INTRODUCTION:

For a biocompatible formulation appropriate for human administration, triglycerides and monoglycerides have been chosen as the biomaterials for LS because of their high biocompatibility, high physicochemical stability, and drug delivery release. Lipospheres formulation is suitable for oral, parenteral and topical drug delivery system. The solid core contains a drug dissolved or dispersed in a solid fat matrix and used as carrier for hydrophobic drugs.¹Lipids are now being studied widely due to their attractive properties namely physicochemical diversity, biocompatibility, biodegradability, ability to increase the oral bioavailability of weakly water soluble drug moieties, hence making them ideal candidates as carriers for problematic drugs.²

Lipospheres are prepared by solid lipid core surrounded by a single unit phospholipids layer that may entrap the drug. The stabilizer is used to form uniform coat around the core material and to aid partition of the drug between the lipid and aqueous phases.³

LS have some advantages over other delivery systems, such as good physical stability, low cost of ingredients, ease of preparation and scale-up, and high entrapment yields for hydrophobic drugs. Because of their large range in particle size, LS can be administered by different routes- such as orally, subcutaneously, intramuscularly, or topically-or they can be used in cell encapsulation, thus allowing them to be proposed for treatment of a number of diseases.^4,5

Therefore, the present research focused on achievements of lipospheres formulation to deliver the drugs in the targeted sites. Polymers as carriers for “difficult to deliver” drugs, “to be targeted drugs” and to achieve a desired release pattern is a popularly known and widely exploited concept in formulation technology.⁶

Recent studies have discovered that discovery and development of new drugs alone are not sufficient to achieve therapeutic excellence but modified formulations of existing drugs are gaining more importance.⁷

The solubility enhancement process of hydrophobic drugs plays an important role in the formulation development in order to achieve the bioavailability and therapeutic action of the drug at the target site. The biopharmaceutics classification system (BCS) reflects that Class II and IV drugs have low water solubility, poor dissolution, and low bioavailability.³

Repaglinide is a water insoluble ant diabetic drug which belongs to the class of medications known as meglitinides. It lowers the blood glucose by stimulating the release of insulin from the pancreatic β cell. Repaglinide is a weakly water-soluble approximately 20 μg/ml, high lipophilicity and comparatively low oral bioavailability (56 %) which is recognized as poor dissolution (BCS-II).⁸^-¹²

Lipospheres formulations also protect active compounds from biological degradation which in turn can lead to an improvement of drug potency, high bioavailability, and controlled drug release and less toxic effect.¹³

MATERIALS AND METHODS:

Materials:

The materials used for preparing lipospheres were obtained as gift sample from torrent research center, Ahmadabad. Stearic acid and cetyl alcohol was obtained from Loba chemie Pvt. Ltd. Mumbai. Bees wax was obtained from S D fine chemicals Ltd, Mumbai. Soybean phosphotidylcholine was obtained from VAV Life sciences Pvt Ltd. Mumbai. Other chemicals were used for analytical grade.

Preparation of lipospheres by solvent evaporation technique:

Correctly weighed the amount of drug, lipid core and phospholipids were dissolved in chloroform. The organic solvents was slowly evaporated under reduced pressure at 50-60c using a rotary evaporator. The resultant was mixed with cold water and stirred using a magnetic stirrer, then emulsified with an external aqueous phase containing the surfactant. The LS formed were recrystallized at room temperature and filtered using 0.45 µm filter paper, and dried using desiccators.^14,15

Table 1: Formulation chart for lipospheres

Si. No	Ingredients Lipid phase	F1	F2	F3	F4	F5	F6	F7
01	Drug	500mg	500mg	500mg	500mg	500mg	500mg	500mg
02	Stearic acid	2gm	1gm	-	2gm	-	-	1gm
03	Bees wax	-	-	3gm	-	-	3gm	1gm
04	Cetyl alcohol	-	1gm	2gm	-	-	-	1gm
05	Soybean phosphatidylcholine	2gm	-	-	-	3gm	-	1gm
06	Aqueous phase tween 80	4ml	5ml	4ml	3ml	3ml	5ml	4ml
07	Water	46ml	95ml	96ml	47ml	47ml	95ml	96ml

Preformulation studies of drug:

Solubility Studies:

The solubility study of repaglinide was done by solubilising the drug in various preferred solvents with an incremental amount by a factor of 10. This test was carried on until the drug was fully solubilised in the respective solvent and shows no signs of cloudiness or precipitation.

The preferred solvent for the solubility study of repaglinide is Phosphate buffer 7.4.¹⁶

Determination of melting point:

Melting point of the drug was determined by capillary method using melting point apparatus. Here, the capillary tube was filled by pressing the open end gently into sample by tapping the bottom of the capillary on a hard surface so that the drug pack down into the bottom of the tube. When the drug packed into the bottom of the tube, the tube was placed into the slot behind the eyepiece on the melt-temperature. Make sure the unit is plugged in and set to zero, and then turn it on. The temperature was noted when the drug start to melt and the drug till complete melt.¹

Scanning for drug absorption (λmax) using double beam spectrophotometer:

Stock solution containing 12 μg/ml concentration of repaglinide in phosphate buffer (7.4 pH) were prepared and scanned between 200-400 nm. Absorption maximum were found at 248 nm.

Development of UV spectrophotometric method for analysis of repaglinide:

Preparation of standard calibration curve:

· Preparation of standard solution.

Solution containing 100μg/ml of drug in 7.4 pH phosphate buffer was prepared and marked as stock solution A (SS-A). From stock solution A 1 ml solution was withdrawn and stock solution B (SS-B) was prepared with dilution to obtain a concentration of 10μg/ml.

· Preparation of working standard solutions.

From SS-B, aliquots containing final concentration of solutions were prepared. Absorbance at 248 nm was measured against blank.

FTIR Study:

Compatibility studies of drug(s) with polymer(s) using Shimadzu FTIR 8400S spectrophotometer by KBr pellet method. Physical mixtures, drugs and polymers were scanned between 4,000-400 cm^-1. IR spectrums of physical mixture obtained were compared with those of drug and polymers to detect the appearance of peaks.⁶,¹⁷

Evaluation of lipospheres:

Particle size determination:

Particle size analysis of repaglinide loaded lipospheres was performed by optical microscopy using a compound microscope. A small amount of dry lipospheres was floating in purified water (10 ml). The suspension was ultrasonicated for 5 sec. A small drop of suspension hence obtained was placed on a clean glass slide. The slide containing lipospheres was mounted on the stage of the microscope and 100 particles were considered using a calibrated ocular micrometer.^13,18

Scanning Electron Microscopical Evaluation (SEM):

Surface morphology of the sample was determined by using a scanning electron microscope (SEM).The samples were prepared by smattering the formulation on double-adhesive tape stuck to aluminum stub. The stub was placed in high-vacuum evaporator. Then the samples were randomly scanned and photomicrographs were taken with a scanning electron microscope.^2,19

Yield of lipospheres:

The lipospheres formed were filtered from the medium, then dried and weighed to get the yield of the lipospheres formulated per batch. The % yield of lipospheres was calculated using the following formula.

Total weight of lipospheres

% Yield = --------------------------------------------------- × 100

Total solid material amount used before emulsification

Determination of Encapsulation efficiency (EE):

The entrapment efficiency of the system was determined by measuring the concentration of free drug in the aqueous phase. The resulting supernatant of each batch obtained after filtration was analysed by using spectrophotometer at 248nm. The absorbance obtained was converted to concentration by using a calibration curve. Then they obtained concentrations were used to calculate the respective entrapment efficiencies. The drug concentration in the aqueous phase and in the whole lipospheres was compared to calculate the entrapment efficiency.^14,20

[Actual drug content]

EE% = ----------------------------------------- × 100

[Theoretical drug content]

Drug content:

The dried lipospheres were accurately weighed. They were added to 5ml of ethyl acetate. After the lipospheres dissolved completely, then 5ml of phosphate buffer (pH-7.4) was added to this solution and mixed thoroughly. The resulting solution was filtered using a whattman filter (0.45μm pore size) and analyzed for repaglinide content by measuring absorbance in UV-spectrophotometer (Shimadzu UV-1700, Pharmaspec, Tokyo, Japan) at 248nm by the first derivative spectrophotometric method using phosphate buffer (pH-7.4) and ethanol mixture in the ratio of 1:1 as blank. Results were spoken as mean (±SD) of 3 experiments.³

In vitro studies:

The in vitro release studies of repaglinide lipospheres were carried out in a US pharmacopoeia dissolution apparatus-II paddle (XVIII) in 900 ml mixture of PBS pH 7.4 with 1% SLS in 9:1 at 37°C±0.5 temperature. The rotational speed of dissolution apparatus was maintained at 100rpm. Each run was carried out in triplicates. At different time intervals, the solution was withdrawn and absorbance was read at 248nm. An equal volume of the medium was replaced into the container after each withdrawal to maintain sink condition.^2,21

Accelerated Stability Studies:

Stability studies for lipospheres were carried out as per ICH guideline; formulations were stored at 40˚C±2˚C/ 75%±5% RH for 90 days. At the end of 0, 1, 2, 3 months, all the selected lipospheres were tested for the change in % yield, % drug content and % drug release. ²⁴

RESULTS AND DISCUSSION:

Pre formulation studies:

Table 2: Solubility of repaglinide in different solvents

Solvent	Solubility
Water	Insoluble
7.4 ph phosphate buffer	Freely soluble
Ethanol	Slightly soluble
Methanol	Very soluble

Repaglinide was found to be freely soluble (1-10 parts of solvent required for 1 part of solute) in phosphate buffer of pH 7.4 and methanol, slightly soluble (100-1000 parts of solvent required for 1 part of solute) in ethanol.

The melting point of repaglinide, was determined by capillary method using Digital Melting Point apparatus was found to be 127°C. The value was comparable with the literature value of 126 to 128°C. The melting point as obtained practically matched the literature value confirms the purity of the sample.

Development of calibration curve of repaglinide:

The calibration curve was determined with absorption maximum (λmax) 248 nm. The concentration ranges and data are reported in table 3. Calibration curve of repaglinide was plotted using this data and shown in the figure 1.

Table 3: Calibration curve for repaglinide in phosphate buffer pH 7.4

SI. No	Concentration (µg/ml)	Absorbance at 248nm --------------------------------- Average± SD*
1	0	0.000+ 0.000
2	3	0.200 +0.013
3	6	0.374 +0.031
4	9	0.602 +0.012
5	12	0.750 +0.043
6	15	0.938 +0.035
7	18	1.112+0.032
8	21	1.293+0.035

*Standard Deviation (n=3)

Figure 1: Standard calibration curve of repaglinide

Table 4: FTIR spectral of pure repaglinide

Wave number (cm^-1)	Functional group
3320	N-H stretching
2947.7	C-H stretching
1728	C=O stretching
1460	C-H deformation

Figure 5: FTIR spectra of stearic acid

Table 5: FTIR spectral comparison of pure drug and physical mixture

Function group	Wave number (cm^-1)
Function group	Pure drug	Physical mixture
N-H stretching	3320	3330
C-H stretching	2947.7	2943.5
C=O stretching	1728 2787.23	1729
C-H deformation	1460	1456

Figure 6: FTIR spectra of physical mixture of drug

The IR spectra of repaglinide and polymers figure IV were compared with the standard spectrum of repaglinide and polymers. The interaction between the drug and the polymers often leads to identifiable changes in the IR profile of spectrum. IR spectra of drug polymer mixture showed no interaction with the repaglinide and prominent peaks of repaglinide were not affected. There was neither shift and nor disappearance of characteristic peaks suggesting that there is no interaction between repaglinide and other polymers.

Evaluation parameters:

Scanning electron microscopy of developed lipospheres:

The surface morphology of the lipospheres prepared by solvent evaporation technique was studied and has been depicted in figure 7. The SEM showed uniformly sized spherical lipospheres with minimum evidence of the crystals of repaglinide.

Figure 7: Scanning electron microscopy (SEM)

Particle size:

As presented in the table 6 the particle size of lipospheres were in the range of 18 – 27.

% Drug Entrapment:

The drug entrapped within the polymer matrices were in the range of 52-94 % as has been shown in the table 6. Entrapment efficiency depends on the drug solubility in the solvent system used for processing. Various co-solvents such as ethanol, has been often used in the formulation of lipospheres since they aid in the higher drug entrapment.

% Yield:

% Yield has been exhibited in the table 6.

Drug content:

As shown in the table 6 the % of drug content was found to be in the range of 3.64-9.65.

Table 6: Evaluation parameters of lipospheres

For.	Particle size	Percentage yield *(±SD)**	*Entrapment efficiency (±SD)**	Drug content *(±SD)**
F1	18	68.80 ± 1.42	72.03 ± 1.25	8.99±2.60
F2	24	68.65 ± 2.05	80.42 ± 1.24	9.30±2.33
F3	27	93.33± 2.07	94.50± 2.60	9.65±2.83
F4	20	55.45± 1.06	76.50± 1.50	6.75±1.43
F5	22	49.81 ± 2.28	71.80 ± 1.62	8.51±1.59
F6	21	50.45 ± 0.93	62.03 ± 1.57	8.78±1.11
F7	20	60.94 ± 2.24	52.31 ± 1.91	3.64±1.03

In-vitro drug release study:

Table 7: In vitro release study of formulations F1-F7 in phosphate buffer pH 7.4

Sl. No	Time (h)	*Cumulative percentage drug release(±SD)**
Sl. No	Time (h)	F1	F2	F3	F4	F5	F6	F7
1	0	0	0	0	0	0	0	0
2	1	9.9±8.7 8.93246213. 27.23312 33.1154749. 54.90196 64.34277	7.0±0.21	8.0±0.81	7.9±9.90	9.9±8.40	6.0±0.21	7.9±9.00
3	2	16.3±7.24	16.4±3.27	18.0±8.8	19.9±9.21	16.3±8.99	20.0±5.87	16.6±9.10
4	3	28.9±3.24	28.1±5.03	27.6±8.6	28.4±2.33	20.9±0.41	21.8±7.65	21.7±1.38
5	4	38.5±0.76	32.9±4.33	35.1±1.5	35.4±3.93	37.3±3.33	24.2±3.38	34.0±5.95
6	5	43.2±3.31	41.2±1.64	43.1±0.3	46.9±7.53	48.1±7.64	36.5±0.69	41.9±0.26
7	6	55.1±1.5	50.3±1.66	52.5±6.7	59.0±1.96	57.5±6.71	43.7±6.90	48.2±9.33
8	8	60.6±7.32	68.9±4.69	65.4±8.2	66.4±9.96	69.4±8.29	59.0±1.96	56.2±0.91
9	10	64.9±0.19	71.3±5.43	79.8±9.0	75.2±8.68	75.4±3.79	65.5±5.55	66.2±0.33
10	12	70.3±4.27	75.7±0.00	92.7±3.8	85.6±1.00	80.1±6.12	77.8±7.36	73.9±8.25

*Standard Deviation (n=3)

Accelerated stability studies were carried out at 40 ± 2 ºC and 75 ± 5 % RH for the optimized formulation F3 for 3 months and monitored for entrapment efficiency, % drug content and % cumulative drug release, which indicated that negligible changes in the result, hence all the lipospheres were stable during storage period.

CONCLUSION:

The present study focused on the development of lipospheres of repaglinide by solvent evaporation technique. The optimized lipospheres performance and in vitro drug release profile were achieved by using cetyl alcohol and bees wax in different ratios i.e.F3. Lipospheres were able to entrap the drug at very high levels and sustained its release over a prolonged time and possessed a very high stability.

ACKNOWLEDGEMENT:

Authors are thankful to the principal, guide of MMU College of pharmacy for providing necessary facilities. The authors’ submission is an original work that reflects research undertaken with integrity and honesty, Further, there is no conflict of interest among the authors.

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Received on 19.04.2021 Modified on 17.11.2021

Res. J. Pharma. Dosage Forms and Tech.2022; 14(2):117-122.

DOI: 10.52711/0975-4377.2022.00018

Temperature and RH 40±2˚C and 75±5%	Parameters	Duration in months
	Parameters	0	1	2	3
	*Entrapment efficiency (±SD) (±SD)*	94.50± 2.60	94.32±2.00	94.40±1.90	94.11±1.03
	%Drug content	9.65±2.83	9.35±2.53	9.20±1.19	9.0±0.15
	*%CDR(±SD)**	92.73±1.50	92.50±1.10	91.70±0.50	90.30±0.19