By Hussam A.S. Murad Department of Pharmacology and Therapeutics Faculty of Medicine, Ain Shams University

. Amphotericin B is a broad spectrum fungicidal used in treatment of life- threatening systemic mycotic infections. It is poorly absorbed and thus given parenterally. The adverse effects of amphotericin B is divided into immediate (infusion-related) as hypotension and slower adverse effects of which nephropathy is the most important one occurring in about 80% of the patients. Amphotericin- induced nephropathy is usually reversible, however, irreversible (permanent) renal tubular damage may occur. Several attempts to decrease this nephrotoxicity were tried as slow infusion, sodium loading and use of the liposomal formulation of the drug (lipid-packaged drug) which is less nephrotoxic but more expensive. Pentoxifylline is a nonspecific phosphodiesterase inhibitor which may exert a protective effect on renal function during taking amphotericin B. N-acetylcysteine is a thiol-containing antioxidant which has a renoprotective activity against several insults but its effect on amphotericin-induced nephropathy has not been fully investigated. Amphotericin B is a broad spectrum fungicidal used in treatment of life- threatening systemic mycotic infections. It is poorly absorbed and thus given parenterally. The adverse effects of amphotericin B is divided into immediate (infusion-related) as hypotension and slower adverse effects of which nephropathy is the most important one occurring in about 80% of the patients. Amphotericin- induced nephropathy is usually reversible, however, irreversible (permanent) renal tubular damage may occur. Several attempts to decrease this nephrotoxicity were tried as slow infusion, sodium loading and use of the liposomal formulation of the drug (lipid-packaged drug) which is less nephrotoxic but more expensive. Pentoxifylline is a nonspecific phosphodiesterase inhibitor which may exert a protective effect on renal function during taking amphotericin B. N-acetylcysteine is a thiol-containing antioxidant which has a renoprotective activity against several insults but its effect on amphotericin-induced nephropathy has not been fully investigated. Introduction The present work was designed to study which of pentoxifylline or N-acetylcysteine is preferred for protection against amphotericin-induced nephropathy in rats. Aim of the Study

Induction of amphotericin-induced nephrotoxicity in rats and effect of the test drugs : A one week period was selected because amphotericin-induced nephrotoxicity does not increase after a second week of treatment. Repeated intravenous injections of amphotericin B in rats cause phlebitis, thus many studies uses the intraperitoneal route (I.P.). Moreover amphotericin B is not metabolized and its I.P. injection is not limited by a first pass hepatic metabolism. 4 groups (6/ group) were used and and given the following: Group I (Control group) : Six I.P. injections of D.W. ( 1 ml /day). Group II (Amp. B group): Six I.P. injections of amphotericin B (10 ug/kg/day). Group III (PTX+ Amp. B group) : as group II + oral pentoxifylline (8 ug/kg/day ) for 7 days, starting 1 day before the amphotericin B injections. Group IV (NAC+ Amp. B group) : as group II + oral N-acetylcysteine (250 ug/kg/day ) for 7 days, starting 1 day before the amphotericin B injections. Induction of amphotericin-induced nephrotoxicity in rats and effect of the test drugs : A one week period was selected because amphotericin-induced nephrotoxicity does not increase after a second week of treatment. Repeated intravenous injections of amphotericin B in rats cause phlebitis, thus many studies uses the intraperitoneal route (I.P.). Moreover amphotericin B is not metabolized and its I.P. injection is not limited by a first pass hepatic metabolism. 4 groups (6/ group) were used and and given the following: Group I (Control group) : Six I.P. injections of D.W. ( 1 ml /day). Group II (Amp. B group): Six I.P. injections of amphotericin B (10 ug/kg/day). Group III (PTX+ Amp. B group) : as group II + oral pentoxifylline (8 ug/kg/day ) for 7 days, starting 1 day before the amphotericin B injections. Group IV (NAC+ Amp. B group) : as group II + oral N-acetylcysteine (250 ug/kg/day ) for 7 days, starting 1 day before the amphotericin B injections. Methods

Before the last (6th ) I.P. injection the bleeding time was measured for all rats, then rats were subjected to direct blood pressure measurement during which the last injection was given. Finally blood was collected for measurement of urea and creatinine. The following was done for rats in all groups : 1) Measurement of the tail bleeding time :   By sectioning the tail and repeated blotting till stop of bleeding 2) Direct measurement of the systolic blood pressure :   The carotid artery at one side was exposed in the urethane- anesthetized rat. Glass cannula was fixed into it and connected via a pressure rubber tube to Harvard pressure transducer. Initial recording of blood pressure was done then amphotericin B was given by I.P. injection (10 ug/kg) and the effects on blood pressure was recorded. 3) Measurement of serum urea and creatinine levels (as an assessment of renal functions) :   Using the enzymatic and end point methods respectively. Before the last (6th ) I.P. injection the bleeding time was measured for all rats, then rats were subjected to direct blood pressure measurement during which the last injection was given. Finally blood was collected for measurement of urea and creatinine. The following was done for rats in all groups : 1) Measurement of the tail bleeding time :   By sectioning the tail and repeated blotting till stop of bleeding 2) Direct measurement of the systolic blood pressure :   The carotid artery at one side was exposed in the urethane- anesthetized rat. Glass cannula was fixed into it and connected via a pressure rubber tube to Harvard pressure transducer. Initial recording of blood pressure was done then amphotericin B was given by I.P. injection (10 ug/kg) and the effects on blood pressure was recorded. 3) Measurement of serum urea and creatinine levels (as an assessment of renal functions) :   Using the enzymatic and end point methods respectively.

There was significant increase in the PTX + Amp. B group in contrast to a non- significant change in the NAC + Amp. B group compared to the Amp. B group. There was a significant increase in the PTX + Amp. B group compared to the NAC + Amp. B group. There was significant increase in the PTX + Amp. B group in contrast to a non- significant change in the NAC + Amp. B group compared to the Amp. B group. There was a significant increase in the PTX + Amp. B group compared to the NAC + Amp. B group. Results 1)Effects of amphotericin B (Amp. B), pentoxifylline + amphotericin B (PTX + Amp. B) and N-acetylcysteine + amphotericin B (NAC + Amp. B) on the tail bleeding time (minutes) in rats : There was significant increase in the PTX + Amp.B in contrast to non-significant changes in the Amp.B and NAC + Amp.B groups compared to the control group.

2)Effects of amphotericin B (Amp. B), pentoxifylline + amphotericin B (PTX + Amp. B) and N-acetylcysteine + amphotericin B (NAC + Amp. B) on the systolic blood pressure ( SBP, mmHg) in rats : There were significant decreases in all groups compared to the control group. There was a significant decrease in PTX + Amp. B group in contrast to a non- significant change in NAC + Amp. B group compared to the Amp. B group. There was a significant decrease in the PTX + Amp. B group compared to the NAC + Amp. B group. There was a significant decrease in PTX + Amp. B group in contrast to a non- significant change in NAC + Amp. B group compared to the Amp. B group. There was a significant decrease in the PTX + Amp. B group compared to the NAC + Amp. B group.

Effects of amphotericin B (Amp. B, 10 ug/kg/day by intraperitoneal injection for 6 days), pentoxifylline + amphotericin B (oral PTX 8 ug/kg/day for 7 days, starting 1 day before the amphotericin B injections) and N-acetylcysteine + amphotericin B (oral NAC 250 ug/kg/day for 7 days, starting 1 day before the amphotericin B injections) on the systolic blood pressure (SBP, mmHg) in rats (n=6).

3)Effects of amphotericin B (Amp. B), pentoxifylline + amphotericin B (PTX + Amp. B) and N-acetylcysteine + amphotericin B (NAC + Amp. B) on serum urea and creatinine levels ( mg/dl ) (as an assessment of the renal functions) in rats : There were significant increases in Amp. B compared to the control group. There were non-significant changes in PTX+ Amp. B and NAC + Amph.B groups compared to control group in contrast to significant decreases compared to Amp. B group. There were non-significant changes in PTX + Amp. B group compared to NAC + Amp. B group. There were significant increases in Amp. B compared to the control group. There were non-significant changes in PTX+ Amp. B and NAC + Amph.B groups compared to control group in contrast to significant decreases compared to Amp. B group. There were non-significant changes in PTX + Amp. B group compared to NAC + Amp. B group. Urea Creatinine

Amphotericin-induced nephropathy is due to vasoconstriction a pre-renal component and direct interaction with epithelial cell membranes causing renal tubular damage. Amphotericin B causes changes in tubular cell permeability to ions and increases delivery and reabsorption of chloride ions in the distal tubule initiating a decrease in the glomerular filtration rate. PGs, cytokines (as TNF-α and IL-6 ) and oxygen free radicals are involved in pathogenesis of amphotericin-induced nephropathy. Pentoxifylline prevents amphotericin B -induced nephrotoxicity preventing the decrease of GFR and the increase in renal sodium and potassium excretions. This protective effect of pentoxifylline is due to decrease of renal vascular resistance, promotion of PG I2 release and inhibition of production of cytokines. Unfortunately, pentoxifylline causes prolonged bleeding time as a result from defects in platelet adhesion, aggregation, or release reaction and acts as antiplatelet increasing bleeding tendency. In addition, pentoxifylline may potentiate the action of antihypertensive drugs necessitating their dose adjustment and it should be given with caution to patients with low or labile blood pressure. Also cerebrovascular disease, arrhythmia, and hypotension have occurred in some patients during treatment with pentoxifylline. Amphotericin-induced nephropathy is due to vasoconstriction a pre-renal component and direct interaction with epithelial cell membranes causing renal tubular damage. Amphotericin B causes changes in tubular cell permeability to ions and increases delivery and reabsorption of chloride ions in the distal tubule initiating a decrease in the glomerular filtration rate. PGs, cytokines (as TNF-α and IL-6 ) and oxygen free radicals are involved in pathogenesis of amphotericin-induced nephropathy. Pentoxifylline prevents amphotericin B -induced nephrotoxicity preventing the decrease of GFR and the increase in renal sodium and potassium excretions. This protective effect of pentoxifylline is due to decrease of renal vascular resistance, promotion of PG I2 release and inhibition of production of cytokines. Unfortunately, pentoxifylline causes prolonged bleeding time as a result from defects in platelet adhesion, aggregation, or release reaction and acts as antiplatelet increasing bleeding tendency. In addition, pentoxifylline may potentiate the action of antihypertensive drugs necessitating their dose adjustment and it should be given with caution to patients with low or labile blood pressure. Also cerebrovascular disease, arrhythmia, and hypotension have occurred in some patients during treatment with pentoxifylline. Discussion

N-acetylcysteine exertes a renoprotective effect preventing deterioration of GFR in amphotericin-induced renal failure. N- acetylcysteine is a thiol-containing antioxidant which scavenge the free radicals involved in pathogenesis of acute renal failure and is commonly used to minimize contrast media-induced nephropathy in high-risk patients. N-acetylcysteine improves the pulmonary antioxidant protection. The beneficial effect of acetylcysteine could be detected even 2 days after its administration. Generally acetylcysteine is well-tolerated and has mild adverse effects. Also it does not alter mean blood pressure or bleeding time. Oral N-acetylcysteine, in contrast to oral pentoxifylline, neither augments the amphotericin-induced hypotension nor increases the bleeding tendency in rats. In addition it is less expensive and has mild adverse effects. Thus N-acetylcysteine is preferred over pentoxifylline for protection against amphotericin-induced nephropathy in rats. Clinical studies are needed to confirm beneficial effects of N-acetylcysteine for patients taking amphotericin B especially elderly or those with renal impairment before a routine administration of N-acetylcysteine with amphotericin B is recommended. Conclusion