Sialorphin Potentiates Effects of [Met5]Enkephalin without Toxicity by Action other than Peptidase Inhibition.

This dose-response study investigated the effects of sialorphin on [Met5]enkephalin (ME)-induced inhibition of contractions in mouse vas deferens and antinociception in male rats. Differences were compared among combinations of three chemical peptidase inhibitors: amastatin, captopril, and phosphoramidon. The ratio of potencies of ME in mouse vas deferens pretreated with both sialorphin (100 µM) and a mixture of the three peptidase inhibitors (1 µM each) was higher than that with the mixture of peptidase inhibitors alone at any dose. Intrathecal administration of sialorphin (100-400 nmol) significantly and dose dependently increased ME (3 nmol)-induced antinociception with the mixture of three peptidase inhibitors (10 nmol each). The degree of antinociception with a combination of any two of the peptidase inhibitors (10 nmol each) in the absence of sialorphin was less than that in the presence of sialorphin (200 nmol). Pretreatment with both sialorphin (200 nmol) and the mixture of three peptidase inhibitors (10 nmol each) produced an approximately 100-fold augmentation in ME (10 nmol)-induced antinociception, but without signs of toxicity such as motor dysfunction in rats. Radioligand receptor binding assay revealed that sialorphin did not affect either binding affinity or maximal binding capacity of [d-Ala2,N-MePhe4,Gly-ol5]enkephalin. These results indicate that sialorphin potentiates the effects of ME without toxicity by a mechanism other than peptidase inhibition and with no effect on its affinity to µ-opioid receptors. SIGNIFICANCE STATEMENT: Sialorphin is regarded as an endogenous peptidase inhibitor that interacts with enkephalin-degrading enzymes. The results of these in vitro and in vivo studies confirm that sialorphin potentiates the effects of [Met5]enkephalin without toxicity by an action other than peptidase inhibition. This suggests that sialorphin offers the advantage of reducing or negating the side effects of opioid drugs and endogenous opioid peptides.

Antinociceptive activity of thiazole-containing cyclized DAMGO and Leu-(Met) enkephalin analogs.

Numerous studies demonstrate the promise of opioid peptides as analgesics, but poor oral bioavailability has limited their therapeutic development. This study sought to increase the oral bioavailability of opioid peptides by cyclization, using Hantzsch-based macrocyclization strategies to produce two new series of cyclized DAMGO and Leu/Met-enkephalin analogs. Opioid receptor affinity and selectivity for compounds in each series were assessed in vitro with radioligand competition binding assays. Compounds demonstrated modest affinity but high selectivity for the mu, delta, and kappa opioid receptors (MOR, DOR and KOR), while selectivity for mu opioid receptors varied by structure. Antinociceptive activity of each compound was initially screened in vivo following intracerebroventricular (i.c.v.) administration and testing in the mouse 55 °C warm-water tail-withdrawal test. The four most active compounds were then evaluated for dose- and time-dependent antinociception, and opioid receptor selectivity in vivo. Cyclic compounds 1924-10, 1936-1, 1936-7, and 1936-9 produced robust and long- lasting antinociception with ED50 values ranging from 0.32-0.75 nmol following i.c.v. administration mediated primarily by mu- and delta-opioid receptor agonism. Compounds 1924-10, 1936-1 and 1936-9 further displayed significant time-dependent antinociception after oral (10 mg kg-1, p.o.) administration. A higher oral dose (30 mg kg-1. p.o.) of all four cyclic peptides also reduced centrally-mediated respiration, suggesting successful penitration into the CNS. Overall, these data suggest cyclized opioid peptides synthesized by a Hantzsch-based macrocyclization strategy can retain opioid agonist activity to produce potent antinociception in vivo while conveying improved bioavailability following oral administration.

Killing effect of methionine enkephalin on melanoma in vivo and in vitro.

Melanoma is a common cutaneous malignancy, that is also found in specific mucosal sites, and is associated with a poor prognosis. The aim of the present study was to investigate the cytotoxicity of methionine enkephalin (MENK) for B16 melanoma cells in vivo and in vitro. The results of the present study allowed our conclusion that MENK regulates the proliferation of B16 cells, causing cell cycle arrest in the G0/G1 phase and a decrease in the percentage of cells in the S and G2/M phases. Reverse transcription-quantitative polymerase chain reaction demonstrated that MENK increased opioid receptor expression in the B16 cells. Furthermore, the tumor volume and weight in the MENK-treated group were lower than those in the control group (NS) and MENK and naltrexone (NTX)-treated groups. MENK exerted both significant antitumor activity on the growth of B16 cells and a longer survival time in mice. The mice treated with MENK exhibited an increased ratio of CD4+ to CD8+ T cells as tested by flow cytometry (FCM), resulting in a ratio of 2.03 in the control group, 3.69 in the MENK-treated group, and 2.65 in the MENK and NTX group. Furthermore, a significant increase in plasma levels of IL-2, IFN-γ and TNF-α was revealed as assessed by ELISA. In conclusion, the results of the present study indicate that MENK has a cytotoxic effect on B16 melanoma cells in vitro and in vivo, and suggest a potential mechanism for these bioactivities. Therefore, we posit that MENK should be investigated, not only as a primary therapy for melanoma, but also as an adjuvant therapy in combination with chemotherapies.

Structure-constrained endomorphin analogs display differential antinociceptive mechanisms in mice after spinal administration.

We previously reported a series of novel endomorphin analogs with unnatural amino acid modifications. These analogs display good binding affinity and functional activity toward the µ opioid receptor (MOP). In the present study, we further investigated the spinal antinociceptive activity of these compounds. The analogs were potent in several nociceptive models. Opioid antagonists and antibodies against several endogenous opioid peptides were used to determine the mechanisms of action of these peptides. Intrathecal pretreatment with naloxone and ß-funaltrexamine (ß-FNA) effectively inhibited analog-induced analgesia, demonstrating that activity of the analogs is regulated primarily through MOP. Antinociception induced by analog 2 through 4 was not reversed by δ opioid receptor (DOP) or κ opioid receptor (KOP) antagonist; antibodies against dynorphin-A (1-17), dynorphin-B (1-13), and Leu5/Met5-enkephalin had no impact on the antinociceptive effects of these analogs. In contrast, antinociceptive effects induced by a spinal injection of the fluorine substituted analog 1 were significantly reversed by KOP antagonism. Furthermore, intrathecal pretreatment with antibodies against dynorphin-B (1-13) attenuated the antinociceptive effect of analog 1. These results indicate that the antinociceptive activity exerted by intrathecally-administered analog 1 is mediated, in part, through KOP with increased release of dynorphin-B (1-13). The chemical modifications used in the present study may serve as a useful tool to gain insight into the mechanisms of endomorphins activity.

Opioid growth factor and low-dose naltrexone impair central nervous system infiltration by CD4 + T lymphocytes in established experimental autoimmune encephalomyelitis, a model of multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS), characterized by infiltrating myelin-reactive T lymphocytes and demyelinating lesions. Experimental autoimmune encephalomyelitis (EAE) is the animal model widely utilized to study MS. EAE is mediated by CD4(+) T cells and can be induced in EAE-susceptible mice through immunization with a myelin antigen, such as proteolipid protein 139-151 (PLP139-151) in SJL mice. In this PLP-induced EAE model, autoreactive CD4(+) T cells migrate from peripheral tissues into the CNS where they are reactivated resulting in CNS damage. Th1 and Th17 cells produce the pro-inflammatory cytokines IFNγ and IL-17, respectively, that have been shown to have pathogenic roles in EAE and MS. Anti-inflammatory Th2, IL-4 secreting cells, have been indicated to inhibit EAE exacerbation. However, given the inflammatory environment of EAE, Th2 effector cells are outnumbered by Th1/Th17 cells. Regulatory CD4(+) T cells suppress immune reactions and have been demonstrated to be dysfunctional in MS patients. Opioid growth factor (OGF), chemically termed [Met(5)]-enkephalin, is a negative growth factor that interacts with the OGF receptor. The OGF-OGFr axis can be activated through exogenous administration of OGF or a low dosage of naltrexone (LDN), an opioid antagonist. We have previously demonstrated that modulation of the OGF-OGFr axis results in alleviation from relapse-remitting EAE, and that CNS-infiltrating CD3(+) T cells are diminished with exogenous OGF or intermittent blockade with LDN administration. In this paper, we aimed to determine whether OGF or LDN alter the Th effector responses of CD4(+) T lymphocytes within the CNS in established EAE. We report in these studies that the numbers of CD4(+) T lymphocytes in the CNS of EAE mice are decreased following treatment with OGF for five days but not LDN. However, modulation of the OGF-OGFr axis did not result in changes to CD4(+) Th effector cell responses in the CNS of EAE mice.

Desempenho e rendimento de carcaça em frangos de corte de um a 46 dias alimentados com dietas contendo diferentes níveis de valina e reduzido teor proteico / Performance and carcass yield of broilers from 1 to 46 days fed diets containing different levels of valine and reduced protein content

Este estudo teve como objetivo avaliar as relações de valina:lisina digestíveis em dietas com teor reduzido de proteína bruta (PB) e os efeitos dessa redução sobre desempenho e rendimento de carcaça em frangos de corte. Foram utilizados 1200 pintos machos seguindo modelo inteiramente ao acaso, com seis tratamentos de seis repetições (exceto controle, com 10 repetições), compostos por 30 aves cada. O tratamento controle (T1) foi formulado conforme os níveis de proteína bruta e aminoácidos (AAs) recomendados por Rostagno et al . (2011), e os demais tratamentos (T2 a T6) tiveram seus níveis de PB reduzidos (4% em relação ao controle) e variaram em função da relação valina:lisina digestíveis, com cinco níveis equidistantes em intervalos de 0,07:1, variando de 0,63:1 e 0,91:1 (dietas até 21 dias) e de 0,64:1 e 0,92:1 (dietas após 21 dias). As seguintes características de desempenho foram avaliadas: ganho de peso, consumo de ração, conversão alimentar, viabilidade criatória e índice de eficiência produtiva. Aos 46 dias de idade, seis animais por repetição foram abatidos para determinação de rendimento de carcaça e de cortes comerciais. As diferentes relações valina:lisina digestíveis não influenciaram o desempenho dos animais (P>0,05) para nenhuma característica avaliada. A redução proteica piorou a conversão alimentar dos animais (P≤0,05) até os 21 dias. Os resultados sugerem que os níveis de valina utilizados não afetam o desempenho dos animais, apenas o rendimento de peito e que, portanto, a redução proteica não é recomendada durante as três primeiras semanas de criação.

This study aimed to evaluate valine:lysine ratios in diets with reduced content of crude protein and the effects of this reduction on the performance of broiler chickens. 1200 male chicks were used following a complete randomized design with six replicates of six treatments (except control, with 10 replicates), each one with 30 chicks. The control treatment (T1) was formulated following levels of crude protein (CP) and the amino acids (AAs) recommended by Rostagno et al. (2011), and the other treatments (T2 to T6) had reduced levels of CP (4 % compared to control) and varied in proportion valine:lysine, with 5 levels at equidistant intervals 0.07:1 ranging from 0.63:1 to 0.91:1 (up to 21 days) and from 0.64:1 to 0.92:1 (after 21 days). The performance characteristics measured were: weight gain, feed intake, feed conversion, viability and productive efficiency index. At 46 days six animals per replicate were slaughtered for evaluation of carcass and commercial cuts. The different valine:lysine ratios did not affect animal performance (P>0.05). Reducing protein impaired feed conversion (P≤.05) up to 21 days. The results suggest that levels of valine used did not affect the broilers' performance, however, breast meat yield and reduced protein are not recommended during the first three weeks.

Orexin type 1 receptor antagonism in rat locus coeruleus prevents the analgesic effect of intra-LC met-enkephalin microinjection.

Long-term administration of opiates leads to development of tolerance to analgesic effects. This in turn compromise clinical use of these drugs for pain management. Although extensive studies have been conducted, the involved cellular mechanisms are still poorly understood. The nucleus locus coeruleus (LC), which is a dense homogenous cluster of noradrenergic neurons in brainstem, has been reported to be involved in mediating opiate effects including analgesia and tolerance. LC neurons express a high density of opioid receptors. On the other hand, orexinergic neurons send widespread projections to the LC region. Among the two types of orexin receptors (OX1R and OX2R), OX1R is highly expressed in LC neurons. It has been shown that orexin-A is involved in modulation of nociceptive behavior. Also, previous studies have demonstrated the involvement of OX1R in the development of morphine induced analgesia and tolerance. In the present study, the involvement of OX1R in development of met-enkephalin (ME) analgesic tolerance was investigated in LC nucleus. The tail flick test was used to evaluate the analgesic effect of intra-LC microinjection of ME in male Wistar rats (250-300g). Analgesic responses were reported as the percentage of maximum possible effect (% of MPE). Also, SB-334867 was used as a selective OX1R antagonist. Results indicate that intra-LC microinjection of ME (5µg/100nL) results in development of analgesic tolerance in 3days. Also, OX1R antagonism in LC nucleus significantly prevents the analgesic effect of intra-LC met-enkephalin microinjection. It appears that the analgesic effect of ME in LC neurons is mediated by orexinergic system.

Methionine enkephalin (MENK) inhibits tumor growth through regulating CD4+Foxp3+ regulatory T cells (Tregs) in mice.

Methionine enkephalin (MENK), an endogenous neuropeptide, plays an crucial role in both neuroendocrine and immune systems. CD4+Foxp3+ regulatory T cells (Tregs) are identified as a major subpopulation of T lymphocytes in suppressing immune system to keep balanced immunity. The aim of this research work was to elucidate the mechanisms via which MENK interacts with Tregs in cancer situation. The influence of MENK on transforming growth factor-ß (TGF-ß) mediated conversion from naïve CD4+CD25- T cells to CD4+CD25+ Tregs was determined and the data from flow cytometry (FCM) analysis indicated that MENK effectively inhibited the expression of Foxp3 during the process of TGF-ßinduction. Furthermore, this inhibiting process was accompanied by diminishing phosphorylation and nuclear translocation of Smad2/3, confirmed by western blot (WB) analysis and immunofluorescence (IF) at molecular level. We established sarcoma mice model with S180 to investigate whether MENK could modulate Tregs in tumor circumstance. Our findings showed that MENK delayed the development of tumor in S180 tumor bearing mice and down-regulated level of Tregs. Together, these novel findings reached a conclusion that MENK could inhibit Tregs activity directly and retard tumor development through down-regulating Tregs in mice. This work advances the deepening understanding of the influence of MENK on Tregs in cancer situation, and relation of MENK with immune system, supporting the implication of MENK as a new strategy for cancer immunotherapy.

Methionine enkephalin (MENK) improves lymphocyte subpopulations in human peripheral blood of 50 cancer patients by inhibiting regulatory T cells (Tregs).

MENK, a penta-peptide is considered as being involved in the regulatory feedback loop between the immune and neuroendocrine systems, with marked modulation of various functions of human immune cells. The aim of the present work was to investigate change of lymphocyte subpopulations in peripheral blood of 50 cancer patients before and after treatment with MENK. Peripheral blood mononuclear cells (PBMCs) of peripheral blood from 50 cancer patients were isolated by density gradient centrifugation using Ficoll-Paque solution and cultured with MENK. We measured proliferation of total nucleated cells, subpopulations of individual CD4+T cells, CD8+T cells, CD4+CD25+ regulatory T cells (Treg), natural killer cells (NK) before and after treatment with 10(-12)M MENK in cell culture by flow cytometry (FCM). Our results indicated that MENK showed a strong inhibiting effect on Treg cells while it stimulated marked proliferation of other lymphocyte subpopulations. All data obtained were of significance statistically. It was therefore concluded that MENK could work as a strong immune booster with great potential in restoring damaged human immune system and we could consider MENK as a drug to treat cancer patients, whose immune systems are damaged by chemotherapy or radiotherapy. Furthermore we could consider MENK as a chemotherapy additive, which would sustain immune system of cancer patients during the process of chemotherapy to get maximized efficacy with minimized side effect.

Hepatoprotective effects of Met-enkephalin on acetaminophen-induced liver lesions in male CBA mice.

Recent histopathological investigations in patients with hepatitis suggested possible involvement of Met-enkephalin and its receptors in the pathophysiology of hepatitis. Consequently, we evaluated the potential hepatoprotective effects of this endogenous opioid pentapeptide in the experimental model of acetaminophen induced hepatotoxicity in male CBA mice. Met-enkephalin exhibited strong hepatoprotective effects in a dose of 7.5 mg/kg, which corresponds to the protective dose reported for several different animal disease models. In this group plasma alanine aminotransferase and aspartate aminotransferase enzyme activities, as well as liver necrosis score were significantly reduced in comparison to control animals treated with physiological saline (p>0.01). The specificity of the peptide hepatoprotection was investigated from the standpoint of the receptor and peptide blockade. It was concluded that Met-enkephalin effects on the liver were mediated via δ and ζ opioid receptors. Genotoxic testing of Met-enkephalin confirmed the safety of the peptide.

Potentiation of [Met5]enkephalin-induced antinociception by mixture of three peptidase inhibitors in rat.

PURPOSE: Previous in vitro studies have shown that degradation of opioid peptides during incubation with cerebral membrane preparations is almost completely prevented by a mixture of three peptidase inhibitors (PIs), namely, amastatin, captopril, and phosphoramidon. In the present in vivo study, we evaluate the effects of intrathecal administration of these PIs on antinociception by [Met(5)]enkephalin (ME) or PIs themselves. METHODS: Drugs were administered into the thoracolumbar level of the spinal cord in the intrathecal space in rat. Induction of antinociception was measured by the tail immersion assay, with 55 °C as the nociceptive stimulus. Effects of PIs on antinociception were evaluated by dose-response study (ME, 1-20 nmol; PIs, 1-20 nmol each), by comparison of differences among two combinations of PIs (amastatin and captopril; captopril and phosphoramidon; amastatin and phosphoramidon) and three PIs (amastatin, captopril, and phosphoramidon), and by using opioid receptor selective antagonists. RESULTS: Intrathecal administration of ME with these three PIs or PIs alone significantly and dose dependently increased antinociception in a µ- and δ-opioid receptor antagonist-reversible manner; moreover, the degree of antinociception with a combination of any two of these was less than that with all three, indicating that any residual single peptidase could inactivate significant amounts of ME. CONCLUSION: The present data, together with those of earlier studies, clearly demonstrate that amastatin-, captopril-, and phosphoramidon-sensitive enzymes play an important role in inactivation of opioid peptides at the spinal level.

Role of bovine adrenal medulla 22 (BAM22) in the pathogenesis of neuropathic pain in rats with spinal nerve ligation.

The opioid peptide bovine adrenal medulla 22 (BAM22) is a cleavage product of proenkephalin and has been shown to be involved in inflammatory pain and morphine tolerance. This study was designed to investigate a role of BAM22 in neuropathic pain. L5 spinal nerve ligation (SNL) significantly reduced BAM22-immunoreactivity in small-sized neurons and depleted IB4 binding in injured L5 dorsal root ganglia (DRG) compared to sham rats. Double labeling study showed that the expression of BAM22-immunoreactivity was decreased mainly in IB4 neurons in the neighboring intact L4 and L6 DRGs following SNL. The nerve injury dramatically increased sensitivity of hindpaw to mechanical stimulation. Intrathecal (i.t.) administration of BAM22 on day 10 post-SNL attenuated mechanical allodynia in a dose-dependent manner (3-30 nmol) and the effect lasted for up to 90 min. Similar treatment with morphine at a dose of 30 nmol produced a mild and brief inhibition on pain hypersensitivity. Furthermore, i.t. administration of 30 nmol of BAM22 suppressed SNL-induced upregulation of interleukin-1ß (IL-1ß) in the spinal dorsal horn. The present study suggests that the reduction of BAM22 expression in small-sized neurons in both injured and the adjacent DRGs may contribute to pain hypersensitivity in peripheral nerve injury as a result of loss of inhibition of IL-1ß upregulation in the spinal dorsal horn. Our results support the hypothesis that a reduction of antinociceptive activity loses the counteraction against activity of pronociceptive mediators, enhancing pain hypersensitivity following peripheral nerve injury.

Antinociceptive effect of [Met5]enkephalin semicarbazide is not affected by dipeptidyl carboxypeptidase-I.

Dipeptidyl carboxypeptidase-I is an enzyme involved in the biological degradation of enkephalins. It has been suggested that C-terminal amidation of enkephalins enhances their resistance to dipeptidyl carboxypeptidase-I-mediated biodegradation. In this study, a novel [Met5]enkephalin amide (MEA) analogue [Met5]enkephalin (ME)-semicarbazide synthesized by another laboratory in our group was assessed for its antinociceptive effects compared with ME-ethylamide, MEA and ME, using tail flick test. To protect the administered drugs from biodegradation, rats were pretreated with peptidase inhibitors including amastatin, phosphoramidon and captopril. Then captopril (dipeptidyl carboxypeptidase-I inhibitor) was deleted from the peptidase inhibitors' combination for evaluating in vivo resistance of the synthetic drugs to dipeptidyl carboxypeptidase-I. According to the results, ME-semicarbazide and MEA were resistant enough to dipeptidyl carboxypeptidase-I to exert their strong antinociception following intrathecal administration even in the absence of captopril, whereas the antinociceptive effects produced by ME-ethylamide (10 nmol) were abolished in rats not pretreated with captopril, indicating that significant amounts of the ME-ethylamide were degraded by dipeptidyl carboxypeptidase-I. Replacement of the amide moiety of MEA with semicarbazide provides a new ME derivative, with high analgesic effects as well as more resistance to dipeptidyl carboxypeptidase-I-mediated biodegradation.

Sympatholytic delta-2 opioid receptors moderate ganglionic vasomotor control.

This study tested the hypothesis that enkephalin increases femoral vascular conductance via the delta-2 phenotype of the opioid receptor (DOR-2) within peripheral sympathetic ganglia. Graded pulses of methionine-enkephalin (ME) were administered (0.03-10 µg/kg) into the terminal aorta of anesthetized dogs proximal to lumbar arteries that perfuse vasomotor ganglia regulating femoral blood flow. Femoral vascular conductance increased sharply (ED50 = 2.6 × 10(-9) mol/kg) accompanied by declines in arterial pressure and femoral vascular resistance. A dose-related increase in arterial pressure preceded each subsequent fall in pressure. The DOR-2 antagonist, naltriben (NTB), abrogated the hyperemic effect of ME (ID50 = 1.4 × 10(-9) mol/kg). DOR-1 blockade (BNTX) was five-fold less effective. The hyperemic effect of ME was also enhanced when sympathetic activity was reflexly increased by bilateral carotid occlusion. The DOR-2 agonist, deltorphin II, produced exaggerated increases in conductance compared with ME that were also reduced by DOR-2 blockade. DOR-1 blockade eliminated the initial pressor responses, exaggerated the subsequent depressor response, increased baseline femoral conductance 10-fold and shifted the ME-mediated hyperemic threshold one dose lower from 0.3 to 0.1 µg/kg, providing indirect support for a competing DOR-1-mediated constriction. Extended exposure to DOR-1 blockade lowered the maximal ME increase in conductance by 30%, suggesting that BNTX reduces the available pool of DOR receptors. In summary, enkephalin mediates a robust hyperemic effect through sympatholytic ganglionic DOR-2 receptors and DOR-1 antagonist studies provide indirect evidence for constituent opposition from a proposed DOR-1-mediated sympathotonic constrictor pathway.

Prevention and diminished expression of experimental autoimmune encephalomyelitis by low dose naltrexone (LDN) or opioid growth factor (OGF) for an extended period: Therapeutic implications for multiple sclerosis.

Endogenous opioids inhibit the onset and progression of experimental autoimmune encephalomyelitis (EAE) with 30days of treatment. This study examined the long term effects of the opioid growth factor (OGF, [Met(5)]-enkephalin) and a low dose of the opioid antagonist naltrexone (LDN) on expression of myelin oligodendrocyte glycoprotein (MOG)-induced EAE. C57BL/6 mice began receiving daily injections of 10mg/kg OGF (MOG+OGF), 0.1mg/kg naltrexone (MOG+LDN), or saline (MOG+Vehicle) at the time of EAE induction and continuing for 60days. In contrast to 100% of the MOG+Vehicle group with behavioral symptoms of EAE, 63% and 68% of the MOG+OGF and MOG+LDN mice expressed disease. Both severity and disease indices of EAE in OGF- and LDN-treated mice were notably decreased from MOG+Vehicle cohorts. By day 60, 6- and 3-fold more animals in the MOG+OGF and MOG+LDN groups, respectively, had a remission compared to MOG+Vehicle mice. Neuropathological studies revealed i) astrocyte activation and neuronal damage as early as day 10 (prior to behavioral symptoms) in all MOG-injected groups, ii) a significant reduction of activated astrocytes in MOG+OGF and MOG+LDN groups compared to MOG+Vehicle mice at day 30, and iii) no demyelination on day 60 in mice treated with OGF or LDN and not displaying disease symptoms. These results indicate that treatment with OGF or LDN had no deleterious long-term repercussions and did not exacerbate EAE, but i) halted progression of disease, ii) reversed neurological deficits, and iii) prevented the onset of neurological dysfunction across a considerable span of time.

Opioids in the hypothalamic paraventricular nucleus stimulate ethanol intake.

BACKGROUND: Specialized hypothalamic systems that increase food intake might also increase ethanol intake. To test this possibility, morphine and receptor-specific opioid agonists were microinjected in the paraventricular nucleus (PVN) of rats that had learned to drink ethanol. To cross-validate the results, naloxone methiodide (m-naloxone), an opioid antagonist, was microinjected with the expectation that it would have the opposite effect of morphine and the specific opioid agonists. METHODS: Sprague-Dawley rats were trained, without sugar, to drink 4 or 7% ethanol and were then implanted with chronic brain cannulas aimed at the PVN. After recovery, those drinking 7% ethanol, with food and water available, were injected with 2 doses each of morphine or m-naloxone. To test for receptor specificity, 2 doses each of the mu-receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-Enkephalin (DAMGO), delta-receptor agonist D-Ala-Gly-Phe-Met-NH2 (DALA), or kappa-receptor agonist U-50,488H were injected. DAMGO was also tested in rats drinking 4% ethanol without food or water available. As an anatomical control for drug reflux, injections were made 2 mm dorsal to the PVN. RESULTS: A main result was a significant increase in ethanol intake induced by PVN injection of morphine. The opposite effect was produced by m-naloxone. The effects of morphine and m-naloxone were exclusively on intake of ethanol, even though food and water were freely available. In the analysis with specific receptor agonists, PVN injection of the delta-agonist DALA significantly increased 7% ethanol intake without affecting food or water intake. This is in contrast to the kappa-agonist U-50,488H, which decreased ethanol intake, and the mu-agonist DAMGO, which had no effect on ethanol intake in the presence or absence of food and water. In the anatomical control location 2 mm dorsal to the PVN, no drug caused any significant changes in ethanol, food, or water intake, providing evidence that the active site was close to the cannula tip. CONCLUSIONS: The delta-opioid receptor agonist in the PVN increased ethanol intake in strong preference over food and water, while the kappa-opioid agonist suppressed ethanol intake. Prior studies show that learning to drink ethanol stimulates PVN expression and production of the peptides enkephalin and dynorphin, which are endogenous agonists for the delta- and kappa-receptors, respectively. These results suggest that enkephalin via the delta-opioid system can function locally within a positive feedback circuit to cause ethanol intake to escalate and ultimately contribute to the abuse of ethanol. This is in contrast to dynorphin via the kappa-opioid system, which may act to counter this escalation. Naltrexone therapy for alcoholism may act, in part, by blocking the enkephalin-triggered positive feedback cycle.

Delivery of neuropeptides from the periphery to the brain: studies with enkephalin.

Many peptides with the potential of therapeutic action for brain disorders are not in clinical use because they are unable to cross the blood-brain barrier (BBB) following peripheral administration. We have developed two potential strategies for the delivery of peptides to the brain and demonstrated their feasibility with enkephalins. In the first approach, designated induced reversible lipophilization, Leu/Met Enkephalins were converted to 9-fluorenylmethoxycarbonyl (Fmoc) derived lipophilic prodrug analogues, which undergo slow, spontaneous hydrolysis under physiological conditions, generating the native agonists. In contrast to Enkephalin, Fmoc-Met-Enkephalin was found to facilitate an analgesic effect following intraperitoneal administration in mice. Fmoc-Leu-Enkephalin was not analgesic. In the second approach, Enkephalin was linked to BBB transport vectors through an Fmoc based linker spacer, forming conjugates that slowly release Enkephalin under physiological conditions. A pronounced antinociceptive response was thus obtained following intraperitoneal administration of either cationized-human serum albumin-Fmoc-Enkephalin or polyethylene glycol(5)-Fmoc-Enkephalin. Derivatives of Enkephalin covalently linked to the same BBB-transport vectors through a stable (nonreversible) chemical bond were not analgesic. In summary, we have demonstrated that lipophilicity can be conferred to hydrophilic peptides to a degree permitting the permeation of the BBB by passive diffusion, without the drawback of agonist inactivation, which is often caused by irreversible derivatization. Similarly, in the second strategy, the conjugation to BBB-permeable vectors overcomes the obstacle of peptide inactivation by releasing the active form in the central nervous system.

Endogenous peptide: Met-enkephalin-Arg-Phe, differently regulate expression of opioid receptors on chronic treatment.

Endogenous peptide, Met-enkephalin-Arg-Phe (Tyr-Gly-Gly-Phe-Met-Arg-Phe; MERF) induces effects like antinociception, inhibit contraction of guinea pig ileum, mouse vas deferens and anti-tussive action. However, results regarding its functional efficiency and selectivity are controversial. Therefore, present study was undertaken to investigate whether MERF on systemic (intra-peritoneal, i.p.) route of administration induce any antinociception or not; to scrutinize the effect of 6 days chronic i.p. treatment of MERF on expression of mu (MOR1), delta (DOR1) and kappa (KOR1) opioid receptors; and finally, the antinociceptive effect of two synthetic peptides, MERFamide and (D-Ala(2))-MERFamide was compared with MERF on intracerebroventricular administration in order to understand the role of FMRF moiety in analgesic effect of MERF. Pharmacological results revealed that only 68.4 and 91.2 micromol/kg dose induce significant antinociception among various doses. Further, on 6 days chronic treatment, MERF induced significant antinociception in comparison to saline. Differential expression of MOR1 and KOR1 showed continuous up-regulation throughout the treatment whereas DOR1 showed down-regulation in initial 3 days followed by subsequently up-regulation during the latter observable period. Moreover, variation in opioid receptors expression had not affected the MERF antinociception. In conclusion, present study discursively demonstrates that MERF during chronic treatment interacts with all three opioid receptors (mu, delta and kappa) in rats and differently regulates their expression. Further, the interaction was such that the induction was mainly observed at molecular/expression level and not at pharmacological level to affect antinociception.

Distribución de la metencefalina en la vía óptica. Estudio experimental inmunocitoquímico / Distribution o met-enkephalin expression in the visual pathway. Experimental study by inmunocytochemstry

Objetivo: Conocer la localización y distribución deneuropéptidos en la vía óptica, concretamente, ladeterminación de inmunorreactividad a met-encefalina,clave para la funcionalidad de dicha vía.Método: Se analizó la vía óptica de ocho gatos.Tras extracción quirúrgica; cortes con microqueratomoy procesamiento mediante inmunocitoquímicaindirecta, utilizando como anticuerpo la antimetencefalina,con el fín de detectar la presencia oausencia del pentapéptido en la vía óptica del gato.Resultados: Se detectaron receptores a met-encefalinaen áreas del encéfalo tanto corticales como subcorticales.Así, se considera la posible implicaciónde dicho pentapéptido en la funcionalidad de la víaóptica.Conclusiones: La presencia de receptores a metencefalinaen áreas del encéfalo tanto corticalescomo subcorticales, muestra la posible implicaciónde dicho neurotransmisor en la funcionalidad de lavía óptica (AU)

Purpose: The localization and distribution of neuropetideexpression in the cat visual pathway can provideinformation about the function of that pathway.Method: Study of optic pathway in eight cats.Following extraction of the brain, slices were preparedusing a microkeratome. The slices were examinedby indirect immunocytochemistry using antimetenkephalinas antibody to determine the presenceor absence of this pentapeptide in the visualpathway.Results: Met-enkephalin receptors in both corticaland subcortical regions of the brain were detected.This suggests that met-enkephalin could be involvedin the visual mechanism.Conclusions: The presence of met-enkephalinreceptors in both cortical and subcortical regions ofthe brain suggests that this pentapeptide could beinvolved in the visual mechanism(AU)

The enhancing effects of peptidase inhibitors on the antinociceptive action of [Met5]enkephalin-Arg6-Phe7 in rats.

Previous in vitro studies have shown that the degradation of [Met(5)]enkephalin-Arg(6)-Phe(7) during incubation with cerebral membrane preparations is largely prevented by a mixture of three peptidase inhibitors: amastatin, captopril, and phosphoramidon. The present in vivo study shows that the inhibitory effect of [Met(5)]enkephalin-Arg(6)-Phe(7) administered intra-third-ventricularly on the tail-flick response was increased more than 1000-fold by the intra-third-ventricular pretreatment with three peptidase inhibitors. The antinociceptive effect produced by the [Met(5)]enkephalin-Arg(6)-Phe(7) in rats pretreated with any combination of two peptidase inhibitors was significantly smaller than that in rats pretreated with three peptidase inhibitors, indicating that any residual single peptidase could inactivate significant amounts of the [Met(5)]enkephalin-Arg(6)-Phe(7). The present data, together with those obtained from previous studies, clearly show that amastatin-, captopril-, and phosphoramidon-sensitive enzymes play important roles in the inactivation of endogenous opioid peptides, such as [Met(5)]enkephalin, [Met(5)]enkephalin-Arg(6)-Phe(7), [Met(5)]enkephalin-Arg(6)-Gly(7)-Leu(8), and dynorphin A (1-8), administered intra-third-ventricularly to rats.