Metabolism of oral contraceptive drugs

Metabolism of oral contraceptive drugs The formation norethindrone administration

1‘.

M.

1’.

J.

W.

kllLI,S, 1~15.

E.

and disappearance of metabolites of and mestranol after intravenous and oral

PH.D M.D.,

F..4.C:.O.G

BRASEI.TON,

,J.

0.

ELLEGOOD,

V.

B.

VAHESH.

PH.D. M.S. PH.D.,

D.PHIL.

.4 ug1t.st11, Grorgic1

PrclG)ns .stndiev jrom thi.c laborato~ wported that 3H-labrled metabolites with hay-1irvj.c of‘ mm thnn 24 h0ltr.r may remain in the plasma of uwrwn wceiving fin intravenous znjcction of ‘H riorc~tlritldro,lp or ‘H mestmnol. To conjrm the presence of these metabolite.r, blood camphs were collected for five days after injrction of ‘H norethindrone or ‘3H mestrnnol; “H representing metabolitcs of no&hindrone disappeared ulith half-l@ zv~luo.~ of 42 to 84 hours (mean 67 hours), ulhile 3H representing metabolitcs oj‘ m~stranol derlined with an awrage halj4j~ of 45 hours (range 37 to 65 hours). It%en the 3H-label~d drtrgs uvre administered orally, metabolites of similar hay-lifl uvw ,Jormc~d. Reca use thew c~omj~~und~ exist for several days after a single administration and ,~in( (1 oral con trareptizle drugs are normally taken daily, the possibility of thp accumulation of’ “H it, the plasma of women rewizGng sezvral consecutiw doses qf ‘H norethindronr ZMS investigated, The results of thi,c study shout a step?crise u.ccumulation thy ‘H metnbo1ito.v u~hvn “H norethindrone urns administered in six daily oral doses. Hoio(‘7ler. the ‘H lerv~lt declinod,from the peak on the sixth and lrzst day of the treatmnt at a ratr equivalent to those prezCousff measured after intravenous or oral admini.stmtion (mran 70 homr). (AM. .J. OBSTET. ~YNECOL. 126: 987, 1976.)

of

RECENT PHARSIACOLOGIC studies from this laboratory demonstrated that norethindronee and mestranol,? two commonly prescribed oral contraceptive

drugs, are rapidly cleared from the plasma following a single intravenous injection of the drug.‘, ’ However, despite this rapid clearance of the native drug, apparent metabolites of norethindrone and mestranol appeared to remain in the plasma at elevated levels for more than 24 hours after the initial in.jection. ‘The present study was designed to further investigate the metabolism of these oral contraceptive drugs and to examine the nature of the metabolites remaining in the plasma after administration of the native drugs. Experiments were performed to determine the half-life ot blood-borne norethindrone and mestranol metabolites following intravenous injection and to determine whether similar metabolites occur after their oral ingestion. Furthermore, the possibility of a progressive accumulation of the metabolites was investigated in

From the Departments (If Endocrinology, Obstetrics and Gynecology and Medicine. and the Center for Population LStudie$, Medical College of Georgia. Supported by National NOI-HD-I-2297. Received for pubhcation RwbedJune

I I, I?76.

Accepted,Juljl

6, 1976.

In.stitutes April

of Health

Contract

No.

19, 1976.

RepwU requests: Dr. Thomas M. Mills, Department of En~docrinology, Medical College of Georgia, Augusta. Georgia 30902. *17fSHydroxy-I t?‘-Methoxy-I

7~-cthvn~l-4-rstren-~-(lne. 7sethyyl-1.3,5(10)-estratrien-I

7p-01.

987

988

Mills

et al.

NORETHINDRONE NORETHINDRONE

MESTRANOL

MESTRANOL

i 0.1I \ \ OSJ

‘a/ \ ’

3H

MESTRANOL

, I

2 Days

after

3

4

5

injection

Fig. 1. Plasma ‘H levels in subjects receiving a single intravenous injection of about 10/&i of either 3H norethindrone (upper panel) or 3H mestranol (lower panel). The solid line through each time point connects the mean of all the determinations of the total 3H levels in the plasma (3H drug plus 3H metabolites of the drug). For comparison, the disappearance curves for 3H norethindrone and 3H mestranol have been included (dashed line). volunteers taking oral doses of 3H norethindrone several consecutive days. Material

2

I

Days

over

and methods

Subjects. Paid volunteers were utilized exclusively in these studies. All subjects had normal menstrual histories, were in apparent good health, and had not taken any form of oral contraceptive drug for at least one month prior to joining the study. Ages ranged from 21 to 28 years. One subject had had a normal pregnancy one year prior to starting the study. Informed consent was obtained for all subjects. Drugs and reagents. 3H-9,1 1 norethindrone and “H-9,1 1 mestranol were purified by paper partition

ofter

oral

3

4

5

ingestlon

Fig. 2. The disappearance of 3H (closed circles) and “H norethindrone or 3H mestranol (open circles) from the plasma following oral administration of 10.2 &i of “H norethindrone (upper panel) or 3H mestranol (lower panel). chromatography prior to use. In some of the studies. subjects took 2.5 mg. of norethindrone per day: other subjects received 0.08 mg. of mestranol per day. All solvents were reagent grade and most were redistilled prior to use. Experimental design. The first series of studies was designed to determine the long-term disappearance rates for metabolites of norethindrone and mestranol following intravenous injection of the drugs. Subjects received a standard dose of norethindrone (2.5 mg.) or mestranol(O.08 mg.) the evening before the start of the study and then again at 9:00 A.M. on the morning of the study. Immediately after this second dose of the drug, each subject received a single intravenous injection ok ‘H norethindrone (10.1 @Zi) or “H mestranol(9.6 /..Ki). Blood samples were collected in heparinized tubes ar several intervals ranging from three minutes to five days after the single injection. The blood samples were centrifuged twice and plasma samples stored at -20” C. until analysis. A second study measured the disappearance of both the native drug’and metabolites of the drug following a

Metabolism

I

2

3

4

5

6

of oral contraceptive

drugs

989

76910

Day

Fig. 3. The accumulation

of 3H in the plasma of four subjects receiving daily drone (5.38 &i per day) for 6 consecutive days. Blood sample collections were after the last capsule. Each bar represents the mean of tour determinations

doses of 3H norerhincontinued

for 4 days

(brackets equal one standard error of the mean). At each time period, the hatched bars are plasma ‘H levels before the “H norethindrone-containing capsule was swallowed and solid bars represent values of 3H in the plasma 3 hours after ingestion. single oral dose of “H norethindrone or 3H mestranol. ,4 gelatin capsule containing norethindrone (10.2 &i “H plus 2.5 mg.) or mestranol (10.2 ,uCi 3H plus 0.08 trig.) was administered at 9:00 A.M. following a light breakfast. Blood samples tvere obtained immediately before treatment and at varying intervals from 15 minutes to :‘, days after ingestion. A third series of’ experiments seas designed to study the buildup of‘ ‘H in the plasma following six consecutive oral doses of “H norethindrone. Gelatin capsules containing :1.38 /.&i of ‘H norethindrone plus 2.5 mg. of norethindrone were ingested daily ai 12 : 00 noon. Blood samples \ver-e obtained immediately before and then again three hours after (3: 00 P.M.) the capsule was taken. The capsules were taken and two dail) blood samples obtained fi)r six consecutive days (Days 1 to 6). Daily collections \cere also continued for four days after the last pill ~vas swallo\ved (Days 7-10). Urine was collected over the entire ten days of the study. Analysis of blood samples. In the studies of longterm disappearance of norethindrone and mestranol following a single intra\,enous injection of the 3Hlabeled drug. blood samples were analyzed for the native drugs as previously described.‘. ’ Briefly, this procedure involved extraction with ether, thin-layer chromatographic separation of the 3H drugs. and

Table I. The half-life (in hours) of 3H fl-om the plasma of’ subjects receiving a single intravenous injection of “H norethindrone (10.1 p(Z) or “H Mestranol (9.6 /Xi)

Suhjec.t Sl S2 D Hl H2 Mean

+ S.E.M.

Half-lif

(hr.)

42.4 53.7 78.1 76.7 83.5 66.9

Sub@ HE1 HE2 HYI HY2 Gl G2

+ 8.0

Half-lifl

(hr.1

37.4 44.7 37.7 44.1 41.1 65.4 45.1 t 4.3

quantification of 3H in a liquid scintillation spectrometer. Plasma levels of total 3H in all studies were yuantified directly by counting an aliquot of plasma in an emulsifier for aqueous samples (Insta-Gel).* “H counting efficiency in Insta-Gel Fvas 20 to 30 per cent and all radioactivity was corrected to 100 per cent efficient,-. Twenty-four-hour urine samples were collected over boric acid and kept refrigerated until analvsis. The “H content of these samples was determined by counting *Insta-Gel, Packard Instrument ville Rd.. Downers Grove, Illinois

Company, 605 1.5.

2200

Warrrn-

990

Mills et al

Day Fig. 4. The excretion of ‘H in the urine of four subjects receiving 5.38 yCi of “H norethindrone per day for 6 consecutive days. Urine collections were continued for 4 days after the last capsule was taken. Each bar is the mean of 4 determinations with brackets representing one standard error of the mean. an aliquot of each urine sample directly in Insta-Gel. Statistical analysis. The results of these studies were analyzed statistically using Student’s t test (unpaired variable) and linear regression analysis by means of a Hewlett Packard 9830A programable calculator. Results are reported as mean & standard error of the mean.

Results Fig. 1 depicts the disappearance of 3H from the plasma of subjects receiving a single intravenous injection of either 3H norethindrone or 3H mestranol. The total radioactivity following 3H norethindrone administration disappeared from the plasma with a half-life averaging 67 hours (Fig. 1, Table I). Plasma radioactivity following 3H mestranol injection was cleared somewhat more quickly with an average halflife of 45 hours (Fig. 1, Table I). The disappearance curves for 3H norethindrone and 3H mestranol have been included in Fig. 1 for comparison. Based on the areas under these disappearance curves, it would appear that less than 5 per cent of the total 3H is cleared as native norethindrone or mestranol with the remainder cleared as metabolites of the drugs. In preliminary studies, it was found that 3H-labeled norethindrone, mestranol, and metabolites of both drugs rapidly appeared in the blood after oral administration of the drugs. Within 15 minutes of ingestion of the 3H norethindrone capsules, measurable drug was present in the peripheral blood. Levels of 3H

norethindrone reached a peak within the first hour and thereafter declined to unmeasurable concentrations over the next 48 hours. Total “H, representing “H norethindrone and 3H metabolites of norethindrone. continued to rise to a maximum at six hours after ingestion and then declined slowly over the next five days with a half-life of about 70 hours (Fig. 2). The results of 3H mestranol ingestion are also presented in Fig. 2. After reaching measurable levels briefly, the “H-labeled native drug rapidly disappeared from the blood. However, radioactivity remained in the blood for more than five days after oral ingestion and declined with a half-life of 30 to 40 hours. Fig. 3 depicts the levels of “H in the plasma of subjects who received daily oral doses of “H norethindrone for six consecutive days. Blood samples for determination of total “H content were obtained immediately before and then again three hours after each capsule was taken. As might have been predicted from the slow rate of decline in the total 3H content of the blood after an oral or intravenous dose of “H norethindrone, ‘H accumulated in the blood in increasing stepwise fashion over the six consecutive days (Fig. 3). The peak blood levels were achieved three hours after the final capsule (123 hours); blood “H levels fell progressively through four days after the last capsule and the half-life of this disappearance averaged about 70 hours. There is no clear indication from the results in Fig. 3 that the blood “H would have reached a maximum or plateau level with subsequent oral doses. On each of the six days of

Metabolism of oral contraceptive drugs

treatment. blood levels were markedly elevated three hours after each capsule when compared to samples obtained before the capsule was swallowed (Fig. 3). The magnitude of the increase appears, however, to have declined somew.hat with subsequent capsules (i.e., Days 4. 5, 6). The urinary excretion of 3H in the subjects receiving six daill, capsules followed a somewhat different pattern (Fig. 4). After an initial rise in urinary 3H output on Day 2 of collection. excretion rates remained essentiall) constant through the six days of treatment. Thereafter, a rapid decline occurred in the excretion of “H. h total of 32.28 &i of “H norethindrone in six capsules was ingested by each of the subjects. The total urinary output of “H averaged 15.5 ? 1.4 &i over the 10 day collection period.

Comment Publications from both the United States and Great Britain have clearly established oral contraceptive drugs t(J be a risk factor in several thromoembolic diseases including myocardial infarctions,3 cerebral and pulmonary embolism.” and others.‘, 6 Oral contraceptives may be diabetogenic,’ alter liver function,R raise serum levels of triglycerides, potentially leading to vascular disease,” and are considered to be hypertensive in some wmicn.‘“~ ” Very recently, certain estrogens have also been linked to endometrial cancer in older women.“. “’ ‘These publications, dealing with adverse effects of many of the steroidal drugs included in or~l contraceptive preparations, should emphasize that tnuch remains unknown about this class of compounds despite their widespread use. For this reason, it is mandatory that basic pharmacokinetic studies of oral contraceptive drugs be performed. Only with extensive knowleclge of the tnetabolistn of these drugs can a potential rcslationship be established between the Untoward effects of oral contraceptive drug therapy and the metabolism of these compounds. The present study was designed to confirm the existence of metabolites of norethindrone and mestranol which had been previouslv reported and found to have half-lives of more than 24 hours.‘. ’ The results presented in Figs. 1 and 2 verif’y the presence of these metabolites of norethindrone and mestranol with half-lives of 50 to 60 hours and 30 to 40 hours, respectively. The metabolites appear to form regardless of whether the drugs are administered orally or intravenously. Preliminary experiments performed in this laboratory have made tentative identification of some of the plasnla rnetabolites of norethindrone. Gas chromatography-mass spectroscopv (GC-MS) has been utilized in the identification of ‘metabolites in both the

991

free form and as glucuronide and sulf;~tc cor+gates. The major blood metabolites pwsent tit! et’ hours after oral ingestion of 2. 5 mg. of norcrhindr~ UK to ;i single supject include free and conjugated wtiucrion products of’ norethindrone. Free !lorethil~tir.o~~~ \\.a~ also found in significant quantities.” Based on pre\-ious determinations of I 11e slow rat.e of’ disappearance of “H-labeled metabolitc, of norethindrone from the blood, the “staircase ’ ac c.umulation phenomenon presented in Fig. 3 I\ as not unexpected. Since the metabolites of norethindrotw wre still prrsent 24 hours after oral ingestion (Fig. 2). a buildup \vould be expected when the pills arc taken e\ery 24 hours. Statistical evaluation of the results presented in Fig. 3 failed to detnonstrate a plateau in IAood “H levels over six consecutive days of oral trcatmcsnt; the levels of “H increased with each claily dose, borh in the prctreatment and three-hour post-treatment wmples. ‘I‘hc urinary excretion of‘ the raclioacti\r cc~tnpounds did. however. appear to plateau after the wcond day of’ treatment. This plateau in urinary “H levels may irtdicate that the kidnev has a maximum capa(,it\ to excrete metabolites of‘ norethindrone. 11’ the pc%aL es< retion rate* was less than the daily intake of the drug and subsequent tnetabolite formation. thtw r !I(’ accumulation of metabolites seen in F’ig. 3 (oulC: he the result. Bl(~~tl “H levels declined in a mat11le1~ \f rhr dt-LIPS, ‘The finding that blood levels of norethindrotw tnetabolites fail to plateau during six consecutive tla!*s of Ireafment raises the possibility that the cotnlwun~ls could a(.cumulate continuously over each 20 ti;i\ i ourse of the drug. Possibly, native norethindrcwc rn,r~ likewise build up in the blood with daily ingcstioll. If’ a considerable amount of the native drug or ;I mc~rabolite persists. it would be of interest to invrsrigaw the relationships between these remaining COI~~OLII~~S md sotne of the untoward effects of oral c.ontrac-t~~~ti\.~~ drugs.

992

Mills

et

Experiments to

are currently

determine

lites

of

monthly

December 15. 1976 Am. J. Obstet. G\necol.

al.

noret.hindrone courses

underway

whether

remain of this

in this

norethindrone

and

in the

plasma

laboratory

The authors nical assistance

metabo-

wish to acknowledge of Mrs. S. C. Lin.

the excellent

between

drug.

REFERENCES 1. Mills, T. M., Lin, T. J., Hernandez-Ayup, S., Greenblatt, R. B., Ellegood, J. O., and Mahesh, V. B.: The metabolic clearance rate and urinary excretion of oral contraceptive drugs. I. Norethindrone, AM. J. OBSTET. GYNECOL. 120: 764, 1974. 2. Mills, T. M., Lin, T. J., Hernandez-Ayup, S., Greenblatt, R. B., Ellegood, J. O., and Mahesh, V. B.: The metabolic clearance rate and urinary excretion of oral contraceptive drugs. II. Mestranol, AM. J. OBSTET. GYNECOL. 120: 773, 1974. 3. Mann, J. I., Vessey, M. P., Thorogood, M., and Doll, R.: Myocardial infarction in young women with special reference to oral contraceptive practice, Br. Med. J. 2: 241. 1975. 4. Inman, W. H.. and Vessey, M. P.: Investigation of deaths from pulmonary, coronary, and cerebral thrombosis and embolism in women of child-bearing age, Br. Med. J. 2: 193, 1968. 5. Astedt, B.: New aspects of the thrombogenic effect of oral contraceptives, Am. Heart J. 90: 1, 1975. 6. Stolley, P. D., Tonascia, J. A., Tockman, M. S., Sartwell, P. E., Rutledge, A. H., and Jacobs, M. P.: Thrombosis with low-estrogen oral contraceptives, Am. J. Epidemiol. 102: 197, 1975. 7. Goldman, J, A.: Effect of ethynodiol diacetate and

8. 9.

10.

11.

12.

13.

14.

combination-type oral contraceptive compounds on carbohydrate metabolism. I. Six month intravenous glucose tolerance study, Diabetologia 11: 45, 1975. Anon.: Oral contraceptives and the liver, Br. Med. J. 4: 430, 1974. Spellacy, W. N., Buhi, W. C., Birk, S. A., and Cabal, R.: The effects of estrogens, progestogen, oral contraceptives, and intrauterine devices on fasting triglyceride and insulin levels, Fertil. Steril. 24: 178, 1973. Clezy. T. M., Foy, B. N., Hodge, R. L., and Lumbers, E. and hypertension, an R.: Oral contraceptives epidemiological survey, Br. Heart J. 34: 1238, 1972. Weir, R. J., Briggs, E., Mack, A., Taylor, L., Browning, L., Naismith, L., and Wilson, E.: Blood pressure in women after one year of oral contraception, Lancet 1: 467, 1971. Smith, D. C., Prentice, R., Thompson, D. J., and Herrmann, W. L.: Association of exogenous estrogen and endometrial carcinoma, N. Engl. J. Med. 293: 1164, 1975. Ziel, H. K., and Finkle, W. D.: Increased risk of endometrial carcinoma among users of conjugated estrogens. N. Engl. J. Med. 293: 1167, 1975. Braselton, W. E., Lin, T. J., Mills, T. M., Ellegood, J. O., and Mahesh, V. B.: Identification and measurement by gas chromatography-mass spectrometry of norethindrone and metabolites in human urine and blood, J. Steroid Biochem. In press.

Fourteenth Annual Rocky Mountsin B)lsanghreering Sympoeium Papers

tech-

are solicited

for

the program

of the

Fourteenth

Annual

Rocky

Mountain

Bioen-

gineering Symposium to be held at Colorado State University, Fort Collins, Colorado, April 25-27, 1977. Papers may be on any aspect of bioengineering. It is expected that sessions will include but not be limited to the following topics: Health Care Delivery, Instrumentation,

Prostheses,

Pattern

Recognition,

Computers

and

Patient

Monitoring,

Grounding and Patient Safety, Environmental Instrumentation Applications, and Biomedical Systems Analysis. Deadline dates are: receipt of abstract (200 words), January 1, 1977; notification of acceptance, January 15, 1977; receipt of completed paper (six pages), February 15, 1977. All abstracts should be sent to: Dr. C. W. Miller, Department of Physiology and Biophysics, Collaborative Radiological Health Laboratory, Colorado State University, Fort Collins, Colorado 8052 1.