Long-acting contraceptive agents: The influence of pharmaceutical formulation upon biological activity of esters of norethisterone

17β-esters of norethisterone (17α-ethynyl-17β-hydroxyestr-4-en-3-one) have been formulated as aqueous microcrystalline suspensions and oily solutionsfor administration to rats to assess the length of progestogenic activity. Results show that, for some of the esters, the rate-controlling step in prolonging activity is the rate of drug release from the injected formulation. For these esters, when formulated as suspensions, it is proposed that crystal size and form will have a critical effect upon duration of estrus suppression. The influence of crystal form has been demonstrated with the 4-(butoxy)phenylacetate ester for which two different crystal forms have been identified. The lower melting point, more soluble crystal form shows marked prolongation of action, whereas the other form is ineffective.     

Long-acting contraceptive agents: Structure activity relationships in a series of norethisterone and levonorgestrel esters

A large number of esters of norethisterone (17α-ethynyl-17β-hydroxyestr-4-en-3-one) and levonorgestrel (D-(-)-13β-ethyl-17α-ethynyl-17β-hydroxygon-4-en-3-one) were synthesized and tested for biological activity. The test employed in these studies was the duration of estrus suppression in cycling mature rats. In the norethisterone series several esters exhibited duration of activity comparable to that of norethisterone enarthate. In the levonorgestrel series the butanoic, cyclobutylcarboxylic and cyclopropylcarboxylic esters were longer acting than medroxyprogesterone acetate (17α-acetoxy-6α-methylpregn-4-ene-3, 20-dione) when prepared as aqueous microcrystalline suspensions.     

Plasma levels of levonorgestrel, gestodene, norethisterone and cyproterone acetate on single-dose subcutaneous administration in oily solution in the rat, beagle and rhesus monkey

This report describes the pharmacokinetics of levonorgestrel, gestodene, norethisterone and cyproterone acetate following subcutaneous administration of oily solutions in the rat, beagle dog and rhesus monkey. The plasma levels of the progestogens were measured by means of specific radioimmunoassays. Half-lives calculated for the disposition process of a particular metabolically unchanged drug in plasma revealed marked differences in different animal species. Furthermore, comparison of the different progestogens showed large variations in this parameter in all the animal species. It became obvious that there are physico-chemical properties as well as metabolic rate limitations effecting the release and elimination of synthetic progestogens administered in oily solution. The results are compared with the half-lives of these progestogens administered intravenously as reported previously. A prolongation of half-life as a result of the depot effect of subcutaneous administration was demonstrated for all the progestogens in the rat, the beagle dog and the rhesus monkey.     

Long-acting contraceptive agents: Bile acid esters of norethisterone

The synthesis of the esters of norethisterone (17α-ethynyl-l7β-hydroxyestr-4-en-3-one) with three bile acids and of the cholesteryl carbonate of norethisterone are described.     

Assessment of the specificity of norethisterone radioimmunoassays

Specifities of 4 different norethisterone (Nor) antisera (coded A,B,C, and D) were evaluated and compared by cross-reaction studies to relate the antiserum specificity to the overall specificity of the radioimmunoassay (RIA), as established by plasma levels measured in women regularly taking the microdose of Nor (300 mcg/day). Using any of the 4 antisera, no significant deviation from parallelism were found among graded doses of authentic Nor and increasing volumes of plasma from women taking Nor for contraception. Cross-reaction studies preceded by chromatography to decrease plasma blanks are described, with each antiserum compared to the others for its efficacy in estimating plasma Nor values. It was concluded that 1) the significance of cross-reaction studies as well as that of a parallelism test for assessing overall specifity of the RIA is limited; 2) a single chromatography before RIA improves assay specificity but may not be sufficient to remove all interfering compounds; and 3) a comparison of direct and chromatographic procedures using several different antisera is useful for selection of the relatively most specific RIA procedure. These study results indicated that either antiserum C or D (preceded by chromatography) will yield better results than A or B.     

Long-acting contraceptive agents: Cyclopropyl and cyclobutyl esters of norethisterone

Several esters of norethisterone (17α-ethynyl-17β-hydroxyestr-4-en-3-one) with carboxylic acids containing a cyclopropyl or cyclobutyl ring have been synthesized and the stereochemistries of the side-chains determined.     

Long-acting contraceptive agents: Esters of norethisterone with alkoxy- and halogeno-substituted carboxylic acids

The chemical synthesis and physical data of several new esters of norethisterone (17α-ethynyl-17β-hydroxyestr-4-en-3-one) are reported, which contain either a chloro- or an alkoxy-group as a substituent in the acid side-chain.     

Long-acting contraceptive agents: norethisterone esters of polyunsaturated acids

Some new derivatives of norethisterone (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one) are described in which the 17 beta-hydroxyl group of the steroid is esterified with polyunsaturated aliphatic acids. The potential of these compounds as long-acting contraceptive agents has been evaluated.     

Long-acting contraceptive agents: carbonates and carbamates of norethisterone

The preparation of three carbonates and two carbamates of norethisterone (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one) are described. Due to instability of the carbonates and the very low solubility of the carbamates these compounds could not be submitted to biological testing.     

Long-acting contraceptive agents: in vitro hydrolysis of esters of norethisterone and levonorgestrel

The hydrolysis of 108 esters of norethisterone (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one) was studied in vitro using a rabbit liver preparation. Introduction of a double or triple bond into a straight-chain ester did not inhibit hydrolysis but a marked reduction in hydrolysis was produced on replacement of a methylene group by an oxygen atom. Hydrolysis was inhibited by substituents at C2 of the ester chain except in short chain esters. Cyclopropylcarboxylate and cyclobutylcarboxylate were readily hydrolysed and introduction of a furan ring into the side-chain did not affect hydrolysis. No hydrolysis occurred with a cholesteryl carbonate ester or with a pentamethyldisilyloxy ether. Forty-nine esters of levonorgestrel (13 beta-ethyl-17 alpha-ethynyl-17 beta-hydroxygon-4-en-3-one) were also studied. In general, the pattern of hydrolysis for these esters was similar to that of the norethisterone esters. However, with few exceptions the levonorgestrel esters were hydrolyzed more slowly. For those esters for which information regarding the biological activity was available, there was no correlation between the potency of the esters and their rate of hydrolysis in vitro.     

Plasma levels of norethisterone measured by radioimmunoassay and gas chromatography-mass fragmentography

Assays designed to quantitate the plasma levels of norethisterone (NET) are compared. Results from radioimmunoassay were compared with those from gas chromatography-mass fragmentography which were established in 5 subjects after a 1-mg oral dose of the steroid. In general, the levels of steroid measured by radioimmunoassay were usually higher than those measured by gas chromatography-mass fragmentography; there was however a correlation coefficient of .96 (P .001). For concentrations of the steroid above 500 pg/ml, the mean overestimate by radioimmunoassay was less than 30%.     

Binding of the gestagens norethisterone and levonorgestrel in blood of various species

This study investigates the binding of 2 widely used contraceptive steroids, levonorgestrel and norethisterone, by plasma from various animal species and compares the results to those obtained with human plasma. Equilibrium dialysis of plasma samples and polyacrylamide gel electrophoresis were performed as previously described. The plasma samples were diluted with phosphate-buffered saline on the percentage of levonorgestrel and norethisterone bound in comparison to human plasma. The concentration of total protein and albumin was measured colorimetrically in each sample. An ammonium sulphate precipitation technique measured the level of sex-hormone binding globulin (SHBG). Results of the equilibrium dialysis show that binding of levonorgestrel and norethisterone in plasma was similar in adult female rhesus monkeys and baboons to that of humans with both high-affinity and low-affinity classes of binding sites. The dissociation constants of the high-affinity class for levonorgestrel was 4-fold lower than that for norethisterone in all 3 primates, indicating levonorgestrel was more tightly bound. Total protein and albumin concentrations were also the same in all 3 primates. SHBG levels in female monkeys and baboons however were 3-4 times those found in normal human females. Although differences exist in the binding of the 2 gestagens between human, baboon, and rhesus monkey plasma, there are no significant differences in the metabolism of the gestagens in the 3 primates. Overall, the results indicate that in the human, baboon, and rhesus monkey, binding of norgestrel and norethisterone occur mainly to SHBG, which had a greater affinity for norgestrel than for norethisterone, and to a lesser extent, albumin. Differences in the binding of gestagens between human and nonprimate species (rat, dog, rabbit) studied suggest that only baboon and rhesus monkeys may be considered appropriate animal models for extrapolation of results of contraceptive studies to humans.     

Microbiological hydroxylation of norethisterone.

Hydroxylation of norethisterone by a large number of fungi has been investigated. 1alpha-Hydroxy-, 6beta-hydroxy-, 10beta-hydroxy-, 10beta,11beta-dihydroxy-15alpha-and 15beta-hydroxy-derivatives were formed from norethisterone. The microbiological dehydrogenation of 10beta-hydroxy-norethisterone resulting in 10beta,17beta-dihydroxy-17-ethynyl-1,4-estradien-3-one was also observed. The structure of transformation products was established by chemical and spectroscopical methods.     

A sensitive solid phase enzymeimmunoassay for norethisterone (norethindrone) in saliva and plasma

A sensitive, solid phase enzymeimmunoassay suitable for determining norethisterone in small aliquots of plasma (10 μl) and saliva (100 μ1) has been developed. A solid phase antiserum raised against a norethisterone-llα-hemi-succinyl/bovine serum albumin conjugate was prepared by coupling to cyanogen bromide activated cellulose. A norethisterone/horseradish peroxidase conjugate was used as enzyme label, o?-phenylenediamine/hydrogen peroxide being the substrate for colour development. The assay had a lower limit of sensitivity of 3 pg/assay tube and satisfied accepted validation criteria. Norethisterone concentrations determined by enzymeimmunoassay and by a well established radioimmunoassay were in excellent agreement in both plasma (r = 0.993, n = 20) and saliva (r = 0.989, n = 15). Plasma and salivary norethisterone concentrations determined in healthy volunteers reached peak values at about 1 hour after administering a norethisterone-containing oral contraceptive preparation. The maximum values achieved in saliva (775–1430 pmol/l) were only approximately 3% of those observed in plasma. Since salivary norethisterone concentrations reflected those in plasma, they may be useful in fertility control programmes and pharmacokinetic studies.     

Evaluation of the lipophilic properties of opioids, amphetamine-like drugs, and metabolites through electrochemical studies at the interface between two immiscible solutions

For the first time, the partition coefficients of the ionized forms of several opioids, amphetamine-like drugs, and their metabolites were determined by studying their ionic transfer process across the bare interface water/organic solvent. The ionic partition coefficients of the monocationic forms of 12 compounds--heroin, 6-monoacetylmorphine (6-MAM), morphine, acetylcodeine, codeine, dihydrocodeine, methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy"), 3,4-methylenedioxyamphetamine (MDA), 3-methoxy-alpha-methyldopamine (3-OMe-alpha-MeDA), and alpha-methyldopamine (alpha-MeDA)-were attained using electrochemical measurements, by cyclic voltammetry, at the interface between two immiscible electrolyte solutions (ITIES). Then the acquired lipophilicity values were correlated to the chemical structure of the compounds and with the metabolic pathways central to each class of drugs. Although the mechanisms of biotoxicity of this type of drugs are still unclear, the data obtained evidence that the lipophilicity of metabolites may be a contributing factor for the qualitative differences found in their activity. In addition, the partition coefficients of the ionic drugs were calculated using three available software packages: ModesLab, Dragon, and HyperChem. As shown by cross-comparison of the experimental and calculated values, HyperChem was the most reliable software for achieving the main goal. The data obtained so far seem to be correlated to the proposed metabolic pathways of the drugs and could be of great value in understanding their pharmacological and/or toxicological profiles at the molecular level. This study may also contribute to gaining an insight into the mechanisms of biotransportation of this type of compounds given that the ionic partition coefficients reflect their ability to cross the membrane barriers.     

Long-acting contraceptive agents: esters of norethisterone with alpha- and/or beta-chain branching

The synthesis of eighteen esters of norethisterone (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one) is described. These all possess some form of alpha- and/or beta-substitution in the ester side-chain. The work was undertaken in order to evaluate any long-acting fertility control effect intrinsic in such compounds. A pentamethyl disiloxy ether was also included in the group of substances prepared for testing because of its similar substitution pattern.     

Radioimmunoassay system for norethisterone using 3H- and 125I-labelled radioligands.

A radioimmunoassay procedure is outlined for norethisterone, a synthetic progestagen. This assay uses both tritiated and iodine-125 labelled radioligands and may serve as a model for assays of synthetic steroids for which no tritiated radioligand exists. Male volunteers took a single oral dose of 10 mg of norethisterone acetate (SH 420). Plasma hormone levels were then measured at various time intervals. The degree of binding of iodine-125 labelled radioligand to antiserum even at low serum dilution was always greater than 80%. Using antinorethisterone-11 alpha-BSA serum, triated norethisterone and norethisterone-3-OCMO-iodine -125-iodohistamine radioligands give comparable results of adequate specificity, precision, accuracy and sensitivity when used to analyze crude ether extracts of the plasma samples. The chromatographic step is unnecessary for specific analysis. Iodine-125 labelled ligands may be useful for the determination of other synthetic steroids.     

Irreversible protein binding of norethisterone (norethindrone) epoxide.

14,15-3H-Norethisterone-4 beta, 5 beta-epoxide, a metabolite of norethisterone, was incubated with several proteins and nucleic acids. After 30 min incubation 0.19 nmol of the epoxide were irreversibly bound per mg albumin which contains free sulfhydryl groups; proteins without SH-groups, such as concanavalin A, gamma-globulin, DNA and RNA, did not irreversibly bind norethisterone epoxide. A superoxide (O2) generating enzyme system comprised of xanthine oxidase and hypoxanthine was capable of catalyzing the irreversible binding of the parent compound, norethisterone, to albumin, indicating that an oxidation product was formed which reacted with the protein. When norethisterone epoxide was incubated for 60 min with hepatic microsomes of rats in absence of NADPH, about 2.0 nmol of the epoxide were irreversibly incorporated per mg microsomal protein. This binding was increased to 5.2 nmol by addition of a NADPH regenerating system. Addition of glutathione and cytosol decreased only the NADPH-dependent protein binding; phenobarbital pretreatment of rats induced this NADPH-dependent binding of norethisterone epoxide to microsomal protein by a factor of 2. In presence of NADPH, binding of the epoxide to microsomal protein depended on substrate concentration used. The results indicate that norethisterone epoxide is able to chemically react with proteins. In addition, hepatic microsomal enzymes convert the epoxide to another metabolite which also can react with proteins.     

Serum progesterone and norethisterone levels following injection of norethisterone enanthate in different sites and doses

Serum progesterone (P) and norethisterone (NET) levels following injection of norethisterone enanthate (NETEN) were studied in 11 regularly menstruating women. In 6 subjects 200 mg NETEN was administered to the gluteal (IG) or deltoid (ID) region sequentially. The serum P levels remained anovulatory (less than 4 ng/mL) up to 12 weeks. Serum NET levels were similar whether injection was given IG or ID (paired t - test, NS). In 5 subjects given a 150-mg dose, serum P levels remained anovulatory up to 11 weeks. Serum NET levels declined faster than with the 200-mg dose, but the difference was not significant (Student's t - test, NS). Wide inter-subject variations were seen in both groups. NETEN therefore can effectively be administered in gluteal or deltoid regions. A lower dose of 150 mg may have contraceptive potential on an 8-week schedule.     

Inhibition of 17β-hydroxysteroid dehydrogenase (17β-HSD) activities of human placenta by steroids and non-steroidal hormone agonists and antagonists

Various naturally occurring steroids, synthetic steroid derivatives and non-steroidal hormone agonists and antagonists were assayed as inhibitors of human placental 17β-HSD activities. Microsomal 17β-HSD was inhibited by C₁₈ -,C₁₉- and C₂₁-steroids. Soluble 17β-HSD was highly specific for C₁₈-steroids. In contrast to the soluble activity, the microsomal enzyme also had a strong affinity for ethinylestradiol (K_I=0.3 μM) and danazol (K_I=0.6 μM); anabolic steroids and norethisterone were weaker inhibitors. Of the non-steroids tested only diethylstilbestrol and o-demethyl CI-680 were inhibitors and they showed a greater affinity for soluble 17β-HSD.K_I-values for estradiol-17β, (0.8 μM), progesterone (27.0 μM) and 20α-dihydroprogesterone (1.5 μM) were comparable to reported tissue levels of these compounds, consistent with a possible competition in vivo among naturally occurring C₁₈-, C₁₉-, and C₂₁-steroids for the active site of microsomal 17β-HSD.