Effect of opioid active therapeutics on the ascending reflex pathway in the rat ileum.

For a long time therapeutic agents that interact with opioid receptors have been used in antidiarrheal therapy. The action of the opioid active substances on motility and transit have already been characterized; however, their effects on myenteric reflexes and their possible luminal action have not yet been investigated. Loperamide, fedotozine and beta-casomorphin-4, as well as the casomorphin-analogue beta-CM-4027, are, or have been, suggested as therapeutic agents and were studied in the isolated rat ileum for their effect on the ascending reflex pathway. beta-CM-4027 > fedotozine > loperamide > beta-casomorphin-4 caused a concentration-dependent inhibition of the ascending contractile reflex response with an IC(50)of 1.4x10(-7)M, 1.5x10(-6)M, 4.1x10(-6)M and 4.5x10(-6)M respectively. At the same time as the oral contractile reflex response was inhibited, all four opioid agonists (CM-4027 > beta-casomorphin-4 > fedotozine > loperamide) increased the latency of the reflex response. Both effects were blocked by naloxone, indicating the involvement of opioid receptors. These results demonstrate that opioid-active drugs and substances modify the peristaltic reflex by reducing the efficacy of the reflex response and modulating the timing of the reflex pathway. In a second series of experiments, luminal application of opioid-active drugs was compared with serosal application. beta-casomorphine-4 caused a concentration-dependent inhibition of the oral reflex response with an IC(50)of 3x10(-3)M which was 750 times higher than after serosal application. In contrast, a stable and highly selective kappa opioid agonist (U-50,488), which caused potent inhibition upon serosal application (IC(50): 2.3x10(-7)M), showed no inhibitory effect after luminal application up to a concentration of 10(-2)M. Thus casomorphins could have a local effect on the gut wall with no need for systemic absorption. This might be used for a possible therapeutic application.

Effects of oral casokefamide on plasma levels, tolerance, and intestinal transit in man.

Food-derived opioid peptides such as beta-casomorphins are of interest for treatment of chronic diarrhea. The beta-casomorphin analog casokefamide was administered orally at doses of 5.5, 8.0, and 16.0 mg to 10 healthy male volunteers, respectively. Dose-dependent increases of plasma levels with a maximum of 350 fmol/l were determined. No side-effects due to casokefamide has been observed. In comparison to placebo, casokefamide showed a trend toward prolongation of oro-caecal transit time. Orally applied casokefamide is well tolerated and may represent a useful tool for treatment of diarrhea in the future.

Milk protein-derived opioid receptor ligands.

Milk is mammalian characteristic and is of particular importance for humans: Mother's milk or its substitutes from cows' milk are absolutely essential nutriments for the neonate and cows' milk also represents a basic foodstuff for adults. However, in addition to their well-known nutritive role, milk constituents apparently are also able to carry specific information from the milk producer's to the milk receiver's organism: Thus, a number of milk protein fragments has been shown to behave like opioid receptor ligands able to address opioidergic systems in the adult's or in the neonate's organism. With respect to the proteins, which they are derived off these peptides have been named alpha-casein exorphins or casoxin D (alpha-casein), beta-casomorphins or beta-casorphin (beta-casein), casoxin or casoxin A, B, or C (k-casein), alpha-lactorphins (alpha-lactalbumin), beta-lactorphin (beta-lactoglobulin) or lactoferroxins (lactoferrin). Only casoxins and lactoferroxins display antagonistic properties; the other peptides behave like opioid receptor agonists. Most of the information available so far has been collected about beta-casomorphins. These peptides obviously can be released from beta-casein in the adult's or in the neonate's organism, where they might elicit opioid effects in the frame of a regulatory role as "food hormones". Several synthetic beta-casomorphin derivatives have been shown to be highly specific and potent mu-type opioid receptor ligands which frequently have been used as standard tools in opioid research.

Solution structure of casokefamide.

Casokefamide (Tyr-D-Ala-Phe-D-Ala-Tyr-NH2) is a synthetic peptide derived from the beta-casomorphin sequence, designed to increase the resistance to gastric proteases. Casokefamide binds to both mu and delta-opioid receptors, while beta-casomorphins and its fragments are typical mu-opioid receptor agonists. Furthermore, casokefamide can affect gastric acid and pancreatic exocrine secretions and also gastrointestinal motility. We have undertaken a conformational study on this peptide based on NMR measurements in a DMSOd6/H2O cryomixture at 265 K and energy calculations. The predominant conformation is characterised by the absence of regular structures and intramolecular hydrogen bonds. The conformation of the message domain is reminiscent of the shape of several peptidic and non peptidic opiates, with the D-Ala2CH3 group sandwiched between Tyr1 and Phe3 aromatic rings.

Absorption of beta-casomorphins from autoperfused lamb and piglet small intestine.

beta-Casomorphins (beta-CMs) derived from milk beta-casein may exert various opiate activities in milk-fed infants. To assess the physiological significance of beta-CMs as a source of circulating opioids in infants, we measured absorption rates of several beta-CMs under near-physiological conditions using in situ autoperfused lamb intestine. The naturally occurring beta-CMs, beta-CM-7 and beta-CM-4-amide, were absorbed readily into blood with no transfer into lymph. Uptake peaked within several minutes of the luminal infusion of peptide but then declined sharply and stopped within a further 10-15 min. The recovery in blood, intestinal contents, and tissue at the end of the 30-min experiment was less than 1% of the infused dose. The low recovery was due to rapid proteolysis based on in vitro studies that demonstrated half-lives of less than 5 min in lamb blood, luminal contents, and lymph. The synthetic dipeptidyl peptidase IV-resistant analogue beta-[D-Ala2]CM- 4-amide was stable during incubation in blood, lymph, or luminal contents and was absorbed into blood at rates that were maximal within several minutes and remained steady for the 30-min period. We conclude that although natural beta-CMs are transferred across the lamb small intestine, rapid degradation within the intestinal lumen, gut epithelium, and blood would prevent entry into the circulation under normal conditions. Val-beta-CM-7, a putative stable precursor, had similar stability and kinetics of absorption to beta-CM-7, results that exclude Val-beta-CM-7 as a stable precursor for delivery of beta-CMs to the circulation. Essentially identical results to those in lambs were obtained in 7-day-old piglets.

Hemorphins, cytochrophins, and human beta-casomorphins bind to antiopiate (TYR-MIE-1) as well as opiate binding sites in rat brain.

Novel peptides with opiate activity, derived from endogenous sources (human and bovine casomorphins from milk, hemorphins from hemoglobin, and cytochrophins from mitochondrial cytochrome b), were tested for their ability to inhibit binding of the brain peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) to its high affinity sites in rat brain. The order of potency in inhibiting binding of 125I-Tyr-MIF-1 was: hemorphin and bovine casomorphins greater than Tyr-MIF-1 greater than cytochrophins greater than human casomorphins. Naloxone and DAMGO were ineffective at inhibiting Tyr-MIF-1 binding. The results provide evidence that, in addition to their ability to bind to mu opiate receptors, these novel endogenous peptides with opiate activity and a peptide (Tyr-MIF-1) with antiopiate properties also bind to a non-opiate site labeled by Tyr-MIF-1. These sites could be involved in a balance between opiate and antiopiate peptides.

Opioid receptor affinities of the blood-derived tetrapeptides hemorphin and cytochrophin.

Hemorphin-4 and cytochrophin-4 displayed affinities for mu- and delta-opioid receptors that were in the same range as those observed for the structurally related beta-casomorphins. However, they showed markedly higher affinities at kappa-opioid binding sites when compared to the beta-casomorphins. These blood-derived peptides could be involved in blood pressure regulation.

Radioreceptor assay for determination of the antimuscarinic drug ipratropium bromide in man.

A radioreceptor assay for the determination of ipratropium bromide in human plasma has been developed, using [3H] N-methyl-scopolamine as a radioligand to label muscarinic cholinergic receptors in a membrane preparation of rat cerebral cortex. There was no interference due to the cross-reactivity of 3 metabolites of ipratropium with the parent compound (5.2, 1.5 and 0.004%, respectively). The validity of the assay was checked between 20 pg/ml and 1000 pg/ml drug. In a pilot study plasma levels following a single oral dose of 30 mg were determined to examine the applicability of the radioreceptor assay to clinical and pharmacokinetic studies, and for measurement of plasma levels after therapeutic oral doses. The peak plasma concentration in three healthy volunteers (means = 322 pg/ml) occurred within 1-3 h.

Effects of a kappa-opioid agonist on adrenocorticotropic and diuretic function in man.

Although kappa-opiate receptors represent an important fraction of the total opiate receptor capacity in human brain their endocrine function is unknown. We determined the effects of a kappa-opiate receptor agonist on the secretion of vasopressin, ACTH and cortisol and on diuresis. The racemic benzomorphan kappa agonist MR 2033 or its opiate active (-)-isomer, MR 2034, inhibited the release of cortisol and ACTH in 12 trials in a naloxone reversible manner; plasma levels of vasopressin were not altered. The (+)-isomer, MR 2035, did not affect the secretion of cortisol or ACTH. Surprisingly, in five other subjects large increases were observed in vasopressin, ACTH and cortisol following the kappa-agonist, which were probably elicited indirectly by aversive effects of the opioid. The subjects in whom vasopressin release was not altered by MR 2033 and MR 2034 displayed large decreases in urine osmolality which were not antagonized by naloxone. The opiate inactive (+)-isomer, MR 2035, caused no diuretic response. Subjects in whom vasopressin release was stimulated did not show decreases in urine osmolality indicating that vasopressin is capable of antagonizing the diuretic action of the kappa-agonist. Our data show that a kappa-agonist inhibits secretion of cortisol and ACTH by acting at stereospecific opiate receptors and elicits diuresis by acting at stereospecific, but naloxone-insensitive non-classical opioid receptors. These data support the concept that different types of kappa-receptors can be distinguished in man.

Psychotomimesis mediated by kappa opiate receptors.

The kappa opioid agonists are analgesics that seem to be free of undesired morphine-like effects. Their dysphoric actions observed with the kappa agonist cyclazocine are thought to be mediated by an action at sigma-phencyclidine receptors. The benzomorphan kappa agonist MR 2033 is inactive at sigma-phencyclidine receptors. In male subjects, the opiate-active (-)-isomer, but not the (+)-isomer, elicited dose-dependent dysphoric and psychotomimetic effects that were antagonized by naloxone. Thus, kappa opiate receptors seem to mediate psychotomimetic effects. In view of the euphorigenic properties of mu agonists, our results imply the existence of opposed opioid systems affecting emotional and perceptual experiences.

Anterior pituitary hormone responses to a kappa-opioid agonist in man.

The present study was designed to investigate pituitary hormone responses to a kappa-opiate receptor agonist in man. Normal men were given the racemic benzomorphan kappa-agonist MR 2033 or its levorotatory isomer MR 2034 iv. Plasma levels of PRL and GH markedly increased after injection of 3.5 micrograms/kg MR 2033 or 1.9 or 3.8 micrograms/kg MR 2034. These effects of MR 2033 were blocked by the opiate antagonist naloxone (10 mg), thereby demonstrating their mediation by opiate receptors. The kappa-agonist did not change plasma levels of LH and FSH. The secretion of TSH was significantly suppressed by MR 2033 and MR 2034, but this action was not antagonized by pretreatment with naloxone. The suppression of plasma TSH was, however, stereospecific since the (+)-isomer, MR 2035, did not affect TSH secretion. These data indicate that kappa-opiate receptors are located on neuronal pathways regulating GH, TSH, and PRL secretion. The pattern of pituitary responses elicited by the kappa-agonist differs from that of mu-opioid agonists, indicating differential endocrine functions for the endogenous agonists.

In vitro effects of beta-casomorphins on ion transport in rabbit ileum.

beta-Casomorphins (beta-CM) represent opioid peptides derived from bovine beta-casein. As opiates are known to decrease short-circuit current (Isc) and stimulate intestinal electrolyte absorption, we tested the effects of natural beta-CM-4-OH, beta-CM-5-OH, and three related analogues on electrolyte transport in rabbit ileum in vitro. At concentrations of 10(-7) to 10(-3) M, the three analogues (beta-[D-Ala2]CM-4-NH2, beta-[D-Ala2,Met5]CM-5-NH2, and beta-[D-Ala2,4,Tyr5]CM-5-NH2) caused a dose-dependent, naloxone-reversible reduction in Isc after addition to the serosal side of the preparation. beta-[D-Ala2,4,Tyr5]CM-5-NH2 also decreased Isc after mucosal addition. Serosal addition of the same analogue stimulated absorption of sodium and chloride (+2.90 +/- 0.95 and +2.12 +/- 0.60 mu eq . h-1 . cm-2, respectively) and inhibited residual flux (-1.80 +/- 0.57 mu eq . h-1 . cm-2). The natural beta-CM tested did not decrease Isc. These results demonstrate that beta-CM analogues stimulate intestinal absorption of electrolytes by an opioid mechanism. The fact that beta-[D-Ala2,4,Tyr5]CM-5-NH2 was effective on the mucosal side favored the hypothesis that certain food-related opioid peptides might be absorbed by the intestine.

Pharmacokinetics of adimolol after single and multiple dose administration in healthy volunteers.

The pharmacokinetic properties of adimolol (MEN 935), a new antihypertensive agents with predominantly beta-receptor blocking and additional alpha-adrenolytic activity were investigated in healthy volunteers. Study A subjects (n = 6) received single intravenous doses of 5 mg adimolol and single oral doses of 200 mg capsules, 200 mg tablets and 100 mg tablets on four occasions separated by at least two weeks. Study B subjects (n = 6) were given single intravenous doses of 5 mg and single oral doses of 100 mg of the 14C-labelled drug on two different occasions. Study C subjects (n = 6) were administered multiple oral doses of 100 mg adimolol daily for five days, and three weeks later 50 mg daily for five days. Adimolol plasma concentrations were assayed over seven days following each single dose using a specific and sensitive high-pressure liquid chromatographic method. The plasma concentration data obtained from the single i.v. dose studies were individually fitted to an open four-compartment model. To describe mathematically the single oral dose plasma level data, two compartments were added to the model to take care of the absorption. Irrespective of the route of administration, the doses and formulations given, all plasma concentration curves could be described with similar pharmacokinetic parameters. Plasma concentration curves predicted by the open four-compartment model were fully confirmed by the actual data obtained after chronic oral administration. The terminal half-life averaged 12 h following intravenous and 15 h after oral administration. The peak plasma concentration was reached on average 4 h following oral administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of beta-casomorphins on somatostatin release in dogs.

In the present study, the effects of orally administered beta-casomorphins (beta-CM) and met-enkephalin on postprandial plasma somatostatin-like immunoreactivity (SLI) were assessed in conscious dogs. The intragastric instillation of a liver extract-sucrose test meal containing 12 mg beta-CM or 10 mg met-enkephalin, respectively, augmented the postprandial rise of peripheral vein plasma SLI levels significantly. This effect was inhibited by the additional administration of the specific opiate-receptor antagonist, naloxone. When liver extract-sucrose was dissolved in fresh bovine milk the increase of plasma SLI levels was significantly greater than liver extract-sucrose dissolved in water. This milk-induced augmentation of SLI levels was also reduced by naloxone. Since these opiate-active compounds have an influence upon insulin release when given iv, the effect of beta-CM-7, beta-CM-5, beta-CM-4 beta-CM-4-amide, and met-enkephalin on SLI levels was assessed during their iv infusion at a rate of 1 nmol/kg . h during an iv background infusion of a glucose-amino acid mixture. The infusion of beta-CM-5 elicited a stimulation of peripheral vein SLI levels, whereas the infusion of met-enkephalin resulted in a significant decrease of SLI levels. beta-CM-7, beta-CM-4, and beta-CM-4-amide had no effect on plasma SLI levels at the dose employed. The present data demonstrate that in dogs the ingestion of opiate-active peptide stimulates postprandial SLI release, indicating that nutrient-contained opiate-active material (exorphins) might participate in the regulation of postprandial gastrointestinal endocrine function.