Development and Evaluation of In-situ gel containing Linezolid and Diclofenac Sodium in the treatment of Periodontitis

 

Inayathulla*, Prakash Goudanavar, Ankit Acharya

Department of Pharmaceutics, Sri Adichunchanagiri College of Pharmacy,

B.G. Nagara, Karnataka -571448, India.

*Corresponding Author E-mail: ankitbaba99@gmail.com

 

ABSTRACT:

Periodontal treatment aims at reduction of infection and inflammation. Linezolid is a synthetic antibacterial agent of a new class of antibiotics, the oxazolidinones, which has clinical utility in the treatment of infections caused by aerobic and anaerobic bacteria susceptible organisms methicillin and vancomycin-resistant in the treatment of periodontitis. Diclofenac sodium is anti-inflammatory agent and in-situ gel containing Linezolid and Diclofenac sodium in this study may have a potential additive effect. Hence, there is broad scope for research in local application of antimicrobial and anti-inflammatory combination drug (s) in periodontitis. Linezolid and  Diclofenac sodium. In-situ gel containing Linezolid in combination with Diclofenac sodium was prepared using combination of carbopol-HPMC and carbopol-NaCMC. The prepared in-situ gel formulation showed satisfactory results for in-vitro gelling capacity, rheology, and other physical properties. In-vitro drug release profile was dependent up on the concentration of polymer, i.e. drug release was decreased with increase in polymer concentration and vice versa. The formulations of batches LDCF1, LDCF2, LDCF3, LDSF1, LD SF2 and LDSF3 failed to extend the drug release up to 12 hours. Thus, the formulation of batches LDCF4 and LDSF4 containing carbopol: HPMC and carbopol: NaCMC in 1:2 ratios were considered as optimum formulation on the basis of optimum viscosity, gelation time, gelation temperature, gelling capacity, and their capacity to extend the in-vitro drug release up to 12 hours. Hence, prepared formulations showed sustain drug release behaviour.

 

KEYWORDS: Linezolid, Diclofenac sodium, carbopol, HPMC, NaCMC.

 

 

 

INTRODUCTION:

Periodontal diseases are mainly of bacterial etiology in initiation and progression of periodontal diseases. Periodontal treatment aims at reduction of infection and inflammation and adjunctive use of drugs such as anti-microbial agents and anti-inflammatory agents1. Linezolid is a synthetic antibacterial agent of a new class of antibiotics, the oxazolidinones, which has clinical utility in the treatment of infections caused by aerobic and anaerobic bacteria susceptible organisms methicillin and vancomycin-resistant2. In situ gels are systems which are applied as solutions and are capable of undergoing rapid sol-to-gel transformation triggered by external stimulus such as pH-triggered system (e.g. cellulose acetate hydrogen phthalate latex and Carbopol), Temperature dependent system (eg. pluronics and tetronics), and. Ion activated system (eg. gelrite).  In situ gel based drug delivery systems consist of pharmaceutical active ingredients, polymer, co-polymer and excipients. The use of carbopol as in situ gel forming system was substantiated by the property to transform into stiff gels when the pH is increased. Hydroxy Propyl Methyl Cellulose (HPMC) is incorporated as a viscosity enhancer to further aid in accomplishment of sustained drug delivery. HPMC is semi synthetic, inert, viscoelastic polymer which is non-ionic nontoxic, a good carrier for pharmaceutical application which exhibits high swelling capacity3. Hence it was a challenge to formulate in-situ periodontal gel containing Linezolid and Diclofenac sodium with rate controlling polymers which provides a longer duration of action and local antibacterial effect without loss of dosage4,5.

 

MATERIALS AND METHODS:

Preparation of in-situ gelling systems:

In-situ gels were prepared by cold method using varying concentration of carbopol 934P and hydroxy propyl methyl cellulose (HPMC) (LDCF1, LDCF2, LDCF3 and LDCF4) and carbopol 934P and sodium carboxy methyl cellulose (NaCMC) (LDSF1, LDSF2, LDSF3 and LDSF4). Optimum batch was selected based on gelling time and gelling capacity. First polymers were prepared by adding the polymers to deionised water and heated to 90° while stirring. After cooling to below 40° appropriate amounts of calcium chloride (0.05% w/v) was added into the solution. Separately drugs were dissolved in propylene glycol. The drug solution was slowly added to the polymer dispersion. To this solution, 2-3 drops triethanolamine was added to adjust the pH. Above mixture was stirred by using a magnetic stirrer to ensure thorough mixing. Finally, methylparaben and propylparaben were added to the above formulation mixture. This solution was allowed to stand for 2 hour for removal of air bubble. The final solution was kept in well closed container refrigerator at 4°C until evaluation.3 The formulation design of in-situ gel formulation was tabulated in Table 1.

 

Drug-polymer compatibility study:

FTIR spectrum of pure drug and mixture of drug and polymers are shown in Fig. 1. From the spectral study, it was observed that there was no significant change in the peaks of pure drug and drug polymer mixture. Hence, no specific interaction was observed between the drug and the polymers used in the formulations.

 

 

Table 1: Formulation of various concentrations

Batch

D

(%w/v)

L

(%w/v)

HPMC

(%w/v)

NaCMC

(%w/v)

Carbopol 934P (%w/v)

Calcium chloride (%w/v)

MP (%w/v)

PP

(%w/v)

Deionized water

LDCF1

0.1

0.5

0.5

--

0.5

0.05

0.15

0.02

q.s

LDCF2

0.1

0.5

0.5

--

1.0

0.05

0.15

0.02

q.s

LDF3

0.1

0.5

0.5

--

1.5

0.05

0.15

0.02

q.s

LDCF4

0.1

0.5

0.5

--

2.0

0.05

0.15

0.02

q.s

LDCSF1

0.1

0.5

--

0.5

0.5

0.05

0.15

0.02

q.s

LDSF2

0.1

0.5

--

0.5

1.0

0.05

0.15

0.02

q.s

LDSF3

0.1

0.5

--

0.5

1.5

0.05

0.15

0.02

q.s

LDSF4

0.1

0.5

--

0.5

2.0

0.05

0.15

0.02

q.s

Note: D; Diclofenac sodium, L; Linezolid, HPMC; Hydroxy propyl methyl cellulose, NaCMC; Sodium carboxy methyl cellulose, MP; methyl paraben, PP; propyl paraben, q.s; quantity sufficient to 100 ml.

 

 

Figure 1: FTIR spectrum of drug linezolid, Diclofenac sodium and physical mixture 

 

Evaluation of in-situ gel:

Appearance:

All prepared formulations were evaluated from the visual inspection.6

 

pH measurement:

pH is one of the most important parameter involved in the in-situ gel formulation and it is measured directly with the help of digital pH meter.

 

Drug content estimation:

For determination of drug content, 1ml of in-situ gel formulation was placed in a vial containing 5 ml of 6.8 pH PBS (simulated salivary pH), equilibrated at 37°C and gel formation was assessed visually. As soon as whole gel body was formed, the whole formulations were taken into separate dialysis tubes. The tubes were kept in a beaker containing 50ml of distilled water and formulations were dialyzed for 30 min at 50rpm and dialysis medium was replaced with fresh quantities of distilled water in between so as to ensure complete removal of unentrapped drug. The concentration of LZ and DS was determined spectrophotometrically at 250 nm and 276 nm, after suitable dilution and filtration using 6.8 pH buffer as blank.7,8

 

Gelling Capacity:

All formulations were evaluated for gelling capacity in order to identify the compositions suitable for use as in-situ gelling systems. The gelling capacity was determined by visual method, in which coloured solution of prepared formulations were prepared. Gelling capacity was estimated by placing 2ml of 6.8 pH phosphate buffer in a 10ml test tube and maintained at 37°C temperature. One millilitre of coloured formulation solution was added to the phosphate buffer. As the formulation comes into contact with phosphate buffer it was immediately converted into a stiff gel-like structure. The gelling capacity of formulation was evaluated on the basis of stiffness of formed gel and time period for which formed gel remains as such. The in-vitro gelling capacity was graded in four categories on the basis of gelation time and the time taken for the gel formed to dissolve. (-) no gelation; (+), gels after few minutes, dispersed rapidly; (++), gelation immediate, remains for few hours; and (+++), gelation immediate, remains for an extended period.7,8

 

Gelation temperature:

10 ml of the sample solution and a magnetic bead were put into a 30ml transparent vial placed in a low temperature digital water bath. A thermometer was placed in the sample solution. The solution was heated at the rate of 1°/min with the continuous stirring. The temperature at which the magnetic bead stopped moving due to gelation was considered as gelation temperature.7

Gelation time:

Gelation time of prepared in-situ gel formulation was measured by placing 2ml of the gel in 15ml borosilicate glass test tube. This test tube was placed in water bath (37oC) and gelation time was noted when there was no flow of the gel when test tube was inverted.7

 

Viscosity and rheological studies:

Brookfield digital viscometer was used for the determination of viscosity and rheological properties of atorvastatin in situ gel using spindle no LC. 50g of the gel was taken in a beaker and the spindle was dipped in it. The viscosity of gel was measured at different angular velocities at a temperature of 25°. A typical run comprised changing of the angular velocity from 10 to 60 rpm.

 

Syringeability:

All prepared formulations were transferred into an identical 5ml plastic syringe placed with 21 gauge needle to a constant volume (1ml). The solutions which were easily passed from syringe was termed as pass and difficult to pass were termed as fail.

 

In-vitro drug release studies:

Drug release profile of in-situ gel formulation was carried out by using Franz diffusion cell (25ml). One gram of linezolid in-situ gel formulation was placed in donor compartment and 25ml of dissolution medium of pH 6.8 (simulated salivary pH) in receptor compartment. Between donor and receptor compartment chicken cheek mucous membrane is placed. Sols were added from the top so that upon contact with the salivary fluid get converted to gel form. The whole assembly was placed on the thermostatically controlled magnetic stirrer. The temperature of the medium was maintained at 37°C ± 0.5°C. 1ml of sample was withdrawn at predetermined time interval for 0.5 hours to 12 hours and same volume of fresh medium was replaced. The withdrawn samples were diluted to 10ml in a volumetric flask with same buffer medium and analyzed by UV spectrophotometer at 250nm and 276nm using reagent blank. The drug content was calculated using the equation generated from standard calibration curve. The % cumulative drug release (% CDR) was calculated.8

 

Accelerated stability studies:

Optimized bathes of in-situ gel were placed in ambient colored vials and sealed with aluminium foil for a short term accelerated stability study at 25±2°C and 60±5% RH and 40±2°C and 75±5% RH as per International Conference on Harmonization states Guidelines. Samples were analyzed every month for clarity, pH, gelling capacity and drug content.8

 

 

RESULTS AND DISCUSSION:

Appearance:

All batches of in-situ gel formulations were clear and there was absence of foreign particles.

 

pH of prepared in-situ gel:

The pH of the formulations was found in the range of 6.2-6.8, which was required pH for periodontal treatment. The pH values of all prepared formulations were within the limit of neutral pH; this indicates formulations can be used without any irritation in the oral cavity.9

 

Syringeability:

Syringeability test showed that prepared in-situ gel formulations were easily injectable through 21 gauge needle at room temperature (25°C), hence passes the syringeability test. This facilitate to injection of sol directly into the periodontal pocket.

 

Gelling time, temperature and capacity:

Optimization of in-situ gel was done based upon its gelling time, temperature and gelling capacity. The formulation should undergo rapid sol-to-gel transition due to ionic interaction. Moreover, to facilitate sustained release of the drug to the periodontal cavity, the formed gel should preserve its integrity without dissolving or eroding for a prolonged period of time. In-situ gels were prepared using various concentration of carbopol (0.5-2.0%) and concentration of NaCMC and HPMC was kept constant. Gelation temperature of solution was observed in the range of desired gelation temperature (35-39oC). Gelation temperature is the temperature at which the liquid phase makes a transition to gel. The gelation temperature of an in-situ gel would be higher than 25°C. If the gelation temperature of the formulation is lower than 25°C, gelation occurs at storage temperature, leading to difficulty in manufacturing, handling, and administering.10

 

Except the formulations of batches LDCF1, LDSF1 and  LDSF2, all the batches showed instantaneous gelation when contacted with gelation fluid (Simulated Saliva). However, the nature of the gel formed depended upon the polymer concentration. Among all batches, formulation LDCF4 (containing 1:4 ratio of HPMC and carbopol) showed shortest gelation time i.e. 2.14 min. Batches LDCF1, LDSF1 and LDSF2 showed the weakest gelation, which could be due to the presence of very low concentration of carbopol. Batches LDCF2, LDCF4, SF3 and SF4 showed immediate gelation but the formed gels did not remain for an extended period of time ranging from 7.21 - 8.3min. This indicates that the formed gel might be less stiff and erodes within a few hours, which could be due to the relatively low concentration of carbopol. Batches LF4 showed immediate gelation (2.14 min) and the gel formed was stiff and remained for an extended period of time.

 

Drug content uniformity:

Drug content estimation was done by using simultaneous estimation of Linezolid (LZ) and for Diclofenac sodium (DS) in 6.8 pH buffer. The percent drug content was found to be in acceptable range for all formulations.  The percent drug content of all in-situ gel formulations was found to be in between 97.88% to 99.91% for Linezolid (LZ) and for Diclofenac sodium (DS) it lies between 96.84% to 98.29%.

 

Viscosity and rheological studies:

Viscosity of prepared in-situ gel formulations were evaluated before and after gelation. It was found that as the shear rate increased the viscosity of gel decreased in both cases. Results of viscosity studies showed that viscosity of prepared formulation was drastically increased after gelation. The viscosity of in-situ gel was increased after gelation. As indicated, a formulation suitable for application to the periodontal pocket should ideally have a low viscosity when applied, and after instillation have a high viscosity in order to stay at the application site. From the phase behaviour, the presently studied formulations seem promising for achieving such behaviour.

 

Viscosity of prepared gel was also dependent on the concentration of the gel based on increasing the concentration of polymer ratio, the viscosity of the gel was increased. These findings indicated the differences between viscosity of gels and their corresponding sols. Rheological evaluation of selected formulation exhibited pseudoplastic flow before and after gelling. It  was  also  reported  that,  in  polymer  solutions,  the  various  interactions between polymer chains regarded as localized junctions are responsible for an increase in viscosity  and exhibit non-Newtonian flow.10 Further, it was also found that, all the formulations were liquid at room temperature and underwent rapid gelation when the pH was raised to 6.8. The viscosity of formulation contributed to the product adhesiveness, reflecting the importance of product rheology on this parameter. Additionally, the gel formed in-situ should maintain its integrity without dissolving or eroding for a prolonged period. Results of rheological studies are shown in Table 2 and 3 and Fig 2 and Fig 3.

 

 

Table 2:

Viscosity before gelation (cps)

Formulation

RPM

LDCF1

LDCF2

LDCF3

LDC

F4

LDSFF1

LDS

F2

LDS

F3

LDSF4

10

880

1115

1288

1327

799

920

948

1020

20

760

926

954

1095

531

810

866

915

30

547

710

756

815

468

665

680

718

40

430

622

645

710

220

419

420

485

50

297

385

460

480

165

258

268

316

60

110

219

352

390

92

103

112

225

 

Table 2: Continued

Viscosity after gelation (cps)

Formulation

R

P

M

LDC1

LDC2

LDC3

LDC4

LDSF1

LDSF2

LDSF3

LDSF4

10

1880

3011

3233

3633

1760

2610

3169

3270

20

1650

2380

2471

2541

1620

1968

2381

2230

30

1270

1375

1878

1944

1235

1244

1775

1850

40

985

1128

1519

1669

951

1076

1334

1690

50

870

892

1236

1250

866

865

1023

1125

60

565

636

680

891

539

560

646

720

 

 

 

Figure 2: Rheological profile of prepared in-situ gel before gelling

 

 

Figure 3: Rheological profile of prepared in-situ gel after gelling

 

In-vitro drug release studies:

The cumulative amount of Linezolid and Diclofenac sodium released vs time profile for the selected formulations is shown in Figure 3. First sampling was done 0.5 hour after the formulation placed on the synthetic cellulose membrane of the donor compartment of Franz Diffusion Cell. Half an hour time for first sampling was selected in order to evaluate the effect of increasing polymer concentration on the cumulative amount of drug released. The results showed that the amount of drug released in the 0.5 hour decreased with increasing polymer concentration, and the trend continued for the entire duration of the study. This might be due to increase in concentration of polymers showed higher viscosity which in term delayed the drug release from the formulation. From the drug release studies we also observed that almost 35% of drug released from all formulations within 2 hours. The initial burst release of the drug from the prepared formulations could be explained by the fact that these systems were formulated in an aqueous vehicle. The matrix formed on gelation was already hydrated and hence hydration and water permeation could no longer limit the drug release, the remaining drug was released at a slower rate (second phase). This bi phasic pattern of release is a characteristic feature of matrix diffusion kinetics. The formulation LDCF1, LDCF2, LDCF3, LDCF1, LDSF2 and LDSF3 completes more than 80% of drug release within 6 hours, whereas formulation LDCF4 and LDSF4 containing 2% carbopolshowed drug release up to 12 hours, which might be due to higher viscosity and gelation capacity of these formulations. Based on the release studies, formulation LDCF4 and LDSF4 was selected for further studies.

 

 

 

Figure 3: The in vitro release profiles of Linozolid (LZ) and Diclofenac sodium (DS) for optimized batches (LDCF4 and LDSF4) of in-situ gels

 

Table 3: In-vitro drug release profile of prepared in-situ gel formulations

Time (hours)

Cumulative percentage drug release (%CDR)

LDCF1

LDCF2

LDCF3

LDCF4

LDSF1

LDSF2

LDSF3

LDSF4

LZ

DS

LZ

DS

LZ

DS

LZ

DS

LZ

DS

LZ

DS

LZ

DS

LZ

DS

0

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.5

19.64

20.44

16.18

18.18

14.92

16.85

10.08

12.08

21.82

22.85

19.12

20.09

15.22

17.74

12.33

15.65

1

35.05

32.62

29.92

30.92

27.54

25.32

25.64

20.64

38.24

34.66

28.54

31.65

29.82

28.92

29.24

24.08

2

59.71

56.78

54.06

52.55

50.44

51.07

36.62

34.62

61.76

52.82

55.88

47.32

53.91

44.01

40.66

32.33

4

81.69

87.92

70.27

74.82

64.78

66.65

44.77

48.82

88.08

84.39

71.33

69.36

68.74

63.85

48.83

41.78

6

98.86

96.75

88.71

85.79

82.32

88.39

50.92

55.92

99.02

97.84

84.09

87.75

83.77

82.62

54.04

58.94

8

--

--

99.05

98.65

96.63

99.85

59.96

62.96

--

--

98.47

99.43

95.11

98.92

61.72

67.39

10

--

--

--

--

--

--

68.02

70.02

--

--

--

--

--

--

70.07

75.73

12

--

--

--

--

--

--

75.19

80.66

--

--

--

--

--

--

79.65

81.07

Note: LZ; linezolid, DS; Diclofenac sodium

 

 

Stability studies:

Stability study of two optimized batches LDCF4 and LDSF4 was performed for 3 months in stability conditions as per ICH guidelines. The formulations were examined for appearance, drug content, pHand gelling capacity for every 1 month for 3 months. It was observed that there was no change in the physical appearance of the formulation. The drug content was analyzed and there was marginal difference between the formulations kept at different temperatures from the result obtained it can be said that the prepared in-situgel formulation was stable as it showed non-significant difference (p<0.05) in appearance, pH, drug content and gelling capacity.

 

CONCLUSION:

In this work in-situ gel containing the drug linezolid and Diclofenac sodium with a combination of carbopol-HPMC and carbopol-NaCMC was developed. The developed formulations showed satisfactory results for in-vitro gelling capacity, rheology, and other physical properties. In-vitro drug release studies showed a polymer concentration-dependent decrease in drug release. All the formulations developed by using different concentrations of Carbopol (0.5-2.0%). The formulations of batches LDCF1, LDSF1, and LDSF2 did not showed gelation up to 45°C, which is not desirable for in-situ gel. The formulations of batches LDCF1, LDCF2, LDCF3, LDSF1, LDSF2 and LDSF3 failed to extend the drug release up to 12 hours. Thus, the formulation of batches LDCF4 and LDSF4 containing carbopol: HPMC and carbopol: NaCMC in 1:2 ratios were considered as optimum formulation on the basis of optimum viscosity, gelation time, gelation temperature, gelling capacity, and their capacity to extend the in-vitro drug release up to 12hours.

 

Further, it was also found that, all the formulations were liquid at room temperature and underwent rapid gelation when the pH was raised to 6.8. The viscosity of formulation contributed to the product adhesiveness, reflecting the importance of product rheology on this parameter. Additionally, the gel formed in-situ maintained its integrity without dissolving or eroding for a prolonged period.

 

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Received on 22.11.2019            Modified on 31.12.2019           

Accepted on 28.01.2020      ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Tech.  2020; 10(1):20-26.

DOI: 10.5958/2231-5713.2020.00005.7