of the potent drugs, reduced drug concentration at the site other than the target site and the protection of the labile compound before and after the administration and prior to appearance at the site of action.The behavior of the drugs in vivo can be manipulated by coupling the drug to a carrier particle.The clearance kinetics, tissue distribution, metabolism and cellular interaction of the drug are strongly influenced by the behavior of the carrier.The exploitation of these changes in pharmacodynamics behavior may lead to enhanced therapeutic effect.The most convenient and commonly employed route of drug delivery has historically been by oral ingestion Drugs that are easily absorbed from the GIT and having a short half-life are eliminated quickly from the blood circulation.To avoid these problems oral controlled drug delivery systems have been developed as they releases the drug slowly into the GIT and maintain a constant drug concentration in the serum for longer period of time.A well designed controlled drug delivery system can overcome some of the problems of conventional therapy and enhance the therapeutic efficacy of a given drug.To obtain maximum therapeutic efficacy, it becomes necessary to deliver the agent to the target tissue in the optimal amount in the right period of time there by causing little toxicity and minimal side effects.

Material and methods
Baricitinib pure drug was a generous gift from Aurobindo Pharma limited, Hyderabad, India.Carbopol 934, Carbopol 940 and Carbopol 974 were brought from Corel Pharm achem, Ahmedabad.Triethanolamine was from Signet Chemicals, Mumbai and PEG 400 was a gift sample from Signet chemicals, Mumbai.All other chemicals used were of Pharma grade.

Preformulation studies of pure drug
Pre-formulation involves the application of biopharmaceutical principles to the physicochemical parameters of drug substance and is characterized with the goal of designing optimum drug delivery system.

Particle size and size distribution
The particle size was measured with microscopic method.The particle size distribution and mean particle size diameter were automatically calculated from the particle size values obtained by using software provided.Average particle size of 500 particles was calculated.Particle size of the powder will influence the powder flow property directly.

Bulk density
It is the ratio of mass to the bulk volume of the powder.It is useful in the determination of compressibility index.Bulk density is significant parameter which influences the pack size of the container.Procedure 1 gm pure drug substance was weighed and placed in a measuring cylinder, the volume occupied by it is noted.

It is defined mathematically as:
Bulk density = mass of powder / Bulk volume

Tapped density
It is the ratio of mass to the tapped volume of the powder.Procedure 1gm pure drug substance was weighed and placed in a measuring cylinder and then it is tapped on a wooden base for about 500 times and finally the volume occupied by it is noted.
Tapped density = mass of powder / tapped volume

Powder flow properties
The wide variety of methods for characterizing the powder flow are generated in the wide spread use of powders in the pharmaceutical industry because no single and simple test method can adequately characterize the flow properties of pharmaceutical powders.Four commonly reported methods for testing powder flow are  Angle of Repose,  Compressibility Index,  Hausner's Ratio  Shear Cell.
In the current research powder flow properties were determined by first three methods

Angle of repose
The angle of repose is a characteristic related to inter particulate friction or resistance to movement between particles.It is the constant, three dimensional angle (relative to the horizontal base) assumed by a cone -like pile of material.The maximum angle which is formed b/w the surface of a pile of powder and horizontal surface is called the angle of repose.

Procedure
The powders were allowed to flow through the funnel fixed to a stand at definite height (h).The angle of repose was then calculated by measuring the height and radius of the heap of granules formed.
It is calculated by using the formula: θ = tan -1 (h/r)

Procedure
The bulk density and tapped density were measured and then compressibility index was calculated using the formula: Tapped density -Poured density x 100 Tapped density

Hausner's ratio
Tapped density and bulk density were measured and the Hausner's ratio was calculated using the formula: The relationship between Carr's index and Hausner's ratio is given below H = 100/100-C

Solubility studies
The solubility of drug is an important physicochemical property because it affects the bioavailability of the drug, the rate of drug release into dissolution medium and consequently, the therapeutic efficiency of the pharmaceutical product.
The solubility of material is usually determined by the equilibrium solubility method, which employs the preparation of a saturated solution of the material, obtained by stirring an excess of material in the solvent for a prolonged period of time [72hrs] until equilibrium is achieved at 25 0 C on mechanical shaker.After this period the solutions were filtered through 0.45µ Millipore filter and analysed spectroscopically at respected wavelength against blank sample (blank sample containing the same concentration of specific solvent used without drug).Three determinations were carried out for each sample to calculate the solubility of drug.Solvents used for solubility determination are:-Water, 4.5pH Acetate Buffer, 6.8 pH Phosphate Buffer, 7.2 pH Phosphate Buffer, 7.4 pH Phosphate Buffer, 0.1 N HCl and 0.1 N NaOH

Standard graph preparation
Accurately weighed 25 mg of drug was transferred to 25 ml volumetric flask.5ml of methanol was added and volume was made up to 25 ml with respective buffer.(1000 micro gram/ml) -I Stock solution.From first stock solution 5ml was taken and diluted to 50 ml with buffer.(100 micro gram/ml) -II Stock solution.From second stock solution various concentrations were prepared and absorbance was taken at lamda max of each drug and a calibration curve was plotted by taking concentration on X-Axis and corresponding absorbance on Y-axis.

Preparation of microspheres
Batches of microspheres were prepared which involved different grades of Carbopol as polymers were dispersed in purified water (10 ml) to form a homogeneous polymer mixture.The API, was added to the polymer premix and mixed thoroughly with a stirrer to form a viscous dispersion.The resulting dispersion was then added through a 22G needle into calcium chloride (4% w/v) solution.The addition was done with continuous stirring at 200 rpm.The added droplets were retained in the calcium chloride solution for 30 min to complete the curing reaction and to produce rigid spherical microspheres.The microspheres were collected by decantation, and the product thus separated was washed repeatedly with purified water to remove excess calcium impurity deposited on the surface of microspheres and then air-dried.

Percentage yield
The percentage of production yield was calculated from the weight of dried microspheres recovered from each batch and the sum of the initial weight of starting materials.The percentage yield was calculated using the following formula: % Yield = Practical mass (Microspheres)/Theoretical mass (Polymer+Drug)×100

Drug entrapment efficiency
Powdered microspheres were suspended in 10 ml of phosphate buffer (pH 6.8).After 24 h, the solution was filtered, the filtrate was centrifuged at 2000 rpm for 3 min, and then analyzed for drug content spectrophotometrically (Shimadzu UV-1800, Japan)), and the concentration of soluble drug was calculated.The amount of drug entrapped in the microspheres was calculated by the following formula, Entrapment efficiency = (Weight of drug added formulation -weight of drug recovered from Microspheres) /Weight of drug added during formulation

Particle size analysis
Samples of the microparticles were analyzed for particle size by optical microscope.The instrument was calibrated and found that 1 unit of eyepiece micrometer was equal to 12.5μm.Nearly about 100 micro particles sizes were calculated under × 45 magnifications.The average particle size was determined using the Edmondson's equation: Dmean= nd/n; Where, n -Number of microspheres observed d -Mean size range

In vitro release studies
The in vitro release of microspheres was done with phosphate buffer pH of 6.8 for 12 h using dissolution apparatus USP I Basket type at a temperature of 37.0±0.5°C.Microsphere equivalent to one dose of drug was taken, and it was inserted in the basket wrapping it with a muslin cloth (900 ml phosphate buffer pΗ 6.8, at 37±2°C and was adjusted to 100 rpm).
The sample was taken at predetermined time intervals for 12 h.To maintain the sink condition, the samples withdrawn were replaced with an equal volume of dissolution medium at different time intervals.After suitable dilution, samples were analyzed at UV visible spectrophotometer.

Formulation of the microsphere-loaded emulgel
The gels with varying concentrations of different grades of Carbopol were prepared by dispersing the required quantity of Carbopol in the required quantity of distilled water with continuous stirring and kept overnight for complete hydration.Further appropriate quantities of triethanolamine were added to the previous polymeric mixture.Optimized formulation of the drug loaded microsphere was added to the above polymeric mixture with constant stirring.Final pH of the preparation was adjusted to4.5 with 0.5 M sodium hydroxide solution.The composition of all the formulation is given in Table.

Visual inspection
The organoleptic properties, such as color, texture, consistency, homogeneity, and physical appearance of gel containing microspheres were checked by visual observation.

Determination of pH
Determination of pH is done using Systronic digital pH meter 335.pH meter was calibrated before use using a standard buffer solution.

Viscosity
The viscosity of the formulated gel is determined by DV-E Brookfield viscometer using spindle no 64

Spread ability
One of the criteria for Emulgel is to meet the ideal quality is that it should possess good spread ability.It is the term expressed to denote the extent of the area to which gel readily spread on application to the skin or affected part.The therapeutic efficacy of a formulation also depends on its spreading value.Spreadability is expressed in terms of time in seconds taken by two slides to slip off from emulgel and placedin between the slides under the direction of a certain load.Lesser the time taken for separation of two slides, better the spread ability It is calculated using the formula: Where, M = wt.tied to upper slide L = length of glass slides.T = time taken toseparate the slides

Drug content determination
Drug concentration in emulgel was measured by spectrophotometer.Drug content in emulgel was measured by dissolving a known quantity of emulgel in solvent (ethanol) by sonication.Absorbance was measured after suitable dilution in UV/visible spectrophotometer.

In vitro release study
Franz diffusion cell was used for the drug release studies.Emulgel (one unit dose) was applied onto the surface of the cellophane membrane evenly.The cellophane membrane was clamped between the donor and the receptor chamber of the diffusion cell.The receptor chamber was filled with freshly prepared PBS solution to solubilize the drug.The receptor chamber was stirred by a magnetic stirrer.The samples(1.0ml aliquots) were collected at a suitable time interval.Samples were analyzed for drug content by UV visible spectrophotometer.Cumulative corrections were made to obtain the total amount of drug release at each time interval.

Solubility Studies
Saturation solubility studies was done in different solvents and results are tabulated below.It was found that the drug is practically insoluble in all the solvents.

Calibration plot of API in different solvents
Calibration curve of API in 7.4pH and 6.8pH buffer was plotted and graphs are shown below along with absorbance values

Physicochemical characteristics of microspheres
The physicochemical characteristics of microspheres like percentage yield, Particle size, content uniformity and % entrapment efficiency were evaluated.All the formulations F1-F9 were evaluated and the results are reported below.From the results it was observed that all formulations have shown the size in micro level with entrapment efficiency of 90%.The prepared formulations (F1-F9) were evaluated for pH, Viscosity (cP), Spreadability (g.cm/s), %Drug content and % yield.The optimized emulgel formulation F7 (drug: Carbopol 974 in 2:1 ratio) is more effective compared to other formulations.The Emulgel formulation F7 showed the acceptable results with a yield of 92.3%, Drug content (100.2%),Spreadability (13.38 g.cm/s), Viscosity (112.3 cP), pH of 6.05 and it as observed that formulation F7 showed 100.2% drug release within 12 hours.From the results, it was observed that all the formulations have shown similar results with similar pH range

In vitro dissolutions of baricitinib emulgels
The results of in vitro dissolution comparisons of Baricitinib Emulgel formulations F1-F9 are summarized in Table below.From the results, it was observed that formulation F7 has shown 100.2% drug release within 12 hours.

Conclusion
Baricitinib emulgel formulations were formulated and evaluated.From this study it was concluded that the optimized formulation of Baricitinibemulgels were formulated using Carbopol 974 in the ratio of 2:1 (Drug loaded Microspheres F8 : Polymer).The formulations F7 showed acceptable results.The optimized Baricitinibemulgel formulation F7 is more effective compared to other formulations.The Emulgel formulation F7 showed the acceptable results with a yield of 92.3%, Drug content (100.2%),Spreadability (13.38 g.cm/s), Viscosity (112.3 cP), pH of 6.05 and it as observed that formulation F7 showed 100.2% drug release within 12 hours.

Figure 1
Figure 1 Saturation solubility profile of baricitinib

Figure 3 3 . 3 .
Figure 3 Calibration curve in 7.4 pH buffer3.3.Preformulation parameters of APIThe preformulation evaluation of Baricitinib API was evaluated for Particle size, Bulk density, Tapped Densit, Hausners ratio, Carrs index and Angle of repose.The reported results are given in the table below.

Table 1
Composition of Baricitinib Microspheres

Table 4
Calibration curve profile in 6.8PH buffer Figure 2 Calibration curve in 6.8 pH buffer

Table 5
Calibration curve profile in 7.

Table 7
Characteristics of Baricitinib MicrospheresThe results of in vitro dissolution comparisons of Baricitinib microspheres formulations F1-F9 are summarized in Tablebelow.The % drug release from Baricitinib microspheres formulation F8 showed was highest (100.1) and faster than other Baricitinib microsphere formulations.From the results, it was observed that formulation F8 has shown 100% drug release within 12 hours and hence has been chosen as best formulation for converting into emulgel.