Formulation and Evaluation of Sustained Release Stavudine Microspheres by Ionotropic Gelation Method

 

Ayanam Vasavi1*, Miriyala Mrunalini1, Ayanam Vasanthi1, G. Raveendra Babu2, M. Sowjanya3

1Department of Pharmaceutics, A.K.R.G. College of Pharmacy, Nallajerla, W.G. Dist., Andhra Pradesh.

2Department of Pharmaceutical Analysis, A.K.R.G. College of Pharmacy, Nallajerla, W.G.

Dist., Andhra Pradesh.

3Department of Chemistry, Vijaya Teja Degree College, Addanki - 523201, Andhra Pradesh.

*Corresponding Author E-mail: vayinam@gmail.com

 

ABSTRACT:

As a novel drug delivery system, microspheres improve the efficacy of a drug, increase the time of action, lower the number of times in which a dosage form needs to be administered, and to increase patient compliance. Oral administration side effects such as gastric irritation are lessened through the use of microspheres. Ionotropic gelation was used to create HPMC K15M, Guar gum, and Carbopol 934 microspheres with different concentrations of Carbopol 934 polymer. Cross-linking was accomplished with the use of calcium chloride. In order to conduct a systematic evaluation of all the preparations, we performed various tests: morphology, FTIR, DSC, entrapment efficiency, size, microsphere size, and in-vitro drug release. The discrete, free-flowing, and spherical particles of prepared Stavudine microspheres were found. In compliance with standards, the mean particle size was in the range of 72.64 to 95.22 percent. In vitro drug release studies were performed in phosphate buffer solution with a pH of 6.8. As the concentration of sodium alginate and calcium chloride increased, the percentage of drug release was reduced. In the case of F9 formulation, which contained Stavudine, the decreased drug release rate was obtained via carbopol 934(1:3), sodium alginate, and calcium chloride. Conclusively, the present study shows that Stavudine microsphere preparation and formulation F9 are successful. Stavudine microspheres must be prepared in order to preserve an effective drug concentration in serum for a long time, while reducing gastrointestinal irritation.

 

KEYWORDS: Microspheres, Controlled Release, stavudine, in-vitro studies.

 

 


INTRODUCTION:

Improved therapeutic effectiveness with controlled delivery, targeting, and sustained delivery is referred to as a novel drug delivery system. The correct dosage and duration of drugs are achieved with the goal of sustaining drug levels and drug action in the body1.

 

Using microencapsulation is a good way to extend the release of a drug from a dosage form and reduce the occurrence of side effects2. Formulation and development of novel drug delivery systems have recently benefitted from technologies that can precisely control the release rates and target specific drug delivery sites. Such novel drug delivery system uses microspheres as an important component3-5. One of the multiparticulate delivery systems is microspheres, which are used to prolong the time the drug is released, to improve bioavailability, or to ensure a particular drug distribution. In addition to their application in the pharmaceutical industry, microspheres may offer benefits like minimising fluctuations within therapeutic range, reducing side effects, decreasing the amount of medication administered, and increasing patient compliance6,7. This medication treats the most common chronic disease on the planet, AIDS. The half-life of biological activity is 0.8–1.5 hours, and 30 milligrammes per day is sufficient to maintain constant levels8-9. Dosing frequency is greater. In order to overcome this issue, we need sustained-release dosage forms to help patients adhere to their treatment regimen. According to the current study, microspheres of stavudine (which might be more effective) will be developed and optimised using the ionotropic gelation method, which may prove to be more productive compared to conventional systems because it gives microspheres a longer period of time in the gastrointestinal tract for absorption. Another factor in favour of microspheres is that the drug itself is delivered using a simple, inexpensive, and less labor-intensive technology than with many other drug delivery systems10-16.

 

MATERIALS AND METHODS:

Materials:

These active ingredients include Stavudine (Glenmark Pharmaceuticals LTD., Mumbai, India), HPMC K15M (Spectrum pharmalabs Hyderabad, India), Guar gum LR (Spectrum pharmalabs Hyderabad), and calcium chloride. The ingredients were of the highest analytical quality.

 

Methods:

Determination of λmax:

A Stavudine solution was prepared with a concentration of 10μg/mL in 0.1N HCL buffer, and the UV spectrum was recorded using a Shimadzu UV-1800 spectrophotometer with dual beams. In the wavelength range of 200-400nm, the solution was scanned.

 

Formulation design:

All the formulations contain Sodium alginate 1gm, Calcium chloride 1gm, and distilled water.

 

Table 1: Formulation design for Stavudine microspheres using different ratios of drug and polymers.

Formulation code

Drug: Polymer (Ratio)

F1

Stavudine: HPMC K15M (1:1)

F2

Stavudine: HPMC K15M (1:2)

F

Stavudine: HPMC K15M (1:3)

F4

Stavudine: Guar gum (1:1)

F5

Stavudine: Guar gum (1:2)

F6

Stavudine: Guar gum (1:3)

F7

Stavudine: Carbopol934 (1:1)

F8

Stavudine: Carbopol934 (1:2)

F9

Stavudine: Carbopol934 (1:3)

 

Preparation of Stavudine microspheres:

Method used: Ionic Gelation Technique

Ionotropic gelation method using Sodium alginate, HPMC K15M, guar gum, Carbopol 934, and calcium chloride were used to form microspheres of Stavudine. a sodium alginate solution was mixed with weighed quantities of drug and polymer, which were then added to the sodium alginate solution with agitation at a speed of 800 rpm. To produce the final solution, the resultant solution was added drop-wise to 100 ml of calcium chloride solution, and the solution was then stirred constantly. For a period of 60 minutes, stirring was kept up. After filtering, washing, and drying, the microspheres were stored at 40°C for 12 hours. Microsphere preparation was optimised to enhance the efficacy of the entrapment and maximise the liberation of the drug.

 

Evaluation of Stavudine Microspheres:

Drug polymer interaction (FTIR) study:

Fourier-transformed infrared spectrophotometer (FTIR spectroscopy) was conducted on (IR-Affinity-1, Shimadzu, Japan). Drug and potassium bromide powder was compressed in a mould to make pellets, which were compressed again for ten minutes at 20 psi on KBr-press to increase their pressure, and the spectra were scanned in the wave number range of 4000-600 cm-1. A FTIR investigation was performed on Stavudine, and various polymers, as well as polymers loaded microspheres and blanks.

 

Percentage yield:

It helps in selection of the most effective production method by determining the percentage yield of Stavudine microspheres. It was found that the yield of Stavudine microspheres after each fermentation was calculated as the weight of microspheres recovered from each batch in relation to the amount of starting material.

 

Determination of percentage drug entrapment (PDE):

 

 

Efficiency of drug entrapment was calculated according to the following formula: the percentage of drug entrapment.

 

Preparation of standard calibration curve of Stavudine in 0.1N HCl:

Scanning of Stavudine by UV-spectrophotometer in 0.1N HCl

I Stock: Stavudine, a chemotherapeutic used to treat HIV, was accurately weighed into a 10 ml volumetric flask, mixed with 0.1 N hydrochloric acid, and made up to volume with 0.1 N hydrochloric acid.

II Stock: Prepare 10 ml of this solution by adding 1 ml of the above solution to another 10 ml volumetric flask. The final solution's concentration is 0.1 N HCl.

 

Procedure for calibration of Stavudine in 0.1N HCl at λ max 292nm:

Concentrations of 100µg/ml were obtained by diluting 1ml of the Stavudine standard stock solution (1000 µg/ml) with 0.1N HCL solution to obtain a final volume of 10ml. This solution was diluted to 10 ml using 0.1N HCL solution, and 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, and 1.2ml aliquots were also prepared from the standard drug solution to achieve concentrations of 10µg/ml solution, as well as 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, and 1.2ml aliquots of, 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0 ml, and 1.2ml. This solution had an absorbance of 292 nm at a value of 0.1 N HCL as a blank.

 

Theoretical drug content:

To determine the theoretical content of a drug in a polymer solution, it was assumed that the entire Stavudine was entrapped in Stavudine microspheres, and no loss occurred throughout the entire process of making Stavudine microspheres.

 

Practical drug content:

The amount of Stavudine microspheres dissolved in 100 ml of 0.1N HCL was used to determine the practical drug content. After one night, the Stavudine microsphere was completely dissolved in 0.1N HCL. The solution was filtered and diluted, with a conc of 10µg/ml, to yield a final solution of 10µg/ml. The UV-visible spectrophotometer measured the solutions' absorbance at 292nm against a 0.1N HCL solution that served as a blank and used the measured value to calculate the percentage of drug in the sample.

 

In vitro dissolution studies:

USP XXIII apparatus was used to calculate the release rate of Stavudine microspheres. To perform the dissolution test, 900ml of 0.1N HCL was added to 37.5°C with 50rpm. The solution was then replaced with 6.8 ph phosphate for the next two hours. To prevent the capsules from floating, the Stavudine microspheres were injected into a basket. The samples were replaced hourly for 12 hours and replaced with fresh dissolution medium. To obtain a more accurate measurement of the solutions' absorbance, the solutions were passed through Whatman filter paper, which was then used to determine the absorbance at 292nm. A plot of drug release versus time showed dissolution profiles of the formulations. Information was also evaluated using a kinetic approach to determine the release mechanism.

 

Drug Kinetics:

To investigate the drug release kinetics and mechanism, the cumulative release data was fitted to models representing zero order (Q v/s t), first order [Log(Q0-Q) v/s t], Higuchi's square root of time (Q v/s t1/2 ), and Korsemeyer Peppas double log plot (log Q v/s log t), respectively, where Q is the cumulative percentage of drug released at time t and (Q0-Q) is In summary, the results of in vitro release studies were plotted in four kinetics models of data treatment as follows.

·       Cumulative percentage drug release vs. time (zero order ratekinetics).

·       Graph of the cumulative percentage of drug retained vs. time (first order ratekinetics).

·       Cumulative percentage drug release as a function of T (Higuchi's classical diffusion equation).

·       Graph of cumulative percentage drug release versus graph of time (Peppas exponential equation).

 

RESULTS:

Preformulation Studies:

Solubility study:

When compared to 0.1 N HCl and water, stavudine was found to be more soluble in 6.8 phosphate buffer.

 

Flow Properties:

Table 2: Flow Properties of Stavudine (pure drug)

Parameter

Values

Angle of Repose

34˚44’

Bulk Density

0.41 g/ml

Tapped Density

0.48 g/ml

Hausner’s Ratio

1.21

Carr’s Index

17.26%

 

UV spectrum of Stavudine:

The standard calibration curve demonstrated linearity, demonstrating that the drug obeys Beers and Lamberts law in the concentration range of 2 to 12g/mL. A standard graph was drawn, with the known concentration on the X – axis and the obtained absorbance on the Y – axis. The values of the Stavudine calibration curve with 0.1 N HCL are listed below.

 

Stavudine's maximum absorbance was discovered to be at 292nm. As a result, a wavelength of 292nm was chosen for drug analysis in dissolution media.

 

Evaluation of stavudine microspheres:

Drug polymer interaction (FTIR) study:

According to the observations made on the spectra of Stavudine, the physical mixture of Stavudine and polymer was seen to have all of the characteristic peaks of Stavudine. This demonstrates that the Stavudine and polymer are mutually compatible. IR spectra below,

 

Fig. 1: IR spectra of pure Stavudine

 

 

The pure drug (Stavudine) did not exhibit any interactions with optimised formulation (Stavudine+excipients) in the drug excipient compatibility studies, which means there were no changes in the drug itself.

 

 

Fig. 2: IR spectra of optimized formulation

 

Flow properties:

Material flow was excellent, as the angle of repose of all formulations (F1- F9) was ≤ 34.44. Thus, it has been proven that blends have no restrictions on their flow. The blend density varied between 0.36 grammes per cubic centimetre to 0.41 grammes per cubic centimetre. The density range that was found between 0.44g/cm3 to 0.48g/cm3 was referred to as "tapped density." The flow property of the blends can be deduced from these values. The formula index, calculated to be between 11.12-17.02, was determined to be consistent with Hausner's flow ratio of 1.12-1.20, indicating the blend has good flow character.

 


 

Table 3: Flow properties and their results

Formulation Code

Derived properties

Flow properties

Bulk density (mean±SD)

Tapped density (mean±SD)

Angle of repose (mean±SD)

Carr’s index (mean±SD)

Hausner’s ratio (mean±SD)

F1

0.36±0.04

0.42±0.012

25.02±0.30

14.28±1.96

1.16±0.02

F2

0.39±0.012

0.47±0.04

26.25±0.39

17.02±1.94

1.20±0.06

F3

0.38±0.012

0.44±0.06

27.81±0.64

13.63±3.92

1.15±0.05

F4

0.39±0.016

0.46±0.012

26.36±0.96

15.21±1.76

1.17±0.02

F5

0.39±0.06

0.46±0.06

31.74±0.73

15.21±2.22

1.17±0.03

F6

0.41±0.05

0.47±0.008

28.96±0.36

12.76±3.18

1.14±0.05

F7

0.40±0.025

0.46±0.021

30.72±0.29

11.12±1.16

1.15±0.02

F8

0.40±0.06

0.45±0.014

32.80±0.40

11.12±3.64

1.12±0.05

F9

0.41±0.04

0.48±0.022

34.44±0.34

14.58±2.78

1.17±0.04

 

Table 4: Percentage drug entrapment efficiency, drug content, percentage yield

Formulation Code

Percentage yield

Drug Content (%)

Entrapment efficiency (%)

F1

72.64

86.28

78.16

F2

74.42

92.62

86.22

F3

84.19

94.62

93.78

F4

76.41

92.96

88.26

F5

80.46

96.82

92.82

F6

84.62

98.83

97.68

F7

80.72

90.16

92.62

F8

84.74

96.72

96.29

F9

95.22

98.58

98.28

 

 

In-vitro dissolution studies:

Table 5: In Vitro release data of Stavudine microspheres

Time (hrs.)

F1

F2

F3

F4

F5

F6

F7

F8

F9

0

0

0

0

0

0

0

0

0

0

1

26.42

22.42

14.26

24.82

20.12

19.24

20.24

18.56

12.76

2

34.24

30.02

21.92

36.26

34.81

26.78

36.64

24.71

20.72

3

48.92

38.24

30.68

49.02

42.86

38.86

48.81

36.81

29.42

4

55.18

46.36

44.84

51.84

51.84

44.68

56.60

48.87

36.46

5

64.69

54.26

53.62

66.86

62.89

52.96

68.98

54.82

48.58

6

72.26

68.61

66.86

79.92

74.02

68.48

74.89

65.46

56.82

7

90.28

74.65

78.42

88.86

82.46

74.18

88.21

78.84

69.77

8

94.12

88.96

80.98

99.86

94.18

86.64

97.64

86.76

78.21

9

 

92.26

89.63

 

97.28

92.98

 

92.92

82.48

10

 

94.62

92.76

 

 

99.14

 

96.28

88.46

11

 

96.01

 

 

 

 

 

94.49

96.01

12

 

 

 

 

 

 

 

 

98.22

 


 

Fig. 3: % CDR profile of Stavudine microspheres F1-F9


 

Drug release kinetics studies:

Through the drug release kinetics, which optimised formulation (F9) exhibited zero order drug release, it was observed that the Non-Fickian diffusion process was employed. The n value was less than 0.89.

 

Table 6: Regression co-efficient (r2) values Stavudine microspheres

Formulation

Zero order

First order

Higuchi matrix

Peppa’s plot

R2 value

N value

F9

0.986

0.872

0.946

0.993

0.869

 

DISCUSSION:

Bioavailability, reduced drug release, and improved solubility for drugs that are less soluble in a high pH environment are all achieved with sustained-release dosage forms. It has other applications, such as administering local drugs to the small intestine. The study aimed to formulate and characterise the microspheres of Stavudine, a lipophilic prodrug of deoxythetracycline, using an ionotropic gelation technique that utilises HPMC K15M, Guar gum, and Carbopol 934 as polymers at different polymer concentrations. Each formulation was prepared and the components are shown in the table below. Following the FTIR, percent yield, drug content, entrapment efficiency, in vitro dissolution, and scanning electron microscopy, the prepared Stavudine microspheres were ready for in vivo administration. HPMC K15M, Carbopol 934, and Guar gum were studied in conjunction with Stavudine. Here, the unique spectral signatures of Stavudine were compared to those of drug and polymers. Stavudine microspheres produced a percentage yield of between 72.64 and 95.22 percent. A higher polymer concentration entraps more material. The data shows that the Stavudine distribution in the microspheres is correct, with deviations no greater than the acceptable range. A range of 86.28% to 98.58% of the drug content was found in the formulations. The entrapment efficiency was determined to be between 78.16% and 98.28%. The results are tabulated below. Stavudine microspheres, which were made using carbopol 934, produced a maximum of 98.28% drug entrapment efficiency. In order to increase the encapsulation efficiency, the polymer concentration was increased. Figure and table were used to draw and represent the plots of cumulative percentage drug release V/s. versus time, cumulative percent drug retained V/s. versus root time, and log cumulative percentage drug retained V/s. versus time and root time. Co-determinations were listed in Table alongside the slopes and regression coefficient of determinations (r2). In addition, the determination coefficient demonstrated that the data points were best described by zero-order kinetics. The Higuchi equation, which describes the diffusion-controlled release mechanism, provides further detail. For the Stavudine microspheres prepared with the drug and Carbopol 934, ‘n' value of 0.86 was found for the Korsemeyer-Peppas model, indicating Non-Fickian diffusion mechanism of the drug through the Stavudine microspheres.

 

CONCLUSION:

F9 showed a good sustained release profile, due to a high polymer concentration and a high entrapment efficiency. According to the coefficient of determination, the data suggested that zero-order kinetics best described the release rate. Korsemeyer-Peppas model diffusion exponent ‘n' has been determined to be between 0.869 and 0.871 for the stavudine microspheres prepared with and Carbopol 934. This indicates the stavudine microspheres' non-Fickian diffusion mechanism, whereby the drug diffuses in the form of microspheres. Ion-gelation of stavudine microspheres with polymers like HPMC K 100M, Carbopol 934, and guar gum yields microspheres with controlled release, as can be seen from the above data.

 

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Received on 17.07.2021         Modified on 04.01.2022

Accepted on 15.03.2022   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Tech. 2022; 12(2):119-124.

DOI: 10.52711/2231-5713.2022.00020