Comparison of two gravimetric methos for water flux determination in single-pass intestinal perfusion

MORA MJ, LONGHI MR, GRANERO GE

Rosario

Congreso; 2º Reunión Internacional de Ciencias Farmacéuticas. RICifa 2010; 2012

COMPARISON OF TWO GRAVIMETRIC METHODS FOR WATER FLUX DETERMINATION IN SINGLE-PASS INTESTINAL PERFUSION STUDIES IN RAT. Mora MJ, Longhi MR, Granero, GE  Departamento de Farmacia, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba Haya de la Torre y Medina Allende, Ciudad Universitaria, X5000HUA, Córdoba, Argentina. Introduction: The oral route is the path of choice for delivering most drugs into the body. Before reaching the bloodstream, drugs administered orally are usually absorbed from small intestine (1). Various methods like Everted sac, Intestinal rings, Ussing chamber, Single-pass intestinal perfusion technique (SPIP) and Caco-2 cells have been used to study intestinal drug permeability (2). Among these methods, the SPIP technique is the most frequently used, which provides conditions closer to what is faced following oral administration, preserved microclimate above intestinal membrane which makes it less sensitive to pH variations (3). Since water absorption and secretion during the perfusion may cause errors in the calculated intestinal permeability values, Peff, ?nonabsorbabel? markers like phenol red and radiolabeled (14C) polyethylene glycols are utilized for net water flux (NWF) calculation. However, phenol red may interfere with the transport and/or analytical measurements, and radiolabeled isotopes may raise safety concerns. To overcome these issues, the gravimetric method has been identified as a simpler alternative. The present study was aimed at comparing two alternative gravimetric methods that is, using intestinal perfusate density as 1.0 g/ml (method 1) and using the actual density (method 2) for calculation of NWF in an SPIP study. The impact of these NWF values on the calculation of Peff of model drugs was also assessed. Materials and methods: The SPIP studies in rats were performed using male Wistar rats weighing 250-300 g. Animals were fasted overnight with free access to water. Anesthesia was induced by an intraperitoneal injection of ketamine/xylazin (1mg/kg). A midline incision was made on the abdomen and an approximated 10 cm of intestine was cannulated with a plastic tubing on both sides The experiment started by delivering the perfusion solution containing the test compounds at flow rate of 0,2 ml/min. The first sample was taken 15 min after start of a continuous outlet flow from the intestine and the outflow perfusate was collected in pre-weighed vials. Samples were collected every 7 min during 75 min and stored until analysis by HPLC. Results: The NWF values were calculated by two methods according to the following Equations: Method 1: NWF = [1- (Qout / Qin)] / Qin/L and Method 2: NWF´ = [1- (Q´out / Qin)] / Qin/L where Qin is the flow rate of entering intestinal perfusate, Qout is the flow of exiting intestinal perfusate and L is the lengh of intestinal segment perfused. The NWF values obtained in rats using the SPIP technique are listed in a table at the end. Conclusions: Assumption of intestinal perfusate density as 1,0 g/ml for calculation of the NWF can introduce an error in the calculated Peff values due to the possibility of abrasion of the intestinal mucosal surface caused by cannulation and the flow of perfusion solution may also lead to entry of certain soluble and insoluble components in the exiting perfusate, which may alter its density. It was found that the gravimetric method employing ?density corrections? for drug intestinal permeability was more reliable. References: 1) Escribano E, García Sala X, Salamanca J, Roig Navarro C, Regué JQ. Single-pass intestinal perfusión to establish the intestinal permeability of model drugs in mouse. Int. J. Pharm 2012; 436:472-477. 2) Issa Ch, Gupta P, Bansal AK. Implicarions of density correction in gravimetric method for water flux determination using rat single-pass intestinal perfusion technique: A technical note. AAPS Pharm Sci Tech 2003; 4(2): 1-6. 3) Zakeri-Milani P, Valizadesh H, Tajerzadeh H, Azarmi Y, Islambolchilar Z, Barzegar S, Barzegar-Jalali M. Predicting human intestinal permeability using single-pass intestinal perfusión in rat. J. Pharm Pharmaceut Sci 2007; 10(3):368-379. Method 1 Method 2 P > 0,05 Drug NWF NWF Verapamil 0,0006 ± 0,0002 -0,0021 ± 0,0002 0,03 Propranolol -0,0003 ± 0,0002 -0,0018 ± 0,0001 0,03 Atenolol -0,0007 ± 0,0001 -0,0018 ± 0,0001 0,04 Phenol Red 0,0006 ± 0,0002 -0,0002 ± 0,0001 0,03 Table: Comparison of intestinal Net Water Flux (ml/min/cm) by two different gravimetric methods.