Studies on a series of milnacipran analogs containing a heteroaromatic group as potent norepinephrine and serotonin transporter inhibitors

A series of milnacipran analogs containing a heteroaromatic group were synthesized and studied as monoamine transporter inhibitors. Many compounds exhibited higher potency than milnacipran at NET and NET/SERT with no significant change in lipophilicity. For example, compound R-26f was about 10-fold more potent than milnacipran with IC(50) values of 8.7 and 26nM at NET and SERT, respectively.     

Identification of 1S,2R-milnacipran analogs as potent norepinephrine and serotonin transporter inhibitors

A series of milnacipran analogs were synthesized and studied as monoamine transporter inhibitors, and several potent compounds with moderate lipophilicity were identified from the 1S,2R-isomers. Thus, 15l exhibited IC(50) values of 1.7nM at NET and 25nM at SERT, which were, respectively, 20- and 13-fold more potent than 1S,2R-milnacipran 1-II.     

Functional expression of the norepinephrine transporter in cultured rat astrocytes

We assessed the functional expression of the norepinephrine (NE) transporter (NET) in cultured rat cortical astrocytes. Specific [3H]NE uptake increased in a time-dependent manner, and this uptake involves temperature- and Na+-sensitive mechanisms. The Na+-dependent [3H]NE uptake was saturable, and the Km for the process was 539.3 +/- 55.4 nm and the Vmax was 1.41 +/- 0.03 pmol/mg protein/min. Ouabain, a Na+-K+ ATPase inhibitor, significantly inhibited Na+-dependent [3H]NE uptake. The selective NE uptake inhibitor nisoxetine, the tricyclic antidepressants desipramine and imipramine, and the serotonin and NE reuptake inhibitor (SNRI) milnacipran very potently inhibited Na+-dependent [3H]NE uptake. On the other hand, GBR-12935 (a selective dopamine uptake inhibitor), fluvoxamine (a selective serotonin reuptake inhibitor), venlafaxine (a SNRI) and cocaine had weaker inhibitory activities. RT-PCR demonstrated that astrocytes expressed mRNA for the cloned NET protein, which was characterized as neuronal NET. Western blots indicated that anti-NET polyclonal antibody recognized a major band of 80 kDa in astrocytes. These data indicate that the neuronal NET is functionally expressed in cultured rat astrocytes. Glial cells may exert significant control of noradrenergic activity by inactivating NE that escapes neuronal re-uptake in sites distant from terminals, and are thus cellular targets for antidepressant drugs that inhibit NE uptake.     

Determination of milnacipran, a serotonin and noradrenaline reuptake inhibitor, in human plasma using liquid chromatography with spectrofluorimetric detection

Milnacipran is an antidepressant drug belonging to the class of serotonin and noradrenaline reuptake inhibitors. A sensitive high performance liquid chromatographic during the development method coupled with a fluorimetric detection was set up, validated and then used routinely of the drug. After liquid-liquid extraction, milnacipran and its internal standard were analyzed by reversed-phase liquid chromatography (LC). The drug was derivatized with fluorescamine for fluorescence detection. The identity of the liquid chromatography peaks was controlled using mass spectrometry. The assay linearity was validated up to 1000 ng/ml. The limit of quantification was set at 5 ng/ml. Precision values (relative standard deviations) were lower than 5.4%, whereas the mean accuracy was higher than 95%. The extraction recoveries were higher than 70% for both milnacipran and the internal standard. In clinics, the LC-fluorescence method was routinely used to investigate the pharmacokinetics of milnacipran in patients and proved to be robust and capable of quantifying milnacipran in plasma for at least 36 h (four- to five-fold the elimination half-life).     

Effects of Milnacipran and Pindolol on Extracellular Noradrenaline and Serotonin Levels in Guinea Pig Hypothalamus

Abstract: Milnacipran, a dual noradrenaline (NA) and serotonin (5-hydroxytryptamine, 5-HT) uptake inhibitor, increased extracellular levels of NA and 5-HT in hypothalamus of freely moving guinea pigs as measured by microdialysis. The basal levels of both monoamines, which were tetrodotoxin sensitive, were increased in a dose-dependent manner and to a similar extent after the intraperitoneal administration of milnacipran (10 and 40 mg/kg i.p.). Levels of the NA metabolite 4-hydroxy-3-methoxyphenylglycol (MHPG) were decreased by milnacipran at 10 and 40 mg/kg i.p., whereas those of the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) showed no effect. Subcutaneous injection of 5-HT_1A and β-adrenergic receptor antagonist (−)-pindolol alone, at 10 mg/kg, had no effect on the extracellular levels of NA or 5-HT. The concomitant administration of (−)-pindolol (10 mg/kg s.c.) with milnacipran (10 mg/kg i.p.) increased severalfold the effect of milnacipran on the extracellular levels of NA and 5-HT. These results indicate that milnacipran, by blocking the uptake of NA and 5-HT, increases virtually equipotently the extracellular levels of NA and 5-HT, confirming previous in vitro studies. In addition, the antagonism of 5-HT_1A autoreceptors by (−)-pindolol potentiates the action of milnacipran on both NA and 5-HT systems, without modifying the ratio of these activities.     

Effects of Milnacipran in Animal Models of Anxiety and Memory

Serotonin (5-HT) and noradrenaline (NA) are involved in both pathogenesis and recovery from depression and anxiety. We examined the effects of acute and chronic treatment with milnacipran, a serotonin/noradrenaline reuptake inhibitors (SNRIs) antidepressant, on anxiety and memory retention in rats. Male Wistar rats received acute or chronic administration of milnacipran (12.5, 25 or 50 mg/kg) or saline (control group). The animals were separately submitted to elevated plus-maze, inhibitory avoidance and open-field tasks 1 h after injection, in the acute group, or 23 h after last injection, in the chronic group. Our results showed an anxiolytic-like effect after chronic administration of milnacipran at doses of 25 and 50 mg/kg. The treatment does not interfere in memory retention and habituation to a novel environment at any doses studied. These findings support that milnacipran, an established SNRIs antidepressant, can also be useful in the treatment of anxiety disorders.     

Continuing efficacy of milnacipran following long-term treatment in fibromyalgia: a randomized trial

Introduction

Previous studies of long-term treatment response in fibromyalgia and other chronic pain states have generally been limited to approximately one year, leaving questions about the longer-term durability of response. The purpose of this study was to demonstrate continuing efficacy of milnacipran by characterizing changes in pain and other fibromyalgia symptoms after discontinuing long-term treatment. The mean length of milnacipran treatment at the time of randomized withdrawal was 36.1 months from initial exposure to milnacipran (range, 17.9 to 54.4 months).

Methods

After completing a long-term, open-label, lead-in study of milnacipran (which followed varying periods of exposure in previous studies), adult patients with fibromyalgia entered the four-week open-label period of the current study for evaluation of ongoing treatment response. After the four-week period to confirm new baseline status, 151 patients taking milnacipran ≥100 mg/day and reporting ≥50% improvement from pre-milnacipran exposure in Visual Analogue Scale (VAS) pain scores were classified as responders. These responders entered the 12-week, double-blind withdrawal period in which they were randomized 2:1 to continue milnacipran or switched to placebo. The prespecified primary parameter was loss of therapeutic response (LTR), defined as increase in VAS pain score to <30% reduction from pre-milnacipran exposure or worsening of fibromyalgia requiring alternative treatment. Adverse events and vital signs were also monitored.

Results

Time to LTR was shorter in patients randomized to placebo than in patients continuing milnacipran (P < 0.001). Median time to LTR was 56 days with placebo and was not calculable for milnacipran, because less than half of the latter group of patients lost therapeutic response by study end. Additionally, 81% of patients continuing on milnacipran maintained clinically meaningful pain response (≥30% improvement from pre-milnacipran exposure), compared with 58% of patients switched to placebo (sensitivity analysis II; P < 0.001). The incidences of treatment-emergent adverse events were 58% and 47% for placebo and milnacipran, respectively. Mean decreases in blood pressure and heart rate were found in both groups, with greater decreases for patients switched to placebo.

Conclusions

Continuing efficacy of milnacipran was demonstrated by the loss of effect following withdrawal of treatment in patients who received an average of three years of milnacipran treatment.

Trial registration

ClinicalTrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT01014585","term_id":"NCT01014585"}}NCT01014585     

Brain Region-Specific Effects of Short-Term Treatment with Duloxetine,Venlafaxine, Milnacipran and Sertraline on Monoamine Metabolism in Rats

We examined brain region-specific changes in monoamines and metabolites, and their ratios, after short-term administration of antidepressants to rats. Serotonin noradrenaline reuptake inhibitors (SNRIs; duloxetine, venlafaxine, milnacipran) and a serotonin-selective reuptake inhibitor (SSRI; sertraline) elevated serotonin (5-HT) levels in the midbrain (MB). Duloxetine and venlafaxine increased 5-HT levels in the brainstem and 5-HT terminal areas, whereas milnacipran and sertraline increased levels in the brainstem only. Significant reductions in 5-HT turnover were observed in various forebrain regions, including the hippocampus and hypothalamus, after treatment with all of the tested drugs except for milnacipran. In addition, there was reduced 5-HT turnover in the dorsolateral frontal cortex (dlFC), the medial prefrontal cortex (mPFC), and both the dlFC and the mPFC after treatment with duloxetine, sertraline, and venlafaxine, respectively. Venlafaxine significantly increased dopamine (DA) levels in the nucleus accumbens (NAc) and the substantia nigra and decreased DA turnover in the NAc. Similar changes were observed after treatment with duloxetine and sertraline in the NAc, whereas milnacipran increased DA levels in the mPFC. Limited increases in noradrenaline levels were detected after treatment with duloxetine, venlafaxine, or sertraline, but not after treatment with milnacipran. These results show that SNRIs and SSRIs induced region-specific monoaminergic changes after short-term treatment.     

Synthesis and pharmacological evaluation of (Z)-9-(heteroarylmethylene)-7-azatricyclo[4.3.1.0(3,7)]decanes: thiophene analogues as potent norepinephrine transporter inhibitors

To further explore the structure-activity relationships (SARs) of certain tropanes, and to gain insights into the structural features required for high activity and selectivity at norepinephrine transporters (NET), we have introduced both five- and six-membered heteroaromatic moieties such as substituted pyridyl, pyrazinyl, pyrimidyl, thiazolyl, and mono- or disubstituted thienyl groups into conformationally constrained, tricyclic tropane analogues. A number of (Z)-9-(heteroarylmethylene)-7-azatricyclo[4.3.1.0(3,7)]decanes were synthesized, and their abilities to block dopamine, serotonin, and norepinephrine reuptake by their respective transporters were evaluated. It was found that the five- or six-membered N-containing aromatics are too basic to display high NET activity, while some of the thiophene analogues were identified as potent and selective NET inhibitors.     

Mitochondrial DNA Released by Trauma Induces Neutrophil Extracellular Traps

Neutrophil extracellular traps (NETs) are critical for anti-bacterial activity of the innate immune system. We have previously shown that mitochondrial damage-associated molecular patterns (mtDAMPs), including mitochondrial DNA (mtDNA), are released into the circulation after injury. We therefore questioned whether mtDNA is involved in trauma-induced NET formation. Treatment of human polymorphoneutrophils (PMN) with mtDNA induced robust NET formation, though in contrast to phorbol myristate acetate (PMA) stimulation, no NADPH-oxidase involvement was required. Moreover, formation of mtDNA-induced NETs was completely blocked by TLR9 antagonist, ODN-TTAGGG. Knowing that infective outcomes of trauma in elderly people are more severe than in young people, we measured plasma mtDNA and NET formation in elderly and young trauma patients and control subjects. MtDNA levels were significantly higher in the plasma of elderly trauma patients than young patients, despite lower injury severity scores in the elderly group. NETs were not visible in circulating PMN isolated from either young or old control subjects. NETs were however, detected in PMN isolated from young trauma patients and to a lesser extent from elderly patients. Stimulation by PMA induced widespread NET formation in PMN from both young volunteers and young trauma patients. NET response to PMA was much less pronounced in both elderly volunteers’ PMN and in trauma patients’ PMN. We conclude that mtDNA is a potent inducer of NETs that activates PMN via TLR9 without NADPH-oxidase involvement. We suggest that decreased NET formation in the elderly regardless of higher mtDNA levels in their plasma may result from decreased levels of TLR9 and/or other molecules, such as neutrophil elastase and myeloperoxidase that are involved in NET generation. Further study of the links between circulating mtDNA and NET formation may elucidate the mechanisms of trauma-related organ failure as well as the greater susceptibility to secondary infection in elderly trauma patients.     

N-Linked Oligosaccharides Are Required for Cell Surface Expression of the Norepinephrine Transporter but Do Not Influence Substrate or Inhibitor Recognition

Abstract: The contribution of N-linked carbohydrates to the function of the human norepinephrine transporter (NET) was investigated using site-directed mutagenesis to inactivate the two most carboxy-terminal (NQQ mutant) or all three (QQQ mutant) sites for N -glycosylation within the extracellular loop between transmembrane domains 3 and 4. In HeLa cells transiently expressing the NET, two glycosylated forms of the transporter at 90 and 60 kDa are immunoprecipitated by NET antisera. A single 50-kDa species is observed in cells expressing the QQQ mutant, and it likely represents the NET core protein. Analyses of substrate transport kinetics showed rank order V _max of 19:9:1 for NET/NQQ/QQQ without a change in the apparent affinity of the wild-type and mutated carriers for either substrates or transport inhibitors. Cell surface biotinylation indicates that all NET, NQQ, and QQQ transporter species are detected at the plasma membrane but that glycosylated forms are selectively enriched. The transport activities exhibited by each of the carriers correlate well with cell surface content. Subcellular localization of transporters using immunofluorescence microscopy shows that reductions in surface expression and transport are associated with a corresponding increase in the intracellular retention of mutated carriers. Thus, N-linked glycosylation does not alter the apparent affinity of NET for either substrates or inhibitors of transport but, instead, appears to influence the abundance of carriers at the cell surface.     

Effect of long-term administration of the antidepressant drug milnacipran on serotonergic and noradrenergic neurotransmission in the rat hippocampus

The effect of a long-term administration of the antidepressant milnacipran on the function of the serotonergic (5-HT) and noradrenergic (NE) systems was studied using single cell recording of CA3 hippocampal pyramidal cells in chloral hydrate-anesthetized male Sprague-Dawley rats, and in vitro [3H]5-HT release measurement from hippocampal slices. The sensitivity of neither the extrasynaptic nor that of the postsynaptic 5-HT1A receptors of the pyramidal neurons was altered, as indicated by their unchanged responsiveness to the microiontophoretic application of 5-HT, and by the unchanged effect of the electrical stimulation at low frequency of the ascending 5-HT bundle, respectively. Increasing the frequency of stimulation (from 1 to 5 Hz) decreased its efficacy in control rats; the milnacipran treatment abolished this phenomenon. This cannot be attributed to a desensitisation of the terminal 5-HT1B autoreceptor, since the suppressive effect of 5-HT agonist 5-carboxyamidotryptamine on [3H]5-HT release was enhanced in milnacipran-treated rats. As for the NE system, the unchanged suppressing effect of microiontophoretic applications of NE and that of the 5 Hz stimulation in the locus coeruleus (LC) on the firing activity of pyramidal neurons indicates that the milnacipran treatment not altered the sensitivity of extrasynaptic alpha2- and postsynaptic alpha1-adrenergic receptors on pyramidal cells, as well as that of the presynaptic alpha2-autoreceptor on NE terminals. The decreased inhibitory effect of NE on the [3H]5-HT release in milnacipran-treated rats revealed that this treatment results in a desensitisation of the presynaptic alpha2-heteroreceptor located on serotonergic terminals. Taken together with the decreased suppressive effect of a low frequency of stimulation of the NE tract, the present results suggest that long-term milnacipran treatment enhances the efficacy of the 5-HT and reduces that of the NE neurotransmission.     

Poly(DL-lactide-co-glycolide)/norethisterone microcapsules: an injectable biodegradable contraceptive

Microcapsules made from a biocompatible, biodegradable polymeric excipient, poly(DL-lactide-co-glycolide) (DL-PLGA) that contained 22 weight percent (wt %) norethisterone (NET), were prepared by a solvent-evaporation microencapsulation process. The effects of changing both the lactide-to-glycolide ratio of the DL-PLGA and the size of the microcapsules on the rate of NET release and the rate of excipient biodegradation were determined in vivo. NET release rates were determined in baboons after injecting the microcapsule formulations intramuscularly. Serum samples obtained at various times following treatment were analyzed for NET, progesterone, and estrogen by radioimmunoassay (RIA). Biodegradation kinetics were determined by injecting NET microcapsules made from radiolabeled DL-PLGA intramuscularly into the hind legs of rats. Residual radioactivity at the injection site was determined at various times after treatment by combustion analysis of the muscle tissue. Changing the ratio of the comonomers to include more glycolide (DL-lactide:glycolide-96:4, 92:8, 87:13, 74:26) increased the rate of NET release and accelerated the biodegradation of the copolymer excipient. Decreasing the size of the microcapsules increased the rate of NET release. On the basis of these studies a NET microcapsule formulation has been identified for clinical testing which releases NET for 3 months and biodegrades completely within 6 months.     

Evidence that a non-aromatizable metabolite of norethisterone induces estrogen-dependent pituitary progestin receptors

Neutral reduced metabolites of norethisterone (NET) specifically interact with intracellular estrogen receptors in target organs. To determine if this interaction can effectively initiate estrogen-dependent cellular responses, the effects of an A-ring-reduced NET derivative upon the induction of cytosol-located pituitary progestin receptors (PR) and uterine growth were studied in adult castrated female rats. Different doses of 17 alpha-ethynyl-5 alpha-estran-3 beta, 17 beta-diol (3 beta, 5 alpha-NET) were s.c. administered to ovariectomized animals for 6 days. 17 beta-Estradiol (E2) and oil-treated rats served as experimental controls. Pituitary PR were labeled in vitro by a post-gradient technique using [3H]ORG-2058 as the ligand. PR binding specificity was determined by the use of an excess of radioinert steroids. The results demonstrated that administration of 3 beta, 5 alpha-NET induced specific 8-9S pituitary cytosol PR in a dose-dependent manner. Binding properties of the 3 beta, 5 alpha-NET-induced progestin binding sites (Kd = 1.0 X 10(-9) M; NBS = 1.2 X 10(-9) M) appear indistinguishable from those induced by E2. In addition, 3 beta, 5 alpha-NET administration resulted in a significant increase in uterine weight at the expense of myometrium and endometrium growth in a similar fashion to that observed in the E2-treated group. When 3 alpha, 5 alpha-epimeric alcohol (3 alpha, 5 alpha-NET) was administered, induction of pituitary PR and uterine growth were also observed although to a lesser extent. Inasmuch as the results demonstrate that neutral non-aromatizable NET metabolites induce biochemical and morphological estrogenic responses, they offer an alternative explanation for the mechanism of estrogen-like action of this synthetic contraceptive progestin.     

Targeting peptidylarginine deiminase reduces neutrophil extracellular trap formation and tissue injury in severe acute pancreatitis

Recent evidence suggests that neutrophil extracellular traps (NETs) play an important role in the development of acute pancreatitis (AP). Herein, we examined the role of peptidylarginine deiminase (PAD), which has been shown to regulate NET formation, in severe AP. AP was induced by retrograde of taurocholate infusion into pancreatic duct in C57BL/6 mice. PAD was pharmacologically inhibited using Cl-amidine, a pan-PAD inhibitor. Pancreata were collected, and histones, citrullinated histone 3, chemokines, myeloperoxidase, and NETs were quantified. Chemokines, matrix metalloproteinase-9 (MMP-9), interleukin-6 (IL-6), and DNA-histone complexes were determined in plasma samples. Infusion of taurocholate induced formation of NETs in pancreatic tissues of mice. Pretreatment with Cl-amidine markedly reduced the NET formation in the inflamed pancreas. Moreover, inhibition of PAD decreased the levels of blood amylase as well as edema, acinar cell necrosis, hemorrhage, and neutrophil infiltration in the pancreas of animals with AP. Administration of Cl-amidine attenuated the myeloperoxidase levels in the pancreas and lung of mice exposed to taurocholate. In addition, Cl-amidine decreased pancreatic levels of CXC chemokines, plasma levels of IL-6, and MMP-9 in mice with severe AP. This study shows that Cl-amidine is a potent inhibitor of NET formation in severe AP. Also, our results suggest that PAD regulates pathological inflammation and tissue damage in the inflamed pancreas. Thus, targeting PAD might be a useful strategy to treat patients with severe AP.     

Periodontal therapy increases neutrophil extracellular trap degradation

In periodontitis, polymorphonuclear leucocytes (PMNs) are activated. They entrap and eliminate pathogens by releasing neutrophil extracellular traps (NETs). Abnormal NET degradation is part of a pro-inflammatory status, affecting co-morbidities such as cardiovascular disease. We aimed to investigate the ex vivo NET degradation capacity of plasma from periodontitis patients compared to controls (part 1) and to quantify NET degradation before and after periodontal therapy (part 2). Fresh NETs were obtained by stimulating blood-derived PMNs with phorbol 12-myristate 13-acetate. Plasma samples from untreated periodontitis patients and controls were incubated for 3 h onto freshly generated NETs (part 1). Similarly, for part 2, NET degradation was studied for 91 patients before and 3, 6 and 12 mo after non-surgical periodontal therapy with and without adjunctive systemic antibiotics. Finally, NET degradation was fluorospectrometrically quantified. NET degradation levels did not differ between periodontitis patients and controls, irrespective of subject-related background characteristics. NET degradation significantly increased from 65.6 ± 1.7% before periodontal treatment to 75.7 ± 1.2% at 3 mo post periodontal therapy, and this improvement was maintained at 6 and 12 mo, irrespective of systemic usage of antibiotics. Improved NET degradation after periodontitis treatment is another systemic biomarker reflecting a decreased pro-inflammatory status, which also contributes to an improved cardiovascular condition.     

Carp neutrophilic granulocytes form extracellular traps via ROS-dependent and independent pathways

Neutrophil extracellular traps (NETs) have recently been described as an important innate defense mechanism that leads to immobilization and killing of invading pathogens. NETs have been identified in several species, but the mechanisms involved in NET formation and their role in infection have not been well determined yet. Here we show that upon in vitro stimulation with different immunostimulants of bacterial, fungal or viral origin, carp neutrophilic granulocytes rapidly release NET structures. We analyzed the composition of these structures and the kinetics of their formation by confocal microscopy, by quantifying the levels of extracellular DNA and the release of enzymes originating from neutrophilic granules: myeloperoxidase, neutrophil elastase and matrix metalloproteinase 9 (MMP-9). Profiles of NET release by carp neutrophils as well as their enzyme composition are stimulus- and time-dependent. This study moreover provides evidence for a stimulus-dependent selective requirement of reactive oxygen species in the process of NET formation. Collectively the results support an evolutionary conserved and strictly regulated mechanism of NET formation in teleost fish.     

Variable expression of the uteroglobin gene following the administration of norethisterone and its A-ring reduced metabolites

Enzyme-mediated A-ring reduction of norethisterone (NET) results in the transformation of a molecule with potent intrinsic progestational activity into neutral derivatives with estrogen-like effects. To ascertain whether these structural modifications of NET are able to modify the uteroglobin (U) gene (G) expression, a series of experiments assessing the UG products after the administration of NET and its reduced A-ring metabolites were conducted in prepubertal female rabbits. Synthesis of endometrial uteroglobin and its specific mRNA were studied in animals following the administration of NET, 5 alpha-dihydro NET,3 beta,5 alpha-tetrahydro NET and progesterone. Animals treated with either estradiol or vehicle alone served as controls. The uteroglobin content in uterine flushings and cytosols was determined by immunodiffusion and polyacrilamide gel electrophoresis techniques and by a specific double-antibody radioimmunoassay, while the U mRNA synthesis was assessed by its molecular hybridization to [alpha 32P]d-ATP uteroglobin cDNA. NET induced a significant increase of the uterine content of uteroglobin similar to that observed with progesterone with a simultaneous increase on U mRNA synthesis. On the contrary, 5 alpha-NET and 3 beta,5 alpha-NET induced very little, if any uteroglobin synthesis with a concomitantly low U mRNA production as compared with NET; thus exhibiting a similar effect to that observed in estradiol-treated animals. The overall results were interpreted as demonstrating that the enzyme mediated structural changes of NET which occur at the target organs induce variable expression of the uteroglobin gene. The data indicate that the rabbit uteroglobin gene products are suitable molecular markers to evaluate the hormonal potency of contraceptive synthetic progestins and their derivatives.     

Stereospecificity of the intracellular binding of norethisterone and its A-ring reduced metabolites

The interaction of norethisterone (NET) and four A-ring reduced metabolites of NET with cytosol receptors for progesterone (PR), androgen (AR), and estrogen (ER) was investigated. Cytosol preparations from: uteri of adult estrogen-primed castrated rats, ventral prostates of adult castrated rats and uteri of immature rats were used as the source of PR, AR, and ER respectively. 3H-Labeled ORG-2058, R-1881, and 17 beta-estradiol were used as the radioligands. The results of competitive studies disclosed that: the most efficient competitor for PR binding sites was NET (Ki = 1.1 X 10(-7) M) followed by 5 alpha-dihydro NET (5 alpha-NET), whereas the 3 alpha,5 alpha; 3 beta,5 alpha and 3 alpha,5 beta-tetrahydro NET derivatives were ineffective the most efficient competitor for AR binding sites was 5 alpha-NET (Ki = 1 X 10(-8), immediately followed by NET, while the three tetrahydro NET derivatives were not competitors and remarkable competition for ER binding sites was only exhibited by the 3 beta,5 alpha-tetrahydro NET derivative (Ki = 4.6 X 10(-8) M) and to a lesser extent by its 3 alpha,5 alpha-epimeric alcohol, while NET and 5 alpha-NET were completely ineffective. These findings demonstrate the stereospecificity of the intracellular binding of NET and its reduced metabolites with cytosol steroid putative receptors, and provide biochemical support to the understanding of the variety of hormone-like effects observed after the in vivo administration of NET.