Pharmacokinetics of Sebacic Acid in Rats
Total Page：16
File Type：pdf, Size：1020Kb
European Review for Medical and Pharmacological Sciences 1999; 3: 119-125 Pharmacokinetics of sebacic acid in rats A.M.R. FAVUZZI, G. MINGRONE, A. BERTUZZI*, S. SALINARI**, A. GANDOLFI*, A.V. GRECO Istituto di Medicina Interna e Geriatria, Catholic University - Rome (Italy) *Istituto di Analisi dei Sistemi ed Informatica, CNR - Rome (Italy) **Dipartimento di Informatica e Sistemistica, Facoltà di Ingegneria, “La Sapienza” University - Rome (Italy) Abstract. – The pharmacokinetics of disodi- enteral nutrition (TPN) as an alternate ener- um sebacate (Sb) was studied in Wistar rats of gy source1-3. The advantage of these diacids both sexes. Sebacate was administered either as over conventional lipid substrates (both long intra-peritoneal (i.p.) bolus (six doses ranging from 10 mg to 320 mg) or as oral bolus (two doses: 80 and medium chain triglycerides) is related to and 160 mg). Plasma and urinary concentrations the immediate availability of their com- of Sb and urinary concentrations of Sb and its pounds. The salts of dicarboxylic acids are products of β-oxidation (suberic and adipic acids) highly water soluble and thus can be directly were measured by an improved method using administered through a peripheral venous gas-liquid chromatography/mass-spectrometry. route. Unlike long chain triglycerides (LCT), A single compartment with two linear elimi- or medium chain triglycerides (MCT), which nation routes was selected after no increase in significance was shown by an additional com- are available under emulsion form for clinical partment and after a saturable mechanism was use, they do not require complex and expen- found to be unsuitable. Both renal and non-re- sive production procedures. In addition, DA nal elimination parameters were obtained by are readily beta-oxidized at the level of Marquardt non linear fitting of plasma concen- mithocondria, through a carnitine indepen- trations together with urinary elimination. dent mechanism, and peroxisomes. The data reported are calculated from the analy- sis on the whole population of rats and referred to DA provide a means of increasing the net an average body weight (bw) of 100 g. The Sb half- oxidation of nutritional fuel without increas- time was 31.5 min. The tissue elimination rate was ing the carbon dioxide production. This could 0.0122 min-1. The overall volume of distribution be a significant advantage in critically ill pa- was found to be 26.817 ml/100 g bw. The renal tients, where different degrees of pulmonary clearance was 0.291 ml/min/100 g of bw, which is insufficiency are usually present. However, much less than the value of GFR reported in litera- odd carbon atom DA (e.g. azelaic acid) (Az), ture (about 1 ml/min/100 g bw), suggesting the β presence of Sb reabsorption from the ultrafiltrate. are incompletely oxidized since the -oxida- The value of Sb renal clearance was found to be a tion is terminated at the level of malonic acid. concentration-independent function, suggesting Malonic acid is then the starting substrate in the presence of a passive back-diffusion. the synthesis of free fatty acids, thus creating The relative bioavailability of the oral form the so-called futile metabolic circles. Azelate compared to the i.p. form was 69.09%, showing oxidation is associated with a low cost of a good absorption of the drug. ATP synthesis in terms of CO2 production; Key Words: however, the major problem is that more than 50% of the administered dose of this Sebacic acid, Dicarboxylic acids, Pharmacokinetics. diacid is excreted in the urine4. Thus, we turned our attention to sebacic acid (Sb), the superior homologous, with 10 carbon atoms, which in preliminary studies3, appeared to be excreted in the urine at low Introduction levels (only 16% vs 50% of Az). Like Az5, Sb In the last years we proposed the use of the has been safely used in laboratory animals, salts of dicarboxylic acids (DA), in total par- and did not show any toxic effect during 119 A.M.R. Favuzzi, G. Mingrone, A. Bertuzzi, S. Salinari, A. Gandolfi, A.V. Greco chronic administration6. Similarly, no terato- washed twice with 0.25 ml ethylacetate. The genic effect was observed, and the develop- extracts were combined together and dried ment of the fetuses appeared to be normal6. under nitrogen stream until a final volume of The present study was undertaken to ob- 200 ml was reached. The extracts were cro- tain a pharmacokinetics basis for pharmaco- matographed on silica gel thin-layer plates (20 dynamic studies of disodium sebacate in the × 20) cm, 0.25 mm thickness, (Carlo Erba, rats. The disposition of sebacate was studied Italy) in exan-diethylether-acetic acid (6:4:0.4, after three different doses (40, 80 and 160 v:v:v). The area of silica gel which corre- mg) were administered, either as i.p. or as sponded to the area of dicarboxylic acids (a oral bolus. The urinary excretion modality mixture of adipic, suberic, azelaic and sebacic was evaluated giving i.p. different doses (10, acids were used as a standard) was scraped of 40, 80, 160, 240, 320 mg) of sebacate. the plates and extracted three times with eth- ylacetate. The combined extracts were dried over anhydrous Na2SO4. The extracted solutes were recovered in conical tubes after evapora- Materials and Methods tion of the solvent by N2 stream and methylat- ed with diazomethane in diethylether. The di- Animals carboxylic acids derivatives were dissolved in Wistar rats of both sexes, weighing 130-250 500 µl exane and 2 ml of the solution were in- g, were used throughout the study. They had jected in to the gas chromatograph. free access to water and a standard pellet diet Urine samples (0.5-1 ml) were treated with containing 20% protein, 4% fat and 50% car- cation-exchange resin (Dowex 50 W-X4, 100- bohydrate. The rats which received oral dos- 200 µ M mesh. H+) to remove salts, concen- es of sebacate, were fasted previously for 18 trated under reduced pressure and finally fil- hours but provided with water “ad libitum”. tered trough and a Millipore HV (0.45 µm). Single i.p doses of DA salts (DAS), were ad- The samples were exstracted three times with ministered to 8 groups of 5-11 rats (about half 0.5 volumes of ethylacetate. The combined males and half females) and the animals were exstracts were dried under N2 and thin layer kept in a metabolism cage. The doses ranged chromatograph (TLC) was performed to pu- from 10 to 320 mg (10, 40, 80, 160, 240, 320 rify the dicarboxylic acids prior to gas liquid mg). Urine was collected in containers with 1 chromatograph (GLC). The GLC analysis ml 0.1% sodium azide to prevent bacterial was performed as described below. growth and cooled in solid CO2 during three different periods: 0-4, 4-24 and 24-48 hours. Gas Liquid Chromatography-Mass Urine samples were stored at – 20° C prior Spectrometry to analysis. Verification of the identity of the di- Fourteen rats (7 males, 7 females), were in- methylesters DAS was carried out by mass jected i.p. with three different doses of seba- spectrometry (MS). A Hewlett-Packard 589A cate, i.e. 40, 80, 160 mg. Heparinized blood GLC, equipped with a split/splitless capillary samples were taken pre-dose and at 5, 10, 20, injector and supplied with a 5970 MS detec- 40, 80, 160, 320 minutes post-dose. Optimal tor was used. The GLC inlet system was blood sampling times were suggested by a pi- equipped with a 30 m fused silica capillary lot study in 2 rats (1 male, 1 female). Plasma column Supelcowax 10 (0.32 mm I.D., 0.25 µ was separated from the blood cells. film thickness from Supelco, Bellefonte, PA, USA). Helium was used as a carrier gas and Separation of Samples maintained at a flow rate of 2 ml/min (col- Samples of plasma (500 µl) were added to umn pressure 40 psi). A flow of 30 ml/min of azelaic acid (10 µg), which acted as an internal helium was used as make-up gas. The injec- standard. The samples were then treated with tion and interface temperatures were main- 10% trichloroacetic acid solution (50 µl) and tained at 240°C. For the analysis of DA the thoroughly mixed. Within 1 our after sample column temperature was maintained initially collection the mixture was then centrifuged at 50°C. Immediately after injection the tem- and the supernatant extracted three times perature was programmed to increase at a with 0.5 ml ethylacetate. The residue was rate of 10°C to a final temperature of 240°C. 120 Pharmacokinetics of sebacic acid in rats A repetitive scanning mode (300 AMU/sec) The second objective was to ascertain was used for compound identification. The whether the urinary data supported a mecha- eluent from the capillary column was repeti- nism of non-linear excretion (due to carrier tively scanned from 49 to 550 m/z. mediated secretion or reabsorption) at the level of the nephron. To do so, stepwise re- Calibration Curve gression of total drug recovered in the urine 100 µg of the following standard dicar- versus administered dose was performed, in boxylic acids were esterified with dia- which polynomials up to fifth degree in the zomethane: adipic (C6), suberic (C8), azelaic independent variable (administered dose) (C9), sebacic (C10) and dodecanedioic (C12). were tested. Both forward and backward al- The solvent was then evaporated by N2 gorithms yielded significance for the linear stream and the residue dissolved in 1 ml term only (Table I). This was taken as an in- hexane. Two ml of each solution at different dication that a single linear relationship exist- concentrations were injected into GLC.