Drug â€excipient Interaction of Anti-tubercular Drugs

do drugs excipient Interaction of Anti-tubercular DrugsDrug excipient interaction of anti-tubercular doses and its in-silico evaluationAbstractisoniazid and Pyrazinamide are the first line anti tubercular drugs. Lactose is mainly used as the excipient in truehearted window pane potpourris of isoniazid and pyrazinamide. These drugs contains primary and secondary a minuteo functional group which interacts with lactose by maillard chemical reaction and form draw. The maillard reaction forces of isoniazid and pyrazinamide with lactose were synthesized at 60oC in saltlike borate buffer pH 9.2 and characterized by UV, FT-IR, DSC, HPLC and MS. Docking think everyplace for in-sillico evaluation of isoniazid-lactose adduct and pyrazinamide-lactose adduct was performed to study its effect on pharmacological activity. The present study shows the figurehead of incompatibility amongst isoniazid and pyazinamidewith lactose which leads to loss the therapeutic effect of isoniazid and pyr azinamide.Keywords isoniazid, pyrazinamide, lactose, maillard reaction, excipient, incompatibility, dosage form.IntroductionExcipients are traditionally better known as promoters of degradation than as stabilizers of drug substances (Crowley 1999). Physicochemical and physiological process e.g. stability, physiological pH, gastrointestinal get over time, disintegration, dissolution, permeability and bioavailability can be altered by drug excipient interaction (Jackson, Young et al. 2000). The interactions of drug with excipients can leads to changes in the chemical, carnal and therapeutic properties can be termed as incompatibilities (Chadha and Bhandari 2014) and it may cause the drug degradation (Narang, Desai et al. 2012) and loss of pharmacological activity (Patil and Patil 2013). Lactose is most widely used as the excipient in the solid dosage forms. Lactose is available in different form and different grade with different physical characteristics. Lactose is very popular exc ipient because of low make up and inertness but in other hand lactose have interaction drug with amino functional group i.e. lactose undergoes maillard Monajjemzadeh, 2009The maillard reaction is named Louis Maillard who reported over 80 years ago that some amine and reducing sugars interact each other and forms brown pigments. The first product of this reaction is simple glycosamine (Wirth, Baertschi et al. 1998).In this study, we attempted to explore the modes of interaction and energy binding of the different isomers of isoniazid adduct, pyrazinamide adduct and also study the biological activity of isoniazid adduct and pyrazinamide adduct compare with the help of various molecular modelling techniques.In treatment of tuberculosis, isoniazid and pyrazinamide are key comp adeptnts of first line regimen (Hemanth, Sudha et al. 2012). Isoniazid is chemically isonicotohydrazide and pyrazinamide is chemically pyrazine-2-carboxamide. Isoniazid and pyrazinamide is susceptible for hydroly sis and oxidation interact with excipient particularly carbohydrate and reducing sugars to form hydrazones. The hydrazone is mainly form by the interaction of isoniazid with lactose. there are also reported incompatibilities between lactose and other drugs containing primary and secondary amino functional group (Haywood, Mangan et al. 2005). In this study we were investigated the interaction between lactose with isoniazid and pyrazinamide for that different analytical technique were used and also done the in-sillico evaluation of isoniazid and pyrazinamide.Materials and methodsMaterialsIsoniazid and Pyrazinamide was generously supplied as a stage sample by Macleods pharmaceuticals Ltd., Wapi (Gujarat), India. Lactose monohydrate was purchased from Merck, Merck specialtiesPvt.Ltd. Mumbai, India. All other chemicals were of high-performance liquid chromatography (HPLC) and analytical grade.MethodsAnalytical methodsUV- viewable spectrophotometryThe Ultraviolet-visible spectra of Iso niazid, Pyrazinamide and the Isoniazidlactose adduct, Pyrazinamide-lactose adductwere recorded on a echo beam UV-visible spectrophotometer (UV-1700 Shimadzu, Japan).An accurately weighed quantity of about 10 mg of isoniazid, 10 mg of pyrazinamide, 11.66 mg isoniazid-lactose adduct (equivalent to 10 mg isoniazid), 13.33 mg of pyrazinamide-lactose adduct (equivalent to 10 mg pyrazinamide) each dissolved separately in 100 ml of distilled water. From this, one ml of solution was diluted to 10.0 mL with of distilled water to obtain parsimony of 10 ppm. All solutionswere scanned in UV-Visible range at 420 and 490 nm (Yates, Jones et al. 2003).Fourier-Transform infrared spectroscopyThe Fourier-transform infrared spectroscopy (FTIR) spectra of isoniazid, pyrazinamide, lactose, a isoniazidlactose physical diverseness, pyrazinamide-lactose physical mixture and the isoniazidlactose adduct, pyrazinamide-lactose were recorded. The spectra were obtained using the diffuse reflectance scan metho d using KBr on an FT-IR spectrophotometer (IR Affinity 1 Shimadzu, Japan). The scanning range was four hundred4000 cm-1. Each sample was scanned 45 times consecutively to obtain FT-IR spectrum.HPLC analysisThe HPLC (Gradient) system used for analysis consisted of Agilent Technologies 1200 series equipment, a G1315D quaternary pump, a G1315D junction rectifier array detector and a rheodyne injector fitted with a 20 L loop. Data were recorded and evaluated using the EZChrome Elite software package. Samples were analyzed using LunaC18 column (250 4.6 mm i.d. 5 m) (Phenomenex) as stationary phase. The mobile phase was water methanol (9505, v/v), flow rate of 0.8 mL/min with detection at 266 nm for isoniazid and 269 nm for pyrazinamide.Differential scanning calorimetryThermal analysis of Isoniazid, pyrazinamide, isoniazidlactose adduct and pyrazinamide-lactose was performed by differential scanning calorimetry (DSC) using a TA 6000 Mettler toledo thermal analyzer. Individual samples a s well as the Maillard adduct (about 2 mg) were weighed in the DSC aluminum pan and were scanned in the temperature range of 25300C. A heating rate of 10C/min was used. The thermograms were reviewed for evidence of interaction.Mass SpectrometryThe Mass spectrometry was performed using 410 Prostar binary LC with 500 MS with Electro disperse Positive ionization and Negative Ionization mode and Mass range is 50-2000 amu. The Isoniazid-lactose, Pyrazinamide-lactose adduct solution dissolved in mobile phase to obtain parsimoniousness about 100g/mL. In the affirmative ion mode with electrospray ionization technique, the sample was analyzed.Determination of lactose in pharmaceutical tablet dosage formsThe presence of lactose in DOTs tablets was initially examined according to Indian Pharmacopoeia 2007 by taking 5ml saturated solution of tablet powder and then add 5ml 1 M NaOH, Heat and cool at room temperature finally add potassium cupri tatatarate the solution becomes red color shows p resence of lactose.Preparation of adductSample inclined(p) in alkaline borate bufferAccurately weighed quantity of Isoniazid 300 mg (equivalent to window pane of isoniazid) and 50 mg lactose monohydrate dissolve in alkaline borate buffer pH 9.2 by stirring and ultrasound in 100 ml round bottom flask. In similar way 750 mg pyrazinamide (equivalent to dose of pyrazinamide) was dissolve with 250 mg lactose monohydrate in alkaline borate buffer pH 9.2 in 100 ml round bottom flask. The cleared solutions were refluxed at 600C for 12 hour on water bath. The reaction mixture filtered was diluted with menthol water (11). The adduct was subjected to HPLC analysis (gradient and isocratic run) and Mass spectrometry (LC-MS) analysis. The intensity of brown color was primed(p) was spectrophotometrically after dissolving weighed quantity in distilled water.Docking studyThe molecular docking tool, GLIDE (Schrodinger Inc., USA) (2006) was used for ligand docking study. The protein training was c arried out using protein preparation wizard in Maestro 9.0.ResultUV-Visible spectroscopyThe UV-visible absorption spectrum of the isoniazidlactose adduct and pyrazinamidelactose adduct had shown an increase in absorption in the visible range as compared with isoniazid and pyrazinamide in distilled water as the solvent. The increased absorption the visible region (brown color) is due to Melanoidins production as the fetch up products of the Maillard reaction as reported earlier (Shen, Tseng et al. 2007).FT-IR spectroscopyThe FT-IR absorption patterns of Isoniazid, Pyrazinamide, lactose, Isoniazidlactose physical mixture immediately after mixing and pyrazinamide-lactose physical mixture immediately after mixing as well as Isoniazidlactose adduct, Pyrazinamide-lactose adduct were recorded.The lead at 1678 cm1 in the IR spectrum of Isoniazid-lactose adduct, 1614 cm1 Pyrazinamide-lactose adduct can be attributed to the imines organic law. The peak of NH bending is present at 1552 cm1 and 1583 cm-1 in the IR spectrum of Isonizid and Pyrazinamide and its physical mixture respectively. The peak present in spectrum of Isonizid and Pyrazinamide and its physical mixture are absent in Isoniazid-lactose adduct and Pyrazinamide-lactose adduct both these observations support the formation of adduct. The NH stretch band of secondary amine appears at 3302 cm1 and at 3292 cm-1 for Isonizid and Pyrazinamide respectively. The peak for the lactose OH appears at 3522 cm1 in the infrared spectra of lactose. The peaks for NH and OH stretching appear in the spectrum of the physical mixture, but the peak for NH disappears in the spectrum of the adduct. This may indicate the reaction of the amine with the reducing sugar, or it may be due overlapping of NH stretching peak with that of OH. The FTIR spectra of Isoniazid, Pyrazinamide, Lactose physical mixture, Isoniazid-lactose adduct and Pyrazinamide-lactose adduct shows an interaction between Isoniazid and Pyrazinamide with lactose le ading to the formation of a Maillard product (Pavia et al 2009).Differential scanning calorimetryThe DSC thermograms show the presence of melting oral sexs for isoniazid and pyrazinamide at 171.61C and 189.55 C. The DSC thermogram of lactose shows the peak at 209.83 C. The adduct shows the disappearance of the melting point peak of isoniazid, pyrazinamide, paracetamol and vildagliptine in adduct samples confirms the formation of adduct.Gradient HPLC analysisInitially a gradient run of water and methanol was performed to obtain preliminary instruction regarding the unknown peaks in maillard reaction products (Shen, Tseng et al. 2007).The mobile phase was optimized to separate the Isoniazid-lactose adduct and Pyrazinamide-lactose adduct was water methanol (9505, v/v) with a flow rate 0.8ml/min at ambient temperature. The Isoniazid-lactose adduct and Pyrazinamide-lactose adduct elutes at 3.833min and 1.613 min respectively. The avow samples for isoniazid and pyrazinamide (without la ctose) were also analyzed which proves method selectivity.Isocratic HPLC analysisThe optimized isocratic HPLC analysis of the Isoniazid-lactose adduct and Pyrazinamide-lactose revealed one extra peak that eluted before Isoniazid and Pyrazinamide elution respectively. Performing analysis under same chromatographic parameters, no another peak was observed in control samples.Mass spectrometryThe Isoniazid-lactose and Pyrazinamide-lactose adduct dissolve in mobile phase to obtain drug concentration about 100g/ml.In the positive ion mode with electrospray ionization technique, the sample was analyzed. The MS spectra show the precursor ion for Isoniazid-lactose adduct and Pyrazinamide-lactose adduct was protonated molecule (M+H+) m/z 463.3 and 448.1 respectively. The Isoniazid-lactose adduct and Pyrazinamide-lactose adduct molecular masses was consistent with Isoniazid-lactose adduct and Pyrazinamide-lactose adduct condensation product respectively. The loss of one water molecule from pa rent leads to maillard-type condensation product.Docking studyIsoniazidIn docking study, isoniazid shows binding with ARG-38 amino acid in the selected structure of protein (PDB statute 3I6N) and isoniazid-lactose adduct shows binding with ASN-72, SER-69, SER-173, ALA-134 and PRO-132 amino acid in the selected structure of protein (PDB code 3I6N) as shown in Table No. 1.1.PyrazinamidePyrazinamide shows binding with ALA-131 amino acid in the selected structure of protein (PDB code 3PL1) and pyrazinamide-lactose adduct shows binding with ASP-133 and LEU-131 amino acid in the selected structure of protein (PDB code 3PL1).DiscussionOn the above observation difficulties in the formulating a new pharmaceutical dosage form have often experient because of the interaction between the lactose and active ingredients itself i.e. isoniazid and pyrazinamide. Although the nature and intensity of this interaction may alter the stability, dissolution rate and consequently absorption of the drug an d also affect the pharmacological effect. it indicates that such interactions involving in the formation of the complexes and it studied by different analytical techniques.The UV results shows increased absorption in the visible region (brown color) is due to Melanoidins production as the end products of the Maillard reaction as reported earlier in Shen, Tseng et al. 2007. The FTIR spectra of Isoniazid, Pyrazinamide, Lactose physical mixture, Isoniazid-lactose adduct and Pyrazinamide-lactose adduct shows peak of C=N it shows that formation of a Maillard product. HPLC analysis of the Isoniazid-lactose adduct and Pyrazinamide-lactose revealed one extra peak of impurity or maillard reaction product that eluted before Isoniazid and Pyrazinamide elution respectively. The MS spectra show the precursor ion for Isoniazid-lactose adduct and Pyrazinamide-lactose adduct and it has same molecular weight related to maillard-type condensation product.In the docking study of isoniazid adduct and p yrazinamide adduct shows to a greater extent binding than isoniazid and pyrazinamide but this is pseudo results because this binding present at hydroxyl group and hydroxyl group are trustworthy for the increase excretion of the isoniazid and pyrazinamide and it may be reduces the therapeutic effect of isoniazid and pyrazinamide. In spite of that analytical study confirm the occurrence of maillard reaction product in lactose containing solid dosage forms of amino functional group containing drugs but lactose is still preferred as excipient in the isoniazid and pyrazinamide containing anti-tubercular formulation i.e. DOTs.ConclusionThe present study reports that antitubercular drugs i.e. isoniazid and pyrazinamide undergoes maillard reaction and that confirmed by UV, FT-IR, HPLC and MS. The docking study of isoniazid adduct and pyrazinamide adduct more binding than isoniazid and pyrazinamide but it is pseudo results pharmacologically the excretion of isoniazid and pyrazinamide increa se and it ultimately reduces the therapeutic activity. A drugs- excipient interaction study can be actively used to the advantage of the formulator to increase the bioavailability of the drug. 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