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Drug –excipient interaction of anti-tubercular drugs and its in-silico evaluation
Isoniazid and Pyrazinamide are the first line anti tubercular drugs. Lactose is mainly used as the excipient in solid dosage forms of isoniazid and pyrazinamide. These drugs contains primary and secondary amino functional group which interacts with lactose by maillard reaction and form adduct. The maillard reaction adducts of isoniazid and pyrazinamide with lactose were synthesized at 60oC in alkaline borate buffer pH 9.2 and characterized by UV, FT-IR, DSC, HPLC and MS. Docking study 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 presence of incompatibility between isoniazid and pyazinamidewith lactose which leads to loss the therapeutic effect of isoniazid and pyrazinamide.
Keywords: isoniazid, pyrazinamide, lactose, maillard reaction, excipient, incompatibility, dosage form.
Excipients 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 transit 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, physical 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 excipient because of low cost and inertness but in other hand lactose have interaction drug with amino functional group i.e. lactose undergoes maillard [Monajjemzadeh, 2009]The 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).
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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 components 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 hydrolysis 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 methods
Isoniazid and Pyrazinamide was generously supplied as a gift 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.
The Ultraviolet-visible spectra of Isoniazid, Pyrazinamide and the Isoniazid–lactose adduct, Pyrazinamide-lactose adductwere recorded on a double 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 concentration of 10 ppm. All solutionswere scanned in UV-Visible range at 420 and 490 nm (Yates, Jones et al. 2003).
Fourier-Transform infrared spectroscopy
The Fourier-transform infrared spectroscopy (FTIR) spectra of isoniazid, pyrazinamide, lactose, a isoniazid–lactose physical mixture, pyrazinamide-lactose physical mixture and the isoniazid–lactose adduct, pyrazinamide-lactose were recorded. The spectra were obtained using the diffuse reflectance scan method using KBr on an FT-IR spectrophotometer (IR Affinity 1; Shimadzu, Japan). The scanning range was 400–4000 cm-1. Each sample was scanned 45 times consecutively to obtain FT-IR spectrum.
The HPLC (Gradient) system used for analysis consisted of Agilent Technologies 1200 series equipment, a G1315D quaternary pump, a G1315D diode array detector and a rheodyne injector fitted with a 20 µL 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 µm) (Phenomenex) as stationary phase. The mobile phase was water: methanol (95:05, v/v), flow rate of 0.8 mL/min with detection at 266 nm for isoniazid and 269 nm for pyrazinamide.
Differential scanning calorimetry
Thermal analysis of Isoniazid, pyrazinamide, isoniazid–lactose adduct and pyrazinamide-lactose was performed by differential scanning calorimetry (DSC) using a TA 6000 Mettler toledo thermal analyzer. Individual samples as well as the Maillard adduct (about 2 mg) were weighed in the DSC aluminum pan and were scanned in the temperature range of 25–300°C. A heating rate of 10°C/min was used. The thermograms were reviewed for evidence of interaction.
The Mass spectrometry was performed using 410 Prostar binary LC with 500 MS with Electro spray 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 concentration about 100µg/mL. In the positive ion mode with electrospray ionization technique, the sample was analyzed.
Determination of lactose in pharmaceutical tablet dosage forms
The 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 presence of lactose.
Preparation of adduct
Sample Prepared in alkaline borate buffer
Accurately weighed quantity of Isoniazid 300 mg (equivalent to dose 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 (1:1). The adduct was subjected to HPLC analysis (gradient and isocratic run) and Mass spectrometry (LC-MS) analysis. The intensity of brown color was determined was spectrophotometrically after dissolving weighed quantity in distilled water.
The molecular docking tool, GLIDE (Schrodinger Inc., USA) (2006) was used for ligand docking study. The protein preparation was carried out using ‘protein preparation wizard’ in Maestro 9.0.
The UV-visible absorption spectrum of the isoniazid–lactose adduct and pyrazinamide–lactose 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 end products of the Maillard reaction as reported earlier (Shen, Tseng et al. 2007).
The FT-IR absorption patterns of Isoniazid, Pyrazinamide, lactose, Isoniazid–lactose physical mixture immediately after mixing and pyrazinamide-lactose physical mixture immediately after mixing as well as Isoniazid–lactose adduct, Pyrazinamide-lactose adduct were recorded.
The peak at 1678 cm−1 in the IR spectrum of Isoniazid-lactose adduct, 1614 cm−1 Pyrazinamide-lactose adduct can be attributed to the imines formation. The peak of N–H bending is present at 1552 cm−1 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 N–H stretching band of secondary amine appears at 3302 cm−1 and at 3292 cm-1 for Isonizid and Pyrazinamide respectively. The peak for the lactose O–H appears at 3522 cm−1 in the infrared spectra of lactose. The peaks for N–H and O–H stretching appear in the spectrum of the physical mixture, but the peak for N–H 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 N–H stretching peak with that of O–H. 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 leading to the formation of a Maillard product (Pavia et al 2009).
Differential scanning calorimetry
The DSC thermograms show the presence of melting points for isoniazid and pyrazinamide at 171.61οC and 189.55 οC. The DSC thermogram of lactose shows the peak at 209.83 οC. 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 analysis
Initially a gradient run of water and methanol was performed to obtain preliminary information 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 (95:05, 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 control samples for isoniazid and pyrazinamide (without lactose) were also analyzed which proves method selectivity.
Isocratic HPLC analysis
The 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.
The Isoniazid-lactose and Pyrazinamide-lactose adduct dissolve in mobile phase to obtain drug concentration about 100µg/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 mass was consistent with Isoniazid-lactose adduct and Pyrazinamide-lactose adduct condensation product respectively. The loss of one water molecule from parent leads to maillard-type condensation product.
In docking study, isoniazid shows binding with ARG-38 amino acid in the selected structure of protein (PDB code: 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.
Pyrazinamide 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).
On the above observation difficulties in the formulating a new pharmaceutical dosage form have often experienced 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 and also affect the pharmacological effect. it indicates that such interactions involving in the formation of the complexes and it studied by different analytical techniques.
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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 pyrazinamide adduct shows more binding than isoniazid and pyrazinamide but this is pseudo results because this binding present at hydroxyl group and hydroxyl group are responsible 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. DOT’s.
The 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 increase 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. By compiling the data the use of lactose in the formulation of isoniazid and pyrazinamide, secondary amines needs to reconsideration.
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