BIOPHARMACEUTICS & DRUG DISPOSITION, VOL. 16,245250 (1995)

SHORT COMMUNICATION

THE EFFECT OF WATER DEPRIVATION ON THE PHARMACOKINETICS OF

METHOTREXATE IN RATS SO0 J. HWANG*, JEONG M. PARK', WOO I. LEE', OK N. KIM*'

AND MYUNG G. LEE'

*College of Pharmacy, Sookmyung Women's University, Chungpa-Dong 2-Ka. Yongsan-Gu, Seoul 140-742, Korea

'College of Pharmacy, Seoul National University, San 56-1, Shinlim-Dong, Kwanak-Gu, Seoul 151-742. Korea

KEY WORDS methotrexate; pharmacokinetics; water deprivation for 48 h

INTRODUCTION

Dehydration in the body occurs as a result of circumstantial water deprivation, excessive sweating and various disease states, such as polyuria and severe diarrhoea.' A short-term stress from water deprivation could cause significant hormonal, physiological, and biochemical changes in the body. For example, it causes an increase in plasma protein concentration, plasma osmotic pressure and haematocrit,2 packed cell volume, plasma glucose and total lipid c~ncentration,~ and blood vasopressin (antidiuretic hormone) c~ncentration,~ and a decrease in body eight,^ urine output, urinary excretion of electrolytes, extracellular and intracellular fluids: cardiac ~ u t p u t , ~ inulin ~learance,~ and the metabolism of hexobarbital in rats.* Therefore, significant changes in the volume and/or composition of body fluid could result in alteration of pharmacokinetics of drugs.

Studies reported in the literature show that, in dehydration, some pharmacokinetic parameters of drugs such as aspirin,' erythr~mycin,~ antipyrine? acetaminophen,'O gentamicin," and chloramphenicol12 are altered. The urine output of f~rosemide,'~ b~rnetanide,'~ and a~osemide'~ reduced significantly after intravenous (IV) administration of the drugs to 48 h water-deprived rats when compared with that of the control rats. This was due to the significantly reduced amount of the drugs excreted in the urine, and/or the water-deprived state i t ~ e 1 f . l ~ ' ~

'Addressee for correspondence.

CCC 0 142-2782/95/030245-06 01995 by John Wiley & Sons, Ltd.

Received I I July 1994 Accepted 1 November 1994

246 S . J. HWANG ET AL.

The purpose of this communication is to report the effect of water deprivation for 48 h on the pharmacokinetics of methotrexate (MTX) after IV administration of the drug, 10 mg kg-', to rats.

MATERIALS AND METHODS

Sixteen male Sprague-Dawley rats, weighing 240-304 g, were obtained from Korea Laboratory Animal Development (Seoul, Korea). The animals were housed in a clean room, and given food (Sam Yang Company, Seoul, Korea) and water ad libitum. The animals were randomly divided into two groups: the control and water-deprived groups. Water was deprived for 48 h before the experiment in the water-deprived rats, but food was given ad libitum.

After overnight fasting (with water ad libitum for the control rats), the carotid artery and the jugular vein of each rat were catheterized with polythene tubing (PE 50 for the vein and PE 55 for the artery, Beckton, Dickson and Company, Parsippany, NJ, U.S.A.) under light ether anaesthesia. Both cannulae were exteriorized to the dorsal side of the neck where each cannula terminated with a long silastic tubing (Dow Corning Company, Midland, MI, U.S.A.) The silastic tubings were covered with wire to allow free movement of the rat and the exposed areas were sutured. Each rat was housed individually in a rat metabolic cage (Daejong Scientific Company, Seoul, Korea), and allowed to recover from anaesthesia for 4-5 h before study. Heparinized 0.9% NaCl injectable solution (20 U mL-I), 0.3 mL, was used to flush each cannula to prevent blood from clotting.

MTX, 10mgkg-', (MTX IV solution, 500mg per 20mL, was kindly donated by the Choong-Wae Pharmaceutical Company, Seoul, Korea, and was diluted with 0.9% NaCl injectable solution to a final concentration of 5mgmL-') was injected in 1 min via the jugular vein of each rat of both groups (n = 8, each). The total injection volume was approximately 0.5 mL. Approximately 0.12 mL of blood was collected via the carotid artery at appropriate time intervals. The blood samples were centrifuged immediately to minimize any 'blood storage effect'16 (the change in plasma concentration of MTX due to the time elapsed between collection and centrifugation of the blood sample) on the plasma concentrations of MTX. Urine samples were collected up to 24 h. Blood and urine samples were handled similarly to those reported p rev io~s ly .~~ At the end of 24h, as much blood as possible was coliected via the carotid artery and the plasma was stored in the freezer prior to the measurement of plasma protein binding of MTX using the equilibrium dialysis method. 1 8 J 9 At the same time, the whole gastrointestinal (GI) tract (including its content) was removed, transferred to a beaker containing 100 mL of 0.01 M NaOH (to facilitate the extraction of MTX) and cut into small pieces with scissors. After shaking manually and stirring with a glass rod, O.lmL aliquots of the supernatant were collected from the beaker and stored in the

METHOTREXATE PHARMACOKINETICS 247

freezer prior to the HPLC analysis of MTX. the concentrations of MTX in plasma, urine and GI tract were determined by the reported HPLC method.16

Pharmacokinetic parameters, such as the total area under the plasma- concentration-time curve from time zero to time infinity (AUC), the time- averaged total body clearance (CL), the area under the first moment of the plasmaconcentration-time curve (AUMC), the mean residence time (MRT), the apparent volume of distribution at steady state ( Vss), and the time-averaged renal (CL,) and non-renal (CL,,) clearances were estimated according to the standard method.20 The mean values of each clearance, half-life, and V,, were determined by the harmonic-mean method.21 Levels of statistical significance were assessed using a t test between two means for unpaired data. A p value of less than 0.05 was considered statistically significant. All data are expressed in terms of mean f standard deviation.

RESULTS AND DISCUSSION

In this study, water deprivation for 48 h caused a 15.4% decrease in the body weight (from 318 to 269g, n=8). Body weight losses become clinically important if they exceed 5%.3 Thus the weight loss observed in this study indicates that short-term water deprivation may lead to clinically significant changes in water balance. Changes in some physiological parameters, and impaired kidney and liver function (based on glomerular filtration rate estimated by creatinine clearance, blood urea nitrogen, or tissue microscopy) in 48 h water-deprived rats were also reported.14

The mean arterial plasma-concentration-time curves of MTX after IV administration to the control (n = 8) and water-deprived (n = 8) rats are shown in Figure 1, and the relevant pharmacokinetic parameters are listed in Table 1. After IV administration, the plasma levels of MTX appeared to decline in a polyexponential fashion with significantly higher levels in the water-deprived rats. This resulted in a significant increase in AUC (1590 against 2100 pgminmL-') in the water-deprived rats. The MRT (10.4 against 27.1 min) and tllz (54.9 against 138min) also increased significantly in the water-deprived rats, indicating that MTX resided longer and was eliminated more slowly. As expected, the CL per kg body weight (6.29 against 4.77 mL min-I kg-I) decreased significantly in the water-deprived rats. The significant decrease in CL in the water-deprived rats was due to the significant decrease in CL,, (3.83 against 2.90mLmin-' kg-I), since the CL, of the control and dehydrated groups were not significantly different. The CL of antipyrine (antipyrine is completely metabolized by the liver) decreased significantly (by 33%) in the 36h water-deprived rats when compared with the control rats, which could be due to the change in the hepatic intrinsic clearance in the water-deprived rats.g The CL, of f~rosemide,'~ bumetanide,I4 azo~emide,'~ and propranololZ2 were also significantly reduced in the 48 h

S. J. HWANG ET AL.

~ * * T

l 0 O j

0 2 4 6 8 1 0 ** TIME (MINUTES)

***

0.05 1, 0 1 2 3 4 5 6

TIME (HOURS) Figure 1. Mean arterial plasma-concentration-time profiles of MTX after 1 min IV infusion of MTX, 1Omgkg-’, to the control (0, n = 8) and water-deprived (0, n = 8) rats. The bars represent the standard deviation. The inset shows the plasma-level profiles in the first 10min. *, p < 0.05; **,

p<O.Ol, and ***,p<0.001

water-deprived rats compared with the control rats. The percentages of contribution of CL, to CL were not significantly different: the values were 60.9 and 60.8% for the control and water-deprived rats, respectively. The amount of unchanged MTX excreted in 24 h urine (Xu,,,=) by the control and dehydrated groups were also not significantly different. The mean values of V, in the water-deprived rats were 83% higher than the control rats,

METHOTREXATE PHARMACOKINETICS 249

Table 1. Mean (f standard deviation) pharmacokinetic parameters of MTX after IV administration of MTX, lOmg kg-I, to the control and water-deprived rats

Parameters Control rats Water-deprived rats

(n = 8) (n = 8)

Body weight (g) AUC (pgmin mL-') MRT (min) v s s (mLkg-') 4 1 2 @in) XUWTX ( w ) CL (mL min-' kg-I) CL, (mLmin-I kg-') CL,, (mLmin-'kg-') Protein binding (%) MTX recovered from whole GI tract (X of

IV dose)

286 f 9.42 1590 f 239 10.4f2.33 62.2 f 104 54.9 f 14.3 1100f268 6.29f 1.16 2.24 f 0.967 3.83f0.615 40.4 f 3.94

2.95f0.811

269 f 18.9 2100f 264* 27.1 f 10-6** 114f55.0 138 f 385**

1090f 237 4.77 f 0-679* 1.78 f 0.701 2.90f 0.566* 27.5+2.09**

*, p < 0.01; **, p < 0.001

although they were not significantly different (p < 0-2403) due to the large intersubject variation in the control rats. This could be at least partly due to the significant increase in free (unbound) fraction of MTX in the plasma of the water-deprived rats; the percentages of MTX bound to plasma protein (40.4 against 27.5%) decreased significantly in the water-deprived rats. The above data, i.e., the significant decrease in CL, CL,,, and protein binding in the water-deprived rats and not significantly different V,, between the two groups of rats, may explain the significant increase in plasma concentrations of MTX, AUC, MRT, and tl12 (54.9 against 138min) in the water-deprived rats.

The percentages of IV dose recovered from the whole GI tract as unchanged MTX at 24h after IV administration increased significantly (2.95 against 18.1%) in the water-deprived rats. This could be due to the increase in the biliary MTX excretion and/or decrease in subsequent reabsorption of the excreted MTX. The enterohepatic recycling of MTX in human studies could be proved by the significantly lower serum concentration of MTX beginning at 18 h after a 6 h MTX IV infusion (1 g per m2 body surface area) in patients treated with a single dose of 25 g of charcoal.23 Generally, MTX has side-effects on the GI tract, such as mucositis, diarrhoea, and GI desquamation. The above data suggest that MTX-treated water-deprived patients might have more severe GI side-effects than those who are not dehydrated. Since MTX-treated patients have diarrhoea and are in a malnutrition state, i.e., a water-deprived state, they might suffer from severe GI side-effects with MTX. On the other hand, the amount of unchanged f~rosemide'~ and azosemidelS recovered from the whole GI tract were not significantly different between the control and 48 h water-deprived rats when the drugs were intravenously administered to rats. Our present animal data might suggest (if they can be extrapolated and applied

250 S. J. HWANG ET AL.

to humans) that the dose of MTX for water-deprived patients may require some modifications.

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