Development of Metadoxine Lipid Microspheres for Alcohol intoxication

 

Abinaya M*, Gayathri R, Ramya Devi A, Mohan S

Department of Pharmaceutics, Karpagam College of Pharmacy-Coimbatore-32, Tamilnadu, India.

 

ABSTRACT:

Objective: The need of the study is to develop Controlled release Metadoxine tableted microsphere in order to extend the action of the drug so that it can release medicament in a controlled manner and maintain its concentration constantly at the alcohol metabolising site for the treatment of alcohol intoxication. Methods: The present study involves the use of Congealable Dispersion phase Encapsulation technique to formulate Metadoxine microspheres. Hydrogenated cottonseed oil (HCSO) and stearic acid were employed as the lipid matrix materials and Tween 60 as the droplet stabilizer. Drug to lipid ratio was taken as 1:2. The prepared microspheres were characterized to assess their behaviour, optimized and compressed into a tablet using various release modifiers such as Eudragit L, PEG 4000, Sodium alginate and Cross-linked Sodium carboxymethyl cellulose. Further the compressed tablets were evaluated to predict the controlled release behaviour.
Results: The drug release from Stearic acid-lipid microspheres was rapid with sustained action. Tablets formulated using release modifiers like Eudragit L and PEG 4000 (S6A and S6B) showed release retardation whereas formulations containing Sodium alginate and Cross-linked carboxymethylcellulose (S6C and S6D) produced a controlled release. Release obtained from formulation S6C was observed to be pH-dependent. With the tablet of a Stearic acid microsphere containing cross-linked sodium carboxymethylcellulose (S6D), the controlled release had been achieved due to gradual disintegration from tablet to aggregates and to individual microspheres.

 

 

INTRODUCTION:

Alcohol intoxication, also known as drunkenness or alcohol poisoning, is the negative behavior and physical effects due to the continuous drinking of ethanol (alcohol)^1,2. Alcohol intoxication typically begins after two or more alcoholic drinks³.

 

Ethanol (CH3CH2OH) is a water-soluble compound that rapidly crosses cell membranes, resulting in ready equilibration between intra and extracellular concentrations. Its absorption occurs mainly in the proximal intestinal tract. Gastric alcohol dehydrogenase (ADH) is responsible for 10% of alcohol metabolism ("first pass effect") and has important gender-related differences.

 

The remaining 90% of ingested ethanol is metabolized in liver as acetaldehyde along three liver enzymatic pathways in different percentages: (1) liver ADH (90%), (2) microsomal ethanol oxidizing system (MEOS; 8–10%), and (3) catalase (0–2%) [4]. on consuming a higher quantity of alcohol results in elation. Subjects with acute and chronic alcohol intoxication require an integrated approach to detoxication⁴.

 

Metadoxine is a drug of choice for abrupt alcohol withdrawal and alcoholic fatty liver that results from chronic alcohol intoxication. It is a selective antagonist of 5HT 2B and accelerates the metabolic degradation of ethanol and acetaldehyde into less toxic higher ketones and to improve their urinary clearance^5,6.

 

The present study, aimed to prepare controlled release lipid microspheres of Metadoxine by the congealable disperse phase encapsulation technique, which utilizes fats of animal or vegetable origin as the matrix and to investigate the effects of dispersant or droplet stabilizer concentration, drug-lipid ratio, type of lipid material, and different additives on the microsphere formation and drug release. Furthermore, the drug release was also tested after microspheres were compressed in the tablet form. The microsphere formulation from which the drug was released at the predetermined rate according to zero order kinetics⁷.

 

MATERIALS AND METHODS:

Materials:

Stearic acid, Eudragit L, PEG 4000 and Sodium alginate were obtained from Himedia, Mumbai, India. Metadoxine was obtained from BSA Pharma, Ambala, India. Hydrogenated Cotton Seed Oil was obtained from KS Industries, Jaipur, India. Tween 60, Cross-Linked Sodium Carboxy Methyl Cellulose and all other materials used were of analytical grade and purchased from Merck Chemicals & Reagents.

 

Method:

Formulation of Microspheres:

Microspheres containing Metadoxine were prepared using Congealable disperse phase encapsulation procedure. Formulations were given in Table 1 and 2.

 

Metadoxine was dispersed in the melted lipid, using Tween 60 as the dispersant agent /droplet stabilizer. The fatty phase was poured into this solution while stirring at 800rpm by a mechanical stirrer (Remi) to form an o/w emulsion. During emulsion formation, the temperature of the fatty phase and aqueous phase was maintained at about 10°C above the melting point of lipid. The resulting emulsion was agitated again for five minutes. The droplets of oily internal phase were solidified for the formation of microspheres by pouring an equal volume of cold water (4°C). Then obtained microsphere suspension was filtered, washed three times with cold water and dried under vacuum⁷.

 

Characterization of Microspheres:

Scanning electron microscopy (SEM):

The Shape and Surface morphology of the dried microspheres was determined by SEM [JOEL JSM - 6480 LV]. The formulations were analyzed at different magnifications 290X and 210X^8,9.

 

Percentage yield analysis:

Percentage yield analysis was calculated by weighing the formulated microspheres using the following formula^10,11.

 

                                  Theoretical yield

Percentage yield = ––––––––––––––––×100

                                   Practical yield

 

Entrapment efficiency:

Drug-loaded microsphere (100 mg) were powdered and dissolved in Dichloromethane and then sonicated for about 30 minutes. The mixture was filtered through a 0.45μm membrane filter (MILLIPORE). The drug content was determined by using Spectrophotometer at 291nm. The entrapment efficiency was calculated using the formula.

                            Actual drug content

EE (%) = –––––––––––––––––––––––––× 100.

                        Theoretical drug content

 

All the batches were prepared in triplicate^12,13,14.

 

In vitro drug release studies:

The in vitro release studies was performed using USP apparatus paddle type with 900 ml of phosphate buffer solution as a dissolution medium at 100rpm at 37°C± 0.5°C temperature. About 500mg of microspheres equivalent to 300mg drug was filled inside a muslin bag and suspended inside the hemispherical basket. 5ml of the sample was withdrawn at regular intervals and sink condition is maintained. The absorbance was measured at 291 nm using UV spectrophotometer and cumulative percentage drug release was calculated^15,16,17.

 

Table 1: Optimization for Hydrogenated cotton seed oil

Formula tion	Metadoxine	HCSO	Tween 60	Microsphere formation
F1	-	6	0.2	Not formed
F2	-	6	0.6	Not formed
F3	-	6	1.0	Formed
F4	10	10	1.0	Not formed
F5	10	15	1.0	Not formed
F6	10	20	1.0	Formed

 

Table 2: Optimization for Stearic acid

Formula tion	Metadoxine (g)	Stearic acid (g)	Tween 60 (%w/v)	Microsphere formation
S1	-	7	0.2	Not formed
S2	-	7	0.6	Not formed
S3	-	6	1.0	Formed
S4	10	10	1.0	Not formed
S5	10	15	1.0	Not formed
S6	10	20	1.0	Formed

 

Formulation of tableted Microspheres:

The optimized Metadoxine loaded microspheres were compressed to form a tablet of 500 mg using release modifiers such as Eudragit L, PEG 4000, Sodium alginate and Cross-linked carboxymethylcellulose. Each tablet contains 300 mg drug^18,19.

 

Evaluation of tablets:

Thickness:

The thickness of the tablets was measured using Digital Vernier caliper. Three tablets were selected randomly from all the batches.

 

Hardness test:

The hardness of tableted microspheres was determined using Monsanto Hardness tester. Three tablets were randomly picked from each batch and analysed for hardness.

 

Weight variation:

From each batch, about 20 tablets were selected at random and weight was determined. Then, the tablets were weighed individually, and each weight was compared with an average weight.

 

 

In vitro dissolution studies:

The in vitro dissolution was performed using the USP Type II apparatus (Lab India Dissolution Apparatus). Four replicates of tablets were placed in the dissolution medium. The dissolution medium used in this case was buffer solution of pH 7.2 containing 0.02% Tween 80. Samples of 5ml were taken at various time intervals up to 12 hrs. The dissolution medium was maintained at 37ºC±0.5ºC throughout the study. The dissolution samples were filtered through 10μ inline filters. The samples were analysed by UV spectrophotometer at 291 nm against blank^20,21,22.

 

Release Kinetics:

Data obtained from in vitro release studies were fitted to various kinetics equations (zero-order, first-order, Higuchi model and Korsemeyer Peppas model) to determine the mechanism of drug release from microspheres²³.

 

RESULT:

Table 3: Formulations for tableted microsphere

 

Fig. 1: Scanning electron microscopy view of microspheres f6 and s6

 

Table 4:  Evaluation of Microsphere

Formulation Code	Particle size (µm)	Drug content (%)	Entrapment efficiency (%)	% yield (%)
F5	400	65.2	75.2	60.31
F6	260	91.32	81.32	93
S3	300	68.51	71.42	71.23
S6	220	96.21	83.33	95.12

 

Table 5: Evaluation of the tablet

Formulation Code	Thickness (mm)	Hardness (kg/cm2)	Weight variation (%)
S6A	2.0±0.01	6.0±0.03	1.2±0.01
S6B	2.01±0.02	6.27±0.01	2.0±0.01
S6C	2.0±0.01	6.0±0.01	1±0.02
S6D	2.02±0.02	6.25±0.02	1.5±0.02

 

Table 6: Kinetic data for optimized formulation

Formulation Code	Zero Order Model		First Order Model		Higuchi Model		Korsemeyer Peppa’s Model
S6D	Slope	R2	Slope	R2	Slope	R2	Slope	R2
	7.553	0.9909	0.0818	0.9149	27.13	0.9431	0.7271	0.4986

 

Fig. 2: Invitro release profile of HCSO containing tableted microsphere

Fig. 3: Invitro release profile of Stearic acid containing tableted microsphere

 

Fig. 4: Zero order, First order, Higuchi and Korsemeyer-peppas kinetics for formulation S6D

 

DISCUSSION:

Metadoxine lipid microspheres were formulated by Congealable disperse phase encapsulation technique in which Metadoxine was encapsulated in the lipid matrix of fatty phases such as HCSO and Stearic acid with 1% concentration of tween 60 as a droplet stabilizer to produce smooth spherical particles. The formulated microsphere showed release retardation up to 12 hrs in a sustained manner. The drug-lipid ratio at 1:2 (F6 and S6) formed rigid microspheres compared to that of other formulations shown in Table 1 and 2.

 

The formed microspheres showed particle size range of 260 and 220µm for F6 and S6 with drug content of 91.321 and 96.21% respectively. The entrapment efficiencies for the optimized microspheres were 81.321 and 83.33% with percentage yield of 93 and 95.12% as indicated in Table 4.

 

The release profile of formulation F6 (Microsphere) and F6A (microsphere compressed to tablet) containing HCSO produced slower release but the drug released from the lipid matrix did not achieve an effective concentration level. On addition of Eudragit L, PEG 4000, sodium alginate and cross-linked Sodium carboxymethyl cellulose as release modifier to HCSO (F6A, F6B, F6C, F6D) retarded drug release was observed but controlled release was not obtained to the optimum level shown in Fig. 2.

 

 

 

Formulation containing an alternative lipid material Stearic acid was chosen to prepare microspheres (S6). The drug release in this formulation was very rapid initially and showed a sustained effect in the later time. Though the compressed microspheres to tablet formulation retarded drug release, but controlled release was not observed to an effective range.

 

The tablet form of stearic acid microspheres was designed to produce a gradual disintegration using release modifiers such as Eudragit L, PEG 4000, Sodium alginate and cross-linked Sodium carboxy Methyl Cellulose as mentioned in Table 3.

 

In-vitro drug release profile for the formulation S6A and S6B did not show release retarding effect to the expected level. S6C showed a better release profile in a controlled manner but the release was found to be pH- dependent.

 

So another formulation with Cross-linked sodium CMC at concentration of 5.0% was formulated (S6D). The tablets disintegrated gradually to aggregates then to individual microspheres resulting in controlled drug release (shown in Fig. 3).

 

In vitro drug release data for the optimized formulation fitted to kinetic models as shown in table 6 and fig. 4 indicates that the optimised formulation follows, zero order kinetics with R2 value0.99 which is significance of controlled release formulation.

 

 

CONCLUSION:

Thus Metadoxine encapsulation with Stearic acid and 5% Cross-linked sodium carboxymethyl cellulose as release modifier produced a controlled release of medication up to 12hours thereby eliminating the frequency of dose administration, reducing the symptoms of alcohol intoxication in a controlled manner throughout the release.

 

Further, this formula can be optimized for the formulation of tablets and extended for in vivo studies to ensure bioavailability.

 

ABBREVIATIONS:

HCSO- Hydrogenated cotton seed oil; PEG- Polyethylene glycol; ADH- Alcohol dehydrogenase; 5HT- 5 Hydroxy tryptamine; SEM- Scanning Electron Microscopy; E.E- Entrapment efficiency.

 

AUTHOR’S CONTRIBUTION:

Gayathri R took keen interest in selection of drug for the selected topic.

 

Abinaya M designed the methodology for this work and contributed in the computation of data for the manuscript.

 

ACKNOWLEDGEMENT:

I thank my Principal for supporting me in completing the research work. I further thank the institution for providing me facilities throughout the study.

 

CONFLICT OF INTEREST:

All the authors have none to declare.

 

REFERENCES:

1.      Luisa Vonghia, Lorenzo Leggio, Anna Ferrulli, Marco Bertini, Giovanni Gasbarrini and Giovanni Addolorato. Acute alcohol intoxication. European journal of internal medicine. 2008;19(8):561–67.

2.      Martinez M D, Martinez AD, Salcedo V V and Fuentes C C. Efficacy of metadoxine in the management of Alcohol intoxication. Journal of International Medical Research. 2002; 30(1):44-51.

3.      Shpilenya Leonid S, Muzychenko Alexander P, Gasbarrini Giovanni, Addolorato and Giovanni. Metadoxine in Acute Alcohol Intoxication: A Double- Blind, Randomized, Placebo-Controlled Study. Alcoholism: Clinical and Experimental Research. 2002;26(3):340-46.

4.      MCLR Diaz Martinez, A Diaz Martinez, V Villamil Salcedo and C Cruz Fuentes. Efficacy of Metadoxine in the Management of Acute Alcohol Intoxication. The Journal of International Medicinal Research. 2002;30(1):44-51.

5.      Nilufer Yuksel, Ahmet Aydmli, Yildiz Ozalp and Nurten Ozdemir. Evaluation and preparation of controlled release lipid microspheres of Sulphamethizole by a Congealable disperse phase encapsulation method. Journal of Acta Polonica Pharmaceutics- Drug Research. 2000;57(3):187-92.

6.      Manosroi A, Wongtrakul P, Manosroi J, Sakai H, Sugawara F, Yuasa M, et al. Characterization of vesicles prepared with various non-ionic surfactants mixed with cholesterol. Colloids Surf B Biointerfaces. 2003;30:129-38.

7.      Subbiah Ganesh, Sudheer Kumar D, Sandeep Kumar B, Abhilash R,  Bharadwaj P S, Prudhvi Raj KVS, Imran Mohammed and Pravalika T. Controlled release formulation and evaluation of Idarubicin Microsphere using Biodegradable Hydrophilic and Hydrophobic polymer mixtures. Asian Journal of Pharmaceutical and Clinical Research. 2010; 3(3):179-82.

8.      Jamini M and  Rawat S. A review on microsphere. Research Journal of Pharmaceutical, Biological and Chemical Sciences. 2013;4(1):1223-33.

9.      Patel N. R, Patel D. A, Bharadia P.D, Pandya V and Modi D. Microsphere as a novel drug delivery. International  Journal of  Pharmaceutical Life Sciences. 2011;2(8):992-97.

10.   Singh C, Purohit S, Singh M and Pandey B.L. Design and evaluation of microspheres. A Review, Journal of Drug Delivery Research. 2013;2(2):18-27.

11.   Prasanth V.V, Moy A. C, Mathew S. T and Mathapan R. Microspheres An overview.  International Journal of  Research in Pharmaceutical and  Biomedical Sciences. 2011;2(2):332-38.

12.   Das MK and Senapati PC. Evaluation of Furosemide loaded alginate microspheres prepared by ionotropic external gelation technique. Acta Poloniae Pharmaceutical Drug Research. 2007;64(3):253-62.

13.   Miyazaki Y, Onuki Y, Yakou S and Takayama K. Effect of temperature increase rate on drug release characteristics of dextran microspheres prepared by emulsion solvent evaporation process. International Journal of Pharmaceutics. 2006;324(2):144-51.

14.   Tao Y, Lu Y, Sun Y, Gu B, Lu W and Pan J. Development of mucoadhesive microspheres of Acyclovir with enhanced bioavailability. International Journal of Pharmaceutics. 2009; 378(2):30-36.

15.   Shoaib MH, Tazeen J, Merchant HA and Yousuf RI. Evaluation of drug release kinetics from Ibuprofen matrix tablets using HPMC. Pakistan Journal of Pharmaceutical Sciences. 2006;19:119-24.

16.   Gursoy A and Cevik S. Sustained release properties of alginate microspheres and tableted microspheres of Diclofenac sodium. Journal of Microencapsulation. 2000;17(5):565-75.

17.   Presumey J, Salzano G, Courties G, Shires M, Ponchel F, Jorgensen C, et al. PLGA microspheres encapsulating siRNA anti-TNFalpha. Efficient RNAi-mediated treatment of arthritic joints. European Journal of Pharmaceutics and Biopharmaceutics. 2012;82(3):457-64.

18.   Shivangi Singh, Nithya Shanthi C and Arun K Mahato. Formulation and Evaluation of Metronidazole tableted microspheres for Colon drug delivery. Asian Journal of Pharmaceutical and Clinical Research. 2016; 9(3):398-403.

19.   Mohsin Khan, Vaseem A Ansari, Poonam Kushwaha, Arun Kumar and Juber Akhtar. Mucoadhesive microspheres for controlled delivery of drugs. Asian journal of Pharmaceutical and Clinical Research. 2015; 8(4):1-20.

20.   Biplab Kumar Chakra, Saumen Karan, Biswajit Das, Souvik Debnath and Tapan Kumar Chatterjee. A controlled release microsphere formulation of an Anti-Diabetic drug and characterization of the microsphere. International Journal of Pharmacy and Pharmaceutical Sciences. 2018; 10(10):30-38.

21.   Mohanraj Palanisamy, Jasmina Khanam, Arun Kumar N, Rani C. Chitosan Microspheres Encapsulated with Metoprolol Succinate: Formulation and In- Vitro Evaluation. Research Journal of Pharmacy and Technology. 2009; 2(2):349-52.

22.   Chandrasekaran N, Balamurugan M. Formulation, Characterization and In-vitro Evaluation of Abacavir Sulphate Loaded Microspheres. Research Journal of Pharmacy and Technology. 2013; 6(7): 731-35.

23.   Virag Shah, Madhuri L.Shinde,  Sandeep Patil and M.B. Nikam. Impact of selected  variables on the preparation of aceclofenac microspheres by spray drying using full factorial design. International Journal of PharmTech Research. 2011; 3(2):1066-72.

 

 

 

 

 

Received on 06.06.2019           Modified on 10.07.2019

Accepted on 01.08.2019         © RJPT All right reserved