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Effects of gender on the pharmacokinetics of drugs secreted by the renal organic anions transport systems in the rat
Pharmacological Research, Vol. 45, No. 2, 2002 doi:10.1006/phrs.2001.0912, available online at http://www.idealibrary.com on
EFFECTS OF GENDER ON THE PHARMACOKINETICS OF DRUGS SECRETED BY THE RENAL ORGANIC ANIONS TRANSPORT SYSTEMS IN THE RAT JORGELINA A. CERRUTTI, NORA B. QUAGLIA, ANABEL BRANDONI and ∗ ˙ ADRIANA M. TORRES Farmacolog´ıa, Facultad de Ciencias Bioqu´ımicas y Farmac´euticas, Universidad Nacional de Rosario, CONICET (2000) Rosario, Argentina Accepted 5 November, 2001
The importance of considering sex differences in drug handling studies was admitted recently. The present work evaluates the sex influences on the pharmacokinetics of para-aminohippuric acid (PAH), the reference substance for the renal organic anion transports systems, and furosemide (FS), a standard loop diuretic which is also a substrate for this transport system. Female rats displayed a lower PAH and FS systemic clearance, and a lower value of the elimination rate microconstant from the central compartment for both drugs. These results may be explained by the diminution of the renal clearance of both PAH and FS observed in females. In summary, sex modifies the pharmacokinetics of organic anions. Although additional experimental work must be done to bridge the gap between studies using animals and humans, the reported experimental observations may have potentially important pharmacological implications. So, caution must be exercised in c 2002 Elsevier Science Ltd administering drugs like organic anions to females.
K EY WORDS : organic anions, sex, pharmacokinetics, renal clearance, systemic clearance.
INTRODUCTION In drug development, therapeutic advantages should be demonstrable in the entire target patient population, and hence dose regimens should be tailored to the needs of the individual patient [1]. Pharmacokinetic studies are usually performed in young, healthy male volunteers [2]. However, gender can affect drug pharmacokinetics, and hence possibly influence efficacy [3–5]. Therefore, it is important, that gender effects on drug pharmacokinetics are clearly defined, to allow appropriate dosage recommendations to be made. Most drugs are excreted into the urine, either as unchanged form or as metabolites. The secretion of organic anions by the renal proximal tubule is very important in the elimination of drugs such as antibiotics, diuretics, hippurates, and nonsteroidal anti-inflammatory agents [6–8]. Because kidneys are sensitive to several hormones [7, 9], it is expected that the kinetics of organic anions will show significant differences related to individual’s gender. In this regard, in vitro studies agreed that accumulation of para-aminohippurate is significantly ∗ Corresponding author. Suipacha 531, Rosario, Postcode: 2000,
Argentina. E-mail: [email protected] 1043–6618/02/020107–06/$35.00/0
greater in renal cortical slices from male than that from female rats and that testosterone is involved in this event [10–13]. Thiazides and loop diuretics are secreted through the organic anion systems [14]. The capacity of the organic anion transport systems to secrete a diuretic determines its intraluminal concentration, which is critical for the diuretic activity [7]. In the present study, we have analysed the influence of sex on the pharmacokinetics of para-aminohippuric acid (PAH, the reference substance for the renal organic anion transport system) and furosemide (FS, a standard loop diuretic).
MATERIALS AND METHODS
Experimental animals Male and female Wistar rats aged from 110–130 days old were used throughout the study. Animals were allowed free access to a standard laboratory chow and tap water, and housed in a constant temperature and humidity environment with regular light cycles (12 h). All procedures were in accordance with institutional guidelines for the care and use of laboratory animals. c 2002 Elsevier Science Ltd
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Pharmacokinetic studies These studies were performed in a manner similar to that previously described [15, 16]. Animals were anaesthetized with sodium thiopental (70 mg kg−1 b.w., i.p.). Both the femoral artery and the vein were both catheterized to obtain samples and to administer test compound, respectively. A single bolus of PAH (3 mg/100 g b.w., aqueous solution, i.v.) or FS (1 mg/100 g b.w., aqueous solution, i.v.) were administered. These doses do not saturate the transport system [11, 17], therefore, the kinetics can be adequately estimated. Blood samples were collected at 0–15 min time range for PAH and at 0–120 min range for FS from the femoral artery cannula. An equivalent volume of isotonic saline solution was infused to restore the amount extracted in the blood samples. Samples were centrifuged at 3000 rpm for 3 min, and the extracted plasmas were frozen as −20 ◦ C until analyzed. Concentration of PAH in plasma was measured using the method described by Waugh and Beall [18] and FS was assayed by the Bratton Marshall technique [19]. The concentration versus time curves for PAH and FS, for each individual animal were fitted with the PKCALC computer program [20]. The choice of the best fit was based on the determination coefficient values (R 2 ) and F test. The following equation was used to describe the biexponential concentration–time curves: Cp = Ae−αt + Be−βt where Cp is PAH plasma concentration (mg%) or FS plasma concentration (µg ml−1 ) at time t (min) after administration; constants α and β represent the disappearance rates for the distribution and elimination components, respectively, and the slopes of each of the adjusted curves give their values. A and B represent the initial concentrations of the distribution and elimination components, respectively, extrapolated from the y-axis intercept. The estimated parameters (α, β, A, B) were used to solve the first-order rate microconstants of transfer from central to peripheral compartments (k12 , k21 ) and the elimination rate microconstant from the central compartment (k10 ) with classical equations. Derived parameters: area under the concentration– time curve (AUC), total volume of distribution (VdT ), volume of the central compartment (VdC ), volume of the peripheral compartment (VdP ), steady-state volume of distribution (Vdss ), plasma clearance (Clp) and elimination half-life (t1/2 ) were calculated according to standard procedures for compartmental analysis.
Renal clearance studies These studies were performed as previously described [21, 22]. Briefly, the animals were anaesthetized with sodium thiopental (70 mg kg−1 b.w., i.p.). Both the femoral artery and vein were cannulated (P.E.50. Intramedic, USA.) and a bladder catheter (3 mm i.d.) was inserted through a suprapubic incision. Animals were maintained in restraining cages throughout the experiment to facilitate urine collection. A priming dose
Table I Pharmacokinetic parameters of p-aminohippurate in male and female rats after a single dose (3 mg/100 g b.w., i.v.) Males (n = 4) AUC (mg × sec/dl) A (mg%) B (mg%) α (min−1 ) β (min−1 ) k1−0 (min−1 ) k1−2 (min−1 ) k2−1 (min−1 ) t1/2 (k) (min) VdT (ml/100 g) VdC (ml/100 g) VdP (ml/100 g) Vss (ml/100 g)
5176 ± 453 19.88 ± 1.45 8.10 ± 0.70 1.40 ± 0.18 0.12 ± 0.01 0.328 ± 0.020 0.69 ± 0.11 0.49 ± 0.08 128 ± 8.72 30.81 ± 1.80 10.83 ± 0.62 20.54 ± 2.21 25 ± 0.40
Females (n = 4) 8411 ± 902∗ 20.45 ± 2.13 12.37 ± 1.30∗ 1.16 ± 0.07 0.10 ± 0.01 0.240 ± 0.020∗ 0.53 ± 0.06 0.51 ± 0.07 182 ± 20∗ 21.99 ± 2.43∗ 9.17 ± 0.33 13.72 ± 3.42∗ 17 ± 0.8∗
Results are expressed as means ± SE. ∗ P< 0.05. AUC = area under curve; constants α and β represent the disappearance rates for the distribution and elimination components, respectively; A and B represent the initial values of the distribution and elimination components, respectively; k1−2 , k2−1 = first-order rate microconstants of transfer from central to peripheral compartments; k1−0 = elimination rate microconstant from the central compartment; t1/2 (k) = elimination half-life (t1/2 ); VdT = total volume of distribution; VdC = volume of the central compartment; VdP = volume of the peripheral compartment; Vss = steady-state volume of distribution.
of PAH (3 mg/100 g b.w.) or FS (1 mg/100 g b.w.) in 1 ml of saline solution was administered through the venous catheter. Then, a solution containing PAH (0.6 g%) or FS (0.20 g%) and saline solution (0.9 g%) was infused through the venous catheter employing a constant infusion pump (Pump 22; Harvard Apparatus, USA) at a rate of 1 ml h−1 /100 g b.w. After equilibration for 60 min, urine was collected during two 30 min periods. Blood from the femoral artery was obtained at the midpoint of each clearance period. Arterial blood pressure was estimated throughout the experiments with a manometer inserted in the femoral artery. Renal clearance of PAH and renal clearance of FS were calculated by conventional formulae for each animal. PAH and FS concentrations in serum and urine were determined as described above. Volume of urine was determined by gravimetry.
Materials Chemicals were purchased from Sigma (St. Louis, MO, USA) and were analytical grade pure.
Statistical analysis Significant differences between groups were determined using Student’s t-test. The probability level chosen was P< 0.05. RESULTS Plasma PAH and FS concentrations in male and female adult rats are shown in Fig. 1. For both drugs, male rats displayed a significant lower area under the curve as compared with females.
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(a)
(b)
Fig. 1. (a) Mean plasma concentration–time profiles and area under the curve of: (a) p-aminohippurate (PAH) in male (n = 4, R 2 = 0.989, F = 7.9) and female (n = 4, R 2 = 0.993, F = 8.4) rats; (b) furosemide (FS) in male (n = 9, R 2 = 0.991; F = 11.5) and female (n = 8, R 2 = 0.998, F = 11.2) rats. Results are expressed as means ± SD. (#) P< 0.05.
Pharmacokinetic parameters obtained in both sexes for PAH and FS are listed in Table I and II, respectively. Females showed a lower peripheral volume of distribution for PAH as compared with male rats. On the contrary, no sex differences were observed in the distribution volumes for FS. Females showed a significant lower systemic clearance and elimination rate microconstants (k1−0 ) for both PAH and FS as illustrated in Fig. 2. Figure 3 shows the hybrid rate constants for slopes in distribution and elimination phase (α and β) for PAH and FS. Both first-order hybrid rate constants for FS were statistically lower in females as compared with males. No gender differences were observed in α and β for PAH. We have also evaluated both the systemic and renal clearance of PAH and FS. As shown in Table III, in both cases a statistically significant decrease in values of female rats as compared with males was observed.
Moreover, a greater diminution in FS renal clearance as compared with FS systemic clearance was observed in females. Instead, a similar decrease in the renal and systemic clearance of PAH was shown by female rats. DISCUSSION In the past, women were under-represented as participants in clinical drug studies [23, 24]. In the majority of cases when women and men were both studied, the results were grouped together and there was no analysis of sex differences [23]. Recently, it has been admitted the importance of considering sex in drug handling studies. The results of animal studies should be used to illustrate the fact that significant sex differences in drug metabolism and elimination can occur in mammals, and should provide impetus for sex-based research in humans.
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Fig. 2. Systemic clearance and elimination rate microconstant from the central compartment (k1−0 ) of p-aminohippurate (PAH) and furosemide (FS) in male and female rats. Results are expressed as means ± SE. (#) P< 0.05.
Table II Pharmacokinetic parameters of furosemide in male and female rats after a single dose (1 mg/100 g b.w., i.v.)
AUC (ug × min/ml) A (ug/ml) B (ug/ml) α(min−1 ) β(min−1 ) k1−0 (min−1 ) k1−2 (min−1 ) k2−1 (min−1 ) t1/2 (k) (min) VdT (ml/100 g) VdC (ml/100g) VdP (ml/100 g) Vss (ml/100 g)
Males (n = 9)
Females (n = 8)
2991 ± 211 67.73 ± 5.38 51.33 ± 5.87 0.592 ± 0.128 0.01809 ± 0.00166 0.0407 ± 0.0035 0.2420 ± 0.0650 0.2630 ± 0.0830 18.09 ± 1.62 19.88 ± 1.80 8.68 ± 0.46 11.21 ± 1.63 17.91 ± 1.29
3709 ± 68∗ 74.42 ± 6.28 38.03 ± 2.91 0.221 ± 0.031∗ 0.01181 ± 0.00065∗ 0.0304 ± 0.0014∗ 0.1184 ± 0.0175 0.0878 ± 0.0160 23.16 ± 1.15∗ 23.96 ± 1.08 8.99 ± 0.36 15.03 ± 1.18 19.90 ± 1.11
Results are expressed as means ± SE. ∗ P< 0.05. AUC = area under curve; constants α and β represent the disappearance rates for the distribution and elimination components, respectively; A and B represent the initial values of the distribution and elimination components, respectively; k1−2 , k2−1 = first-order rate microconstants of transfer from central to peripheral compartments; k1−0 = elimination rate microconstant from the central compartment; t1/2 (k) = elimination half-life (t1/2 ); VdT = total volume of distribution; VdC = volume of the central compartment; VdP = volume of the peripheral compartment; Vss = steady-state volume of distribution.
Renal excretion is a major route of elimination for many drugs and especially for their hydrophilic metabolites. The processes by which a drug is excreted may be any combination of glomerular filtration, tubular secretion and tubular re-absorption. The net excretion resulting from these processes can be modulated by numerous factors, like renal blood flow, protein binding, and urine pH and flow [7]. Carrier-mediated transport of
drugs is confined to the proximal tubule, and separate carrier systems exist for the active secretion of organic cations and anions. The organic anions transport systems play a critical role in the elimination of a large number of drugs (e.g. antibiotics, chemotherapeutics, diuretics, non-steroidal anti-inflamatory drugs, radiocontranst agents, cytostatics). Previous studies have suggested that testosterone might modulate these organic anion transport systems [10, 12, 13]. The present study was undertaken to evaluate whether sex influences the pharmacokinetics of organic anions. We selected PAH, the reference substance for the organic anion transport systems, and FS, a standard loop diuretic that is also a substrate for this system. Our data reveal that male rats have a higher systemic depuration of both PAH and FS than female rats. The lower absolute value observed for the systemic clearance of FS as compared with systemic clearance of PAH in both sexes may be explained by their different binding to the plasma proteins: 91–99% of FS and only a fraction (lower than 25%) of PAH are bound to plasma proteins [7]. Moreover, sex differences in plasma protein binding of FS might account for the gender differences observed in the disappearance rates for the distribution and elimination components. In this sense, sex differences in plasma protein binding have been observed for several drugs [25–28]. On the other hand, we did not observe sex differences in PAH disappearance rate for the distribution phase. In this connection, no sex difference in PAH plasma protein binding have been reported [13]. Central volume of distribution for PAH was similar in both sexes. By contrast, the peripheral and steady state volumes of distribution were higher in males. The gender differences in these volumes may be explained by the
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Fig. 3. Disappearance constants rates for the distribution (α) and elimination (β) components of p-aminohippurate (PAH) and furosemide (FS) in male and female rats. Results are expressed as means ± SE. (#) P< 0.05. Table III Absolute and percentage values of systemic and renal clearance of p-aminohippurate and furosemide in male and female rats p-Aminohippurate Males
Furosemide Females
Males
Females
Systemic clearance (ml/min/100 g)
4.10 ± 0.36 (n = 4) 100 ± 8.78%
(n = 4) 61.98 ± 4.89∗ %
0.344 ± 0.021 (n = 9) 100 ± 6.10 %
0.265 ± 0.004# (n = 8) 77.27 ± 1.39# %
Renal clearance (ml/min/100 g)
2.73 ± 0.477 (n = 6) 100 ± 17.95%
1.49 ± 0.22∗ (n = 6) 53.85 ± 8.05∗ %
0.295 ± 0.031 (n = 6) 100 ± 10.51%
0.165 ± 0.030# (n = 6) 55.94 ± 4.17#,& %
2.54 ± 0.20∗
Results are expressed as means ± SE. ∗ P< 0.05 vs the respective value in males. # P< 0.05 vs the respective value in males. & P< 0.05 vs the percentage value of systemic clearance in females.
fact that females tend to have a greater proportion of their body weight to be of fat than males and so PAH, which is an hydrosoluble drug, has a lesser space of distribution [4]. No sex related differences were observed in volumes of distribution for FS, which is a lesser hydrosoluble drug than PAH. The elimination rate microconstant from the central compartment (k1−0 ) decreased approximately of 25% in females as compared with male rats for both PAH and FS. This might indicate a sex related difference in the biotransformation and in the excretion of the drugs. PAH is subject to negligible metabolism, and is principally eliminated by renal tubule secretion. Approximately more than half the dose of FS is eliminated unchanged by the kidneys and the remainder is uptaken and metabolized by the liver. So we evaluated sex influence on the renal clearance of both organic anions. Our results demonstrated a diminution of PAH and FS renal clearance for females which explain the sex related difference observed in the systemic clearance of both organic anions. An important role in this sex
difference must be played by the organic anion transport systems which are involved in proximal tubular secretion of several drugs. Organic anions transporters have been recently cloned [6, 8, 30]. ROAT1 represents the classical basolateral PAH/dicarboxylate exchanger and has a broad substrate specificity [8, 31–34]. Uwai et al. [14] have recently demonstrated that ROAT1 contributes to the renal tubular secretion of FS. The role of ROAT1 in the sex related difference observed in the renal clearance of PAH and FS remains to be determined. FS renal clearance decreased in females as compared with males in a higher percentage than systemic clearance: 44 and 22%, respectively. On the contrary, the sex related difference observed for PAH systemic clearance was similar in percentage to that obtained for PAH renal clearance. This different behavior of PAH and FS might be account for the hepatic clearance of FS. In this sense, it has been reported that females have a higher organic anions hepatic clearance than males [15, 29].
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In summary, sex modifies the pharmacokinetics of organic anions. Female rats display a lower PAH and FS systemic clearance as consequence of its lower renal clearance. Although additional experimental work must be done to bridge the gap between studies using animals and humans, the reported experimental observations may have potentially important pharmacological implications. So, caution must be exercized in administering negatively charge organic drugs to females.
ACKNOWLEDGEMENTS This study was supported by the following grants: FONCYT (PICT 05-06160), Fundaci´on Antorchas, CONICET, Beca ‘Ram´on Carrillo-Arturo O˜nativia 2001’ and Universidad Nacional de Rosario (PID-UNR). The authors also thank Wiener Lab Argentina for analytical kits.
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EFFECTS OF GENDER ON THE PHARMACOKINETICS OF DRUGS SECRETED BY THE RENAL ORGANIC ANIONS TRANSPORT SYSTEMS IN THE RAT JORGELINA A. CERRUTTI, NORA B. QUAGLIA, ANABEL BRANDONI and ∗ ˙ ADRIANA M. TORRES Farmacolog´ıa, Facultad de Ciencias Bioqu´ımicas y Farmac´euticas, Universidad Nacional de Rosario, CONICET (2000) Rosario, Argentina Accepted 5 November, 2001
The importance of considering sex differences in drug handling studies was admitted recently. The present work evaluates the sex influences on the pharmacokinetics of para-aminohippuric acid (PAH), the reference substance for the renal organic anion transports systems, and furosemide (FS), a standard loop diuretic which is also a substrate for this transport system. Female rats displayed a lower PAH and FS systemic clearance, and a lower value of the elimination rate microconstant from the central compartment for both drugs. These results may be explained by the diminution of the renal clearance of both PAH and FS observed in females. In summary, sex modifies the pharmacokinetics of organic anions. Although additional experimental work must be done to bridge the gap between studies using animals and humans, the reported experimental observations may have potentially important pharmacological implications. So, caution must be exercised in c 2002 Elsevier Science Ltd administering drugs like organic anions to females.
K EY WORDS : organic anions, sex, pharmacokinetics, renal clearance, systemic clearance.
INTRODUCTION In drug development, therapeutic advantages should be demonstrable in the entire target patient population, and hence dose regimens should be tailored to the needs of the individual patient [1]. Pharmacokinetic studies are usually performed in young, healthy male volunteers [2]. However, gender can affect drug pharmacokinetics, and hence possibly influence efficacy [3–5]. Therefore, it is important, that gender effects on drug pharmacokinetics are clearly defined, to allow appropriate dosage recommendations to be made. Most drugs are excreted into the urine, either as unchanged form or as metabolites. The secretion of organic anions by the renal proximal tubule is very important in the elimination of drugs such as antibiotics, diuretics, hippurates, and nonsteroidal anti-inflammatory agents [6–8]. Because kidneys are sensitive to several hormones [7, 9], it is expected that the kinetics of organic anions will show significant differences related to individual’s gender. In this regard, in vitro studies agreed that accumulation of para-aminohippurate is significantly ∗ Corresponding author. Suipacha 531, Rosario, Postcode: 2000,
Argentina. E-mail: [email protected] 1043–6618/02/020107–06/$35.00/0
greater in renal cortical slices from male than that from female rats and that testosterone is involved in this event [10–13]. Thiazides and loop diuretics are secreted through the organic anion systems [14]. The capacity of the organic anion transport systems to secrete a diuretic determines its intraluminal concentration, which is critical for the diuretic activity [7]. In the present study, we have analysed the influence of sex on the pharmacokinetics of para-aminohippuric acid (PAH, the reference substance for the renal organic anion transport system) and furosemide (FS, a standard loop diuretic).
MATERIALS AND METHODS
Experimental animals Male and female Wistar rats aged from 110–130 days old were used throughout the study. Animals were allowed free access to a standard laboratory chow and tap water, and housed in a constant temperature and humidity environment with regular light cycles (12 h). All procedures were in accordance with institutional guidelines for the care and use of laboratory animals. c 2002 Elsevier Science Ltd
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Pharmacological Research, Vol. 45, No. 2, 2002
Pharmacokinetic studies These studies were performed in a manner similar to that previously described [15, 16]. Animals were anaesthetized with sodium thiopental (70 mg kg−1 b.w., i.p.). Both the femoral artery and the vein were both catheterized to obtain samples and to administer test compound, respectively. A single bolus of PAH (3 mg/100 g b.w., aqueous solution, i.v.) or FS (1 mg/100 g b.w., aqueous solution, i.v.) were administered. These doses do not saturate the transport system [11, 17], therefore, the kinetics can be adequately estimated. Blood samples were collected at 0–15 min time range for PAH and at 0–120 min range for FS from the femoral artery cannula. An equivalent volume of isotonic saline solution was infused to restore the amount extracted in the blood samples. Samples were centrifuged at 3000 rpm for 3 min, and the extracted plasmas were frozen as −20 ◦ C until analyzed. Concentration of PAH in plasma was measured using the method described by Waugh and Beall [18] and FS was assayed by the Bratton Marshall technique [19]. The concentration versus time curves for PAH and FS, for each individual animal were fitted with the PKCALC computer program [20]. The choice of the best fit was based on the determination coefficient values (R 2 ) and F test. The following equation was used to describe the biexponential concentration–time curves: Cp = Ae−αt + Be−βt where Cp is PAH plasma concentration (mg%) or FS plasma concentration (µg ml−1 ) at time t (min) after administration; constants α and β represent the disappearance rates for the distribution and elimination components, respectively, and the slopes of each of the adjusted curves give their values. A and B represent the initial concentrations of the distribution and elimination components, respectively, extrapolated from the y-axis intercept. The estimated parameters (α, β, A, B) were used to solve the first-order rate microconstants of transfer from central to peripheral compartments (k12 , k21 ) and the elimination rate microconstant from the central compartment (k10 ) with classical equations. Derived parameters: area under the concentration– time curve (AUC), total volume of distribution (VdT ), volume of the central compartment (VdC ), volume of the peripheral compartment (VdP ), steady-state volume of distribution (Vdss ), plasma clearance (Clp) and elimination half-life (t1/2 ) were calculated according to standard procedures for compartmental analysis.
Renal clearance studies These studies were performed as previously described [21, 22]. Briefly, the animals were anaesthetized with sodium thiopental (70 mg kg−1 b.w., i.p.). Both the femoral artery and vein were cannulated (P.E.50. Intramedic, USA.) and a bladder catheter (3 mm i.d.) was inserted through a suprapubic incision. Animals were maintained in restraining cages throughout the experiment to facilitate urine collection. A priming dose
Table I Pharmacokinetic parameters of p-aminohippurate in male and female rats after a single dose (3 mg/100 g b.w., i.v.) Males (n = 4) AUC (mg × sec/dl) A (mg%) B (mg%) α (min−1 ) β (min−1 ) k1−0 (min−1 ) k1−2 (min−1 ) k2−1 (min−1 ) t1/2 (k) (min) VdT (ml/100 g) VdC (ml/100 g) VdP (ml/100 g) Vss (ml/100 g)
5176 ± 453 19.88 ± 1.45 8.10 ± 0.70 1.40 ± 0.18 0.12 ± 0.01 0.328 ± 0.020 0.69 ± 0.11 0.49 ± 0.08 128 ± 8.72 30.81 ± 1.80 10.83 ± 0.62 20.54 ± 2.21 25 ± 0.40
Females (n = 4) 8411 ± 902∗ 20.45 ± 2.13 12.37 ± 1.30∗ 1.16 ± 0.07 0.10 ± 0.01 0.240 ± 0.020∗ 0.53 ± 0.06 0.51 ± 0.07 182 ± 20∗ 21.99 ± 2.43∗ 9.17 ± 0.33 13.72 ± 3.42∗ 17 ± 0.8∗
Results are expressed as means ± SE. ∗ P< 0.05. AUC = area under curve; constants α and β represent the disappearance rates for the distribution and elimination components, respectively; A and B represent the initial values of the distribution and elimination components, respectively; k1−2 , k2−1 = first-order rate microconstants of transfer from central to peripheral compartments; k1−0 = elimination rate microconstant from the central compartment; t1/2 (k) = elimination half-life (t1/2 ); VdT = total volume of distribution; VdC = volume of the central compartment; VdP = volume of the peripheral compartment; Vss = steady-state volume of distribution.
of PAH (3 mg/100 g b.w.) or FS (1 mg/100 g b.w.) in 1 ml of saline solution was administered through the venous catheter. Then, a solution containing PAH (0.6 g%) or FS (0.20 g%) and saline solution (0.9 g%) was infused through the venous catheter employing a constant infusion pump (Pump 22; Harvard Apparatus, USA) at a rate of 1 ml h−1 /100 g b.w. After equilibration for 60 min, urine was collected during two 30 min periods. Blood from the femoral artery was obtained at the midpoint of each clearance period. Arterial blood pressure was estimated throughout the experiments with a manometer inserted in the femoral artery. Renal clearance of PAH and renal clearance of FS were calculated by conventional formulae for each animal. PAH and FS concentrations in serum and urine were determined as described above. Volume of urine was determined by gravimetry.
Materials Chemicals were purchased from Sigma (St. Louis, MO, USA) and were analytical grade pure.
Statistical analysis Significant differences between groups were determined using Student’s t-test. The probability level chosen was P< 0.05. RESULTS Plasma PAH and FS concentrations in male and female adult rats are shown in Fig. 1. For both drugs, male rats displayed a significant lower area under the curve as compared with females.
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(a)
(b)
Fig. 1. (a) Mean plasma concentration–time profiles and area under the curve of: (a) p-aminohippurate (PAH) in male (n = 4, R 2 = 0.989, F = 7.9) and female (n = 4, R 2 = 0.993, F = 8.4) rats; (b) furosemide (FS) in male (n = 9, R 2 = 0.991; F = 11.5) and female (n = 8, R 2 = 0.998, F = 11.2) rats. Results are expressed as means ± SD. (#) P< 0.05.
Pharmacokinetic parameters obtained in both sexes for PAH and FS are listed in Table I and II, respectively. Females showed a lower peripheral volume of distribution for PAH as compared with male rats. On the contrary, no sex differences were observed in the distribution volumes for FS. Females showed a significant lower systemic clearance and elimination rate microconstants (k1−0 ) for both PAH and FS as illustrated in Fig. 2. Figure 3 shows the hybrid rate constants for slopes in distribution and elimination phase (α and β) for PAH and FS. Both first-order hybrid rate constants for FS were statistically lower in females as compared with males. No gender differences were observed in α and β for PAH. We have also evaluated both the systemic and renal clearance of PAH and FS. As shown in Table III, in both cases a statistically significant decrease in values of female rats as compared with males was observed.
Moreover, a greater diminution in FS renal clearance as compared with FS systemic clearance was observed in females. Instead, a similar decrease in the renal and systemic clearance of PAH was shown by female rats. DISCUSSION In the past, women were under-represented as participants in clinical drug studies [23, 24]. In the majority of cases when women and men were both studied, the results were grouped together and there was no analysis of sex differences [23]. Recently, it has been admitted the importance of considering sex in drug handling studies. The results of animal studies should be used to illustrate the fact that significant sex differences in drug metabolism and elimination can occur in mammals, and should provide impetus for sex-based research in humans.
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Fig. 2. Systemic clearance and elimination rate microconstant from the central compartment (k1−0 ) of p-aminohippurate (PAH) and furosemide (FS) in male and female rats. Results are expressed as means ± SE. (#) P< 0.05.
Table II Pharmacokinetic parameters of furosemide in male and female rats after a single dose (1 mg/100 g b.w., i.v.)
AUC (ug × min/ml) A (ug/ml) B (ug/ml) α(min−1 ) β(min−1 ) k1−0 (min−1 ) k1−2 (min−1 ) k2−1 (min−1 ) t1/2 (k) (min) VdT (ml/100 g) VdC (ml/100g) VdP (ml/100 g) Vss (ml/100 g)
Males (n = 9)
Females (n = 8)
2991 ± 211 67.73 ± 5.38 51.33 ± 5.87 0.592 ± 0.128 0.01809 ± 0.00166 0.0407 ± 0.0035 0.2420 ± 0.0650 0.2630 ± 0.0830 18.09 ± 1.62 19.88 ± 1.80 8.68 ± 0.46 11.21 ± 1.63 17.91 ± 1.29
3709 ± 68∗ 74.42 ± 6.28 38.03 ± 2.91 0.221 ± 0.031∗ 0.01181 ± 0.00065∗ 0.0304 ± 0.0014∗ 0.1184 ± 0.0175 0.0878 ± 0.0160 23.16 ± 1.15∗ 23.96 ± 1.08 8.99 ± 0.36 15.03 ± 1.18 19.90 ± 1.11
Results are expressed as means ± SE. ∗ P< 0.05. AUC = area under curve; constants α and β represent the disappearance rates for the distribution and elimination components, respectively; A and B represent the initial values of the distribution and elimination components, respectively; k1−2 , k2−1 = first-order rate microconstants of transfer from central to peripheral compartments; k1−0 = elimination rate microconstant from the central compartment; t1/2 (k) = elimination half-life (t1/2 ); VdT = total volume of distribution; VdC = volume of the central compartment; VdP = volume of the peripheral compartment; Vss = steady-state volume of distribution.
Renal excretion is a major route of elimination for many drugs and especially for their hydrophilic metabolites. The processes by which a drug is excreted may be any combination of glomerular filtration, tubular secretion and tubular re-absorption. The net excretion resulting from these processes can be modulated by numerous factors, like renal blood flow, protein binding, and urine pH and flow [7]. Carrier-mediated transport of
drugs is confined to the proximal tubule, and separate carrier systems exist for the active secretion of organic cations and anions. The organic anions transport systems play a critical role in the elimination of a large number of drugs (e.g. antibiotics, chemotherapeutics, diuretics, non-steroidal anti-inflamatory drugs, radiocontranst agents, cytostatics). Previous studies have suggested that testosterone might modulate these organic anion transport systems [10, 12, 13]. The present study was undertaken to evaluate whether sex influences the pharmacokinetics of organic anions. We selected PAH, the reference substance for the organic anion transport systems, and FS, a standard loop diuretic that is also a substrate for this system. Our data reveal that male rats have a higher systemic depuration of both PAH and FS than female rats. The lower absolute value observed for the systemic clearance of FS as compared with systemic clearance of PAH in both sexes may be explained by their different binding to the plasma proteins: 91–99% of FS and only a fraction (lower than 25%) of PAH are bound to plasma proteins [7]. Moreover, sex differences in plasma protein binding of FS might account for the gender differences observed in the disappearance rates for the distribution and elimination components. In this sense, sex differences in plasma protein binding have been observed for several drugs [25–28]. On the other hand, we did not observe sex differences in PAH disappearance rate for the distribution phase. In this connection, no sex difference in PAH plasma protein binding have been reported [13]. Central volume of distribution for PAH was similar in both sexes. By contrast, the peripheral and steady state volumes of distribution were higher in males. The gender differences in these volumes may be explained by the
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Fig. 3. Disappearance constants rates for the distribution (α) and elimination (β) components of p-aminohippurate (PAH) and furosemide (FS) in male and female rats. Results are expressed as means ± SE. (#) P< 0.05. Table III Absolute and percentage values of systemic and renal clearance of p-aminohippurate and furosemide in male and female rats p-Aminohippurate Males
Furosemide Females
Males
Females
Systemic clearance (ml/min/100 g)
4.10 ± 0.36 (n = 4) 100 ± 8.78%
(n = 4) 61.98 ± 4.89∗ %
0.344 ± 0.021 (n = 9) 100 ± 6.10 %
0.265 ± 0.004# (n = 8) 77.27 ± 1.39# %
Renal clearance (ml/min/100 g)
2.73 ± 0.477 (n = 6) 100 ± 17.95%
1.49 ± 0.22∗ (n = 6) 53.85 ± 8.05∗ %
0.295 ± 0.031 (n = 6) 100 ± 10.51%
0.165 ± 0.030# (n = 6) 55.94 ± 4.17#,& %
2.54 ± 0.20∗
Results are expressed as means ± SE. ∗ P< 0.05 vs the respective value in males. # P< 0.05 vs the respective value in males. & P< 0.05 vs the percentage value of systemic clearance in females.
fact that females tend to have a greater proportion of their body weight to be of fat than males and so PAH, which is an hydrosoluble drug, has a lesser space of distribution [4]. No sex related differences were observed in volumes of distribution for FS, which is a lesser hydrosoluble drug than PAH. The elimination rate microconstant from the central compartment (k1−0 ) decreased approximately of 25% in females as compared with male rats for both PAH and FS. This might indicate a sex related difference in the biotransformation and in the excretion of the drugs. PAH is subject to negligible metabolism, and is principally eliminated by renal tubule secretion. Approximately more than half the dose of FS is eliminated unchanged by the kidneys and the remainder is uptaken and metabolized by the liver. So we evaluated sex influence on the renal clearance of both organic anions. Our results demonstrated a diminution of PAH and FS renal clearance for females which explain the sex related difference observed in the systemic clearance of both organic anions. An important role in this sex
difference must be played by the organic anion transport systems which are involved in proximal tubular secretion of several drugs. Organic anions transporters have been recently cloned [6, 8, 30]. ROAT1 represents the classical basolateral PAH/dicarboxylate exchanger and has a broad substrate specificity [8, 31–34]. Uwai et al. [14] have recently demonstrated that ROAT1 contributes to the renal tubular secretion of FS. The role of ROAT1 in the sex related difference observed in the renal clearance of PAH and FS remains to be determined. FS renal clearance decreased in females as compared with males in a higher percentage than systemic clearance: 44 and 22%, respectively. On the contrary, the sex related difference observed for PAH systemic clearance was similar in percentage to that obtained for PAH renal clearance. This different behavior of PAH and FS might be account for the hepatic clearance of FS. In this sense, it has been reported that females have a higher organic anions hepatic clearance than males [15, 29].
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In summary, sex modifies the pharmacokinetics of organic anions. Female rats display a lower PAH and FS systemic clearance as consequence of its lower renal clearance. Although additional experimental work must be done to bridge the gap between studies using animals and humans, the reported experimental observations may have potentially important pharmacological implications. So, caution must be exercized in administering negatively charge organic drugs to females.
ACKNOWLEDGEMENTS This study was supported by the following grants: FONCYT (PICT 05-06160), Fundaci´on Antorchas, CONICET, Beca ‘Ram´on Carrillo-Arturo O˜nativia 2001’ and Universidad Nacional de Rosario (PID-UNR). The authors also thank Wiener Lab Argentina for analytical kits.
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