scholarly journals Pre-clinical Anti-Addictive and Side-Effect profiles of Novel Kappa-opioid Agonists

2021 ◽  
Author(s):  
◽  
David Young
Keyword(s):  
Side Effects ◽  
Side Effect ◽  
Salvinorin A ◽  
The Novel ◽  
Self Administration ◽  
Kappa Opioid ◽  
Drug Seeking ◽  
Hek 293 ◽  
293 Cells ◽  
Clinical Models

<p>Background: Drug addiction is a chronic, relapsing disorder with great socioeconomic and morbidity costs. An estimated 27 million people worldwide suffer from drug dependence, with over 180,000 drug abuse-related deaths reported annually (UNODC, 2015). Currently, there are no FDA-approved pharmacotherapies for psychostimulant addiction, limiting the efficacy of treatment for cocaine and amphetamine abuse. Kappa-opioid receptor (KOPr) agonists can act as inhibitors of reward, and have been investigated in pre-clinical models of drug abuse for potential anti-addictive properties, but display undesirable side-effects such as dysphoria and sedation. A naturally-occurring KOPr agonist, Salvinorin A (SalA), has been explored as a lead for new KOPr-based anti-addictive medications. SalA is a short-acting but potent non-nitrogenous KOPr agonist with known anti-cocaine effects, and chemical alterations to this structure have produced novel agonists with comparable or greater potency at the KOPr. This thesis compares two novel SalA analogues, 16-ethynyl Salvinorin A (Ethy-SalA) and 16-methyl Salvinorin A (Me-SalA), in pre-clinical models of addiction and side-effect tests.  Methods: Sprague-Dawley rats were used to model the behavioural effects of acute KOPr treatment upon cocaine self-administration and drug-seeking behaviour, natural reward-seeking, cocaine-induced and spontaneous locomotion, and pro-depressive forced-swim testing. Transiently co-transfected HEK-293 cells were used to model the influence of KOPr activation upon dopamine transporter (DAT) function in an in vitro model of dopamine uptake, using confocal microscopy to detect internalisation of the fluorescent DAT substrate ASP+.  Results: Acute pre-treatments of Ethy-SalA significantly attenuated cocaine-reinstatement of drug-seeking behaviour (at 0.1 and 0.3 mg/kg) and progressive ratio (PR) self-administration of cocaine (at 2.0 mg/kg). The less potent agonist Me-SalA did not attenuate cocaine-reinstatement or PR self-administration at the doses tested (0.3-2.0 mg/kg). Despite apparent anti-cocaine effects, Ethy-SalA (0.3 mg/kg) was not found to effectively reduce cocaine-induced locomotor hyperactivity or sensitisation in rats. Side-effect screens were carried out on the novel compounds using the doses tested in cocaine-primed reinstatement. Ethy-SalA (0.3 mg/kg) and Me-SalA (1.0 mg/kg) did not significantly affect spontaneous locomotor behaviour 0.3 mg/kg, or reduce self-administration of the natural reward sucrose at a dose of 0.3 mg/kg in rats. Depression-like effects caused by acute Ethy-SalA treatment (0.3 mg/kg) were also not detected in the Forced Swim Test. Treatment with Ethy-SalA (10 µM) significantly increased uptake of the fluorescent ASP+ in co-transfected DAT/KOPr HEK-293 cells.   Conclusions: A single treatment of the novel KOPr agonist Ethy-SalA, but not the novel agonist Me-SalA, was found to attenuate drug-seeking behaviours in models of cocaine administration with greater potency than SalA, and without detectable sedative or depression-like effects at a dose of 0.3 mg/kg. The cellular mechanism-of-action by which Ethy-SalA depresses cocaine reward is at least in part due to positive regulation of DAT, which would act to reduce extracellular dopamine within the brain. The lack of significant side-effects and the apparent improved potency of the compound support further exploration of Ethy-SalA as a lead for the development of an anti-addictive pharmacotherapy.</p>

scholarly journals Pre-clinical Anti-Addictive and Side-Effect profiles of Novel Kappa-opioid Agonists

2021 ◽  
Author(s):  
◽  
David Young
Keyword(s):  
Side Effects ◽  
Side Effect ◽  
Salvinorin A ◽  
The Novel ◽  
Self Administration ◽  
Kappa Opioid ◽  
Drug Seeking ◽  
Hek 293 ◽  
293 Cells ◽  
Clinical Models

<p>Background: Drug addiction is a chronic, relapsing disorder with great socioeconomic and morbidity costs. An estimated 27 million people worldwide suffer from drug dependence, with over 180,000 drug abuse-related deaths reported annually (UNODC, 2015). Currently, there are no FDA-approved pharmacotherapies for psychostimulant addiction, limiting the efficacy of treatment for cocaine and amphetamine abuse. Kappa-opioid receptor (KOPr) agonists can act as inhibitors of reward, and have been investigated in pre-clinical models of drug abuse for potential anti-addictive properties, but display undesirable side-effects such as dysphoria and sedation. A naturally-occurring KOPr agonist, Salvinorin A (SalA), has been explored as a lead for new KOPr-based anti-addictive medications. SalA is a short-acting but potent non-nitrogenous KOPr agonist with known anti-cocaine effects, and chemical alterations to this structure have produced novel agonists with comparable or greater potency at the KOPr. This thesis compares two novel SalA analogues, 16-ethynyl Salvinorin A (Ethy-SalA) and 16-methyl Salvinorin A (Me-SalA), in pre-clinical models of addiction and side-effect tests.  Methods: Sprague-Dawley rats were used to model the behavioural effects of acute KOPr treatment upon cocaine self-administration and drug-seeking behaviour, natural reward-seeking, cocaine-induced and spontaneous locomotion, and pro-depressive forced-swim testing. Transiently co-transfected HEK-293 cells were used to model the influence of KOPr activation upon dopamine transporter (DAT) function in an in vitro model of dopamine uptake, using confocal microscopy to detect internalisation of the fluorescent DAT substrate ASP+.  Results: Acute pre-treatments of Ethy-SalA significantly attenuated cocaine-reinstatement of drug-seeking behaviour (at 0.1 and 0.3 mg/kg) and progressive ratio (PR) self-administration of cocaine (at 2.0 mg/kg). The less potent agonist Me-SalA did not attenuate cocaine-reinstatement or PR self-administration at the doses tested (0.3-2.0 mg/kg). Despite apparent anti-cocaine effects, Ethy-SalA (0.3 mg/kg) was not found to effectively reduce cocaine-induced locomotor hyperactivity or sensitisation in rats. Side-effect screens were carried out on the novel compounds using the doses tested in cocaine-primed reinstatement. Ethy-SalA (0.3 mg/kg) and Me-SalA (1.0 mg/kg) did not significantly affect spontaneous locomotor behaviour 0.3 mg/kg, or reduce self-administration of the natural reward sucrose at a dose of 0.3 mg/kg in rats. Depression-like effects caused by acute Ethy-SalA treatment (0.3 mg/kg) were also not detected in the Forced Swim Test. Treatment with Ethy-SalA (10 µM) significantly increased uptake of the fluorescent ASP+ in co-transfected DAT/KOPr HEK-293 cells.   Conclusions: A single treatment of the novel KOPr agonist Ethy-SalA, but not the novel agonist Me-SalA, was found to attenuate drug-seeking behaviours in models of cocaine administration with greater potency than SalA, and without detectable sedative or depression-like effects at a dose of 0.3 mg/kg. The cellular mechanism-of-action by which Ethy-SalA depresses cocaine reward is at least in part due to positive regulation of DAT, which would act to reduce extracellular dopamine within the brain. The lack of significant side-effects and the apparent improved potency of the compound support further exploration of Ethy-SalA as a lead for the development of an anti-addictive pharmacotherapy.</p>

scholarly journals The effect of novel kappa opioid peptide receptor agonists on learning and memory in rats

2021 ◽  
Author(s):  
◽  
Susan Adele Welsh
Keyword(s):  
Recognition Memory ◽  
Side Effect ◽  
Memory Impairment ◽  
Perirhinal Cortex ◽  
Opioid Peptide ◽  
Salvinorin A ◽  
The Novel ◽  
Kappa Opioid ◽  
The Brain

<p>Kappa opioid peptide receptors (KOPrs) are a class of opioid receptors which shown analgesic and anti-addictive properties. Nonaddictive analgesics would be beneficial as morphine, one of the most commonly prescribed opioids for chronic pain, activates the brain reward system and can lead to addiction. Although medical research is progressing rapidly, there is still no treatment for psychostimulant abuse. KOPr agonists show promise in this regard but display undesirable side effects and could negatively affect memory. Salvinorin A (Sal A), a structurally unusual KOPr agonist, has a reduced side effect profile compared to the more traditional KOPr agonists such as U50,488. The effect of Sal A and U50,488 on memory is controversial as they have both been shown to induce a memory impairment and also to improve memory impairments. Sal A also has a poor pharmacokinetic profile with a short duration of action. Structural analogues of Sal A have improved pharmacokinetic and side effect profiles compared to Sal A yet retain the analgesic and anti-addiction properties. This thesis will investigate whether Sal A analogues, namely Ethynyl Sal A (Ethy Sal A), Mesyl Salvinorin B (Mesyl Sal B), and Bromo Salvinorin A (Bromo Sal A), produce a memory impairment.  Male Sprague-Dawley rats were evaluated in the novel object recognition (NOR) task to determine whether novel Sal A analogues impair long term recognition memory. The degree of novelty was also investigated on a cellular basis through quantifying c-Fos immunoreactive neurons within the perirhinal cortex, an area of the brain shown to respond to novelty.  Acute administration of Sal A (0.3 and 1 mg/kg) and novel analogues Ethy Sal A (0.3 and 1 mg/kg), Mesyl Sal B (0.3 and 1 mg/kg), and Bromo Sal A (1 mg/kg) showed no significant differences compared to vehicle when tested in the NOR task. The prototypical KOPr agonist, U50,488 (10 mg/kg), produced a significant decrease in recognition index compared to vehicle when tested in the same task as the novel analogues. Correlating the recognition indices calculated from U50,488 in the NOR to c-Fos counts in the perirhinal cortex showed a strong positive correlation with an increase in RI relating to an increase in c-Fos activation. U50,488 (10 mg/kg) showed a non-significant trend compared to vehicle in the number of c-Fos immunoreactive cells within the perirhinal cortex.  Neither Sal A nor novel analogues affected NOR, suggesting no impairment of long term recognition memory. The lack of this side-effect, among others, demonstrates that the development of potent KOPr agonists with reduced side-effect profiles is feasible. These novel analogues show improvement over the traditional KOPr agonists.</p>

scholarly journals The effect of novel kappa opioid peptide receptor agonists on learning and memory in rats

2021 ◽  
Author(s):  
◽  
Susan Adele Welsh
Keyword(s):  
Recognition Memory ◽  
Side Effect ◽  
Memory Impairment ◽  
Perirhinal Cortex ◽  
Opioid Peptide ◽  
Salvinorin A ◽  
The Novel ◽  
Kappa Opioid ◽  
The Brain

<p>Kappa opioid peptide receptors (KOPrs) are a class of opioid receptors which shown analgesic and anti-addictive properties. Nonaddictive analgesics would be beneficial as morphine, one of the most commonly prescribed opioids for chronic pain, activates the brain reward system and can lead to addiction. Although medical research is progressing rapidly, there is still no treatment for psychostimulant abuse. KOPr agonists show promise in this regard but display undesirable side effects and could negatively affect memory. Salvinorin A (Sal A), a structurally unusual KOPr agonist, has a reduced side effect profile compared to the more traditional KOPr agonists such as U50,488. The effect of Sal A and U50,488 on memory is controversial as they have both been shown to induce a memory impairment and also to improve memory impairments. Sal A also has a poor pharmacokinetic profile with a short duration of action. Structural analogues of Sal A have improved pharmacokinetic and side effect profiles compared to Sal A yet retain the analgesic and anti-addiction properties. This thesis will investigate whether Sal A analogues, namely Ethynyl Sal A (Ethy Sal A), Mesyl Salvinorin B (Mesyl Sal B), and Bromo Salvinorin A (Bromo Sal A), produce a memory impairment.  Male Sprague-Dawley rats were evaluated in the novel object recognition (NOR) task to determine whether novel Sal A analogues impair long term recognition memory. The degree of novelty was also investigated on a cellular basis through quantifying c-Fos immunoreactive neurons within the perirhinal cortex, an area of the brain shown to respond to novelty.  Acute administration of Sal A (0.3 and 1 mg/kg) and novel analogues Ethy Sal A (0.3 and 1 mg/kg), Mesyl Sal B (0.3 and 1 mg/kg), and Bromo Sal A (1 mg/kg) showed no significant differences compared to vehicle when tested in the NOR task. The prototypical KOPr agonist, U50,488 (10 mg/kg), produced a significant decrease in recognition index compared to vehicle when tested in the same task as the novel analogues. Correlating the recognition indices calculated from U50,488 in the NOR to c-Fos counts in the perirhinal cortex showed a strong positive correlation with an increase in RI relating to an increase in c-Fos activation. U50,488 (10 mg/kg) showed a non-significant trend compared to vehicle in the number of c-Fos immunoreactive cells within the perirhinal cortex.  Neither Sal A nor novel analogues affected NOR, suggesting no impairment of long term recognition memory. The lack of this side-effect, among others, demonstrates that the development of potent KOPr agonists with reduced side-effect profiles is feasible. These novel analogues show improvement over the traditional KOPr agonists.</p>

scholarly journals Kappa Opioid Receptor Agonist Mesyl Sal B Attenuates Behavioral Sensitization to Cocaine with Fewer Aversive Side-Effects than Salvinorin A in Rodents

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2602 ◽  
Author(s):  
Bronwyn Kivell ◽  
Kelly Paton ◽  
Nitin Kumar ◽  
Aashish Morani ◽  
Aimee Culverhouse ◽  
...  
Keyword(s):  
Side Effects ◽  
Learning And Memory ◽  
Behavioral Sensitization ◽  
Forced Swim Test ◽  
Salvinorin A ◽  
Self Administration ◽  
Kappa Opioid ◽  
Swim Test ◽  
Forced Swim ◽  
Antinociceptive Effects

The acute activation of kappa opioid receptors (KOPr) produces antinociceptive and anti-cocaine effects, however, their side-effects have limited further clinical development. Mesyl Sal B is a potent and selective KOPr analogue of Salvinorin A (Sal A), a psychoactive natural product isolated from the plant Salvia divinorum. We assessed the antinociceptive, anti-cocaine, and side-effects of Mesyl Sal B. The anti-cocaine effects are evaluated in cocaine-induced hyperactivity and behavioral sensitization to cocaine in male Sprague Dawley rats. Mesyl Sal B was assessed for anhedonia (conditioned taste aversion), aversion (conditioned place aversion), pro-depressive effects (forced swim test), anxiety (elevated plus maze) and learning and memory deficits (novel object recognition). In male B6.SJL mice, the antinociceptive effects were evaluated in warm-water (50 °C) tail withdrawal and intraplantar formaldehyde (2%) assays and the sedative effects measured with the rotarod performance task. Mesyl Sal B (0.3 mg/kg) attenuated cocaine-induced hyperactivity and behavioral sensitization to cocaine without modulating sucrose self-administration and without producing aversion, sedation, anxiety, or learning and memory impairment in rats. However, increased immobility was observed in the forced swim test indicating pro-depressive effects. Mesyl Sal B was not as potent as Sal A at reducing pain in the antinociceptive assays. In conclusion, Mesyl Sal B possesses anti-cocaine effects, is longer acting in vivo and has fewer side-effects when compared to Sal A, however, the antinociceptive effects are limited.

MEDICAL METHODS OF ABORTION (MMA), HOW SAFE?

2021 ◽  
pp. 51-52
Author(s):  
Anita Pathak
Keyword(s):  
Side Effects ◽  
Side Effect ◽  
Unsafe Abortion ◽  
Self Administration ◽  
Mortality And Morbidity ◽  
Incomplete Abortion ◽  
Referral Practices ◽  
Maternal Mortality And Morbidity ◽  
Insight Into

Unsafe abortion is important and preventable cause of maternal mortality and morbidity. Medical method of abortion is a safe efcient and affordable method of abortion. However incomplete abortion is known side effect. An insight into the referral practices in cases of incomplete abortion following medical method of abortion will give an idea of the safety prole of medical methods of abortion. 150 women with incomplete abortion following medical method of abortion were administered a questionnaire which included information regarding onset of bleeding, treatment received, use of medication for abortion, its prescription, and administration. 90% of incomplete abortion following medical method of abortion were due to self-administration or prescription by unregistered practitioners, lack of examinations and lack of follow up. Complications such as collapse, blood requirement and fever were signicantly higher in these patients. The side effects of incomplete abortion following medical method of abortion can be minimized by following the standard guidelines.

scholarly journals Ca2+-ATPases in non-failing and failing heart: evidence for a novel cardiac sarco/endoplasmic reticulum Ca2+-ATPase 2 isoform (SERCA2c)

2006 ◽  
Vol 395 (2) ◽  
pp. 249-258 ◽  
Author(s):  
Saoussen Dally ◽  
Raymonde Bredoux ◽  
Elisabeth Corvazier ◽  
Jens P. Andersen ◽  
Johannes D. Clausen ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Turnover Rate ◽  
Normal Subjects ◽  
The Novel ◽  
Hek 293 ◽  
Transfected Cells ◽  
293 Cells ◽  
Mrna Variant ◽  
Close Proximity

We recently documented the expression of a novel human mRNA variant encoding a yet uncharacterized SERCA [SR (sarcoplasmic reticulum)/ER (endoplasmic reticulum) Ca2+-ATPase] protein, SERCA2c [Gélébart, Martin, Enouf and Papp (2003) Biochem. Biophys. Res. Commun. 303, 676–684]. In the present study, we have analysed the expression and functional characteristics of SERCA2c relative to SERCA2a and SERCA2b isoforms upon their stable heterologous expression in HEK-293 cells (human embryonic kidney 293 cells). All SERCA2 proteins induced an increased Ca2+ content in the ER of intact transfected cells. In microsomes prepared from transfected cells, SERCA2c showed a lower apparent affinity for cytosolic Ca2+ than SERCA2a and a catalytic turnover rate similar to SERCA2b. We further demonstrated the expression of the endogenous SERCA2c protein in protein lysates isolated from heart left ventricles using a newly generated SERCA2c-specific antibody. Relative to the known uniform distribution of SERCA2a and SERCA2b in cardiomyocytes of the left ventricle tissue, SERCA2c was only detected in a confined area of cardiomyocytes, in close proximity to the sarcolemma. This finding led us to explore the expression of the presently known cardiac Ca2+-ATPase isoforms in heart failure. Comparative expression of SERCAs and PMCAs (plasma-membrane Ca2+-ATPases) was performed in four nonfailing hearts and five failing hearts displaying mixed cardiomyopathy and idiopathic dilated cardiomyopathies. Relative to normal subjects, cardiomyopathic patients express more PMCAs than SERCA2 proteins. Interestingly, SERCA2c expression was significantly increased (166±26%) in one patient. Taken together, these results demonstrate the expression of the novel SERCA2c isoform in the heart and may point to a still unrecognized role of PMCAs in cardiomyopathies.

scholarly journals Exploring the Aversive and Anxiogenic Effects of Novel Kappa Opioid Receptor Agonists in Rats

2021 ◽  
Author(s):  
◽  
Aimee Culverhouse
Keyword(s):  
Side Effects ◽  
Opioid Receptor ◽  
Kappa Opioid Receptor ◽  
Prime Model ◽  
The Novel ◽  
Negative Consequences ◽  
Sprague Dawley ◽  
Initial Attempt ◽  
Kappa Opioid ◽  
Duration Of Action

<p>Drug addiction is characterised by uncontrolled, compulsive drug use despite negative consequences. As this disease has a high social and economic cost, greater attention is required in finding an effective treatment for individuals suffering addiction. Kappa opioid receptor (KOPr) agonists demonstrate anti-addiction effects in the rodent cocaine drug-prime model of reinstatement. Salvinorin A (Sal A), a novel non-nitrogenous KOPr agonist, has demonstrated reduced side-effects compared to traditional agonists. However, its short half-life and duration of action limit clinical development. The design of novel Sal A analogues with improved pharmacokinetics, anti-addiction effects, and reduced side-effects is an important step towards the pharmaceutical development of KOPr agonists. β-Tetrahydropyran Sal B (β-THP Sal B), Mesyl Sal B, ethoxymethyl salvinorin B ether (EOM Sal B), and Ethynyl Sal A (Ethy Sal A) have demonstrated anti-addiction effects by reducing cocaine-seeking behaviour in rats, but their aversive and anxiogenic properties have yet to be examined. Here the conditioned place aversion (CPA) paradigm is used to evaluate aversion and the elevated plus maze (EPM), light/dark test, and open field are utilised to measure anxiety in male Sprague-Dawley rats.  EOM Sal B (0.1 mg/kg, i.p) and Ethy Sal A (0.3 mg/kg, i.p) did not produce aversive effects, whereas the traditional KOPr agonist U50,488 (10 mg/kg, i.p), Sal A (0.3 mg/kg, i.p), and the novel analogue β-THP Sal B (1 mg/kg, i.p) produced significant aversion using the CPA protocol.  In the EPM all the novel analogues, β-THP Sal B, EOM Sal B, Mesyl Sal B, and Ethy Sal, A did not show a reduction in time spent on the open arm. In addition, EOM Sal B showed a significant increase in time spent on the open arm compared with Sal A (0.3 mg/kg, i.p). Sal A (0.3 and 1 mg/kg, i.p) showed significant anxiogenic effects, but the traditional agonist U50,488 did not. In the light/dark test Sal A (1 mg/kg, i.p) showed significant dose dependent anxiogenic effects with significant effects observed at 1 but not 0.3 mg/kg dose. This is in contrast to results observed in the EPM. The novel analogues EOM Sal B and β-THP Sal B demonstrated a non-significant trend toward anxiogenic behaviour in the light/dark test, but U50,488, Mesyl Sal B, and Ethy Sal A did not show significant reductions in time spent in the light box.  KOPr stimulation activates its associated G-proteins, allowing them to interact with several intracellular effectors. Activation of cAMP response element binding protein (CREB) can occur downstream of the KOPr signalling cascade. The phosphorylation of CREB is associated with dysphoria and stress-induced reinstatement of drug-seeking behaviour. An initial attempt to validate CREB assays was made.  The lack of behavioural anxiogenic and aversive side-effects with EOM Sal B, Mesyl Sal B and Ethy Sal A treatment demonstrates that the development of KOPr agonists with desirable effects and reduced side-effects is possible. These novel Sal A agonists provide promising candidates for pharmacotherapy development.</p>

scholarly journals MON-185 ACTH-Derived Peptide Antagonists as an Alternative Treatment Strategy for Congenital Adrenal Hyperplasia

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Tina Schubert ◽  
Lennart Nicke ◽  
André Schanze ◽  
Nicole Reisch ◽  
Armin Geyer ◽  
...  
Keyword(s):  
Side Effects ◽  
Congenital Adrenal Hyperplasia ◽  
Alternative Treatment ◽  
Treatment Strategy ◽  
Steroid Hormone ◽  
Adrenal Hyperplasia ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Alternative Treatment Strategy ◽  
293 Cells

Abstract Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by different enzyme deficiencies in the steroid hormone synthesis leading to a disturbed cortisol biosynthesis. The medical treatment of CAH includes suboptimal ACTH-suppressing high glucocorticoid doses to reduce adrenal hyperplasia and overproduction of androgens. These inappropriate corticoid substitutions are often associated with undesirable side effects such as arterial hypertension, growth failure and obesity. Since the current therapy of patients with CAH is often unsatisfactory, innovative treatment options are required. The aim of our study was to specifically block the melanocortin 2 receptor (MC2R) signaling pathway as an alternative treatment strategy for CAH. We tested ACTH-derived selective peptides with incorporation of various synthetic non-natural amino acids in the activation motif of ACTH. To study the antagonistic activity of the peptides, cAMP production of MC2R/MRAP stably transfected human embryonic kidney (HEK) 293 cells were measured. All new synthesized peptide antagonists reduced ACTH-stimulated MC2R activity as competitive inhibitors indicated by a reduced in vitro cAMP response. Cells pre-incubated with peptide LNP009 showed the most efficient blockade of the MC2R and the highest shift of EC50 of ACTH (33.8 nM ± 0.08 vs. 7.3 nM ± 0.09). LNP009 was additionally tested for specificity concerning the other known melanocortin receptors and showed no antagonistic effect up to 1 μM on MC3, MC4 or MC5 receptor transiently transfected HEK 293 cells. To further investigate the inhibitory effect of our most potent antagonist peptide LNP009 on the steroid hormone response, we assessed steroidogenic enzyme expression of the human adrenocortical tumor cell line NCI-H295RA and performed mass spectrometry analyzes of steroids in the cell culture supernatant. Pre-incubation with LNP009 reduced the expression of the genes CYP21A2, CYP11B1 and HSD3B2 in NCI-H295RA cells and significantly reduced the synthesis of aldosterone (P=0.046; n=3), cortisol (P=0.020; n=3) and corticosterone (P=0.035; n=3). With the successful blocking of the ACTH binding and signal transduction by our antagonistic peptides, we anticipate an alternative approach for optimizing the treatment of CAH patients lacking the side effects of the currently used ACTH-suppressing corticoid therapy.

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