IN VITRO INHIBITION OF SULTAMICILIN AND CEFDINEX USED

IN VITRO INHIBITION OF SULTAMICILIN AND CEFDINEX USED AS ANTIINFECTIVE DRUG ON HUMAN SERUM PARAOXANASE 1 ENZYME ACTIVITY Serhat Yıldırım 1, Sultan Mehtap Akın 1, Esra DİLEK 1* 1 Erzincan University Faculty of Pharmacy, Erzincan-TURKEY *Corresponding Author: edilek@erzincan. edu. tr RESULTS INTRODUCTION Paraoxonase 1 (PON 1) enzyme has important functions in metabolism. PON 1 is a calcium-dependent ester hydrolase with a glycoprotein structure with both arylesterase (EC 3. 1. 1. 2) and paraoxonase (EC 3. 1. 8. 1) activities. PON 1 is synthesized in the liver and given to the blood. The PON 1 enzyme is linked to HDL in serum and prevents the lipoprotein oxidation with its antioxidant property by hydrolyzing the lipid peroxides. A protein with about 43 k. Da molecular mass and 354 amino acids. Due to the hydrophobic termination at the amino terminal, the lipoprotein and lipid containing materials are tightly bound. Through this hydrophobic region has been found to bind to Figure 1. Structures of Sultamicilin and Cefdinex phospholipids in HDL. One of the 3 cysteine amino acids found in the structure is found to be free (284) and the disulfide bond is found between the We studied in vitro effects of two different drugs (Sultamicilin and Cefdinex ) other two (42 -352). There are 2 calcium atoms in the space at the center of (Figure 1) which are often used as anti-infective drug on human serum the three-dimensional structure of the enzyme. The Ca atom at the top of the paraoxanase 1 (PON 1) enzyme activity. The drugs decreased the in vitro PON 1 cavity, "Catalytic Calcium", is involved in the direct catalytic reaction, or activity. The inhibition mechanism of Sultamicillin was noncompetitive whereas serves to protect the active site by ensuring that the active site is held in the Cefdinex was a uncompetitive inhibitor (Table 1). The IC 50 values for were proper conformation. The other is called "Structural Calcium". The removal of Sultamicillin and Cefdinex calculated to be 34. 7 m. M, and 0. 59 µM (Figure 2), the structural Ca atom from the structure causes the unconverted respectively, and the Ki constants were calculated to be 38. 3 m. M, and 0. 86 µM denaturation of the enzyme. (Figure 3), respectively. These results showed that Cefdinex is more effective than Sultamicillin. Our results showed that these drugs in vitro inhibit the activity of the enzyme with different inhibition mechanisms at low doses. PURPOSE Purification of PON 1 isoenzyme from human serum by DEAE-Sephadex A-50 and Sephadex G-200 gel filtration chromatography. On purified enzyme activity, investigation of in vitro inhibition effects of drugs in medicine. 120 100 R 2 = 0, 9722 % Aktivite 80 MATERİALS AND METHODS 60 40 80 40 20 0 0 20 40 [Sultamisilin] m. M 60 R 2 = 0, 9956 60 20 0 The inhibitory effects of Sultamicillin and Cefdinex drugs were tested in vitro. % Aktivite vancomicyn and colistin, the drug active ingredients, used as anti-infective 120 0 80 0, 5 1 [Sefdinir] m. M 1, 5 2 Figure 2. % Activity-[I] graph for Sultamicillin and Cefdinex Control activity was considered to be 100% in the absence of complexes. A graph was drawn for each complex concentration against percent activity for each complex. Ki values of each drug were determined by selecting three drawn for identifying Ki values and inhibition type Paraoxonase activity of 1/V different drug concentrations showing inhibition. Lineweaver-Burk curves were Kontrol 15, 2 m. M 40, 5 m. M 60, 6 m. M 0, 1 1/V 0, 6 Kontrol 0, 31 m. M 0, 62 m. M 0, 93 m. M 0, 3 PON 1 has been identified with paraoxone (1 m. M) in 50 m. M glycine-Na. OH (p. H 10. 5) buffer including 1 m. M Ca. Cl 2 at 25 °C. 0 0 -1 1 3 1/[S] m. M-1 5 7 -3 -1 1 3 1/ [S] m. M-1 5 7 Figure 3. Determination of inhibition types and Ki values for Sultamicillin and Cefdinex using Lineweaver-Burk curves DETERMINATION OF HUMAN SERUM PON 1 ACTIVITY Enzyme activity assay is based on spectrophotometric measurement of pnitrophenol at 412 nm. The molar extinction coefficient of paranitrophenol (ε = 18. 290 M− 1 cm− 1 at p. H 10. 5) is used for calculation of the activity. One enzyme unit is defined as the amount of enzyme that catalyzes hydrolysis of 1 μmol of paraoxon at 25 °C. Table 1. IC 50, Ki values and inhibition types for Sultamicillin and Cefdinex Inhibitory Sultamicillin Cefdinir IC 50 (m. M) The average Ki (m. M) 34, 7 0, 59 REFERENCES 1. Canales, A. , Sanchez-Muniz, F. J. , 2003. Paraoxonase something more than an enzyme. Med Clin (Barc), 121, 537– 48. 2. Renault, F. , Chabri`ere, E. , Andrieu, J. P. , Dublet, B. , Masson, P and Rochu, D. , Tandem purification of two HDL-associated partner proteins in human plasma, paraoxonase (PON 1) and phosphate binding protein (HPBP) using hydroxyapatite chromatography, Journal of Chromatography B, 836 (2006) 15– 21 38, 3 0, 86 Inhibition type Noncompetetive Uncompetetive