INFLUENCE OF THIOLS TO DECOMPOSITION OF PENTAERYTHRITYL TETRANITRATE AND ISOSORBIDE DINITRATE AT NONENZIMATIC IN VITRO CONDITION

• Main Author: Reuben Suwitono, Marvel
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/45892
• Institution: Institut Teknologi Bandung

Several compounds such as glyceryl trinitrate (GTN), isosorbide dinitrate (ISDN), isosorbide mononitrate (ISMN), and pentaerythrityl tetranitrate (PETN) are organic nitrate compound used in the therapy as general vasodilator, for instance in the treatment of angina pectoris. After metabolized, the mentioned compound will release nitric oxide (NO) as active component. Therefore, organic nitrate compound constitute a prodrug and its vasodilator function depends on the ability of biological system to bioactivate it to produce NO. Clinically, it is known a tolerance phenomenon to organic nitrate, that is quickly decreasing of efficacy with the result that its vasodilator effect to become ineffective. This phenomenon depends on its frequency and dose. Literature has been reported two type of tolerance to nitrate, which is functional and metabolic tolerance. Functional tolerance occurs because of the change in reductiveoxidative state that causes oxidative stress condition which decrease the ability to do chemically reduction. Metabolic tolerance happens because of decreasing in response of tissue and organ where NO metabolism occurs. One of the causes of decreasing in the ability to do chemically reductive in functional tolerance is upregulation in endogenic tiol system. It has been proved that supplementation of thiol is able to overcome tolerance to nitrate, nevertheless there is no comprehensive reports on interaction between organic nitrate and thiol compounds. The goal of this research is to study decomposition of organic nitrate PETN and ISDN under influence of thiol in the condition of physiological resembled, with the result of decomposition profile data, intermediate and decomposition product, along with its decomposition reaction kinetics will be obtained. The result will give scientific contribution about tolerance phenomena in organic nitrate, particularly in the role of thiol compound to overcome nitrate tolerance. The research is started by an analytical method development and validation to quantify PETN and ISDN by using high performance liquid chromatography (HPLC) analytical technique. On the main research, incubation of PETN dan ISDN has been done without and with influence of thiol compounds N-acetyl cystein (NAC) and thiosalicylic acid (TSA), by in vitro non-enzymatic physiological condition, using phosphate buffer pH 7.4 at 37.0 ºC. Incubation has been done with molar ratios of nitrate and thiol are 1:1, 1:2, 1:5, and 1:10, and 4 groups of decomposition reaction is designed, which are reactions between PETN and NAC, PETN and TSA, ISDN and NAC, and ISDN and TSA. Each group was incubated to 48 hours, and at certain time sample is taken. As treatment control, PETN and ISDN were incubated at the same research condition without addition of neither NAC nor TSA. To each samples, concentration of PETN or ISDN is determined. Analysis of decomposition reaction product of organic nitrate and thiol is conducted qualitatively by mass spectroscopy analysis technique. Nitrite as one of nitrate decomposition product is analyzed qualitatively and quantitatively by using Griess spectrophotometric method. To obtain decomposition profile of PETN and ISDN, concentration of each compound along the incubation, without and with influence of thiol, is plotted in the function of time. Through the decomposition profile, kinetic model is arranged to drawn reaction rate of PETN and ISDN decomposition, by curve approximation. As the result of initial research, it is obtained analytical method to determine organic nitrate by using HPLC analytical technique, with mobile phase is acetonitrile:water in 1:1 ratio, at 1 ml/min flowrate, by using reverse phase column C-18 (300 mm x 3,9 mm, partikel size 10 µm) at 215 nm wavelength. The mentioned method fulfill all requirements of stability indicating method in validation of HPLC system, therefore it could be used to determine PETN and ISDN concentration. The result shows that PETN and ISDN concentration, without neither NAC nor TSA after incubated to 48 hours, were slightly decomposed which is less than 5%. Under NAC influence at several molar ratios, PETN is decomposed as much as 84.7% to 92.7%, and under influence of TSA is decomposed as much as 86.0% to 92.9%. ISDN decomposition under influence of NAC is occurs as much as 18.4% to 25.1%, and under influence of TSA is decomposed as much as 20.0% to 23.6%. Kinetic calculation by using curve approximation to decomposition reaction data of PETN and ISDN under influence of NAC and TSA shows that PETN and ISDN decomposition were occurs in equilibrium first order composite reaction. Identification to the mixture of decomposition reaction solution by using mass spectroscopy analytical technique shows PETN decomposition product, which include pentaerythrityl trinitrate (PETriN), pentaerythrityl dinitrate (PEDN), pentaerythrityl mononitrate (PEMN) dan pentaerythritol (PE), while decomposition products of ISDN are ISMN and isosorbide. Decomposition product of NAC is detected as 3,3'-disulfanediylbis(2-acetamidopropanoic acid), and decomposition product of TSA is 2,2'-disulfanediyldibenzoic acid, where both of them are having disulphide bond. Identification and measurement of nitrite concentration in the decomposition reaction solution of both PETN and ISDN show that it contains nitrite, although the result show insignificant correlation between nitrate decomposed and nitrite formed. vi Based on the research result, it could be expressed that is without influence of neither NAC nor TSA, PETN and ISDN shows a good stability. NAC and TSA influences and able to increase decomposition rate of PETN and ISDN. Under influence of either NAC or TSA, PETN is decomposed four times higher than ISDN. Interaction between PETN as well as ISDN with either NAC or TSA is a redox reaction which is proved by the presence of nitrite as nitrate reduction product, and disulphide bond containing compound as product of thiol oxidation. However, the mentioned reaction is not undergoing stoichiometrically. This result is on the same track with analytical result of reaction kinetic which is PETN and ISDN decomposition under influence of either NAC or TSA is equilibrium first order composite reaction.