BIOPHARMACEUTICS & DRUG DISPOSITION, VOL. 13, 703-709 (1992)
PHARMACOKINETICS OF PROGESTERONE IN OVARIECTOMIZED RATS AFTER SINGLE DOSE INTRAVENOUS ADMINISTRATION NUTAN K. GANGRADE', F. DOUGLAS BOUDINOT and JAMES C. PRICE Department of Pharmaceutics, College of Pharmacy, The University of Georgia, Athens, Georgia 30602, U.S.A.
ABSTRACT The pharmacokinetics of progesterone were characterized in ovariectomized female rats. Progesterone was administered intravenously at a dose of 500 pg kg-'. Serum progesterone concentrations were determined by radioimmunoassay. Serum concentrations of progesterone were best described by a two-compartment model with elimination from the central compartment. The distribution and elimination phase half-lives were 0.13 ? 0-024 (mean 5 SD) and 1.21 k 0.21 h, respectively. Elimination of the steroid was rapid with a total clearance of 2.75k0.421 h-I kg-'. Progesterone was widely distributed in the rat with a steady state volume of distribution of 2.36kO-23 1 kg-I, a volume of the central compartment of 0-86+0.24 1 kg-' and a volume of the peripheral compartment of 1.50+0.19 1 kg-I. The results of this study suggest that the ovariectomized female rat is a suitable animal model for examining the pharmacokinetics of progesterone. KEY WORDS
Progesterone Pharmacokinetics Ovariectomized female rats
INTRODUCTION
Progesterone is a steroid hormone secreted mainly by mammalian ovary and placenta. Small amounts of the hormone are also secreted by the testis and adrenal cortex. Clinically, progesterone is used for contraception and for the treatment of clinical conditions such as dysfunctional uterine bleeding, dysmenorrhea, premenstrual tension, endometriosis, habitual abortion, suppression of post-partum lactation, and endometrial carcinoma. Use of progesterone has been limited mainly because of its low bioavailability after oral administration. Few studies have investigated the disposition of progesterone.2-6 In humans, progesterone is rapidly cleared, primarily by hepatic metabolism. Progesterone *Presentaddress: American Cyanamid Company, Marketed Product Research, Pearl River, NY 10965. Correspondence to: Dr James C. Price, Department of Pharmaceutics, College of Pharmacy, The University of Georgia, Athens, Georgia 30602, U.S.A.
0142-2782/92/090703-07$08.50 0 1992 by John Wiley & Sons, Ltd.
Received 6 January 1992 Accepted 20 April 1992
704
N. K . GANGRADE ET A L .
is readily absorbed from the gastro-intestinal tract, however, the steroid is rapidly biotransformed during passage through the intestinal mucosa and transit through the liver. Due to the extensive first pass metabolism of the hormone, bioavailability of progesterone after oral administration is very low. Most of the studies eximining the disposition of progesterone have employed nonintravenous administration, and thus assessment of pharmacokinetic parameters is confounded by drug absorption. A further complication in characterizing progesterone pharmacokinetics is the presence of endogenous hormone in the body and the cyclical nature of its secretion. Clinical studies necessitate either male subjects, ovariectomized female subjects or administration of radioactive hormone to study the pharmacokinetics of progesterone. Complete pharmacokinetic characterization of progesterone in humans or animals is not currently available. A laboratory animal model will be useful for providing insight into the disposition of progesterone. The objective of this study was to characterize the pharmacokinetics of progesterone and to assess the suitability of the ovariectomized female rat as an animal model. This will facilitate further elucidation of the pharmacokinetics and pharmacodynamics of progesterone and also studies pertaining to improvement of steroid dosing using formulation approaches. METHODS Experimental design
Five adult ovariectomized female Sprague-Dawleyrats (Charles River Breeding Laboratories, Wilmington, MA) weighing 250-300 g were used for the pharmacokinetic studies. The rats were housed in a 12 h light/l2 h dark cycle with controlled temperature (22 "C). Food (Purina Lab Chow 5001, Ralston Purina Co., St. Louis, MO) and water were provided ad libitum. The animals were allowed an acclimation period of 1 week before starting the experiment. One day before the experiment, cannulas were surgically implanted in the right exterior jugular vein of the rats. An intramuscular injection of ketamine :acepromazine: xylazine (40 :2.7 :2.7 mg kg - l) solution was used to induce anesthesia. Each rat received a single intravenous dose of 500pg kg- progesterone dissolved in 150 pl of a 1 : 1 mixture of propylene glycol and physiologic saline. The drug was administered over a period of 1 min through the cannula. Pure propylene glycol, when injected intravenously in doses up to 2m1, was well tolerated by rats and did not have any observable toxic effects on the rats. Blood samples (100 pl) were collected from the cannula prior to the injection of the dose and at 0.08, 0.25, 0-50, 0-75, 1, 1-5, 2, 3, 4, 5 , and 6 h after the dose. Blood volume was replaced with an equal volume of normal saline. After 30 min, clotted blood samples were centrifuged and serum (10 pl) was diluted with 990pl water and frozen at -20 "C until analysis.
PROGESTERONE IN OVARIECTOMIZED RATS
705
The progesterone concentrations in duplicate serum samples were determined by a radioimmunoassay (RIA) using antiserum developed in rabbits (Sigma Chemical Co., St. Louis, MO). Briefly, lop1 serum sample was placed in polypropylene tubes, diluted to 1 ml with distilled deionized water and 2ml anhydrous ethyl ether was added. Tubes were vigorously mixed for 20 s, allowed to settle, and the aqueous phase was frozen in a dry icelacetone bath. The ether phase was decanted into a polypropylene tube and evaporated to dryness at 45 "C under a stream of nitrogen gas. Antiserum (0.5ml) was added, tubes were vigorously mixed and incubated at 22 "C for 30 min. Tritiated progesterone (0-1 ml) was added, tubes were mixed, incubated at 37 "C for 60 min and then cooled at 4 "C for 15 min. Ice cold dextran-coated charcoal (0.2 ml) was rapidly added, tubes were mixed and incubated at 0 "C in an ice/water bath for 10 min. Tubes were centrifuged at 2000~ g at 4 "C for 15 min. The supernatant was transferred to scintillation vials, 5 ml scintillation cocktail was added, and radioactivity was determined using a Beckman LS 7500 liquid scintillation counter (Beckman Instruments, Inc., Irvine, CA). Concentrations of progesterone were calculated from standard curves (loglogit plots) prepared daily. The limit of quantitation of the assay was 5 ng ml- I . Cross-reactivity of the assay for endogenous hormones and known metabolites of progesterone was less than 6 per cent. The coefficients of variation of the assay determined at 10 ng ml- and 400 ng ml- were 14- 8 per cent and 5 -49 per cent, respectively.
Pharmacokinetic analysis Noncompartmental (area/moment) analysis of serum progesterone concentration data was used initially to determine pharmacokinetic parameters. The area under the serum progesterone versus time curve (AUC) and the first moment (AUMC) were calculated by Lagrange polynomial interpolation and integration from time zero to the last sample time7**with extrapolation to time infinity using the nonlinear least-squares terminal slope.9 Total systemic clearance (CL,) was calculated from Dose/AUC, mean residence time (MRT) from from MRT*CLT. AUMC/AUC, and steady state volume of distribution ( Vss) Serum progesterone concentration data were further analyzed by compartmental analysis to calculate micro-rate constants and volumes of distribution. The Akaike's information criterion (AIC)Io and lack of systematic deviations around fitted curves were used to select the best exponential equation to fit the data. Concentrations of progesterone were best characterized by a biexponential decline, therefore, a two-compartment model with elimination from the central compartment was fitted to the drug disposition data. According to this model, serum progesterone concentrations (C,) as a function of time ( t ) can be described by:
706
N. K. GANGRADE ET A L .
where A and B are the hybrid ordinate intercepts corresponding to the distribution and elimination phases, respectively, and a and /3 are the hybrid rate constants (slope values) representing the distribution and elimination phases. The two-compartment model was fitted to the serum progesterone concentration versus time data by NONLIN least-squares regres~ion.~ Reciprocal C, values were found to be acceptable weighting factors for generating a normal distribution of weighted residuals in NONLIN. Values of coefficients A and B and rate constants a and /3 were generated by NONLIN fitting of the serum progesterone concentration data. Distribution (t%,J and elimination ( t % , J half-lives were calculated from 0 - 693/a and 0*693/&respectively. The intercornpartrnental transfer rate constant from the peripheral compartment (k2J was calculated from (A*p+ B*a)/(A+ B), the elimination rate constant from the central compartment (klo)from a*/3/k21, and the transfer rate constant from the central compartment (k12)from a + /3 - klo- k2,. l l AUC, AUMC, and MRT were calculated from A / a + B / P , A / a 2+ B / p 2 , and AUMC/AUC, respectively. Total clearance (CLT) was calculated from Dose/AUC, V,, from CLT*MRT, volume of the central compartment (V,) from Dose/(A + B), volume of the peripheral compartment (V,) from V,, - V,, and intercompartmental distributional clearance (CLd) from VC*kl2.ll RESULTS AND DISCUSSION Serum progesterone concentrations as a function of time following single dose intravenous administration of 500 pg kg- progesterone to a representative rat are shown in Figure 1. No progesterone was detected in serum samples taken prior to the administration of the dose, indicating that progesterone secretion from the adrenal cortex was negligible. Initial concentrations of progesterone in the rat were approximately five-fold higher than observed clinically after intramuscular administration of 50 mg; however, by 30 min, progesterone levels had declined to values similar to those reported in clinical ~ t u d i e s .Serum ~ concentrations were measurable up to 3 h after administration of the steroid. The area covered by the sampling time was more than 90 per cent of the total AUC, indicating the accuracy of the pharmacokinetic analysis. Similar reliable characterization of the disposition of progesterone could not be attained using lower doses of the hormone as serum progesterone concentrations rapidly fell below the limit of quantitation of the analytical methodology. Estimates of CLT, V,, and MRT provided by noncompartmental analysis of the data were 2.70fO-301h-1kg-1, 2-49+0.371kg-1 and 0-93_+0-11h, respectively. Serum progesterone concentrations declined in a biexponential manner (Figure 1) and were best described by a twocompartment model. Pharmacokinetic parameters of progesterone in ovariectomized female rats after intravenous administration of 500 pg kg- derived from compartment analysis are presented
707
PROGESTERONE IN OVARIECTOMIZED RATS
E
\ (r
C
z-
E ! c4
!x t Z W 0
Z 0 V W
Z
0
IY W
t
m w
I
u
0 !x
a
TIME, h
Figure 1 . Representative serum progesterone concentrationsas a function of time following a single intravenous dose of 500pg kg-' to a rat. Symbols are experimental data and the curve is the leastsquares line fitted by a two-compartment model
in Table 1. Values for pharmacokinetic parameters, CLT and Vss, generated from the compartmental and noncompartmental analyses were virtually identical. Distribution of progesterone was rapid with a tfi,aof approximately 8 min. The elimination half-life of progesterone in ovariectomized female rats was 1 -2h. In reports on investigations of progesterone disposition in humans, estimates of half-life ranged from 0.5 to 1.6 h.4,5Estimates of half-life varied in these studies probably due to differences in subjects and in experimental Table 1. Pharmamkinetic parameters determined by compartmental analysis of progesterone after intravenous administration of 500 pg kg-I to ovariectomized female rats Parameter
Mean
SD
a (h-l) tvl..a (h)
5.46 0.13
P m-9
0.60
t E , @(h)
1.21 2.75 2.36 0.86
0.90 0.024 0.12 0.21 0.42 0.23 0.24
CL, (1 h-I kg-')
v,, (1 kg - 9 v, (1 kg-') v, (1 kg- 9
kio (h-9 k12 (h - 9 kZl (h-9 CLd (1 h-' kg-')
1-50 0.19 0-36 0.60
3.30 1.80 1-02 1.49
0.30 0.42
708
N. K. GANGRADE ET A L .
procedures. The half-life obtained in rats in the present study is comparable to the values observed in clinical studies. Progesterone is rapidly eliminated from the rat with a total clearance of 2.75 1 h-I kg-'. Similar to other lipophilic steroid hormones, progesterone is eliminated from the body primarily by hepatic m e t a b ~ l i s m . ~Several . ~ . ~ metabolites of progesterone including many pregnane derivatives and isomers conjugated with glucuronide and sulfate have been identified.576The total clearance of progesterone in the rat approaches hepatic blood flow (3.53 1 h-' kg-l)'2 suggesting that the hormone can be considered a high clearance compound. Indeed, assuming that total clearance of progesterone is essentially hepatic clearance, the intrinsic clearance (CL,) of the hormone is 12-51 h-I kg-I in the rat. A similar high clearance of the steroid is observed in humans.' Certainly, the low bioavailability of progesterone after oral administration is primarily the result of the high first pass metabolism of the steroid owing to this high intrinsic clearance. The volume of distribution of the central compartment (0.86 1 kg-') is 11-fold greater than the volume of blood (0.0761 kg-I)l2 indicating that the drug is widely distributed in extravascular fluids and tissues. The steady state volume of distribution value of 2.41 kg-I is greater than total body water (0.7 1 kg-')12 of the rat, further confirming intracellular distribution of progesterone. This is not unexpected owing to the lipophilic nature of the drug that enables it to cross cell membranes and partition into tissue^.^ Distribution parameter values in human subjects have not been reported. In conclusion, the disposition of progesterone in the rat can be described by a two-compartment model. Both compartmental and noncompartmental methods of data analysis are reliable for determining the pharmacokinetic parameters of progesterone. The steroid is widely distributed and rapidly cleared in the rat. The results of this study suggest that the ovariectomized female rat is a suitable animal model for examining the pharmacokinetics of progesterone. REFERENCES 1. F. Murad and R. C. Haynes, Jr., Estrogens and progestins, in Goodman and Gilman's The
2. 3. 4.
5. 6. 7.
Pharmacologic Basis of Therapeutics, A. G. Gilman, L. S. Goodman and A. Gilman (Eds), 6th edn, Macmillan, New York, 1986, pp 1420-1447. M. I. Whitehead, P. T. Townsend, D. K. Gill, W. P. Collins and S. Campbell, Absorption and metabolism of oral progesterone, Br. Med. J., 280, 825-827 (1980). S. J . Nillius and E. D. B. Johansson, Plasma levels of progesterone after vaginal, rectal or intramuscular administration, Amer. J. Obsrer. Gynecol., 110,470-477 (1971). P. Fylling, Disappearance rate of progesterone following simultaneous removal of the corpus luteum and the foeto-placental unit in woman. Acta Endocrinologica, 65,284-294 (1970). J. H. H. Thijssen and J. Zander, Proge~terone-4-'~C and its metabolites in the blood after intravenous injection into woman, Acta Endocrinologica, 51, 563-577 (1966). H. Adlercreutz and F. Martin, Biliary and intestinal metabolism of progesterone and estrogens in man, J. Steroid Biochem., 13, 231-244 (1980). M. L. Rocci and W. J. Jusko, LAGRAN Program for area and moments in pharmacokinetic analysis, Comp. Prog. Biomed., 16, 203-216 (1983).
PROGESTERONE IN OVARIECTOMIZED RATS
709
8. K. C. Yeh and K. C. Kwan, A comparison of numerical integrating algorithms by trapezoidal, Lagrange and Spline Approximation, J. Phurmucokinet. Biopharm., 6 , 79-98 (1978). 9. C. M. Metzler and D. L. Weiner, PCNONLIN, Statistical Consultants, Inc., Edgewood, KY, 1989. 10. K. Yamaoka, T. Nakagawa and T. Uno,Application of Akaike’s Information Criterion (AIC) in the evaluation of linear pharmacokinetic equations, J. Phurmucokinet. Biophurm., 1978, 6, 165-175. 11. M. GibaIdi and D. Perrier, Pharmucokinetics, 2nd edn, Marcel Dekker, New York, 1982, pp. 45-111. 12. H. Harashima, Y. Sawada, Y. Sugiyama, T. Iga and M. Hanano, Analysis of nonlinear tissue distribution of quinidine in rats by physiologically based pharmacokinetics, J. Phurmucokiner. Biophurm., 13, 425-440 (1985).
PHARMACOKINETICS OF PROGESTERONE IN OVARIECTOMIZED RATS AFTER SINGLE DOSE INTRAVENOUS ADMINISTRATION NUTAN K. GANGRADE', F. DOUGLAS BOUDINOT and JAMES C. PRICE Department of Pharmaceutics, College of Pharmacy, The University of Georgia, Athens, Georgia 30602, U.S.A.
ABSTRACT The pharmacokinetics of progesterone were characterized in ovariectomized female rats. Progesterone was administered intravenously at a dose of 500 pg kg-'. Serum progesterone concentrations were determined by radioimmunoassay. Serum concentrations of progesterone were best described by a two-compartment model with elimination from the central compartment. The distribution and elimination phase half-lives were 0.13 ? 0-024 (mean 5 SD) and 1.21 k 0.21 h, respectively. Elimination of the steroid was rapid with a total clearance of 2.75k0.421 h-I kg-'. Progesterone was widely distributed in the rat with a steady state volume of distribution of 2.36kO-23 1 kg-I, a volume of the central compartment of 0-86+0.24 1 kg-' and a volume of the peripheral compartment of 1.50+0.19 1 kg-I. The results of this study suggest that the ovariectomized female rat is a suitable animal model for examining the pharmacokinetics of progesterone. KEY WORDS
Progesterone Pharmacokinetics Ovariectomized female rats
INTRODUCTION
Progesterone is a steroid hormone secreted mainly by mammalian ovary and placenta. Small amounts of the hormone are also secreted by the testis and adrenal cortex. Clinically, progesterone is used for contraception and for the treatment of clinical conditions such as dysfunctional uterine bleeding, dysmenorrhea, premenstrual tension, endometriosis, habitual abortion, suppression of post-partum lactation, and endometrial carcinoma. Use of progesterone has been limited mainly because of its low bioavailability after oral administration. Few studies have investigated the disposition of progesterone.2-6 In humans, progesterone is rapidly cleared, primarily by hepatic metabolism. Progesterone *Presentaddress: American Cyanamid Company, Marketed Product Research, Pearl River, NY 10965. Correspondence to: Dr James C. Price, Department of Pharmaceutics, College of Pharmacy, The University of Georgia, Athens, Georgia 30602, U.S.A.
0142-2782/92/090703-07$08.50 0 1992 by John Wiley & Sons, Ltd.
Received 6 January 1992 Accepted 20 April 1992
704
N. K . GANGRADE ET A L .
is readily absorbed from the gastro-intestinal tract, however, the steroid is rapidly biotransformed during passage through the intestinal mucosa and transit through the liver. Due to the extensive first pass metabolism of the hormone, bioavailability of progesterone after oral administration is very low. Most of the studies eximining the disposition of progesterone have employed nonintravenous administration, and thus assessment of pharmacokinetic parameters is confounded by drug absorption. A further complication in characterizing progesterone pharmacokinetics is the presence of endogenous hormone in the body and the cyclical nature of its secretion. Clinical studies necessitate either male subjects, ovariectomized female subjects or administration of radioactive hormone to study the pharmacokinetics of progesterone. Complete pharmacokinetic characterization of progesterone in humans or animals is not currently available. A laboratory animal model will be useful for providing insight into the disposition of progesterone. The objective of this study was to characterize the pharmacokinetics of progesterone and to assess the suitability of the ovariectomized female rat as an animal model. This will facilitate further elucidation of the pharmacokinetics and pharmacodynamics of progesterone and also studies pertaining to improvement of steroid dosing using formulation approaches. METHODS Experimental design
Five adult ovariectomized female Sprague-Dawleyrats (Charles River Breeding Laboratories, Wilmington, MA) weighing 250-300 g were used for the pharmacokinetic studies. The rats were housed in a 12 h light/l2 h dark cycle with controlled temperature (22 "C). Food (Purina Lab Chow 5001, Ralston Purina Co., St. Louis, MO) and water were provided ad libitum. The animals were allowed an acclimation period of 1 week before starting the experiment. One day before the experiment, cannulas were surgically implanted in the right exterior jugular vein of the rats. An intramuscular injection of ketamine :acepromazine: xylazine (40 :2.7 :2.7 mg kg - l) solution was used to induce anesthesia. Each rat received a single intravenous dose of 500pg kg- progesterone dissolved in 150 pl of a 1 : 1 mixture of propylene glycol and physiologic saline. The drug was administered over a period of 1 min through the cannula. Pure propylene glycol, when injected intravenously in doses up to 2m1, was well tolerated by rats and did not have any observable toxic effects on the rats. Blood samples (100 pl) were collected from the cannula prior to the injection of the dose and at 0.08, 0.25, 0-50, 0-75, 1, 1-5, 2, 3, 4, 5 , and 6 h after the dose. Blood volume was replaced with an equal volume of normal saline. After 30 min, clotted blood samples were centrifuged and serum (10 pl) was diluted with 990pl water and frozen at -20 "C until analysis.
PROGESTERONE IN OVARIECTOMIZED RATS
705
The progesterone concentrations in duplicate serum samples were determined by a radioimmunoassay (RIA) using antiserum developed in rabbits (Sigma Chemical Co., St. Louis, MO). Briefly, lop1 serum sample was placed in polypropylene tubes, diluted to 1 ml with distilled deionized water and 2ml anhydrous ethyl ether was added. Tubes were vigorously mixed for 20 s, allowed to settle, and the aqueous phase was frozen in a dry icelacetone bath. The ether phase was decanted into a polypropylene tube and evaporated to dryness at 45 "C under a stream of nitrogen gas. Antiserum (0.5ml) was added, tubes were vigorously mixed and incubated at 22 "C for 30 min. Tritiated progesterone (0-1 ml) was added, tubes were mixed, incubated at 37 "C for 60 min and then cooled at 4 "C for 15 min. Ice cold dextran-coated charcoal (0.2 ml) was rapidly added, tubes were mixed and incubated at 0 "C in an ice/water bath for 10 min. Tubes were centrifuged at 2000~ g at 4 "C for 15 min. The supernatant was transferred to scintillation vials, 5 ml scintillation cocktail was added, and radioactivity was determined using a Beckman LS 7500 liquid scintillation counter (Beckman Instruments, Inc., Irvine, CA). Concentrations of progesterone were calculated from standard curves (loglogit plots) prepared daily. The limit of quantitation of the assay was 5 ng ml- I . Cross-reactivity of the assay for endogenous hormones and known metabolites of progesterone was less than 6 per cent. The coefficients of variation of the assay determined at 10 ng ml- and 400 ng ml- were 14- 8 per cent and 5 -49 per cent, respectively.
Pharmacokinetic analysis Noncompartmental (area/moment) analysis of serum progesterone concentration data was used initially to determine pharmacokinetic parameters. The area under the serum progesterone versus time curve (AUC) and the first moment (AUMC) were calculated by Lagrange polynomial interpolation and integration from time zero to the last sample time7**with extrapolation to time infinity using the nonlinear least-squares terminal slope.9 Total systemic clearance (CL,) was calculated from Dose/AUC, mean residence time (MRT) from from MRT*CLT. AUMC/AUC, and steady state volume of distribution ( Vss) Serum progesterone concentration data were further analyzed by compartmental analysis to calculate micro-rate constants and volumes of distribution. The Akaike's information criterion (AIC)Io and lack of systematic deviations around fitted curves were used to select the best exponential equation to fit the data. Concentrations of progesterone were best characterized by a biexponential decline, therefore, a two-compartment model with elimination from the central compartment was fitted to the drug disposition data. According to this model, serum progesterone concentrations (C,) as a function of time ( t ) can be described by:
706
N. K. GANGRADE ET A L .
where A and B are the hybrid ordinate intercepts corresponding to the distribution and elimination phases, respectively, and a and /3 are the hybrid rate constants (slope values) representing the distribution and elimination phases. The two-compartment model was fitted to the serum progesterone concentration versus time data by NONLIN least-squares regres~ion.~ Reciprocal C, values were found to be acceptable weighting factors for generating a normal distribution of weighted residuals in NONLIN. Values of coefficients A and B and rate constants a and /3 were generated by NONLIN fitting of the serum progesterone concentration data. Distribution (t%,J and elimination ( t % , J half-lives were calculated from 0 - 693/a and 0*693/&respectively. The intercornpartrnental transfer rate constant from the peripheral compartment (k2J was calculated from (A*p+ B*a)/(A+ B), the elimination rate constant from the central compartment (klo)from a*/3/k21, and the transfer rate constant from the central compartment (k12)from a + /3 - klo- k2,. l l AUC, AUMC, and MRT were calculated from A / a + B / P , A / a 2+ B / p 2 , and AUMC/AUC, respectively. Total clearance (CLT) was calculated from Dose/AUC, V,, from CLT*MRT, volume of the central compartment (V,) from Dose/(A + B), volume of the peripheral compartment (V,) from V,, - V,, and intercompartmental distributional clearance (CLd) from VC*kl2.ll RESULTS AND DISCUSSION Serum progesterone concentrations as a function of time following single dose intravenous administration of 500 pg kg- progesterone to a representative rat are shown in Figure 1. No progesterone was detected in serum samples taken prior to the administration of the dose, indicating that progesterone secretion from the adrenal cortex was negligible. Initial concentrations of progesterone in the rat were approximately five-fold higher than observed clinically after intramuscular administration of 50 mg; however, by 30 min, progesterone levels had declined to values similar to those reported in clinical ~ t u d i e s .Serum ~ concentrations were measurable up to 3 h after administration of the steroid. The area covered by the sampling time was more than 90 per cent of the total AUC, indicating the accuracy of the pharmacokinetic analysis. Similar reliable characterization of the disposition of progesterone could not be attained using lower doses of the hormone as serum progesterone concentrations rapidly fell below the limit of quantitation of the analytical methodology. Estimates of CLT, V,, and MRT provided by noncompartmental analysis of the data were 2.70fO-301h-1kg-1, 2-49+0.371kg-1 and 0-93_+0-11h, respectively. Serum progesterone concentrations declined in a biexponential manner (Figure 1) and were best described by a twocompartment model. Pharmacokinetic parameters of progesterone in ovariectomized female rats after intravenous administration of 500 pg kg- derived from compartment analysis are presented
707
PROGESTERONE IN OVARIECTOMIZED RATS
E
\ (r
C
z-
E ! c4
!x t Z W 0
Z 0 V W
Z
0
IY W
t
m w
I
u
0 !x
a
TIME, h
Figure 1 . Representative serum progesterone concentrationsas a function of time following a single intravenous dose of 500pg kg-' to a rat. Symbols are experimental data and the curve is the leastsquares line fitted by a two-compartment model
in Table 1. Values for pharmacokinetic parameters, CLT and Vss, generated from the compartmental and noncompartmental analyses were virtually identical. Distribution of progesterone was rapid with a tfi,aof approximately 8 min. The elimination half-life of progesterone in ovariectomized female rats was 1 -2h. In reports on investigations of progesterone disposition in humans, estimates of half-life ranged from 0.5 to 1.6 h.4,5Estimates of half-life varied in these studies probably due to differences in subjects and in experimental Table 1. Pharmamkinetic parameters determined by compartmental analysis of progesterone after intravenous administration of 500 pg kg-I to ovariectomized female rats Parameter
Mean
SD
a (h-l) tvl..a (h)
5.46 0.13
P m-9
0.60
t E , @(h)
1.21 2.75 2.36 0.86
0.90 0.024 0.12 0.21 0.42 0.23 0.24
CL, (1 h-I kg-')
v,, (1 kg - 9 v, (1 kg-') v, (1 kg- 9
kio (h-9 k12 (h - 9 kZl (h-9 CLd (1 h-' kg-')
1-50 0.19 0-36 0.60
3.30 1.80 1-02 1.49
0.30 0.42
708
N. K. GANGRADE ET A L .
procedures. The half-life obtained in rats in the present study is comparable to the values observed in clinical studies. Progesterone is rapidly eliminated from the rat with a total clearance of 2.75 1 h-I kg-'. Similar to other lipophilic steroid hormones, progesterone is eliminated from the body primarily by hepatic m e t a b ~ l i s m . ~Several . ~ . ~ metabolites of progesterone including many pregnane derivatives and isomers conjugated with glucuronide and sulfate have been identified.576The total clearance of progesterone in the rat approaches hepatic blood flow (3.53 1 h-' kg-l)'2 suggesting that the hormone can be considered a high clearance compound. Indeed, assuming that total clearance of progesterone is essentially hepatic clearance, the intrinsic clearance (CL,) of the hormone is 12-51 h-I kg-I in the rat. A similar high clearance of the steroid is observed in humans.' Certainly, the low bioavailability of progesterone after oral administration is primarily the result of the high first pass metabolism of the steroid owing to this high intrinsic clearance. The volume of distribution of the central compartment (0.86 1 kg-') is 11-fold greater than the volume of blood (0.0761 kg-I)l2 indicating that the drug is widely distributed in extravascular fluids and tissues. The steady state volume of distribution value of 2.41 kg-I is greater than total body water (0.7 1 kg-')12 of the rat, further confirming intracellular distribution of progesterone. This is not unexpected owing to the lipophilic nature of the drug that enables it to cross cell membranes and partition into tissue^.^ Distribution parameter values in human subjects have not been reported. In conclusion, the disposition of progesterone in the rat can be described by a two-compartment model. Both compartmental and noncompartmental methods of data analysis are reliable for determining the pharmacokinetic parameters of progesterone. The steroid is widely distributed and rapidly cleared in the rat. The results of this study suggest that the ovariectomized female rat is a suitable animal model for examining the pharmacokinetics of progesterone. REFERENCES 1. F. Murad and R. C. Haynes, Jr., Estrogens and progestins, in Goodman and Gilman's The
2. 3. 4.
5. 6. 7.
Pharmacologic Basis of Therapeutics, A. G. Gilman, L. S. Goodman and A. Gilman (Eds), 6th edn, Macmillan, New York, 1986, pp 1420-1447. M. I. Whitehead, P. T. Townsend, D. K. Gill, W. P. Collins and S. Campbell, Absorption and metabolism of oral progesterone, Br. Med. J., 280, 825-827 (1980). S. J . Nillius and E. D. B. Johansson, Plasma levels of progesterone after vaginal, rectal or intramuscular administration, Amer. J. Obsrer. Gynecol., 110,470-477 (1971). P. Fylling, Disappearance rate of progesterone following simultaneous removal of the corpus luteum and the foeto-placental unit in woman. Acta Endocrinologica, 65,284-294 (1970). J. H. H. Thijssen and J. Zander, Proge~terone-4-'~C and its metabolites in the blood after intravenous injection into woman, Acta Endocrinologica, 51, 563-577 (1966). H. Adlercreutz and F. Martin, Biliary and intestinal metabolism of progesterone and estrogens in man, J. Steroid Biochem., 13, 231-244 (1980). M. L. Rocci and W. J. Jusko, LAGRAN Program for area and moments in pharmacokinetic analysis, Comp. Prog. Biomed., 16, 203-216 (1983).
PROGESTERONE IN OVARIECTOMIZED RATS
709
8. K. C. Yeh and K. C. Kwan, A comparison of numerical integrating algorithms by trapezoidal, Lagrange and Spline Approximation, J. Phurmucokinet. Biopharm., 6 , 79-98 (1978). 9. C. M. Metzler and D. L. Weiner, PCNONLIN, Statistical Consultants, Inc., Edgewood, KY, 1989. 10. K. Yamaoka, T. Nakagawa and T. Uno,Application of Akaike’s Information Criterion (AIC) in the evaluation of linear pharmacokinetic equations, J. Phurmucokinet. Biophurm., 1978, 6, 165-175. 11. M. GibaIdi and D. Perrier, Pharmucokinetics, 2nd edn, Marcel Dekker, New York, 1982, pp. 45-111. 12. H. Harashima, Y. Sawada, Y. Sugiyama, T. Iga and M. Hanano, Analysis of nonlinear tissue distribution of quinidine in rats by physiologically based pharmacokinetics, J. Phurmucokiner. Biophurm., 13, 425-440 (1985).
