Formulation of Monodisperse Alginate (Micro) Spheres Containing Tenofovir for Extended Release Dosage Form

 

Pathuri Raghuveer*, A. Prameela Rani, Madhuri Desavatu, A. Sai Chaitanya, G. Srikar

University College of Pharmaceutical Sciences, Acharay Nagarjuna University, Guntur-522510

 

ABSTRACT:

Tenofovir (TF) belongs to a class of antiretroviral drugs known as nucleotide analogue reverse transcriptase inhibitors (NRTIs) used in the treatment of human immunodeficiency virus 1 (HIV-1) and hepatitis B. Despite its safety and effectiveness, TF oral administration limited by several factors : low bioavailability (25%), adverse effects like decrease in bone mineral density and severe renal adverse events, including fanconis syndrome due to frequent dosing, and high circulating plasma levels. By developing particulate drug carriers it is possible to achieve effective plasma concentration without significant fluctuation, to avoid sub-therapeutic or toxic plasma concentrations, to achieve an effective therapy with a low dosage and to reduce the dosage frequency. TF loaded floating particulate drug carriers for control release medication were designed and constructed to prolong the retention in the stomach and to facilitate drug absorption over a prolonged period of time using Extrusion ionic gelation technique employing sodium alginate alone and in combination with MC, HPMC K100M, HPMC K15M. The drug release for the optimized formulation, F10, followed first order kinetics and Higuchi plot of F10 formulation showed an R² value of 0.9582. The data were fitted to the Korsmeyer-Peppas equation and the values of diffusion exponent ‘n’ for the batch F10 was 0.623 which indicated the drug release by Non-Fickian diffusion, suggesting that the diffusion along with erosion/swelling plays an important role in extending the drug release. Surface morphology of microspheres was found to be Smooth. These results suggested that the TF microbeads are promising and should be investigated further in the near future as an effective oral delivery system.

 

 

 

INTRODUCTION:

Tenofovir (TF) belongs to a class of antiretroviral drugs known as nucleotide analogue reverse transcriptase inhibitors (NRTIs), which blocks reverse transcriptase, a crucial viral enzyme in human immunodeficiency virus 1 (HIV-1) and hepatitis B virus infections. It is on the World Health Organization's List of Essential Medicines, the most important medications needed in a basic health system.

 

Despite its safety and effectiveness, TF oral administration limited by several factors : low bioavailability (25%), adverse effects like decrease in bone mineral density and severe renal adverse events, including fanconis syndrome due to frequent dosing, and high circulating plasma levels. The pharmacokinetic human studies showed that administration of conventional immediate release oral formulations of TF characterized by limited bioavailability due to poor cell membrane permeability. To overcome these limitations, the use of various sustained release carriers, such as tablets, polymeric cellulose resins, or microparticles of various shapes, have been studied ⁽¹⁾. By developing particulate drug carriers it is possible to achieve effective plasma concentration without significant fluctuation, to avoid sub-therapeutic or toxic plasma concentrations, to facilitate the drug release in a controlled manner, to achieve an effective therapy with a low dosage of the drug, to reduce the dosage frequency, thus to improve patient compliance, and to prevent interference to the therapy with day-to-day lifestyle. Bio-adhesive and floating particulate drug carriers were designed to prolong the retention in the stomach and to facilitate drug absorption over a prolonged period of time. Hence, combinations of both sustained release and floating properties in the micro carriers system would further enhance therapeutic efficacy ⁽²⁾. The Ability of the alginate gel based micro beads to release molecules has enabled their use as a carrier of active substances for a variety of applications. This also poses certain advantages such as hydrophilicity, biocompatibility and non-toxic, have become very popular for use in the pharmaceutical and nutraceutical industry ⁽³⁾

 

Natural polysaccharides or gums represents one of the most abundant industrial raw materials and have been the subject of intensive research to prepare multi-unit microbeads for sustained drug delivery, over comparable synthetic materials due to their sustainability, biodegradability and safety ⁽⁴⁾. The advantage of these natural polymers were that they will be broken down into biologically acceptable molecules which will be metabolized and removed from the body via normal metabolic pathways. These natural polymers are hydrophilic and are more amenable to physical and chemical modifications using simpler processes than the widely used synthetic polymers ⁽⁵⁾.

 

Over the last few years, a great deal of attention has paid to the development of polysaccharide-based hydrogel beads through ionic gelation technique useful as potential carriers in controlled drug delivery ⁽⁶⁾. Their porous structure allows encapsulation of active substances, within the hydrogel matrix allow the sustained release of active ingredients.

 

Alginate hydrogels have proven to be attractive for use in biomedical, pharmaceutical and (functional) food purposes due to their biocompatibility and drug retentive properties⁽¹⁾. Alginate particles have been employed as a carriers for controlled drug release Moreover, alginate hydrogel properties lead to an increase in the residence time and reduction of drug metabolism⁽⁷⁾

 

Sodium alginate is a water soluble anionic polysaccharide, mainly found in the cell walls of brown algae and can be isolated from the bacteria Pseudomonas ⁽⁸⁾. This natural polymer possesses several attractive properties such as good biocompatibility, wide availability, low cost, and may be used for the formulation of microbeads under mild conditions. Alginate composition is variable and consists in homo polymeric blocks alternating 1,4-linked b-Dmannuronic acid (M) and A-L-guluronic acid (G) residues. Physical properties of alginate are dependent on the composition, sequence and molecular weight. Gel formation is driven by interactions between G-blocks which associate to form firmly held junctions due to divalent cations. In addition to the G-blocks, MG blocks also participate by forming weaker junctions⁽⁹⁾. Calcium chloride is commonly used as gelling agent for the synthesis of alginate hydrogels⁽¹⁾.

 

Although alginate/tenofovir systems could be very promising as a drug delivery systems, to our knowledge up to now there has been no study on entrapment of TF into alginate microbeads prepared by particularly crosslinking technique. In addition, in the existing studies incorporation of TF in alginate micro/nano-particles has performed by ionic gelation. Thus, in this study an attempt was made to load TF into the alginate microbeads. Since TF has toxic effects above a certain level, it was important to know how to design the most efficient formulations with the minimum amount of crosslinking molecules in order to enhance the safety of these systems.

 

MATERIALS AND METHODS:

Sodium Alginate was obtained from marine chemicals (India) and. The crosslinking agent, calcium chloride was obtained from Ace industries. Methyl cellulose (paramount chemicals), HPMC K15 and HPMC K100 are obtained from alpha chemicia. All chemicals were used as received, without purification. Tween80 (Polyoxyethylene sorbitan mono oleate) obtained from (Sigma-Aldrich, Spain). Distilled water was used for the preparation of the water phase, as well as for the preparation of the buffer solutions. Potassium dihydrogen phosphate, sodium hydroxide, hydrochloric acid (Sigma-Aldrich, India) were used for drug release studies.

 

Preparation of blank alginate microbeads:

Aqueous solution of the sodium alginate solution alone and in combination with polymers Methyl cellulose, HPMC K15M, HPMC K100M was prepared in double distilled water and the resulting solution was added drop wise through an 18-gauge flat-tipped needle into100ml of 5% aqueous calcium chloride solution containing 0.08% (w/v)Tween80 being stirred at 100rpm. Following an incubation period of 10min, the ability of different polymer compositions to form isolatable, self-standing beads were examined. The hydrogel beads were formed instantaneously in the presence of polymers and sodium alginate alone at all concentrations. The beads were filtered off, washed with 50ml of double distilled water thrice and air-dried. .

Table:1 Formulation design

 

 

 

Preparation of Tenofovir loaded microbeads

The batches of drug loaded microbeads (F1 TO F12) prepared by using SA alone and with different concentrations of MC, HPMC K15M, HPMC K100M. TF was dissolved into10ml dispersion of polymer. The dispersion was added drop wise through 18-gauze flat tipped needle into slightly agitated 100ml of 5% (w/v) CaCl₂ solution containing 0.08% (w/v) Tween80. Following an incubation period of 10min, the beads were isolated by filtration, washed with 50ml of double distilled water and air-dried to constant weight. Keeping the concentration of CAcl2 fixed at 5% (w/v), following processing variables were investigated:

1. Concentration of sodium alginate

2. Type of floating Polymer

3. Ratio of sodium alginate to floating polymer

 

Evaluation tests for micro-particulate unit like, micrometric properties, percentage yield value, Bead size, Drug Drug-polymer interaction study, Estimation of drug entrapment efficiency, Equilibrium swelling study, In vitro TF release study, drug release mechanism study was done according to (Battacharya et al) . Each sample was tested and analyzed in triplicate⁽¹⁰⁾.

 

Drug-polymer interaction study:

From the spectrum of TF, physical mixture of TF and polymers observed that all characteristic peaks of TF were present in the combination spectrum, thus indicating compatibility TF and polymer ⁽¹¹⁾

 

Preparation of Floating Microbeads:

Immediately after separation from Calcium chloride solution, the alginate spheres were found to be very sticky to touch. The texture was smooth, but the spheres appeared soft, indicating that the interaction with calcium chloride may not have hardened the alginate spheres sufficiently. Upon drying, they shrunk as well as became relatively harder. Totally, 12 different formulations of TF were prepared by using the above polymers

 

Post formulation studies:

Percentage Yield

The percentage yield of placebo microbeads was found to be 73% to 81%. The prepared placebo microbeads have an average particle size of 1110 mm-1662 mm. Drug loaded spheres were found to be discrete, spherical, free-flowing, and of the monolithic matrix type. All the formulations demonstrated good flow property.

 

Table 2: mean particle size and angle of repose of formulations

 

Table :3  Percentage Drug Loading and Percentage Drug Entrapment Efficiency

Buoyancy:

Microbeads were found floating for 4 hrs over the surface of simulated gastric fluid (pH 1.2). The Percentage buoyancy of the microbeads was in the range of 75% (batch F11) to 98% (batch F1, F7). The good floating behavior of the microbeads may be attributed the hollow nature of the micro beads. Formulations F1, F2, F3 showed extended floating time, whereas the formulations (F4-F12) containing floating polymer has shown less floating lag time, among them F10 has shown very less floating lag time this may be attributed to viscous nature of HPMC K150M^(12).

 

Dissolution Study:

TF release from the drug loaded spheres was studied in the acidic buffer (pH 1.2) for 6 hours. The dissolution profiles of the different batches of the spheres are depicted in Figure 1. The dissolution parameters of the different formulations are indicated in Table 4. TF release from the spheres was slow and depended on the composition of the coat. Based on the T₅₀ (hrs) values (time taken for 50% of drug release), the drug release from the different formulations can be arranged as:

F7>F8>F9>F4=F6>F5>F3>F2>F1>F10>F11>F12

 

Similarly, based on the T₇₅ (hrs) values (time taken for 75% of drug release), the drug release from the different batches can be arranged as:

F7>F8>F9>F4>F5>F6>F3>F2>F1>F10>F11>F12

 

The batches, F2, F3, F4, F5, F6, F7, F8 and F9 did not attain T75 values even after 10 hours of the dissolution study. The formulations: F11 and F12 achieved 90% of drug release in 8.8 and 8.5 hours. All other formulations did not attain T₉₀ (hrs) values (time taken for 90 % of drug release) even after 10 hours of the dissolution study. The differences in the drug release characteristics of the various spheres are due to the differences in the porosity of the coat formed and its solubility in the dissolution fluid. Between all the batches, the F10 (Drug: Sod. Alginate: HPMC K15M = 1:1:1), F11 (Drug: Sod. Alginate: HPMC K15M = 1:1:2), F12 (Drug: Sod. Alginate: HPMC K15M = 1:1:3) batches are considered to be the optimized formulations {(T₅₀ = 3 h, T₇₅ = 6 hrs, T₉₀ = 10 hrs), (T₅₀ = 2.6 hrs, T₇₅ = 5.3 hrs, T₉₀ = 8.8 hrs), (T₅₀ = 1.5 hrs, T₇₅ = 5 hrs, T₉₀ = 8.5 hrs)} because among all the batches they shows better extent of drug release, good entrapment efficiency (95%, 82%, 89%), and good buoyancy (96, 79, 85) respectively.

 

TF release from the optimized formulations (F10) was slow and extended over a period of 10 hrs and these drug loaded spheres were found suitable for the oral controlled release.

 

The initial release of TF appears to depend on the concentration of alginate in the spheres. Smaller concentration of alginate may have produced more cracked and rough surface spheres which released TF more quickly due to more liquid penetration ⁽¹²⁾. Moreover, spheres containing smaller alginate concentration may have produced a relatively weaker network, which broke down faster than the relatively stronger network formed into spheres containing a larger concentration of alginate. In most release studies dealing with multi- particulate systems, an initial burst effect is reported due to migration of drug to the surface of the particles ⁽¹³⁾. In this investigation, a burst effect was exhibited by the spheres containing a low concentration of the alginate. Good solubility of TF and insufficient crosslinking between the sodium alginate and CaCl2 may have contributed to the fast release from the beads. So sodium alginate as a carrier system is not suitable to sustain the release of highly soluble drugs like TF. The initial slow release was followed by a linear rate of release until almost 90% of the drug release was obtained with beads produced with alginate and rate retarding polymers ⁽¹⁴⁾. The decrease in release rate of the drug from the alginate spheres (compared to almost instantaneous dissolution of pure drug in pH 1.2 acidic buffer) is because of an increase in the degree of cross-linking with the increase in the concentration of sodium alginate⁽¹⁵⁾. As the degree of cross-linking increases, the porosity decreases and the reduced porosity will further retard the release of drug from the alginate spheres ⁽¹⁶⁾. Also, drug release from a hydrophilic matrix is controlled by the formation of a hydrated viscous layer around the matrix which acts as a barrier to drug release by opposing penetration of water in to the matrix and also movement of dissolved solutes out of the matrix⁽¹⁷⁾. In case of spheres containing the highest concentration of the polymer, the hydrophilic property of the polymer may bind better with water to form the viscous gel structure, which may block the pores on the sphere surfaces and prolong the drug release. As the polymer concentration of the prepared spheres was increased, the release rate was decreased⁽¹⁸⁾. An inverse relationship was observed between the polymer concentration and the drug release from the prepared spheres. The release of the drug was considered to occur mostly by diffusion, but could be accelerated by the weight loss of the polymers⁽¹⁹⁾. The structure of the spheres changed significantly over time, indicating that there was substantial hydration and swelling of the polymeric matrix. The alginate-polymeric gel might have acted as a barrier to the penetration of the dissolution medium, thereby suppressing the diffusion of the drug from the swollen alginate-polymeric matrix. The release of the drug was modulated by the diffusion of the drug through the swollen polymeric matrix⁽²⁰⁾.

Table 4: Percentage Drug Release

Time (min)

Percentage Drug Release

F1

F2

F3

F4

F5

F6

F7

F8

F9

F10

F11

F12

0

0

0

0

0

0

0

0

0

0

0

0

0

30

13.74± 3.56

7.72±7.22

12.27±7.06

13.02±3.20

9.53±6.02

7.53±5.95

4.26±2.87

3.97± 7.39

8.40± 6.23

15.31± 4.54

12.33±2.67

28.11± 6.76

60

24.23± 7.97

18.34±7.56

22.30±5.23

16.28±9.82

17.43±5.24

8.79±6.59

5.43±6.47

4.55± 2.60

9.80± 4.43

26.48± 3.14

27.55±2.25

31.31± 2.59

120

29.95± 5.63

21.61±6.92

27.80±7.94

20.95±2.25

21.77±4.70

13.77±1.83

7.80±1.45

6.97± 5.84

12.98± 5.73

31.60± 4.63

37.43±5.38

40.00± 7.02

180

39.55± 3.88

30.65±7.99

34.98±4.41

26.46±3.37

25.12±3.27

19.55±4.14

10.51±6.90

9.18± 7.31

19.38± 7.26

42.51± 2.54

48.34±3.65

45.86± 9.74

240

45.19± 7.27

41.35±9.14

40.91±3.38

30.34±4.71

29.44±4.74

23.36±7.97

15.69±1.76

13.27±2.02

23.18± 5.83

53.82± 8.81

59.60±7.14

56.05± 6.32

300

56.64± 5.66

48.60±3.15

45.01±5.99

34.27±6.00

33.32±4.82

28.08±2.35

22.16±7.25

19.99±5.86

26.77± 5.76

68.51± 7.40

70.34±3.35

67.26± 3.97

360

58.23± 5.21

53.67±6.86

49.55±5.31

38.93±3.41

40.40±9.04

42.79±7.72

26.38±3.46

29.80±5.93

30.22± 7.56

77.89± 5.99

82.45±5.94

87.50± 4.65

 

Release Kinetics:

The regression coefficient (R²) values of the different formulations are indicated in Table 6. Based on the values of R², the drug release from the formulations (F1, F2, F3, F4, F5, F9, and F11) exhibited first order kinetics, whereas the drug release from the batches (F6, F7, F8, F10, F12) demonstrated zero order kinetics.

 

The values of the release rate constants, K (for first order) and the diffusion exponent (n) are represented in table. The values of K1 (min^-1), the first order release rate constant of the different batches ranged from 0.0006 (batch F7) to 0.0046 (batch F12).

 

All the formulations obeyed Higuchi equation (R2> 0.9), indicating that the drug release mainly depends on diffusion and erosion. The values of n (diffusion exponent) for all the formulations ranged from 0.427 (batch F12) to 0.776 (batch F8).

 

Based on the n values, the drug release from the formulations: F4, F12 followed Fickian diffusion (n< 0.45), whereas the drug release from the batches: F1, F2, F3, F5, F6, F7, F8, F9, F10, F11 exhibited non-Fickian diffusion (n> 0.45) mechanism controlled by swelling and relaxation of the polymeric matrix.

 

Table : 5  Highchi Plot of Formulations F1-F12

 

Table: 6 Percentage buyoyancy and krosmayer constant

 

The drug release for the optimized formulation, F10, followed zero order kinetics and the R² value was 0.9765. The Higuchi plot of F10 formulation showed an R² value of 0.9605. The data were fitted to the Korsmeyer-Peppas equation and the values of diffusion exponent ‘n’ for the batch F10 was 0.623 which indicated the drug release by Non-Fickian diffusion, suggesting that the diffusion along with erosion/swelling plays an important role in extending the drug release.

Figure 1: First order graph

 

Figure 2: Higuchi Plots

 

Figure3: Korsemeyer-Peppas plots

 

SEM: Morphology:

Morphology of the prepared floating microbeads was examined by scanning electron microscopy (SEM). Results show that microbeads were predominantly smooth & spherical in appearance with some visible surface irregularity. The porous nature and spherical shape of the microbeads are evident from their SEM photomicrographs.

 

Figure 4: SEM Analysis

 

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Accepted on 31.03.2017           © RJPT All right reserved