The synthetic progestin norgestrel acts to increase LIF levels in the rd10 mouse model of retinitis pigmentosa.

PURPOSE: Retinal degenerative conditions affect thousands of people worldwide. Retinitis pigmentosa (RP) is among the most common, but it is currently incurable. It is characterized by the progressive death of photoreceptor cells, eventually leading to blindness. Neurotrophic factors play an important role in such retinopathies, and much research has been performed on their use as treatments. Our group previously demonstrated the ability of the synthetic progestin norgestrel to rescue photoreceptors from cell death, the mechanism of which is believed to include upregulation of the neurotrophic factor basic fibroblast growth factor (bFGF). The objective of the present study was to investigate whether the protection provided by norgestrel is likely to be mediated by other neurotrophins. METHODS: The 661W photoreceptor cells and retinal explants from P30 to P40 wild-type (wt) C57BL/6 mice were treated with norgestrel over time. Homozygous rd10/rd10 mice that mimic the human form of RP were fed either a control or a norgestrel-containing diet. Changes in neurotrophic factor expression in response to norgestrel were detected with real-time PCR, western blotting, or immunofluorescence staining. Using specific siRNA, leukemia inhibitory factor (Lif) expression was knocked down in 661W photoreceptor cells that were stressed by serum starvation. Cells were treated with norgestrel followed by measurement of cell viability with (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay. RESULTS: LIF, a potent neuroprotective cytokine, was found to be upregulated in response to norgestrel in vitro and in vivo. Upregulation of LIF in degenerating rd10 retinas coincided with preservation of the photoreceptor layer. We also found LIF was necessary for the norgestrel-mediated rescue of stressed photoreceptor cells from cell death in vitro. CONCLUSIONS: LIF was upregulated in response to norgestrel in all models studied and is necessary for the protective effects of norgestrel in vitro. The increase in LIF expression in rd10 mice undergoing retinal degeneration was concurrent with rescue of the photoreceptor cell layer. These results highlight the ability of norgestrel to induce prosurvival molecules in the compromised retina, underlining norgestrel's potential as a viable drug for treatment of RP.

( )-6,6-Difluoronorgestrel, a new synthetic hormonal steroid.

PIP: The total synthesis of (q)-6,6-difluoronorgestrel (DFN) from (q)-17b eta-hydroxy-13beta-ethyl-4-gonen-3-one, using NOF and SF4 chemistry, is reported. The substitution of 6,6-difluoro and 18-methyl upon norethindrone enhanced its progestational activity by 2- and 7-fold, respectively, as determined by the Clauberg assay. However, in combination, DFN has about the same activity as (q)-norgestrel. The findings indicate that 6,6-difluoro substitution is not detrimental to the progestational activity of 17alpha-ethynly-19-norsteroids.^ieng

Norethisterone and levonorgestrel esters: a novel synthetic method.

Aliphatic, alicyclic and arylcarboxylic esters of norethisterone and levonorgestrel were prepared in a one-step synthesis and in near-quantitative yield using trifluoroacetic anhydride.

Long-acting contraceptive agents: aliphatic and alicyclic carboxylic esters of levonorgestrel.

Esters of levonorgestrel (13 beta-ethyl-17 alpha-ethynyl-17 beta-hydroxygon-4-en-3-one) with a variety of aliphatic and alicyclic carboxylic acids have been prepared and characterised. In tests for the suppression of estrus in rats, esters with short-chain aliphatic acids and with cyclobutane-carboxylic acid were considerably more active than the standard, norethisterone enanthate (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one). Such esters show great promise for development as long-acting progestogens.

Development and use of a radioimmunoassay for D-(-)-norgestrel 17 beta-cyclopentanecarboxylate.

The synthesis of the 6 alpha-carboxymethylmercapto BSA and homologous histamine conjugate of D-(-)-norgestrel 17 beta-cyclopentanecarboxylate is reported. Using the BSA conjugate as an immunogen for the development of antibody in the rabbit and the 125I-histamine conjugate as the radioligand, a radioimmunoassay (RIA) for the ester was developed. Serum profiles of the free alcohol and ester were determined following IV or IM injection in macaques. Peak values for the ester (about 12 ng/mL) were observed 2 min following an IV bolus of 0.5 mg in one rhesus monkey. Blood levels dropped rapidly within the first 30 min and were barely detectable at 24 h. Serum levels of the free alcohol rose to a peak at 30 min and then declined slowly to very low values by 24 h. Following IM injection of 20 mg in cynomolgus monkeys, peak levels of the ester were observed within a few days while the free alcohol reached a maximum about day 30. Serum concentrations of D-(-)-norgestrel had fallen to about 0.4 ng/mL 160 days post-injection when levels of the ester fell below 0.2 ng/mL.

Long-acting contraceptive agents: levonorgestrel esters of unsaturated acids.

Esters of levonorgestrel (13 beta-ethyl-17 beta-ethynyl-17 beta-hydroxygon-4-en-3-one) with a variety of unsaturated carboxylic acids have been synthesized for evaluation as potential long-acting, injectable contraceptive agents.

PIP: This paper describes the synthesis of esters of levonorgestrel (13 beta-ethyl-17 beta-ethynyl-17 beta-hydroxygon-4-en-3-one) with a variety of unsaturated carboxylic acids for evaluation as potential longacting injectable contraceptive agents. 1-Cyclohexenylacetic acid was prepared by the hydrolysis of 1-cyclohexenylacetonitrile. The synthesis of E-penta-2,4-dienoic acid was achieved by the condensation of acrolein with malonic acid. Reformatsky reaction between crotonaldehyde and ethyl 2-bromopropionate followed by dehydration of the condensation product was used for the synthesis of E,E-2-methylhexa-2,4-dienoic acid. In the preparation of 5-methyl-2-furylacetic acid, 5-methylfurfural was subjected to condensation reaction with rhodanine followed by hydrolysis. The levonorgestrel esters were synthesized by reaction of the appropriate acid chloride with the thallim salt of levonorgestrel, which was obtained by use of thallous ethoxide. The esters prepared were levonorgestrel 1-cyclohexenylacetate; levonorgestrel 1-cyclopentenylacetate; levonorgestrel E-penta-2,4-dienoate; levonorgestrel E,E-2methylhexa-2,4-dienoate; levonorgestrel 5-methyl-2-furylethaoate; levonorgestrel 3-(5'-methyl-2'-furyl)propanoate; levonorgestrel 3-(5'-ethyl-2'-furyl)propanoate; leveonorgestrel 4-(5'-methyl-2'-furyl)butanoate; levonorgestrel E-non-2-en-4-ynoate; 1-cyclohexenylacetic acid; 1-cyclopentenylacetic acid; E-penta-2,4-dienoic acid; E,E-2-methylhexa-2,4-dienoic acid; 5-methyl-2-furylacetic acid; and E-non-2-en-4-ynoic acid.

Long-acting contraceptive agents: analysis and purification of steroid esters.

More than 200 samples of esters of norethisterone (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one) and levonorgestrel (13 beta-ethyl-17 alpha-ethynyl-17 beta-hydroxygon-4-en-3 -one) have been analysed by a combination of techniques, including high performance liquid chromatography (HPLC). Compounds having a purity below the required limit (99.5%) were purified, mainly by preparative HPLC, prior to formulation and biological evaluation as long-acting progestogens.

Estimation of impurity profiles of drugs and related materials. Part 16: Identification of the side-products of the ethinylation step in the synthesis of contraceptive gestogens.

A new apolar impurity (3,17 alpha-diethinyl-13-ethyl-3,5-gonadiene-17-ol, IIb) was detected and identified in norgestrel with the aid of thin-layer and high-performance chromatography and spectroscopic techniques. IIb is the product of the acid-catalysed dehydration of an overethinylated side product (Ib) of the ethinylation step in the synthesis of norgestrel. IIb can be determined by thin-layer densitometry and high-performance liquid chromatography. Another impurity (17 alpha-ethinyl-13-ethyl-4-gonene-17-ol, IV), originating from a side product of the Birch reduction step in the synthesis of norgestrel was also detected and identified. The spot of IV overlaps with that of IIb in the TLC system of USP XXIII but can be separated and quantification by more selective TLC systems and by gas chromatography.