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Development of a low-dose monthly injectable contraceptive system: II. Pharmacokinetic and pharmacodynamic studies
CONTRACEPTION
II.
DEVELOPMENT OF A LOW-DOSE MONTHLY INJECTABLE CONTRACEPTIVE SYSTEM: PHARMACOKINETIC AND PHARMACODYNAMIC STUDIES V. Diaz-Skchezl J. Garza-Flares' S. Jimen&-Tho as2 H.W. Rude1 ?
1Department of Reproductive Biology, Instituto National la Nutrici6n "Salvador Zubir&", Mexico City
de
2Department of Biochemistry, School of Medicine, Universidad National Autonoma de Mgxico, Mgxico City 3Fertility and Sterility Research Center, Me'xico City
Abstract A drug delivery system which provides a sustained release of norethindrone (NET) and mestranol (ME) for one month after a single intramuscular injection was assessed as a long-acting injectable contraceptive. The system is based upon well defined particle size crystals of the synthetic steroids maintained in suspension with saline solution. Eight healthy ovulating women volunteered for the study; they received a combination of 10 mg of NET plus 1 mg of ME in 1 ml of vehicle by intramuscular injection on day five of their menstrual cycle. Blood samples were drawn at 0, 1, 5, 10, 13, 17 and 21 days after drug administration. The immunoreactive serum levels of estradiol, progesterone, NET and ethinylestradiol were measured specific by radioimmunoassay procedures to assess ovarian function and the kinetic parameters of the synthetic steroids. This newly developed contraceptive system proved to be both effective, and long-lasting as well as devoid of side effects. Address correspondence to: V. Dlhz-Sgnchez, Department of Reproductive Biology, Institute National de la Nutrici6n "Salvador Zubirdn'!, Vasco de Quiroga No. 15, Tlalpan 14000 Mkxico, D.F.
Submitted for publication November 24, 1986 Accepted for publication December 9, 1986
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1987 VOL. 35 NO. 1
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CONTRACEPTION
Introduction Most of the information available from family planning programs in developing countries indicates an increase in acceptability for long-acting contraception These (1) observations have created the need for the development of a highly effective monthly injectable contraceptive exhibiting a low incidence of side effects. Maintenance of the contraceptive potency over a period of one month injectable hormonal formulation is possible; however, most of the preparations currently available cause profound disruptions of the endometrial bleeding pattern, particularly prolonged and heavy endometrial flow in early phases with a tendency toward periods of amenorrhea later on (2). The concept of a combined low-dose hormonal injectable given monthly to inhibit ovulation appears to be an attractive alternative. Accordingly a progestogen-estrogen formulation was recently developed (3). In an attempt to avoid the estrogen-related side effects observed following its administration, the formulation has been modified. The new formulation is a microcrystalline aqueous suspension in which ethinylestradiol (EE-2) has been replaced by mestranol. The aim of the present investigation was to assess the pharmacokinetics of the two synthetic steroids in the formulation and their effects upon the ovarian function during the treatment cycle. Materials and Methods Norethindrone (17 (J -ethinyl-17-hydroxy-4-estren-3-one), ethinylestradiol (17 Q -ethinyl-1,3,5(10)-estratrien-3,17diol) and mestranol (17a-ethinyl-1,3,5(10)-estratrien-3,17diol-3-methylester) were a gift of Syntex S.A. (Mexico). Chemical purity of the compounds (>99%) was determined by at infrared and least two of the following procedures: ultraviolet absorption and gas-liquid chromatographic (GLC) All the reagents used were of the highest purity behaviour. available and were used without further purification. In vitro dissolution studies -obtained as previously Crystals of EE-2 and ME were described (3). Dissolution rates of both synthetic steroids were analyzed following the same methodology (3) and the dissolution, expressed as a percentage of the total steroid against time on a added to the system, was plotted log/linear scale.
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Clinical data Eight healthy women aged 23.5 + 3.0 years, with a history of regular menstrual periods (30 + 2 days) in the preceding three months, volunteered for the clinical trial. None of them used any form of steroidal medication for a minimum of six months prior to admission to the study, and none of them All had been pregnant during the previous twelve months. subjects were informed of the purpose of the study and a written consent was obtained. Before admission to the study a full medical history and a complete physic1 examination All women were advised to was performed on each subject. use a barrier method of contraception. The body surface area (BSA) was calculated and the value was used as a correction factor in expressing the hormonal serum concentrations (4). On day five of their menstrual cycle each subject received by intragluteal injection a combination of 10 mg NET plus 1 mg ME in 1 ml saline solution as diluent. Residual active ingredients not delivered were measured in the needles and syringes using a GLC method. The actual quantity of steroid injected was recorded in each case. Blood samples were taken from an antecubital vein at: 0, 1, 5, 10, 13, 17 and 21 days after drug administration. Blood samples were allowed to clot at room temperature, centrifuged within one hour after sampling and the serum separated and kept at-20°C until analyzed. The concentrations of immunoreactive estradiol (E-Z), (P-4), NET and EE-2 in each sample were measured as described below. Radioimmunoassay
procedures
Ethinvlestradiol Ethinylestradiol, 17 c~6,7-~-H(N), specific activity 41.5 Ci/mmol and anti-ethinylestradiol-7-(3-thiopropionic acid) bovine serum albumin serum were obtained from New England Nucler (Boston, Mass.) and Research Triangle Institute (Research Triange Park, N.C.), respectively. Immunoreactive unconjugated EE-2 in serum samples was measured after solvent extraction the method following described by Stanczyk & al. (5) The sensitivity of the assay at a coefficient of variation (C.V.) of 15% was 21 pmol/L and a precision profile over a range of 42 to 676 pmol/L was ~10% C.V. Bias was assessed after addition of 169 and 845 pmol/L of EE-2 to blank serum samples, the recovery values were 109 2 9% and 91 + 12%, respectively.
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Norethindrone A direct method of unextracted serum samples was followed. The combined use of a high specific unpurified antiserum and heat treatment of diluted serum samples obviated both extraction and chromatographic procedures. The direct NET assay has been previously validated (6) and fulfilled all the quality control parameters, Estradiol and nroqesterone The naturally occurring steroids were measured according to the method manual of the Programme for the Provision of Matched Assay Reagents for Radioimmunoassay of Hormones in Reproductive Physiology of the World Health Organization, Special Programme of Research, Development and Research Training in Human Reproduction (Geneva, Switzerland). The within and between batches C.V.s were 10% and 12% for both hormones. RIA data reduction The binding of the labelled analyte to the specific antiserum was expressed as a relative percentage of the total binding using a logit-log transformation program for a HP-97 desk top programmable calculator. Results Comparative dissolution rates for the synthetic estrogens In the dissolving system used (ethanol 35 : water 65, v/v), the two particle size ranges tested (<50 urn and 125 - 177 urn) exhibited different dissolution behaviours. As shown in Figure 1, the micronized material (<50 urn) dissolved faster In order to establish a than the 125 - 177 urn particles. comparison point between the solubilities of both synthetic estrogens, the time to obtain 50% dissolution (TD 50) was analysis of the after a linear regression calculated The calculated functions and the individual observed data. values are shown in Table I. It can be seen from the data that the dissolution ratio ME/EE-2 for the 125 - 177 urn particle size is 2.9%; thus indicating the relative lower solubility of ME and therefore making it more suitable for a sustained release system. Clinical trial The B A mean value for the eight subjects studied was The mean 1.50 mS2 (range, 1.26 - 1.79) with a C.V. of 12%. values for the real dose of NET and ME administered to each
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E THINYLESTAADIOL
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Figure 1. Comparative in vitro dissolution of two synthetic estrogens in?GGGGi : water (35 : 65). Five milligrams of the steroids in crystals of the particle size indicated were added to 100 ml of solvent mixture. Constant agitation and temperature were maintained for 60 min.
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Table
I
Calculated time to obtain 50% dissolution (TD 501 of two different particle size ranges of ethinylestradiol and mestranol in ethanol 35 : water 65, v/v
Compound
Particle
EE-2
< 50 um
EE-2
size
TD 50
(min)
Function
9.23
~=0.72(X)+42.72
125 - 177 urn
39.46
Y=o.91(x)+14.05
MR
< 50 Lull
78.43
~=0.41(X)+17.56
ME
125 - 177 um
115.77
Table
~=0_35(X)+
8.54
II
Real dose administered of NET and ME expressed as mg/m2 BSA after a single i-m. injection of 10 mg NET plus 1.0 mg ME to eight normal women
Subject
NET
ME
Ratio
1
6.65
0.61
10.90
2
5.60
0.62
9.03
3
5.56
0.62
8.96
4
5.24
0.56
9.35
5
4.89
0.51
9.58
6
4.41
0.49
9.00
7
4.40
0.47
9.36
8
4.03
0.47
8.57
Mean
5.09
0.54
9.34
S.D.
0.84
0.06
0.70
16.64
12.24
7.49
C.V.
62
%
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1987 VOL. 35 NO. 1
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woman expressed as mg/m2 BSA were 5.09 and 0.54 with C.V. of The mean ratio for the 16.6% and 12.2%, respectively. Individual values injected NET/ME was calculated as 9.34. are presented in Table II. Clinical findings None of the subjects experienced early side effects such as breast vomiting. Neither engorgement or nausea or oedema, headaches, dizziness, bloating or tenderness, evidence of fluid retention, mental depression/anxiety, vaginal discharge, midcycle pain, local pain from injection nor skin changes were reported during the treatment cycle. Bleeding patterns The mean for the treatment cycle length was 29.5 + 2.03 days and was not statistically different from the control cycle, treatment 30 + 2 days (p bO.5). The duration of flow was: cycle 4.0 + 1.2 days and control cycle 4.6 + 1.4 days (p Seven out of eight subjects presented with spotting >0.5). at midcycle, starting at 10.2 + 4 days after injection and lasting for 6.4 + 3.2 days. Progesterone serum levels Progesterone concentrations at or above 16 nmol/l for five consecutive days were considered as an indication for a normal ovulatory cycle (7). During the treatment cycle, all the subjects studied had progesterone levels consistently below 2 nmol/l. It was therefore assumed that ovulation and subsequent luteal function were inhibited. Figure 2 (right panel, bottom) shows the mean + s.d. of the progesterone profile throughout the study. Estradiol serum levels The mean E-2 profile (mean + s.d.) is presented in Figure 2 of the (right panel, upper). After the administration combined NET-ME formulation, the maximum level of E-2 was present 17 days after injection and was significantly higher (p ~0.05) than values considered the normal range for the pre-ovulatory surge (7). The entire E-2 profile throughout the treatment cycle revealed a cyclic variation, suggesting follicular activity. Norethindrone
and ethinylestradiol
serum levels
The levels of NET and EE-2 (mean + sem) for the eight subjects studied presented as a group are shown in Figure 2 The ME release from the site of injection was (left panel). assessed by the measurement of its active metabolite EE-2 (S,9). In all the subjects NET and EE-2 were detectable 21 Serum levels are expressed in days after injection.
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Figure 2. Peripheral serum levels of synthetic (left panel) and natural (right panel) steroids, after i-m. injection of norethindrone and mestranol to eight normal women.
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ng/ml/m2 and pg/ml/m2, respectively. The levels represent Statistical methods included steroids. unconjugated calculation of mean and standard error by the method for The area under the serum small numbers of samples (10). curve estimated by the concentration-time (ADC) was trapezoidal rule: the maximum serum concentration value (peak value): the time required to reach the peak value (TP); the time taken to absorb 50% of the administered dose (T1/2 ABS) (11); and the mean rate of release (MPP) were calculated and are presented in Table III. The observed NET p?ak value and the TP are almost half (13.2 and twice (8.3 vs. 4.3 vs. 24 nmol/l/m ) days), respectively, of the values reported by Sang et al. (11) in a group of nine women receiving 200 mg of NET-enanthate. For ME, there are no previous reports following parenteral administration; however, the available information on plasma levels and pharmacokinetics of EE-2 derived from ME after oral administration in various populations indicates that in Thai womqn the peak values after 100 ug of ME are around 700 pmol/l/m at two hours (4). With the present formulation containing five times more ME, although given by a different route and in a different population, the peak value was only slightly higher (905 pmol/l/m) but with a considerable delay in the time taken to reach the maximum value (12.2 days). Discussion that The data presented demonstrate the combined administration of 5.09 + 0.84 mg of NET + 0.54 + 0.06 mg ME inhibited ovulation and corpus luteum formation for one month in all subjects studied. The endogenous E-2 rise observed 17 days after injection reflected that although follicular maturation occurred, it was not however followed by a subsequent progesterone rise. This observation is in parallel to that reported in users of low-dose progestinonly contraceptives (12) and corresponds to the B-type ovarian reaction described by Landgren and Diczfalusy (7). Whether follicular activity without subsequent ovulation may lead to an ovarian morphophysiologic alteration has not been ascertained from our data and deserves further studies. The analysis of serum levels of NET revealed that the mean peak value obtained is significantly lower than that previously reported for the long-acting 200 mg NET-enanthate preparation (13) and for the 1 mg oral ethynodiol diacetate (14). The "burst effect" described after the intramuscular injection of progestins as a parenteral mode of hormonal contraception, in which the release rate is initially higher than the steady-state, was not observed. Even though there were variations in drug release and absorption among
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Table III Pharmacokinetic parameters of NET and EE-2 in eight normal women following a single i.m. injection of NET 10 mg and ME 1 mg Parameter
NET
EE-2
AUC
1.7x10 nmol/day/l/m2
92x10 pmol/day/l/m2
Peak value
13.2k7.6 nmol/l/m2
905f217 pmol/l/m2
TP
8.37+5.6 days
12.25+5.82 days
T1/2 ABS
7.79+2-l days
9.49f2.27 days
MRR*
242.4 ug/day
25.75 ug/day
*Calculated by dividing the duration of release by the total amount of steroids injected.
subjects, the concentration-time curve follows a near zero order release pattern. Since serum concentrations of the steroids synthetic were detectable after 20 days of injection, the possibility of a llbuilding upI' effect with repeated injections at one month intervals is being investigated. The estimated 24-hour release from the site of injection was calculated as 250 ug of NET and 25 ug of ME, amounts similar daily intake of to the low-dose oral combined Since enterohepatic metabolism of steroids contraceptives. constitutes a major component of their general metabolism, administration would the first parenteral by-pass enterohepatic passage, thus decreasing the incidence of liver-mediated side effects. The observed side effects, which consisted in rather minor estrogen spotting at midcycle, might be attributed to the the formulation. It is expected that component of additional modifications either in dose and/or rate of release would improve the performance of the system. Indeed control of the size of the crystals plays an important role in the system, since one has to consider the maximum-gauge for routine injection as a limiting needle acceptable For adequate drug delivery, we consider that factor. particles larger than 250 urn are not likely to be injected because of the back pressure produced by the muscular tissue, which leads to a particle size sieving effect in the syringe needle.
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results presented herein provide a novel The overall the pharmacological design of parenteral approach to The data are consistent with the hormonal contraception. concept that it is possible to maintain stable circulating levels of synthetic steroids within the range of minimal compromising their contraceptive without intervention, efficacy. Acknowledgements This work received partial support from the World Health Programme in Special Human Reproduction Organization, (Geneva, Switzerland) and the National Council of Science The comments of Ms. B. and Technology, CONACyT (Mexico). Bayramian are greatly appreciated.
References 1.
Diczfalusy, E.: New developments in oral, injectable contraceptives, and implantable vaginal rings, and intrauterine devices. A review. Contraception 33:722, 1986.
2.
Hall, E.P. and Faser, S.I.: Monthly injectable contraceptives. In: Advances in Human Fertility and Reproductive Endocrinology. vol. II, Long-Acting Steroid Contraception (Mishell, D.R., Jr., Ed.). Raven Press, New York, pp 65-88, 1983.
3.
Garza-Flores, J., Diaz-Sanchez, V., Jimenez-Thomas, S. and Rudel, W.H.: Development of a low-dose monthly injectable contraceptive system: I. Choice of compounds, dose and administration route. Contraception 30:371-379, 1984.
4.
Goldzieher, J.W., Dozier, T.S. and de la Pena, A.: Plasma levels and pharmacokinetics of ethynyl estrogens in various populations. II. Mestranol. Contraception 21:17-27, 1980.
5.
Stanczyk, F.Z., Gale, J.A. and Goebelsman, u.: Radioimmunoassay of plasma ethinylestradiol in the presence of circulating norethindrone. Contraception 22:457-470, 1980.
6.
Garza-Flores, J., Diaz-Sanchez, V., Bedolla-Tovar, N. and Lozano-Ruy, S.A.: A rapid and sensitive radioimmunoassay for norethisterone. Steroids 41:693701, 1983.
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68
7.
Landgren , B.M. and Diczfalusy, E.: Hormonal effects of the 300 ug norethisterone (NET) minipill. 1. Daily steroid levels in 43 subjects during a pretreatment and cycle during the second month of NET administration. Contraception 21:87-113, 1980.
a.
Warren, J.R. and Fotherby, K.: Plasma levels ethinylestradiol after administration ethinylestradiol or mestranol to human subjects. Endocr. 5:369-370, 1973.
9.
Kappus, H., Bolt, H.M. and Remmer, H.: Demethylation of mestranol to ethinylestradiol in vitro and in vivo. Acta Endocrinol. 71:374-384, 1972.
10.
Okerholm, A.R., Peterson, E.F., Kecley, J.F., Smith, C.T. and Glazko, J.A.: Bioavailability of norethindrone in human subjects. Europ. J. Clin. Pharmacol. 13:35-39, 1978.
11.
Sang, W.G., Fotherby, K., Howard, B., Elder, M. and Bye, S.P.: Pharmacokinetics of norethisterone oenanthate in humans. Contraception 24:15-27, 1981.
12.
Bassol, S., Garza-Flares, J., Cravioto, M.C., DiazSanchez, V., Fotherby, K., Lichtenberg, R. and PerezPalacios, G.: Ovarian function following a single administration of depo-medroxyprogesterone (DMPA) at different doses. Fertil. Steril. 42:216-222, 1984.
13.
Weiner, E. and Johansson, E.D.B.: Plasma levels of after i.m. injection of 200 mg norethindrone of norethindrone oenanthate. Contraception 11:419, 1975.
14.
Walls, C., Vase, W.C., Horth, E.C. and Palmer, F.R.: norethisterone after Radioimmunoassay of plasma administration. J. Steroid diacetate ethynodiol Biochem. 8:167-171, 1977.
JANUARY
1987 VOL.
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35 NO. 1
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DEVELOPMENT OF A LOW-DOSE MONTHLY INJECTABLE CONTRACEPTIVE SYSTEM: PHARMACOKINETIC AND PHARMACODYNAMIC STUDIES V. Diaz-Skchezl J. Garza-Flares' S. Jimen&-Tho as2 H.W. Rude1 ?
1Department of Reproductive Biology, Instituto National la Nutrici6n "Salvador Zubir&", Mexico City
de
2Department of Biochemistry, School of Medicine, Universidad National Autonoma de Mgxico, Mgxico City 3Fertility and Sterility Research Center, Me'xico City
Abstract A drug delivery system which provides a sustained release of norethindrone (NET) and mestranol (ME) for one month after a single intramuscular injection was assessed as a long-acting injectable contraceptive. The system is based upon well defined particle size crystals of the synthetic steroids maintained in suspension with saline solution. Eight healthy ovulating women volunteered for the study; they received a combination of 10 mg of NET plus 1 mg of ME in 1 ml of vehicle by intramuscular injection on day five of their menstrual cycle. Blood samples were drawn at 0, 1, 5, 10, 13, 17 and 21 days after drug administration. The immunoreactive serum levels of estradiol, progesterone, NET and ethinylestradiol were measured specific by radioimmunoassay procedures to assess ovarian function and the kinetic parameters of the synthetic steroids. This newly developed contraceptive system proved to be both effective, and long-lasting as well as devoid of side effects. Address correspondence to: V. Dlhz-Sgnchez, Department of Reproductive Biology, Institute National de la Nutrici6n "Salvador Zubirdn'!, Vasco de Quiroga No. 15, Tlalpan 14000 Mkxico, D.F.
Submitted for publication November 24, 1986 Accepted for publication December 9, 1986
JANUARY
1987 VOL. 35 NO. 1
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CONTRACEPTION
Introduction Most of the information available from family planning programs in developing countries indicates an increase in acceptability for long-acting contraception These (1) observations have created the need for the development of a highly effective monthly injectable contraceptive exhibiting a low incidence of side effects. Maintenance of the contraceptive potency over a period of one month injectable hormonal formulation is possible; however, most of the preparations currently available cause profound disruptions of the endometrial bleeding pattern, particularly prolonged and heavy endometrial flow in early phases with a tendency toward periods of amenorrhea later on (2). The concept of a combined low-dose hormonal injectable given monthly to inhibit ovulation appears to be an attractive alternative. Accordingly a progestogen-estrogen formulation was recently developed (3). In an attempt to avoid the estrogen-related side effects observed following its administration, the formulation has been modified. The new formulation is a microcrystalline aqueous suspension in which ethinylestradiol (EE-2) has been replaced by mestranol. The aim of the present investigation was to assess the pharmacokinetics of the two synthetic steroids in the formulation and their effects upon the ovarian function during the treatment cycle. Materials and Methods Norethindrone (17 (J -ethinyl-17-hydroxy-4-estren-3-one), ethinylestradiol (17 Q -ethinyl-1,3,5(10)-estratrien-3,17diol) and mestranol (17a-ethinyl-1,3,5(10)-estratrien-3,17diol-3-methylester) were a gift of Syntex S.A. (Mexico). Chemical purity of the compounds (>99%) was determined by at infrared and least two of the following procedures: ultraviolet absorption and gas-liquid chromatographic (GLC) All the reagents used were of the highest purity behaviour. available and were used without further purification. In vitro dissolution studies -obtained as previously Crystals of EE-2 and ME were described (3). Dissolution rates of both synthetic steroids were analyzed following the same methodology (3) and the dissolution, expressed as a percentage of the total steroid against time on a added to the system, was plotted log/linear scale.
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Clinical data Eight healthy women aged 23.5 + 3.0 years, with a history of regular menstrual periods (30 + 2 days) in the preceding three months, volunteered for the clinical trial. None of them used any form of steroidal medication for a minimum of six months prior to admission to the study, and none of them All had been pregnant during the previous twelve months. subjects were informed of the purpose of the study and a written consent was obtained. Before admission to the study a full medical history and a complete physic1 examination All women were advised to was performed on each subject. use a barrier method of contraception. The body surface area (BSA) was calculated and the value was used as a correction factor in expressing the hormonal serum concentrations (4). On day five of their menstrual cycle each subject received by intragluteal injection a combination of 10 mg NET plus 1 mg ME in 1 ml saline solution as diluent. Residual active ingredients not delivered were measured in the needles and syringes using a GLC method. The actual quantity of steroid injected was recorded in each case. Blood samples were taken from an antecubital vein at: 0, 1, 5, 10, 13, 17 and 21 days after drug administration. Blood samples were allowed to clot at room temperature, centrifuged within one hour after sampling and the serum separated and kept at-20°C until analyzed. The concentrations of immunoreactive estradiol (E-Z), (P-4), NET and EE-2 in each sample were measured as described below. Radioimmunoassay
procedures
Ethinvlestradiol Ethinylestradiol, 17 c~6,7-~-H(N), specific activity 41.5 Ci/mmol and anti-ethinylestradiol-7-(3-thiopropionic acid) bovine serum albumin serum were obtained from New England Nucler (Boston, Mass.) and Research Triangle Institute (Research Triange Park, N.C.), respectively. Immunoreactive unconjugated EE-2 in serum samples was measured after solvent extraction the method following described by Stanczyk & al. (5) The sensitivity of the assay at a coefficient of variation (C.V.) of 15% was 21 pmol/L and a precision profile over a range of 42 to 676 pmol/L was ~10% C.V. Bias was assessed after addition of 169 and 845 pmol/L of EE-2 to blank serum samples, the recovery values were 109 2 9% and 91 + 12%, respectively.
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Norethindrone A direct method of unextracted serum samples was followed. The combined use of a high specific unpurified antiserum and heat treatment of diluted serum samples obviated both extraction and chromatographic procedures. The direct NET assay has been previously validated (6) and fulfilled all the quality control parameters, Estradiol and nroqesterone The naturally occurring steroids were measured according to the method manual of the Programme for the Provision of Matched Assay Reagents for Radioimmunoassay of Hormones in Reproductive Physiology of the World Health Organization, Special Programme of Research, Development and Research Training in Human Reproduction (Geneva, Switzerland). The within and between batches C.V.s were 10% and 12% for both hormones. RIA data reduction The binding of the labelled analyte to the specific antiserum was expressed as a relative percentage of the total binding using a logit-log transformation program for a HP-97 desk top programmable calculator. Results Comparative dissolution rates for the synthetic estrogens In the dissolving system used (ethanol 35 : water 65, v/v), the two particle size ranges tested (<50 urn and 125 - 177 urn) exhibited different dissolution behaviours. As shown in Figure 1, the micronized material (<50 urn) dissolved faster In order to establish a than the 125 - 177 urn particles. comparison point between the solubilities of both synthetic estrogens, the time to obtain 50% dissolution (TD 50) was analysis of the after a linear regression calculated The calculated functions and the individual observed data. values are shown in Table I. It can be seen from the data that the dissolution ratio ME/EE-2 for the 125 - 177 urn particle size is 2.9%; thus indicating the relative lower solubility of ME and therefore making it more suitable for a sustained release system. Clinical trial The B A mean value for the eight subjects studied was The mean 1.50 mS2 (range, 1.26 - 1.79) with a C.V. of 12%. values for the real dose of NET and ME administered to each
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E THINYLESTAADIOL
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Figure 1. Comparative in vitro dissolution of two synthetic estrogens in?GGGGi : water (35 : 65). Five milligrams of the steroids in crystals of the particle size indicated were added to 100 ml of solvent mixture. Constant agitation and temperature were maintained for 60 min.
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Table
I
Calculated time to obtain 50% dissolution (TD 501 of two different particle size ranges of ethinylestradiol and mestranol in ethanol 35 : water 65, v/v
Compound
Particle
EE-2
< 50 um
EE-2
size
TD 50
(min)
Function
9.23
~=0.72(X)+42.72
125 - 177 urn
39.46
Y=o.91(x)+14.05
MR
< 50 Lull
78.43
~=0.41(X)+17.56
ME
125 - 177 um
115.77
Table
~=0_35(X)+
8.54
II
Real dose administered of NET and ME expressed as mg/m2 BSA after a single i-m. injection of 10 mg NET plus 1.0 mg ME to eight normal women
Subject
NET
ME
Ratio
1
6.65
0.61
10.90
2
5.60
0.62
9.03
3
5.56
0.62
8.96
4
5.24
0.56
9.35
5
4.89
0.51
9.58
6
4.41
0.49
9.00
7
4.40
0.47
9.36
8
4.03
0.47
8.57
Mean
5.09
0.54
9.34
S.D.
0.84
0.06
0.70
16.64
12.24
7.49
C.V.
62
%
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woman expressed as mg/m2 BSA were 5.09 and 0.54 with C.V. of The mean ratio for the 16.6% and 12.2%, respectively. Individual values injected NET/ME was calculated as 9.34. are presented in Table II. Clinical findings None of the subjects experienced early side effects such as breast vomiting. Neither engorgement or nausea or oedema, headaches, dizziness, bloating or tenderness, evidence of fluid retention, mental depression/anxiety, vaginal discharge, midcycle pain, local pain from injection nor skin changes were reported during the treatment cycle. Bleeding patterns The mean for the treatment cycle length was 29.5 + 2.03 days and was not statistically different from the control cycle, treatment 30 + 2 days (p bO.5). The duration of flow was: cycle 4.0 + 1.2 days and control cycle 4.6 + 1.4 days (p Seven out of eight subjects presented with spotting >0.5). at midcycle, starting at 10.2 + 4 days after injection and lasting for 6.4 + 3.2 days. Progesterone serum levels Progesterone concentrations at or above 16 nmol/l for five consecutive days were considered as an indication for a normal ovulatory cycle (7). During the treatment cycle, all the subjects studied had progesterone levels consistently below 2 nmol/l. It was therefore assumed that ovulation and subsequent luteal function were inhibited. Figure 2 (right panel, bottom) shows the mean + s.d. of the progesterone profile throughout the study. Estradiol serum levels The mean E-2 profile (mean + s.d.) is presented in Figure 2 of the (right panel, upper). After the administration combined NET-ME formulation, the maximum level of E-2 was present 17 days after injection and was significantly higher (p ~0.05) than values considered the normal range for the pre-ovulatory surge (7). The entire E-2 profile throughout the treatment cycle revealed a cyclic variation, suggesting follicular activity. Norethindrone
and ethinylestradiol
serum levels
The levels of NET and EE-2 (mean + sem) for the eight subjects studied presented as a group are shown in Figure 2 The ME release from the site of injection was (left panel). assessed by the measurement of its active metabolite EE-2 (S,9). In all the subjects NET and EE-2 were detectable 21 Serum levels are expressed in days after injection.
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Figure 2. Peripheral serum levels of synthetic (left panel) and natural (right panel) steroids, after i-m. injection of norethindrone and mestranol to eight normal women.
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ng/ml/m2 and pg/ml/m2, respectively. The levels represent Statistical methods included steroids. unconjugated calculation of mean and standard error by the method for The area under the serum small numbers of samples (10). curve estimated by the concentration-time (ADC) was trapezoidal rule: the maximum serum concentration value (peak value): the time required to reach the peak value (TP); the time taken to absorb 50% of the administered dose (T1/2 ABS) (11); and the mean rate of release (MPP) were calculated and are presented in Table III. The observed NET p?ak value and the TP are almost half (13.2 and twice (8.3 vs. 4.3 vs. 24 nmol/l/m ) days), respectively, of the values reported by Sang et al. (11) in a group of nine women receiving 200 mg of NET-enanthate. For ME, there are no previous reports following parenteral administration; however, the available information on plasma levels and pharmacokinetics of EE-2 derived from ME after oral administration in various populations indicates that in Thai womqn the peak values after 100 ug of ME are around 700 pmol/l/m at two hours (4). With the present formulation containing five times more ME, although given by a different route and in a different population, the peak value was only slightly higher (905 pmol/l/m) but with a considerable delay in the time taken to reach the maximum value (12.2 days). Discussion that The data presented demonstrate the combined administration of 5.09 + 0.84 mg of NET + 0.54 + 0.06 mg ME inhibited ovulation and corpus luteum formation for one month in all subjects studied. The endogenous E-2 rise observed 17 days after injection reflected that although follicular maturation occurred, it was not however followed by a subsequent progesterone rise. This observation is in parallel to that reported in users of low-dose progestinonly contraceptives (12) and corresponds to the B-type ovarian reaction described by Landgren and Diczfalusy (7). Whether follicular activity without subsequent ovulation may lead to an ovarian morphophysiologic alteration has not been ascertained from our data and deserves further studies. The analysis of serum levels of NET revealed that the mean peak value obtained is significantly lower than that previously reported for the long-acting 200 mg NET-enanthate preparation (13) and for the 1 mg oral ethynodiol diacetate (14). The "burst effect" described after the intramuscular injection of progestins as a parenteral mode of hormonal contraception, in which the release rate is initially higher than the steady-state, was not observed. Even though there were variations in drug release and absorption among
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Table III Pharmacokinetic parameters of NET and EE-2 in eight normal women following a single i.m. injection of NET 10 mg and ME 1 mg Parameter
NET
EE-2
AUC
1.7x10 nmol/day/l/m2
92x10 pmol/day/l/m2
Peak value
13.2k7.6 nmol/l/m2
905f217 pmol/l/m2
TP
8.37+5.6 days
12.25+5.82 days
T1/2 ABS
7.79+2-l days
9.49f2.27 days
MRR*
242.4 ug/day
25.75 ug/day
*Calculated by dividing the duration of release by the total amount of steroids injected.
subjects, the concentration-time curve follows a near zero order release pattern. Since serum concentrations of the steroids synthetic were detectable after 20 days of injection, the possibility of a llbuilding upI' effect with repeated injections at one month intervals is being investigated. The estimated 24-hour release from the site of injection was calculated as 250 ug of NET and 25 ug of ME, amounts similar daily intake of to the low-dose oral combined Since enterohepatic metabolism of steroids contraceptives. constitutes a major component of their general metabolism, administration would the first parenteral by-pass enterohepatic passage, thus decreasing the incidence of liver-mediated side effects. The observed side effects, which consisted in rather minor estrogen spotting at midcycle, might be attributed to the the formulation. It is expected that component of additional modifications either in dose and/or rate of release would improve the performance of the system. Indeed control of the size of the crystals plays an important role in the system, since one has to consider the maximum-gauge for routine injection as a limiting needle acceptable For adequate drug delivery, we consider that factor. particles larger than 250 urn are not likely to be injected because of the back pressure produced by the muscular tissue, which leads to a particle size sieving effect in the syringe needle.
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results presented herein provide a novel The overall the pharmacological design of parenteral approach to The data are consistent with the hormonal contraception. concept that it is possible to maintain stable circulating levels of synthetic steroids within the range of minimal compromising their contraceptive without intervention, efficacy. Acknowledgements This work received partial support from the World Health Programme in Special Human Reproduction Organization, (Geneva, Switzerland) and the National Council of Science The comments of Ms. B. and Technology, CONACyT (Mexico). Bayramian are greatly appreciated.
References 1.
Diczfalusy, E.: New developments in oral, injectable contraceptives, and implantable vaginal rings, and intrauterine devices. A review. Contraception 33:722, 1986.
2.
Hall, E.P. and Faser, S.I.: Monthly injectable contraceptives. In: Advances in Human Fertility and Reproductive Endocrinology. vol. II, Long-Acting Steroid Contraception (Mishell, D.R., Jr., Ed.). Raven Press, New York, pp 65-88, 1983.
3.
Garza-Flores, J., Diaz-Sanchez, V., Jimenez-Thomas, S. and Rudel, W.H.: Development of a low-dose monthly injectable contraceptive system: I. Choice of compounds, dose and administration route. Contraception 30:371-379, 1984.
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Goldzieher, J.W., Dozier, T.S. and de la Pena, A.: Plasma levels and pharmacokinetics of ethynyl estrogens in various populations. II. Mestranol. Contraception 21:17-27, 1980.
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Stanczyk, F.Z., Gale, J.A. and Goebelsman, u.: Radioimmunoassay of plasma ethinylestradiol in the presence of circulating norethindrone. Contraception 22:457-470, 1980.
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Garza-Flores, J., Diaz-Sanchez, V., Bedolla-Tovar, N. and Lozano-Ruy, S.A.: A rapid and sensitive radioimmunoassay for norethisterone. Steroids 41:693701, 1983.
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Landgren , B.M. and Diczfalusy, E.: Hormonal effects of the 300 ug norethisterone (NET) minipill. 1. Daily steroid levels in 43 subjects during a pretreatment and cycle during the second month of NET administration. Contraception 21:87-113, 1980.
a.
Warren, J.R. and Fotherby, K.: Plasma levels ethinylestradiol after administration ethinylestradiol or mestranol to human subjects. Endocr. 5:369-370, 1973.
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Kappus, H., Bolt, H.M. and Remmer, H.: Demethylation of mestranol to ethinylestradiol in vitro and in vivo. Acta Endocrinol. 71:374-384, 1972.
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Okerholm, A.R., Peterson, E.F., Kecley, J.F., Smith, C.T. and Glazko, J.A.: Bioavailability of norethindrone in human subjects. Europ. J. Clin. Pharmacol. 13:35-39, 1978.
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Sang, W.G., Fotherby, K., Howard, B., Elder, M. and Bye, S.P.: Pharmacokinetics of norethisterone oenanthate in humans. Contraception 24:15-27, 1981.
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Bassol, S., Garza-Flares, J., Cravioto, M.C., DiazSanchez, V., Fotherby, K., Lichtenberg, R. and PerezPalacios, G.: Ovarian function following a single administration of depo-medroxyprogesterone (DMPA) at different doses. Fertil. Steril. 42:216-222, 1984.
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Weiner, E. and Johansson, E.D.B.: Plasma levels of after i.m. injection of 200 mg norethindrone of norethindrone oenanthate. Contraception 11:419, 1975.
14.
Walls, C., Vase, W.C., Horth, E.C. and Palmer, F.R.: norethisterone after Radioimmunoassay of plasma administration. J. Steroid diacetate ethynodiol Biochem. 8:167-171, 1977.
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