This and that: an artefactual alkaloid and its peptide analogs

TiPS - September 2992 [Vol. 231

341

This and That: an artefactual alkaloid and its peptide analogs IT STARTED WITH an inexplicable attraction for a plant alkaloid. It was complicated by the discovery of numerous endogenous opioid peptides having a wider range of activities than the plant alkaloid, although their physiological significance has yet to be clarified. More recently, complexity has been added to complication by the finding of opioid substances in numerous other sources, including foods. _ alkaloid morphine, have been disMorphine and morphinecussed2. Although similar phannalike compounds hold perennial cological ends are achieved, the interest. The narcotic and analpharmacologies of morphine and gesic effects of opium sparked the opioid peptides are not identthe search for the responsible ical. Morphine is active mainly at the u-opioid receptor, while the agent, a search that began +ectrum of activity of the peporganic chemistry a quarter of tides is wider, with at least three a century before Wiihler conreceptors, the lo-, 6- and x-opioid verted the ‘inorganic’ comtypes, being well accepted while pound ammonium cyanate to others still hover beyond the the ‘organic’ compound urea, penumbra of respectability. thereby refuting the vital force Endorphins arise from the theory. products of three genes, each Life is a dream, and dreams themselves are only dreams Calderon Life is a Dream The more that is learned about opiates and opioids, the more intriguing they become. They comprise a classic paradigm of the aphorism that as the circle of our knowledge expands so does the circumference of our ignorance. In the study of these compounds, science continues to stumble into vast areas of study that we were earlier unaware even existed. Before the existence of opioid peptides was suspected, chemists devoted enormous effort to the synthesis of morphine analogs. At first, rigid derivatives were produced, by Die&Alder addition across the double bond in morphinel. Later, with increased understanding of conformational flexibility, ‘floppy’ molecules such as pethidine, methadone and fentanyl were synthesized, molecules at first sight very different from morphine, but capable of assuming a conformation analogous to the rigid configuration of the morphine molecule. In an earlier column, the similarities between the endogenous opioid peptides, the enkephalins and endorphins, and the plant

coding for a precursor ‘prepro’ protein that is cleaved to a propeptide. Proenkephalin gives rise to several copies of [Met]- and [Leulenkephalin; prodynorphin gives rise to [Leulenkephalin plus the dynorphins and neoendorphins; and from proopiomelanocortin come the endorphins as well as the hormones B-lipotropin, melanocyte-stimulating hormone and horadrenocorticotropic mone3A. If any small polypeptide containing the sequence Tyr-Gly-GlyPhe could be expected to have some kind of opioid activity, it should hardly have been surprising to find that hydrolysates of numerous proteins contained opioid peptides. Peptic digestion of bovine casein, a major protein in milk, produces a range of peptides with opioid activity, the morphiceptin, including B-casomorphins, and the lactorphins5. These, and other exogen-

0us1y

formed

have been exorphins

opioid peptides, collectively named by analogy with the

endorphins found in the brain. B-Casomorphin immunoreactivity also develops in high levels in milk contaminated with caseolytic bacteria6. It has been shown, by aspiration of the small intestine of long-suffering volunteers made to drink a liter of milk, that casomorphins are actually formed in humans7. These peptides have all the actions associated with morphine, including analgesia, catalepsy and physical dependences. Their actions are blocked by naloxone, an opiate antagonist. Similar peptides are found in peptic digests of human milk. These peptides vary in activity and potency. In all assays, the most potent are moThiceptin and the B-casomorphins s9.Additionally, a grrup of peptides, the casoxins, have been found to have opioid antagonist activity. One surprise, however, was the structure of these peptides. They contain sequences not found in the enkephalins and endorphins, and thus they extend the structureactivity requirements for opioid activity (Table I). What they have in common with the endogenous opioids are two aromatic amino acids, equivalent to the two planes of the ‘t’-shaped rigid morphine molecule (Fig. 1). The difference is in the nature of the spacer unit. Casomorphins are relatively resistant to further hydrolysislo. They have local activities in the tracP’. These gastrointestinal include an antidiarrheal effect, prolongation of transit time, and increased absorption’,“. There is some, incomplete, evidence that exorphins can be absorbed intact

TABLE I. Amino acid sequences and sources of some exorphins Exorphin

SOUreC

SkXjUb2llCe

Pr-Caseinexorphin p-Casomorphin,, ftCasomorphin,, @Casorphin o-Lactorphin Morphiceptin Cytochrophin Hemorphln Tyr-MIF-1

bovine casein digest bovine casein digest human B_casein human p-casein human p-casein bovine casein bovine oytochrome b bovine hemoglobin bovine brain

Arg-Tyr-Leu-Gly-Tyr-Leu-(Glu) Tyr-Pro-PhsPro-Gly Tyr-Pro-Phe-Val-Glu Tyr-Pro-Ser-PheNY Tyr-Gly-Leu-Phe-NH, Tyr-Pro-Phe-Pro-NH, Tyr-Pro-Phe-Trp-lie Tyr-Pro-Trp-Thr-Gln Tyr-Pro-Leu-Gly-NH, Q 1992. Ekvier

Science Publishers 1 td Rng

TPS - September 1992 fvof.133

Fi 1. Mnphine. Tradifionly drawn sbucfure (fefl) and molecularmodel of nnnpfnk. correspondingdngs of the m&cule are labelleda-d. A, double bond. and may have actions in the central nervous system- &Casomorphin-immunoreactive material is found in lasma of calves following nursing ? 3. Human milk has been shown to contain a &sleep-inducing peptide*4. Rxorphins from milk may be integrally involved in the feeding process. Searching for nutrition seem5 to be innate for newborn mammal5, in that in many species there is an instinctive reaching for the nipple by the neonate. The enlargement and darkening of the areola in nursing women presumably provides a visual cue to the infant in a further example of the biological functions of melanin’5~‘6. However, absorption of opioid peptides generated by peptic digestion of milk may provide a reward to the infant, reinforcing the search for food, increasing mother-infant bonding, and cueing satiation-induced sleep. Perhaps for the infant, .periodic to feeding-acquired exposure casomorphins becomes as much a habit as the adult addict’s needle. Abbott, in her departing editorial in this journal, talks of drugs of abuse ‘hijacking reward pathways that exist to ensure survival of the species’“. it could be that these reward pathways are maintained

ne~~~emically by analogs of agents that we consider to be drugs of abuse. The difference between the two groups of compounds could lie in the absence, in the latter group, of the rewardbenefit correlation that exists in the former group. That is to say, the opioid-food or opioid-analgesia association in the physiological setting has degenerated into seeking the opiate alone in the pharmacological setting. Supporting functionality for nutritionally-derived peptides is the finding that casomorphins, given in~acerebroven~c~arly to chicks, reduce the frequency of the distress calls chicks give when separated from the hen”. Although chicks do not drink milk, this expe~ment~ system does provide a model for warmblooded vertebrates. The finding indicates that these peptides have the ability to modulate social interactions if they reach the brain unhydrolysed. Militating against central actions of dietary exorphins is that although the casomorphins are readily absorbed from the gastrointestinal tract, they undergo rapid proteolysis in the blood, the half-life in plasma being around 5 minutes1gt20, On the other hand, rapid termination of action is considered a criterion

for physiological relevancy”. Conversely, high-molecular-weight casomorphin-immunoreactive material in the blood of pregnant or lactating women was stable in human plasma*‘. speculations the Among spawned by the finding of opioid peptides in milk is a possible connection between these substances and sudden infant death syndrome (SIDS, or crib death)=. In support of such a connection, respiratory depression is a wellknown action of opiates; nearmiss SIDS babies have high circulating levels of endorphins, and oriental children fed on exorphinfree soya bean milk have a lower incidence of SIDS. Perhaps the combination of endogenously increased responsiveness of the brain stem and exposure to exorphins proves deadly to a subpopulation of susceptible babies. But the exorphin story does not stop at the breast, or the various breast substitutes used in Western countries. Hydrolysis of other common foods has also been shown to release opioid peptides. In 1979, pepsin hydrolysis of wheat gluten was shown to release a substance, gluten exorphin, that was as potent as the endogenous ~~et]enkeph~n. Indeed, gluten exorphins are 100 times more potent than morphine in the mouse vas deferens assay, a measure of b-opioid receptor activityz3. These uncharacterized exorphins are resistant to further hydrolysis by proteinases, trypsin and chymotrypsin’O. Their discovery has been used to explain earlier findings linking schizophrenia with the consumption of foods containing wheat gluten. Many schizophrenics had been found to improve when given a gluten-free dietz4,25. In one 1973 study, schizophrenics rando~y assigned to a milk-free, cereal-free diet were discharged twice as rapidly as those not given such a dietz6. ‘Secret’ addition of gluten to the diet abolished the effect. As long ago as 1933, Buscaino found a high incidence of atrophy of small intestinal mucosa in schizophrenic patients (quoted in Ref. 23). As with casein hydrolysates, hydrolysed gluten prolongs gastrointestinal transit by a naloxone-reversible mechanism*‘. However, Morley et al.*’ did not notice any effect of hydrolysed

TiPS - September 1992 [Vol. 131 gluten on the perception of satiety. Exorphins are also generated by digestion of meat. The peptide cytochrophin (Table I) is formed from the breakdown of mitochondrial cytochrome b, and hemorphin (Table I) from the breakdown of bovine hemoglobinz3. In place of the Tyr-Gly N-terminal sequence in the endorphins, these exorphins contain a Tyr-Pro or some other sequence (Table I). Some, such as Tyr-MIF-1, bind to both opioid sites and naloxone-insensitive ‘anti-opioid’ sites in the brainz8. This opens the possibility that there are still other receptor systems interacting with opioid peptides waiting to be elucidated. Dietary opioid peptides affect a number of hormone systems. In one study, intragastric instillation of digested gluten compared with undigested gluten produced a more rapid and greater rise in circulating insulinz5. These peptides also affect post-prandial metabolism by increasing the secretion of somatostatins,*g and pancreatic polypeptide30. Intraperitoneal injection of B-casomorphin raises serum prolactin concentrations in rats31. All of these effects are blocked by naloxone. The prolongation of gastrointestinal transit time may be a consequence of increased somatostatin secretionz7. 0

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AT THIS POINT, we can return to the unfinished mystery of chocolate3’. The addictiveness

Fig 2. A

343 of this substance seems beyond dispute (Fig. 2). Many people are compulsive eaters of chocolate-containing foods but not of candy or other sweet substances. One recovering chocoholic tells me she would eat half a gallon of chocolate chip ice cream a day, but no other sort, even getting up in the night to scoop down hefty helpings straight from the container. Milk chocolate contains a lot of milk. In fact, the percentage of milk protein in chocolate (4.4%) is higher than that in milk itself (3.3%). So assuredly opioid peptides are generated from the milk in milk chocolate. However, this cannot account, at least not in toto, for the addictive attractions of chocolate, as chocolate addicts discriminate between eating chocolate and drinking milk. A clue perhaps lies with the original users of chocolate, the Aztecs. Earlier, I suggested that the fat and sugar content of chocolate made it a hedonically near-ideal substance3*. However, the Aztecs drank it bitter, without milk and without sugar. In such a form, it could have had little hedonic appeal. The high regard in which chocolate was held by the Aztecs was not on account of its taste. Chocolate is prepared by ‘fermenting’ the cacao bean for 3-9 days at temperatures of up to 50°C. This activates enzymes that hydrolyse and oxidize proteins, and pave the way for the development of the chocolate flavor and aroma on subsequent roasting. One consequence of fermentation may be the production of a fairly

specialized literatureexists on the allureof chocolate.

potent exorphin, and that the typical chocoholic is merely indulging in a more socially acceptable form of the craving that drives others to the products of another plant, the poppy. It is perhaps a less acceptable form, however, of the general desire to consume exorphin-containing foodstuffs, be they milk, meat, cheese, wheat products, or even something as non-nutritious as coffee, in which a potent opiate has been detectedJ3. Cl

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IN AN EARLIER discussion of opioids, I had written that plants were more ingenious chemists than mammals, in that the sophisticated biosynthesis of morphine could only be approximated in animals by peptides generated from the breakdown of high-molecularweight proteins’. This is not a completely true picture. Traces of morphine-like morphine and detectable in alkaloids are numerous animal tissues and fluids. Bovine and human milk contain such alkaloids in the 0.20.5 pmol l-’ range34. Eat brain contains around 0.7pmolg-* tissue of free and conjugated morphine and around 1.2pmolg-’ tissue of codeine35. Both morphine and codeine, as well as the tetrahydroisoquinoline tetrahydropapaveroline, are found in low amounts in human urine. The question has been whether these are truly of animal origin, or whether they derive from food sources. A whole range of animal and human foodstuffs, including lettuce and hap, has now been found to contain morphine. Support for an animal origin comes from studies showing that Parkinson’s patients treated with the aromatic amino acid L-DOPA yield much higher levels of the three alkaloids in their urine36. Codeine conccntrations, for example, average compared to 62 pm01 ml-’ 2pmol ml-l in control subjects. Urinary alkaloid levels are also high in patients with severe pain due to Herpes zoster infection. This suggests that morphine and codeine can be synthesized in small amounts in humans by a route analogous to that used in

Tips - September 1992 IVol. 131

Fg. 3. ffqx&c Canaan of reticuiine fo eaiuteridine. Activated positionsottho or para to a pheno/ are numbered. Formation of satutaddine involves oxidative opts Mweef7 positions (1) and (3). Furmationof morphine involves coupling between positions (2) and (3). The threedimensionat model of the fiexibte ret&tin8 mo&cuie in the ~f~~on closest to that of the rig@ saiutaridine shows that, despite the apparent profound differences in the molecules when drawn ~-~ns~~~y, ~stder~ arcssionaily the molecutes are sterica/ly similar.Ring lettering as in Fig. 1.

plants, from L-DOPA

and/or do-

pamine via a tetrahydroisoquinohm?. In confirmation, Weitz and co-workers3’ have demonstrated that rat liver, but not brain, is capable of cyclizing reticuline to salutaridine (Fig. 3). In general, the efficiency of conversion in mammals of phenethyiamines and tetrahydroisoquinolines to rno~~e-be structures is low. Perhaps, however, we should not be unduly surprised that such reactions occur as the underlying chemistry is facile. The ubiquity of morphine-like compounds is an indication of their ready, spontaneous formation. It was shown many years ago by Pictet that mixtures of aldehydes and phenethylamines readily condense and cyclize to isoquinolines. Unlike an enzymatic synthesis, the products are not optically active at the C-l position. Phenolic oxidative coupling to give a mo~hine-me structure is also spontaneous. However, there are many competing oxidations available, so the yield will be low unless the possible combinations that may be formed are reduced by selective methylation of phenols (Fig. 4). The richest animal source found so far is toad skin, in which over 3pmolg-’ tissue of morphine is presents*. In general, the skin contains more opiate alkaloids than

other tissues. It would be interesting to know if the alkaloids are synthesized in the skin, perhaps as a function of phenol radical formation under the high ambient 0, con~~ations, or whether synthesis occurs in the liver. If the latter, concentration into the skin may reflect a relict route of excretion, of more significance to an aquatic existence. Ethanol increases tetrahydropapaveroline formation in rats given L-DOPA, perhaps because of inhibition of dihydroxyphenylacetaldehyde oxidation, allowing more for Schiff’s base formation with dopamine. It appears, therefore, that the proposal of Davis and Walsh in 1970 regarding ethanol and alkaloid formation is, in the main, correct39. They suggested that the increased formation of endogenous opiates in the presence of ethanol provided a Possible basis for alcoholism. As yet, however, the physiological implication of these relatively low levels of endogenously formed alkaloids is unclear. In view of the difference in pharmacology between the alkaloids and the peptides, they carry potential significance. Their spectrum of activity varies, as does their pharmacokinetics. Compared to the alkaloids, the peptides are much more readily degraded and inactivated.

‘CH,

OH

Fg. 4. C2tbr bats pmducts possfbte fmm reticuiline by phenol oxidative coupling behveen numbered positions on Fig. 3. In norlaudanosofine (not il/ustratedJ lacking the three methyl groups of feticufine, the possible ixunbef of beats pmchlcts is greRtar.

TiPS - September 1992 [Vol. 231 These findings have worrying legal and forensic implications. In Japan, the presence of any amount of morphine in the urine is sufficient ‘proof’ of illicit use. In the United States, people have got into trouble because of urinary morphine resulting from the use of poppy seeds. There is a risk that people may unwittingly run foul of the law because of their endogenous biochemistry combined with such factors as pain or infection. 0

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SOME OF THE speculations in this column will be shown to be just that. Others will surely be substantiated. The discoveries of the last two decades have revealed phenomena hitherto unconceived. This suggests that such discoveries will continue, and the true dimensions of the biology of opioid substances still lies beyond the imagination. There are no truly wild popu-

lations of the poppy, Papauer somniferum. It is a cultivar, as surely a creation of man as is the acropolis and the Mona Lisa. In creating such a cultivar, man has fashioned botany to reproduce the essence of the beguiling neurochemistry and pharmacology of his own species. Now it appears as if this is but one facet of man’s love affair with the narcotic opioids, be they peptide, alkaloid or synthetic chemical. Opioids may help provide the appeal of chocolate to the satisfaction of eating. Perhaps, in the form of a glass of hot milk, they contribute to the ease of a good night’s sleep, while adding to the pleasures and protecting from the pains and discomfort of existence. Our days are passed in a mist of opioids. Perhaps opioids do not so much create an illusory world but help create reality itself, comprising a chemical interface between those two strange worlds of being and non-being. In naming morphine for the god of dreams, perhaps Sertiirner was unwittingly glossing that comment in Montaigne’g Essays that ‘those who have compared our life to a dream were right’. B. MAX

345

References 1 Bentley, K.

829-831 19 Kreil, G., Umbach, M., Brantl, V. and Teschemacher, H. (1983) Life Sri. 33 (Suppl. I), 137-140 20 Read, L. C., Lord, A. P. D., Brantl, V. and Koch, G. (1990) Am. J. ikysiol. &9, G44=452 21 Koch, G. et nl. (1988) Regul. Pept. 20,

W. (1954) in The Chemistry of the Morphine Alkaloids (Bentley, K. W., ed.), pp. 289-301, Clarendon Press 2 Max, B. (1988) Trends Pkarmacol. Sci. 9,198-200 3 Levine, A. S. and Atkinson, R. L. (1987) Fed. Proc. 46,159-162 4 Cox, B. M. and Werling, L. L. (1991) in The Biological Basis of Drug Tolerance and Dependence (Pratt, J. A., ed.), uu. 199-229, Academic Press 5 Geisel, H. and Schlimme, E. (1990) Trends Food Sci. Technol. 1,41-43 Kielwein, G. 6 Hamel, U., and Teschemacher, H. (1985)-_1. Dairy _ Res. 52,13%148 7 Svedberg, J., de Haas, J., Leimenstoll, G.. Paul, F. and Teschemacher. H. (1485) Peptides 6,82%830 . 8 Chane, K-l., Su. Y. F.. Brent, D. A. and Cha& J-i<: (1485) 1.. Biol. Chem. 260, 9706-9712 9 Brantl, V. (1985) Eur. J. Phartnncol. 106,213-214 10 Zioudrou, C., Streaty, R. A. and Klee, W. A. (1979) 1. Biol. Chem. 254, 2446-2449 11 Tome, D., Dumontier, ’ A. M., Hautefeuille, M. and Desjeux, J. F. (1987) Am. 1. Physiol. 253, G737-G744 Brantl, V., 12 Hautefeuille, M., Dumontier, A. M. and Desjeux, J. F. (1986) Am. J. Physiol. 250, G92697 13 Umbach, M., Teschemacher, H., Praetorius, K., Hirschhguser, R. and Bostedti H. (1985) Regul. Pept. l2, 223-230 14 Graf, M. V., Hunter, C. A. and Kastin, A. 1. (1984) 1. Clin. Endocrinol. Metab.. 59, i27-132. . 15 Max, B. (1989) Trends Phannncol. Sci. 10,60-63 16 Max, B. (1989) Trends Phartnacol. Sci. 10,220-223 17 Abbott, A. (1992) Trends Phannacof. Sci. 13, 169 18 Panksepp, J., Normansell, L., Siviy, S. and Rossi, J., III (1984) Peptides 5,

107-117

22 Ramabadran, K. and Bansinath. M. (1988) Med. hypotheses 27.181-187 23 Paroli, A. (1988) World Reo. Nutr. Diet. 55; 58-97 24 Sir@, M. M. and Kay, S. R. (1976) Science 191,40142 25 Morley, J. E. (1982) J. Am. Med. Assoc. 247.2379330 26 Dohan, F. C. and Grasberger, J. C. (1973) Am. J. Psychfurry l30,68!%88 27 Morley, J. E. el al. (1983) Gasfroenteroloxy 84,1517-1523 28 Zadina,-1. E., Kastin, A. J., Ge, L-J. and Brantl, v. (1990) Lqe sci. 47, PL25-PL30 29 Schusdziarra, V. et al. (1983) Endocrinology 112,1948-1951 30 Schusdziarra, V. et al. (1983) Pepfides 4,20%210 31 Nedvikovi, J_, Kasafirek, E., Dlabac, A. and Felt, V. (1985) Exp. Clin. Endocrinol. 85,249-252 32 Max, B. (1989) Trends Pkantzacol. ScL 10,390-393 33 Boublii, J_ et nl. (1983) Nature 301, 246-248 34 Hazum, E. ef aI. (1981) Science 213, 1010-1012 35 Kosterlitz, H. W. (1987) Nuture 330, 606 36 Matsubara, K., Fukushima, ‘5.. Akane, A., Kobayashi, S. and Shiono, H. (1992) J. Phannncol. Exp. Ther. 260, %I-978 37 Weib, C. J., FauR, K. F. and Goldstein, A. (1987) Nature 330, 674-677 38 Oka, K., Kantrowib, J. D. and Spector, S. (1985) Proc. N&l Acad. Sd USA 82,1852-1854 39 Davis, V. E. and Walsh, M. J. (1970) Science 167,1005-1007

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