Multiple sclerosis (MS) is the most severe white matter demyelinating events of central nervous system (CNS). Pathological hallmarks of disease are chronic neuro-inflammation and neuro-degeneration of myelin sheaths thus causing axonal damage. This injury was seen in white matter that causes inhibition of axonal transmission. This damage occurred neuro-psychiatric problems and permanent disability in a patient. Glioblastoma (GBM) are the serious, fast growing and lethal type of brain tumor and that has 10th leading cause of death 9,10. Higher proliferation rate was seen in GBM that cause excessive growth rate of mortality in patient. Treatment of GBM are very challenging task to researchers because the growth of tumour have uncontrollable and many therapies was failed in this case. Innumerable treatments are available for GBM such as chemotherapy, surgery and radiation but all therapies have many disadvantages like cytotoxicity, tissue and cell lysis. Thereby effective and safe therapies are required for the treatment of GBM. Recent advantages of dihydroorotate dehydrogenase (DHODH) inhibitors have that a restricted proliferation rate of tumor cells without damage to the normal tissues.
Teriflunomide (TFM) is an effective inhibitor of DHODH as well as the active metabolite of leflunomide. It is shown anti-inflammatory activity also restrict the proliferation and multiplication of tumor cells. TFM was retarded the conversion of dihydroorotate (DHO) to orotate this leads to obstruction of pyrimidine de novo biosynthesis pathway. US Food and Drug Administration (FDA) has been approved the TFM for the treatment of neuro-degenerative disorder like MS. The exact mechanism of a drug in the treatment of MS is not clearly understood but it possibly involves a decrease in the number of activated immune responses that enters in CNS. Moreover, TFM was blocks the proliferation of activated T-cell by blocking the synthesis of immune-modulating agents such as cytokines.
Oral administration is unsuitable for TFM because the drug exhibits high risk of hepatotoxicity. Blood brain barrier (BBB) has been restricted an access of therapeutic agents to the brain. Hence, the sufficient concentration of drug cannot be reached into a targeted site via the oral route. Intranasal (IN) route is highly vascular relatively large surface area and high blood flow available for delivery. The IN administration is proven strategy for rapid achievement of a drug to the targeted site and avoidance of hepatic first pass metabolism is beneficial features of the nose to brain delivery. This is a non-invasive route, reduce dose and dosing frequency, less adverse effects and enhance patient compliance. Targeted nose to brain delivery is the unique solution to enhanced drug concentration at a targeted site as well as enhanced the cure rates of MS. However, the primary barrier of IN route is mucociliary clearance for the delivery of drugs by IN route. Mucoadhesive formulation was a possible solution for this problem of nasal route clearance. To enhance the residence time of formulation in nasal cavity that is a capacity of mucoadhesive agents hence the drug retard for longer period of time into nasal cavity.
Microemulsion (ME) is lipid-based formulations provides a promising strategy for improving solubility and permeability of poorly soluble drugs. This strategy includes incorporation of hydrophilic and hydrophobic drugs into the lipid vehicle possibly for rapid and targeted drug delivery. Many characteristics of ME formulations which make them perfect dosage form for IN delivery. This includes nanometric droplet size (10-200 nm) of formulation and excellent thermodynamic stability which helps in enhances the therapeutic concentration of drug into the targeted site. ME in situ gel was prepared by using Poloxamer 407. It is hydrophilic In situ gel forming polymer used with a mucoadhesive polymer (HPMC K4M) is to enhance the resident time of formulation in a nasal cavity. An in situ gel formulations are liquid aqueous solutions at room temperature but it turns to gel under nasal temperature. The different gel forming mechanisms was produced by gelling agents, a temperature sensitive gelling agents was properly suited with the nasal mucosa. Poloxamer 407 is non-ionic and nontoxic in nature thereby it is safer for human use while poloxamer 407 was used for formulation of mucoadhesive microemulsion (MME).
Quality by design (QbD) approach covers design and optimization of formulation which meets with its pre-determined specifications and quality attributes. This approach determines how formulation factors and independent variables affect on a quality of the product and demonstrate the parameters with respect to final specifications. In this study the TFM ME was prepared by using different ratios of oil, surfactant/co-surfactant mixture (SMix) and water with enhancing the stability of a formulation. Poloxamer 407 was added in order to produce the temperature sensitive in situ gelling as well as HPMC K4M was to enhanced the adhesion of formulation to the nasal mucosa thus avoid loss of drug from the site of application. The mixed gel system including poloxamer 407 and HPMC K4M are useful to enhancing gelling and mucoadhesion properties of the formulation. The major goal of design of experiment (DoE) is to determine the effect of independent variables on dependent variables as well as the quality of a product. The pseudo-ternary phase diagram is demonstrating the larger ME region with effective SMix ratio. The Box-Behnken design was used to determine the effect of oil, SMix and concentration of water on the dependent variables such as particle size, PDI, % transmittance and drug content of the formulation. Finally, the best formulation was selected for pharmacological studies.
Unfortunately, a formulation of the ME containing TFM has been not thoroughly published and demonstrates so far for treatment of a brain tumour and MS. The main purpose of the present investigation is to formulate, optimized and pharmacological studies of TFM ME in situ gel for the treatment of brain tumor and multiple sclerosis.
1. Materials and methods
TFM was received as a gift sample from Glenmark Pharmaceuticals Ltd. (Mumbai, India). Carbopol 970 P was acquired from Lubrizol Advanced Materials (Mumbai, India). Labrafil M 1944 CS, Maisine 35-1, Peceol, Labrasol and Transcutol HP were all procured as a gift samples from Gattefosse (Mumbai, India). Poloxamer 407 was purchased from Yarrow Chem. products (Mumbai, India). HPMC K4M mucoadhesive agent was received from Analab Fine Chemicals (Mumbai, India). Calcium chloride (CaCl2) was acquired from Thomas Baker Chemicals Pvt. Ltd. (Mumbai, India). Potassium chloride (KCl) was purchased from Loba Chemie. (Mumbai, India).Ammonium acetate was obtained from Poona Chemical Lab Reagent (Pune, India). A dialysis membrane (mol. Wt. 12000 Da.) was obtained from HiMedia Labs. (Mumbai, India). Sodium chloride (NaCl), Methanol HPLC grade, Acetonitrile HPLC grade and Ethanol all were procured from Research Lab Fine Chem. industries (Mumbai, India).
1.2. High Performance Liquid Chromatography (HPLC) analysis of TFM
RP-HPLC method was most suitable for estimation of a total amount of TFM in various formulations. The Kromosil C18 (4.6 X 250 mm) column was used for analysis of TFM with Mobile phase considering as Methanol:Ammonium Acetate buffer consisting the ratio of 75:25 (pH 4). The obtained flow rate of the pump was found 0.8 mL/min. The detection of TFM samples was performed at a wavelength 291 nm through UV–vis detector. The defined range (2-10 µg/ml) was used for plotting the calibration curve and R2 for TFM was found 0.999.
1.3. Solubility studies for screening of suitable oils and surfactants system
To find suitable oils, surfactants and co-surfactants for ME, as well as determine the highest solubility of TFM in different oils such as capmul MCM, labrafil M 1944 CS, maisine 35-1, peceol, isopropyl myristate, labrafac PG, oleic acid, acrysol K-150, captex oil, hariol, and castor oil. To determined appropriate solubility of TFM in different surfactants and co-surfactants (S-Cos) like tween 80, labrasol, tween 20, cremophore RH-40, span 80, transcutol HP, polyethylene glycol (PEG), propylene glycol (PG) and ethanol. To add the excess amount of TFM in 5 ml of oil and S-Cos mixture, the prepared mixture was shaking at room temperature for 72 h using an orbital shaker then centrifuged this mixture for 15 min at 5000 rpm through the refrigerated centrifuge (Remi Laboratory instruments). The collected supernatant was filtered by membrane filter paper (0.45 µ) and concentration of drug in the collected filtrate was estimated by HPLC analysis.
1.4. Construction of pseudo-ternary phase diagrams
Highest solubility of TFM was found in maisine 35-1 (oil), labrasol (surfactant) and transcutol HP (co-surfactant). The maximum ME area was assessed by using Pseudo-ternary phase diagrams, which indicates that highest stability of a formulation. The phase diagram was exactly fixed ratios of S-Cos (SMix). The SMix ratio of surfactant, labrasol and co-surfactant transcutol HP were selected as 3:1, 2:1, 1:1, 1:2 and 1:3. The oil to Smix ratio has change for every phase diagram and the selected ratios were taken as 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1 (w/w). Accurately weighing the required quantity of ingredients in the glass vials and titrated by using aqueous titration method where the stepwise addition of water in the mixture of oil to SMix. After equilibrium clarity or turbidity were determined by visual observations, while software CHEMIX VERSION 4 were used for the construction of phase diagrams.