Pre-Formulation Development of Lamivudine 300 mg and Tenofovir Disoproxil Fumarate (TDF) 300 mg Fixed Dose Combination Tablets

Introduction: In this study, physical and chemical characteristics of Lamivudine, Tenofovir Disoproxil Fumarate (TDF) and potential excipients were systematically followed and documented [1]. Objective: The objective of this scientific work was to carry out pre-formulation studies including compatibility studies on Lamivudine and Tenofovir Disoproxil Fumarate with their potential excipients prior a direct compression process [2]. Methodology: The interaction was studied in three set of environments namely uncontrolled room conditions for Zone VI b (30 ̊C ± 2 ̊C), oven conditions in which the oven was set at 50 ̊C and accelerated climatic conditions in which a climatic chamber was set at 40 ̊C ± 2 ̊C/75% ± 5% Relative Humidity (RH %). Sample preparation was done by mixing the amount of formulation excipients to active substances at a ratio of 1:10, whereas active substance to another active substance at a ratio of 1:1, active substance to coating materials at 1:4, coating materials to the whole set of excipients 1:4. The whole set of samples was geometrically mixed and triturated by mortar and pestle to very fine uniform powder to ensure homogeneity of the mixture. HPLC analytical method was used for simultaneous quantitative determination of lamivudine and tenofovir disoproxil fumarate. Transmittance of the mixture was determined by Near Infra-Red (NIR) technique. Results: The amount of Lamivudine as on day 0 was comparable to day 90 for in all tested conditions (Room, Oven and Climatic Chamber), whereas for Tenofovir Disoproxil Fumarate only the amount of the drug at Room (30 ̊C ± 2 ̊C) was comparable to results on day 90. A significant drop of amount of Tenofovir Disoproxil Fumarate (TDF) exposed to moisture (Climatic chamber at 40 ̊C ±


Introduction
A generic fixed dose combinational product containing Lamivudine 300 mg/Tenofovir Disoproxil Fumarate 300 mg has never been produced in Tanzania and east Africa region as a whole though appears to be beneficial in TB/HIV co-infected patients.Most of the local manufacturers in developing countries like most of the countries in the sub-saharan regions have got no Research and Development (R&D) Laboratories, hence quite difficult to undertake formulation development of such kind which can lead into the drug formulation like Lamivudine-300 mg/Tenofivir Disoproxil Fumarate-300 mg.On the other hand, the drug formulations are very expensive and quite difficult for the typical third world pharmaceutical industries to take a step towards buying a full developed drug formulation.In any case, formulation development needs well trained and self motivated personnel, hence the pharmaceutical industries with the Pharmaceutical Research and Development laboratories are faced with the problem of man power which in most cases leads to the closure of the laboratories.
For a pharmaceutical scientist to undertake such a step in pharmaceutical formulation development he/she has to pass through a crucial step of undertaking pre-formulation studies [3].A pre-formulation begins with literature search of similar type of compounds to provide and understand of the degradation process, any adverse conditions relevant to the drug, solubility and hence bioavailability, pharmacokinetics and formulation of similar compound and toxicity [4].Hence a successful pre-formulation study will influence selection of the drug candidate itself, selection of formulation components, API & drug product manufacturing processes, determination of the most appropriate container closure system, and development of analytical methods [5].Pre-formulation studies strengthen the scientific foundation of the guidance, provide regulatory relief and conserve resources in the drug development and evaluation process, improve public safety standards, enhance product quality, facilitate the implementation of new technologies, facilitate policy development and regulatory decision making [6].Pre-formulation studies give directions for development of formulation in choice of drug form, excipients, composition, physical structure, helps in adjustment of pharmacokinetic and biopharmaceutical properties, support for process development of drug substance support for PAT (Process Analytical Technology) (critical process parameters), produce necessary and useful data for development of analytical methods [7].

Sample Preparation
The preparation of samples for compatibility considered the amount of active ingredients and excipients in the specific ratios by mixing excipients to active substances at a ratio of 1:10, active substance to active substance at a ratio of 1:1, active substance to coating materials at 1:4, coating materials to the whole set of excipients 1:4 [8].The samples were geometrically mixed and triturated by mortar and pestle to very fine uniform powder to ensure homogeneity of the mixture [9] [10].

Materials
Acetonitrile HPLC grade was procured from Carlo Elba Spain through financial support from MUHAS/SIDA capacity building programme.Water was in-house prepared at MUHAS Pharm R&D Laboratory.Lamivudine and Tenofovir Disoproxil Fumarate were purchased from Desano, China through financial support from MUHAS/SIDA capacity building programme.All excipients, Microcrystalline Cellulose [8], Hydroxypropyl Methyl Cellulose (HPMC), Cross Carmellose Sodium, Crospovidone, Magnesium Stearate were donated by MUHAS Pharm R&D Laboratory.

Tested Variables
A total of two variables were tested in this study, this include Assay and Identity of the prepared mixtures.All these variables were compared as on day 0 and day 90.

Chromatographic Technique, HPLC
The validated in-house HPLC analytical method was used for quantitative determination (assay) of the amount of drug substance in the mixture.The chromatographic conditions were set with mobile phase-Acetonitrile: Water-55:45% v/v, flow rate-1 ml/min, column-C18, 4.6 mm × 250 mm × 5 µm temperature: ambient temperature, analysis (development) time: 20 minutes, detector: 252 nm DAD (Diode Array Detector).

Near Infra-Red
Transmittance of the mixture was determined by Near Infra-Red (NIR) technique.

Results and Discussion
It can be seen clearly in Table 1 that the amount of active substance as on day 0 was comparable to day 90 for Lamivudine in all tested conditions (Room, Oven and Climatic Chamber), hence no significant change of Lamivudine was observed whereas for Tenofvir Disoproxil Fumarate only the amount of the drug at Room (30˚C ± 2˚C) was comparable to day 90.There was a slight colour change of the materials of which the containers were opened and subjected to accelerated conditions at 40˚C ± 2˚C/75% ± 5% Relative Humidity (RH %).The observed colour change as per Table 2 was clearly depicted by the Near Infra-Red spectra at 400 to 1500 cm −1 (Fingerprint region) as indicated in Figures 1-4.The results indicated a promising compatibility of Lamivudine and Tenofovir Disoproxil Fumarate (TDF), however, the preparation process for a final formulation should be restricted from light, moisture uptake and high temperature above 30˚C.Table 1.Drug content of samples subjected into pre-formulation studies as from day 0 to 90.The acceptance limits for assay 95% -105% (In-house specifications).Regarding the obtained results, it could be clearly noted that excipients including Microcrystalline cellulose, Cross Linked Sodium carboxymethylcellulose, Magnesium Stearate and sodium carboxymethylcellulose could be safely applied into a final formulation of Lamivudine and Tenofovir Disoproxil Fumarate Fixed Dose Combination (FDC).These findings were obtained and verified by first exposing the active substances alone to the harsh conditions as it could be evidenced in Figures 1-3.
The effect of excipients on the active substances, Lamivudine and Tenofovir Disoproxil Fumarate (TDF) was followed by exposing the drug substances to harsh conditions when well mixed together with the potential

Conclusion
It was generally found that Microcrystalline cellulose, Sodium carboxymethyl cellulose cross linked, Magnesium Stearate and sodium carbxymethyl cellulose can be compressed together with Lamivudine and Tenofovir Disoproxil Fumarate (TDF) to produce a pharmaceutically acceptable solid dosage form, tablet.The produced tablets should be protected from moisture, high temperature above 30˚C and oxidation by light as they are found to affect Tenofovir Disoproxil Fumarate (TDF) [11].

Figure 3 .
Figure 3.An overlay plot of Lamivudine and Tenofovir mixture subjected at 40˚C ± 2˚C/75% ± 5%-Climatic chamber, 50˚C-Oven and Room uncontrolled room conditions with 30˚C ± 2˚C, from Day 0 to Day 90.excipients (Figure4).As in Figure4, Lamivudine, Tenofovir Disoproxil Fumarate and Magnesium Stearate were mixed together and the mixture was exposed to harsh condition to allow for plausible basic hydrolysis reaction between Magnesium which is basic in nature and TDF.Generally there was no formation of new substance as indicated by Figure4.This is confirmed by having identical spectra for day 0 and 90.

Table 2 .
Appearance of pre-formulation samples as on day 0 compared to day 90.