Pharmacology IS: 18 24 (1979)
Kinetic Studies in Isolated Organs: Tools to Design Analgesic Peptides and to Analyze Their Receptor Effects A.Z. Ronai. I. Berzetei. J.I. Szekely, /.. Graf and S. Bajusz Research Institute for Pharmaceutical Chemistry. Budapest
Key Words. Fentanyl • Guinea pig ileum • Mouse vas deferens • Naltrexone derivatives • Opioid peptides
Since it has been recognized that amongst the opioid peptides of natural origin with known structure /3-endorphin is the only one having remarkable analgesic activity (7, 12. 36). several explanations were proposed to explain this phenomenon. There is a general agreement that the moiety responsible for the opioid activity is the H-Tyr-Gly-Gly-Phe-Met-OH Nterminal pentapeptide portion (for review see 13). Increasing the chain length would enhance or decrease the opioid agonist activity in vitro. depending on the assay system used whereas in vivo the untriakontapeptide /3-endorphin is the only effective analgesic. The first likely explanation for this discrep ancy is the higher susceptibility of shorter peptides to biodegradation. The finding that
/3-endorphin is relatively resistant against enzy mic degradation in brain homogenates as com pared to shorter peptides (1), and the fact that amongst tire partially protected enkephalin analogues potent analgesics exist (2 -4 . 20, 21. 25), seem to support this interpretation. On the other hand, shorter natural opioid peptides and certain non-analgesic enkephalin analogues may act preferentially on different receptors in the central nervous system serving as binding sites for /3-endorphin, analgesic enkephalin analogues and non-peptide narcotics. The purpose of this study is to compare kinetic data of opioid peptides found in vitro with those obtained in vivo. In the last 2 years several partially protected enkephalin analogues have been synthesized at
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 8/15/2018 7:40:40 PM
Abstract. The opioid activities of peptide and non-peptide narcotics were studied in longitu dinal muscle strip of guinea pig ileum (GP1) and in mouse vas deferens (MVD). The comparison of agonist activities of peptides found in GPI and MVD offered the opportunity to predict the presence but not the magnitude of potential analgesic activity. The kinetics of the antagonism between naltrexone and different types of agonists were also determined in these systems. Using C-6 epimers of naltrexone, it was found that the site of opiate receptors recognizing the information provided by the C-6 substituent of naltrexone are different in GPI and MVD.
Design of Analgesie Peptides
our institute. Some of them are potent anal gesics (2, 3, 25). One of the two in vitro assay systems used tire mouse vas deferens prepara tion was known to differentiate between non peptide and certain peptide narcotics, and also between the peptides themselves (16, 17, 22, 23). Furthermore, comparison of activities of opioid peptides found in vitro (GP1 and MVD) offered the opportunity to predict analgesic activity of the peptide in question. Using fentanyl and C-6 epimers of narcotic antagonist naltrexone, we tried to characterize opiate re ceptors in guinea pig ileum (GPI) and mouse vas deferens (MVD).
19
Kinetic parameters of opioid activities of peptide and non-peptide narcotics were determined according to the method elaborated by Kosterlitz and Watt (15). Opioid agonist potencies were characterized by the IDw/50% inhibitory dose/values (in nmol). Antagonist activities were expressed as Ke values (equilibrium constant) (15). When studying the antagonism between different opioid structures and the specific antagonist naltrex one and its derivatives, a different schedule was ap plied in the two in vitro systems. In GPI the agonist-antagonist interaction was deter mined by the single dose method (15), whereas in MVD complete dose-response curves were constructed for the agonist in presence and absence of the antago nist. In both cases Ke values were calculated for the antagonist according to the equation
Ke “ DR 1 ’
The longitudinal muscle strip of GP! from guinea pigs of either sex (350 500 g) was prepared as de scribed by Pawn and Vizi (19). The experiments were performed in Krebs’ solution bubbled with 5% CO: in oxygen, at 37 "C. The composition of the Krebs’ solution used was as follows (in mAf): NaC'l 118, NaHCO, 2S, KC1 4.7, KILPO, 1.2, CaCl, 2.5, MgSO, 1.2 and glucose 11.5. Field electrical stimulation (9) was applied; the supramaximal (1.5 times the maximal voltage) rectangular stimuli of 1 msec duration were delivered at a rate of 0.1 Hz. Contractions were re corded isometrically by means of a strain gauge coupled to a Hellige amplifier-recorder system. Vasa deferentia, taken from mice of CI-'LP strain weighing 25 35 g, were prepared according to Hughes et at. (14) and bathed at 31 °C in modified Krebs’ solution. The modification consisted of omission of MgS04 front the normal Krebs’ solution. We slightly changed the parameters of the stimulation (22) as compared to the original description (14). Field elec trical stimulation was applied through platinium wire electrodes positioned at the top and the bottom of the organ bath. The upper electrode was a ring of 4 mm inner diameter. Paired shocks with 100 msec delay between supramaximal rectangular stimuli of 1 msec duration were used, delivered at a rate of 0.1 Hz. The recording system was the same as described for the longitudinal muscle strip of GPI.
where ‘a’ is the nanomolar concentration of the antagonist and DR (dose ratio) is the virtual shift of the agonist dose-response curve to the right in presence of a given agonist concentration. In GPI dose ratios were calculated according to Kosterlitz and Watt (15). When the results are numerically listed, mean + SEM are given, the number of experiments are indi cated in parentheses. For statistical comparison Student’s t test was applied. Materials used were: fentanyl1 (Janssen Pharmaeeutica, Belgium), morphine sulphate (Mallinckrodt Co., USA), normorphine (Alkaloida, Hungary), nalox one hydrochloride (Endo Labs., USA) was kindly donated by Prof. F.F. Foldes, Montefiore Hospital and Medical Center, USA. Naltrexone hydrochloride (Endo Labs.. USA) was a gift from J. Fishman and E.F. Hahn. Rockefeller University. USA. The C-6 epimers of naltrexone, i.e. a-naltrexol (N-cyclopropylmethyl-14hydroxy-7,8-dihydronormorphine hydrochloride) and (J-naltrcxol (N-cyclopropylmethyl- 14-hydroxy-7,8dihydronorisomorphine hydrochloride) were synthe sized (24) and kindly donated by E.F. Hahn and J. Fishman. LPH (61 91), LPH (61 79) and 1.PH (61 69 peptides were prepared by Graft 10, 12). Methionine-enkephalin (11) and its analogues (2, 3) were synthesized by S. Bajusz. 1 All in vitro experiments with fentanyl were carried out in siliconized glassware.
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 8/15/2018 7:40:40 PM
Methods and Materials
20
Rônai/Berzétei/Szckely/Graf/Bajusz
Results If one plots the 1DS0 values of opioid pep tides obtained in MVD against those measured in GPI (fig. 1), two well-defined groups of peptides could be differentiated. The points of the first group fit well into a straight line with a slope near to unity. The points of the other group were shifted to the right, but were also gathering along a straight line, which was steep-
pounds lacking analgesic activity at the dose level of 100 nmol/animal, ICV. Enkephalins are specified by displaying abbreviations of the substituent amino acids and also indicating the sequence position, referring to the structure of methionine-enkephalin (H-Tyr'-Gly!Gly3-Phe4-Mets-OH).
er than the former. The point for D-nle2, pro5-enkephalin-ethylamide is above the upper line, whereas that for D-met2, pro5-enkephalin falls on the territory between tire lines. Compounds with analgesic activity, injected intraventricularly (ICV) to rats (2, 3, 12, 25) are indicated by dark symbols, those, displaying no significant analgesic activity at the dose level of 100 nmol/animal (2, 12) ICV are plotted in open symbols. Thus a group of peptides, no-
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 8/15/2018 7:40:40 PM
Fig. 1. ID;„ values of normorphine (*), opioid peptides of natural origin ( or •) and synthetic enkephalin analogues ( or ■) found in electrically stimulated longitudinal muscle strip of GPI (abscissa) and in MVD (ordinate). Dark symbols represent com pounds with analgesic activity in tail-flick test in rats, after ICV administration. Open symbols indicate com-
21
Design of Analgesic Peptides
V
\
V N
ch2 i c
7 y > .
o
;
r
OH
-o
CH2
CH2 N
V ,z -
Table I. The opiate antagonist and agonist activities of naltrexone derivatives in GPI and MVD
\
/ v d
OH
Compound:
Naltrexone
Preparation:
GPI
Agonist activity (IOs0- nM ) Antagonist activity1 ( K e’ rM
)
-71.400 (n = 3) 0.8 t 0.1 (n = 4)
K \
>26.000
0.6 t 0.1 (n = 7)
GPI
a
A
V
oh '
> .
OH
OH
(3-NaltrexoI
a-Naltrexol MVD
OH
MVD
MVD
GPI
33.7 t 3.8 >52,000 (n = 4)
oo
1.2 1 0.2 (n = 4)
1.6 t 0.1 (n = 4)
0.6 i 0.06 (n = 8)
>26.000
0.7 ± 0.03 (n= 7)
1 Ke values were determined against morphine or normorphine.
Table II. Characterization of the opiate activity of fentanyl in GPI and MVD preparation
GPI
MVD
ID!0 (nAf) of fentanyl
3.2 * 0.5 (n = 4)
26.4 t 3.2 (n = 12)
Ke (nM) of naltrexone against fentanyl
1.954 ± 0.432' (n = 61
0.451 * 0.060 (n = 8)
tably those without analgesic properties appear to be mucli more potent as opioid agonists in MVD than in GPI. The agonist character of a morphine-like compound was assumed to be governed mostly by two moieties in a morphinoid structure. The first one is the quality and the relative orienta tion of C-6 substituent in the morphine back bone (8, 24) or that of the parent substituents in synthetic narcotics. The other one is the
presence of the ‘second’ benzene ring in an appropriate position (6). Compounds suitable to check the sensitivity of MVD preparation to the functional moieties mentioned above, were tested. Agonist and antagonist potencies of C-6 epimers of the pure opiate antagonist naltrex one were determined both in GPI and in MVD (table 1). In GPI, all naltrexone derivatives were potent morphine antagonists and, if the C-6-
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 8/15/2018 7:40:40 PM
1 Because fentanyl could not be tested repeatedly with safety in GPI preparation, Ke of naltrexone was estimated from single fentanyl doses acting in the absence or presence of antagonist.
Rónai/Berzétei/Székely/Gráf/Bajusz
hydroxyl group was oriented away from the equipotent in both preparations. The en nitrogen (a-naltrexol) a strong agonist activity kephalin analogues containing a D-amino acid could be detected. In MVD antagonist poten (methionine or alanine) in position 2, bearing cies of the compounds determined against nor- A-nle in position 5, proved to be 60 times more morphine are comparable to those found in potent in MVD than in GPI. However, in rats GPI, indicating that the affinities of structures both compounds were lacking of analgesic ac to the opiate receptors are similar in both tivity at the dose level of 100 nmol/animal 1CV. preparations. However, in MVD preparation It appears from the data published by Baxter et a-naltrexol exhibited no measurable agonist al. (4) that their analgesic pentapeptide would activity at the concentration indicated. closely fit the right-hand line in figure 1, below A synthetic non-peptide narcotic which the dose level of 10“ 10 M in MVD. It is there should be regarded as a representative of those fore tempting to speculate that amongst the opiates with ‘two’ benzene rings, fentanyl was opioid peptides there are at least two groups also tested for agonist activity in GPI and MVD acting differently at the receptor site. The (table II). Fentanyl was found to be approxi members of the first group consisting of (3mately eight times stronger in GPI than in endorphin the pro5-enkephalin analogues and MVD. Furthermore, naltrexone antagonizes Z)-met2, D-mets-enkephalin-ethylamide, which with equal potency the effects of both fentanyl are nearly equiactive in MVD and GPI all and normorphine in MVD preparation (tables I, exhibit moderate to strong analgesic activity (2, II). Regarding, that in GPI only an approximate 3, 25). In the other group the presence of an estimation could be made on the Ke of naltrex extremely high affinity to one type opiate one against fentanyl, the moderately higher receptors (i.e. to those present in MVD) is values found against this compound than that required to engender significant analgesic activ against morphine cannot be interpreted un ity in vivo (4). This may suggest that members of the latter group also in tire CNS would ambiguously. preferentially attack a receptor type different from the hypothetical ‘ju’ receptor (9, 18). Based on kinetic studies (Szekely et al., Discussion submitted for publication) with naloxone in It is known that all natural opioid peptides vivo, it appears that /3-endorphin and £)-met2, with known structure except /5-endorphin, are pro5-enkephalinamide exert their analgesic much more potent in MVD than in GPI (16, 17, action on the same in) type of receptors as 22). There is some indication that the en morphine does in mice, and, at least in part, kephalin analogues hitherto reported (4, 5, 20) also in rats. possessing analgesic activity, appear to share In consequence: (1) the analgesic activity of this property of natural peptides. In contrast to opioid peptides is mostly governed by the the compounds reported above the enkephalin preferential affinity of the peptide to one or analogues synthesized in our institute contained the other type of opiate receptors, and (2) dif /.-proline in position 5. All compounds of this ferential spectra of CNS effects could be ex type including the ¿-gly2 substituted derivative pected in the two subgroups outlined above. (not plotted in figure 1) and also ¿)-met2, Keeping in mind that MVD was the prepara D-met5-enkephalin-ethyl amide, are nearly tion, differentiating best between opioid pep
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 8/15/2018 7:40:40 PM
22
tides (16, 17, 22, 23), we made an attempt to characterize opiate receptors in MVD. The experiments with C-6 naltrexone epimers revealed that moieties of opiate recep tors recognizing the information provided by orientation of the C-6 hydroxyl substituent are different in GPI and in MVD. Since all opioid peptides with known struc ture contain two benzene rings (i.e. ty r1 and phe4) which are substantial for opiate activity, we tested whether presence of the two benzene rings in an opioid structure (6) accounts for (a) the higher activity of an opioid compound in MVD than in GPI, and (b) the harder antagonizability of the bulk of opioid peptides by naloxone or naltrexone in MVD (17, 23) as compared to normorphine. The results obtained for fentanyl suggest that, provided the analogy is relevant, none of the properties described above could be ex plained by the presence of two benzene rings per se.
Acknowledgement Wc are indebted to Professor Dr. K. /•'. Sewing for the valuable help in the preparation of manuscript.
References 1 Austen, B.M. and Smyth, D.G.: The NH,-terminus of ('-fragment is resistant to the action of aminopeptidases. Biochem. biophys. Res. Commun. 76: 477-482 (1977). 2 Bajusz, S.: Ronai, A.Z.; Szckely. J.I.; DunaiKovacs, Zs.: Berzetei, l„ and Graf, L.: Enkephalin analogs with enhanced opiate activity. Acta biochim. biophys. hung. II: 305 309 (1976). 3 Bajusz, S.; Ronai, A.Z.; Szckely, J.I.; Graf, L.; Dunai-Kovacs. Zs.. and Berzetei. I.: A superactive antinociceptive pentapeptidc, (D-met!, pro')enkephalinamidc. EEBS Lett. 76: 91 -92 (19 7 7).
23
4 Baxter, M.G.; Goff, D.; Miller, A.A., and Saunders, I.A.: Effect of a potent synthetic opioid pentapeptide in some antinociceptive and behavioural tests in mice and rats. Br. J. Pharmacol. 59: 455 (1977). 5 Coy. D.H.; Kastin, A.J.; Schally. A.V.; Morin. O.; Caron, N.G.; Labrie, I*'.; Walker, J.M.; Fertcl, R.: Bcrtson, G.G., and Sandman, C.A.: Synthesis and opioid activities of stereoisomers and other /7-amino acid analogs of methionine enkephalin. Biochem. biophys. Res. Commun. 73: 6 3 2 638 (1976). 6 Ecinberg, A.P.; Creese, I., and Snyder, S.H.: The opiate receptor: a model explaining structureactivity relationships of opiate agonists and antago nists. Proc. natn. Acad. Sci. USA 73: 4215 - 4219 (1976). 7 Eeldbcrg, W. and Smyth, D.G.: The C-fragment of lipotropin a potent analgesic. J. Physiol.. Loud. 260: 30 31 (1976). 8 lujimoto, J.M.; Roerig, S.; Wang. R.I.H.; Chattcijie, N., and Inturrisi, C.: Narcotic agonist activ ity of several metabolites of naloxone and naltrex one tested in morphine dependent mice. Proc. Soc. exp. B:ol. Med. 148: 443 448 (1975). 9 Gilbert, P.E. and Martin, W.R.: The effects of morphine- and nalorphine-like drugs in the nondependent. morphine-dependent and cyclazocinedependent chronic spinal dog. J. Pharmac. exp. Thcr. 198: 66-83 (1976). 10 Graf, L. and Li, C.H.: Action of plasmin on ovine-lipotropin: revision of the carboxyl terminal sequence. Biochem. biophys. Res. Commun. 53: 1304-1309 (1973). 11 Graf, I..: Ronai, A.Z.: Bajusz, S.; Csch, G„ and Szckely, J.I.: Opioid agonist activity of d-lipotropin fragments: a possible biological source of morphine-like substances in the pituitary. EEBS Lett. 64: 181 184 (1976). 12 Graf, I..; Szckely, J.I.; Ronai, A.Z.; Dunai-Kovacs, Zs., and Bajusz, S.: Comparative study of analgesic effect of MeO-enkcphalin and related lipotropin fragments. Nature, Lond. 263: 240 242 (1976). 13 Horn, A.S. and Rodgers, J.R.: The enkephalins and opiates: structure-activity relations. J. Pharm. Pharmac. 29: 257 265 (1977). 14 Hughes, J.; Kostcrlitz, H.W., and Leslie, E.M.: Assessment of the agonist and antagonist activities of narcotic analgesic drugs by means of the mouse vas deferens. Br. J. Pharmacol. 51: 139 140 (1974).
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 8/15/2018 7:40:40 PM
Design of Analgesic Peptides
15 Kosterlitz, M.W. and Watt, A.J.: Kinetic parameters of narcotic agonists and antagonists with particular reference to N-allyl noroxymorphonc (naloxone). Br. J. Pharmacol. 33: 266 276 (1968). 16 Lord, J.A.H.: Waterfield, A.A.; Hughes, J., and Kosterlitz, H.W.: Multiple opiate receptors; in Kosterlitz, Opiates and endogenous opioid pep tides, pp. 275 - 280 (North-Holland, Amsterdam 1976). 17 Lord, J.A.H.; Waterfield, A.A.; Hughes. J.. and Kosterlitz, H.W.: Endogenous opioid peptides: multiple agonists and receptors. Nature, Lond. 267: 495 -499 (1977). 18 Martin, W.R.; Eades, C.G.; Thompson, J.G.; llupplcr, R.E., and Gilbert, P.E.: The effects of morphine-and nalorphine-like drugs in the nondependent and morphine-dependent chronic spinal dog. J. Pharmac. exp. Ther. / 97: 517 532 (1976). 19 Paton, W.D.M. and Vizi, E.S.: The inhibitory action of noradrenaline and adrenaline on acetyl choline output on guinea-pig longitudinal muscle strip. Br. J. Pharmacol. 35: 10-28 (1969). 20 Pert. C.B.; Pert, A.; Chang, J.-K., and Long. B.T.: (£>-AlaJ)-met-enkephalinamide: a potent, longlasting synthetic pentapeptide analgesic. Science, N.Y. 194: 330-332 (1976). 21 Roemcr, D.: Bucscher, H.H.; Hill. R.C.; Plcss, J.; Bauer, W.; Cardinaux, E.; Closse, A.; Hauser, D., and Hugucnin, R.: A synthetic enkephalin ana logue with prolonged parenteral and oral analgesic activity. Nature. Lond. 268: 547 549 (1977).
Ronai/Berzetei/Szekely/Graf/Bajusz
22 Ronai, A.Z.; Graf, L.; Szekcly, J.L; Dunai-Kovacs, Zs., and Bajusz, S.: Differential behaviour of LPH(61—91 (-peptide in different model systems: com parison of the opioid activities of LPH-(61-9Dpeptidc and its fragments. EEBS Lett. 74: 182 184 (1977). 23 Ronai, A.Z.; Bcrzetei, L, and Bajusz, S.: Differenti ation between opioid peptides by naltrexone. Eur. J. Pharmacol. 45: 393-394 (1977). 24 Ronai, A.Z.; Foldes, F.F.; Hahn, E.F., and Fish man, J.: Orientation of the oxygen atom atC-6 as a determinant of agonistic activity in the oxymorphone series. J. Pharmac. exp. Ther. 200: 496 500 0977). 25 Szckely, J.L: Ronai, A.Z.; Dunai-Kovacs, Zs.; Miglecz. E.; Berzetei, L; Bajusz, S., and Graf, L.: (£>-met! , pro5)-Enkephalinamide: a potent morphinc-like analgesic. Eur. J. Pharmacol. 43: 293294 (1977). 26 Tseng, L.-F.; Loh, H.H., and Li, C.H.: (5-Endorphin as a potent analgesic by intravenous injection. Nature. Lond. 263: 239-240 (1976).
Received: December 12, 1977 Accepted: March 18. 1978 A.Z. Ronai, Research Institute for Pharmaceutical Chemistry, H 1325 Budapest, PO Box 82 (Hungary)
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 8/15/2018 7:40:40 PM
24
Kinetic Studies in Isolated Organs: Tools to Design Analgesic Peptides and to Analyze Their Receptor Effects A.Z. Ronai. I. Berzetei. J.I. Szekely, /.. Graf and S. Bajusz Research Institute for Pharmaceutical Chemistry. Budapest
Key Words. Fentanyl • Guinea pig ileum • Mouse vas deferens • Naltrexone derivatives • Opioid peptides
Since it has been recognized that amongst the opioid peptides of natural origin with known structure /3-endorphin is the only one having remarkable analgesic activity (7, 12. 36). several explanations were proposed to explain this phenomenon. There is a general agreement that the moiety responsible for the opioid activity is the H-Tyr-Gly-Gly-Phe-Met-OH Nterminal pentapeptide portion (for review see 13). Increasing the chain length would enhance or decrease the opioid agonist activity in vitro. depending on the assay system used whereas in vivo the untriakontapeptide /3-endorphin is the only effective analgesic. The first likely explanation for this discrep ancy is the higher susceptibility of shorter peptides to biodegradation. The finding that
/3-endorphin is relatively resistant against enzy mic degradation in brain homogenates as com pared to shorter peptides (1), and the fact that amongst tire partially protected enkephalin analogues potent analgesics exist (2 -4 . 20, 21. 25), seem to support this interpretation. On the other hand, shorter natural opioid peptides and certain non-analgesic enkephalin analogues may act preferentially on different receptors in the central nervous system serving as binding sites for /3-endorphin, analgesic enkephalin analogues and non-peptide narcotics. The purpose of this study is to compare kinetic data of opioid peptides found in vitro with those obtained in vivo. In the last 2 years several partially protected enkephalin analogues have been synthesized at
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 8/15/2018 7:40:40 PM
Abstract. The opioid activities of peptide and non-peptide narcotics were studied in longitu dinal muscle strip of guinea pig ileum (GP1) and in mouse vas deferens (MVD). The comparison of agonist activities of peptides found in GPI and MVD offered the opportunity to predict the presence but not the magnitude of potential analgesic activity. The kinetics of the antagonism between naltrexone and different types of agonists were also determined in these systems. Using C-6 epimers of naltrexone, it was found that the site of opiate receptors recognizing the information provided by the C-6 substituent of naltrexone are different in GPI and MVD.
Design of Analgesie Peptides
our institute. Some of them are potent anal gesics (2, 3, 25). One of the two in vitro assay systems used tire mouse vas deferens prepara tion was known to differentiate between non peptide and certain peptide narcotics, and also between the peptides themselves (16, 17, 22, 23). Furthermore, comparison of activities of opioid peptides found in vitro (GP1 and MVD) offered the opportunity to predict analgesic activity of the peptide in question. Using fentanyl and C-6 epimers of narcotic antagonist naltrexone, we tried to characterize opiate re ceptors in guinea pig ileum (GPI) and mouse vas deferens (MVD).
19
Kinetic parameters of opioid activities of peptide and non-peptide narcotics were determined according to the method elaborated by Kosterlitz and Watt (15). Opioid agonist potencies were characterized by the IDw/50% inhibitory dose/values (in nmol). Antagonist activities were expressed as Ke values (equilibrium constant) (15). When studying the antagonism between different opioid structures and the specific antagonist naltrex one and its derivatives, a different schedule was ap plied in the two in vitro systems. In GPI the agonist-antagonist interaction was deter mined by the single dose method (15), whereas in MVD complete dose-response curves were constructed for the agonist in presence and absence of the antago nist. In both cases Ke values were calculated for the antagonist according to the equation
Ke “ DR 1 ’
The longitudinal muscle strip of GP! from guinea pigs of either sex (350 500 g) was prepared as de scribed by Pawn and Vizi (19). The experiments were performed in Krebs’ solution bubbled with 5% CO: in oxygen, at 37 "C. The composition of the Krebs’ solution used was as follows (in mAf): NaC'l 118, NaHCO, 2S, KC1 4.7, KILPO, 1.2, CaCl, 2.5, MgSO, 1.2 and glucose 11.5. Field electrical stimulation (9) was applied; the supramaximal (1.5 times the maximal voltage) rectangular stimuli of 1 msec duration were delivered at a rate of 0.1 Hz. Contractions were re corded isometrically by means of a strain gauge coupled to a Hellige amplifier-recorder system. Vasa deferentia, taken from mice of CI-'LP strain weighing 25 35 g, were prepared according to Hughes et at. (14) and bathed at 31 °C in modified Krebs’ solution. The modification consisted of omission of MgS04 front the normal Krebs’ solution. We slightly changed the parameters of the stimulation (22) as compared to the original description (14). Field elec trical stimulation was applied through platinium wire electrodes positioned at the top and the bottom of the organ bath. The upper electrode was a ring of 4 mm inner diameter. Paired shocks with 100 msec delay between supramaximal rectangular stimuli of 1 msec duration were used, delivered at a rate of 0.1 Hz. The recording system was the same as described for the longitudinal muscle strip of GPI.
where ‘a’ is the nanomolar concentration of the antagonist and DR (dose ratio) is the virtual shift of the agonist dose-response curve to the right in presence of a given agonist concentration. In GPI dose ratios were calculated according to Kosterlitz and Watt (15). When the results are numerically listed, mean + SEM are given, the number of experiments are indi cated in parentheses. For statistical comparison Student’s t test was applied. Materials used were: fentanyl1 (Janssen Pharmaeeutica, Belgium), morphine sulphate (Mallinckrodt Co., USA), normorphine (Alkaloida, Hungary), nalox one hydrochloride (Endo Labs., USA) was kindly donated by Prof. F.F. Foldes, Montefiore Hospital and Medical Center, USA. Naltrexone hydrochloride (Endo Labs.. USA) was a gift from J. Fishman and E.F. Hahn. Rockefeller University. USA. The C-6 epimers of naltrexone, i.e. a-naltrexol (N-cyclopropylmethyl-14hydroxy-7,8-dihydronormorphine hydrochloride) and (J-naltrcxol (N-cyclopropylmethyl- 14-hydroxy-7,8dihydronorisomorphine hydrochloride) were synthe sized (24) and kindly donated by E.F. Hahn and J. Fishman. LPH (61 91), LPH (61 79) and 1.PH (61 69 peptides were prepared by Graft 10, 12). Methionine-enkephalin (11) and its analogues (2, 3) were synthesized by S. Bajusz. 1 All in vitro experiments with fentanyl were carried out in siliconized glassware.
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 8/15/2018 7:40:40 PM
Methods and Materials
20
Rônai/Berzétei/Szckely/Graf/Bajusz
Results If one plots the 1DS0 values of opioid pep tides obtained in MVD against those measured in GPI (fig. 1), two well-defined groups of peptides could be differentiated. The points of the first group fit well into a straight line with a slope near to unity. The points of the other group were shifted to the right, but were also gathering along a straight line, which was steep-
pounds lacking analgesic activity at the dose level of 100 nmol/animal, ICV. Enkephalins are specified by displaying abbreviations of the substituent amino acids and also indicating the sequence position, referring to the structure of methionine-enkephalin (H-Tyr'-Gly!Gly3-Phe4-Mets-OH).
er than the former. The point for D-nle2, pro5-enkephalin-ethylamide is above the upper line, whereas that for D-met2, pro5-enkephalin falls on the territory between tire lines. Compounds with analgesic activity, injected intraventricularly (ICV) to rats (2, 3, 12, 25) are indicated by dark symbols, those, displaying no significant analgesic activity at the dose level of 100 nmol/animal (2, 12) ICV are plotted in open symbols. Thus a group of peptides, no-
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 8/15/2018 7:40:40 PM
Fig. 1. ID;„ values of normorphine (*), opioid peptides of natural origin ( or •) and synthetic enkephalin analogues ( or ■) found in electrically stimulated longitudinal muscle strip of GPI (abscissa) and in MVD (ordinate). Dark symbols represent com pounds with analgesic activity in tail-flick test in rats, after ICV administration. Open symbols indicate com-
21
Design of Analgesic Peptides
V
\
V N
ch2 i c
7 y > .
o
;
r
OH
-o
CH2
CH2 N
V ,z -
Table I. The opiate antagonist and agonist activities of naltrexone derivatives in GPI and MVD
\
/ v d
OH
Compound:
Naltrexone
Preparation:
GPI
Agonist activity (IOs0- nM ) Antagonist activity1 ( K e’ rM
)
-71.400 (n = 3) 0.8 t 0.1 (n = 4)
K \
>26.000
0.6 t 0.1 (n = 7)
GPI
a
A
V
oh '
> .
OH
OH
(3-NaltrexoI
a-Naltrexol MVD
OH
MVD
MVD
GPI
33.7 t 3.8 >52,000 (n = 4)
oo
1.2 1 0.2 (n = 4)
1.6 t 0.1 (n = 4)
0.6 i 0.06 (n = 8)
>26.000
0.7 ± 0.03 (n= 7)
1 Ke values were determined against morphine or normorphine.
Table II. Characterization of the opiate activity of fentanyl in GPI and MVD preparation
GPI
MVD
ID!0 (nAf) of fentanyl
3.2 * 0.5 (n = 4)
26.4 t 3.2 (n = 12)
Ke (nM) of naltrexone against fentanyl
1.954 ± 0.432' (n = 61
0.451 * 0.060 (n = 8)
tably those without analgesic properties appear to be mucli more potent as opioid agonists in MVD than in GPI. The agonist character of a morphine-like compound was assumed to be governed mostly by two moieties in a morphinoid structure. The first one is the quality and the relative orienta tion of C-6 substituent in the morphine back bone (8, 24) or that of the parent substituents in synthetic narcotics. The other one is the
presence of the ‘second’ benzene ring in an appropriate position (6). Compounds suitable to check the sensitivity of MVD preparation to the functional moieties mentioned above, were tested. Agonist and antagonist potencies of C-6 epimers of the pure opiate antagonist naltrex one were determined both in GPI and in MVD (table 1). In GPI, all naltrexone derivatives were potent morphine antagonists and, if the C-6-
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 8/15/2018 7:40:40 PM
1 Because fentanyl could not be tested repeatedly with safety in GPI preparation, Ke of naltrexone was estimated from single fentanyl doses acting in the absence or presence of antagonist.
Rónai/Berzétei/Székely/Gráf/Bajusz
hydroxyl group was oriented away from the equipotent in both preparations. The en nitrogen (a-naltrexol) a strong agonist activity kephalin analogues containing a D-amino acid could be detected. In MVD antagonist poten (methionine or alanine) in position 2, bearing cies of the compounds determined against nor- A-nle in position 5, proved to be 60 times more morphine are comparable to those found in potent in MVD than in GPI. However, in rats GPI, indicating that the affinities of structures both compounds were lacking of analgesic ac to the opiate receptors are similar in both tivity at the dose level of 100 nmol/animal 1CV. preparations. However, in MVD preparation It appears from the data published by Baxter et a-naltrexol exhibited no measurable agonist al. (4) that their analgesic pentapeptide would activity at the concentration indicated. closely fit the right-hand line in figure 1, below A synthetic non-peptide narcotic which the dose level of 10“ 10 M in MVD. It is there should be regarded as a representative of those fore tempting to speculate that amongst the opiates with ‘two’ benzene rings, fentanyl was opioid peptides there are at least two groups also tested for agonist activity in GPI and MVD acting differently at the receptor site. The (table II). Fentanyl was found to be approxi members of the first group consisting of (3mately eight times stronger in GPI than in endorphin the pro5-enkephalin analogues and MVD. Furthermore, naltrexone antagonizes Z)-met2, D-mets-enkephalin-ethylamide, which with equal potency the effects of both fentanyl are nearly equiactive in MVD and GPI all and normorphine in MVD preparation (tables I, exhibit moderate to strong analgesic activity (2, II). Regarding, that in GPI only an approximate 3, 25). In the other group the presence of an estimation could be made on the Ke of naltrex extremely high affinity to one type opiate one against fentanyl, the moderately higher receptors (i.e. to those present in MVD) is values found against this compound than that required to engender significant analgesic activ against morphine cannot be interpreted un ity in vivo (4). This may suggest that members of the latter group also in tire CNS would ambiguously. preferentially attack a receptor type different from the hypothetical ‘ju’ receptor (9, 18). Based on kinetic studies (Szekely et al., Discussion submitted for publication) with naloxone in It is known that all natural opioid peptides vivo, it appears that /3-endorphin and £)-met2, with known structure except /5-endorphin, are pro5-enkephalinamide exert their analgesic much more potent in MVD than in GPI (16, 17, action on the same in) type of receptors as 22). There is some indication that the en morphine does in mice, and, at least in part, kephalin analogues hitherto reported (4, 5, 20) also in rats. possessing analgesic activity, appear to share In consequence: (1) the analgesic activity of this property of natural peptides. In contrast to opioid peptides is mostly governed by the the compounds reported above the enkephalin preferential affinity of the peptide to one or analogues synthesized in our institute contained the other type of opiate receptors, and (2) dif /.-proline in position 5. All compounds of this ferential spectra of CNS effects could be ex type including the ¿-gly2 substituted derivative pected in the two subgroups outlined above. (not plotted in figure 1) and also ¿)-met2, Keeping in mind that MVD was the prepara D-met5-enkephalin-ethyl amide, are nearly tion, differentiating best between opioid pep
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 8/15/2018 7:40:40 PM
22
tides (16, 17, 22, 23), we made an attempt to characterize opiate receptors in MVD. The experiments with C-6 naltrexone epimers revealed that moieties of opiate recep tors recognizing the information provided by orientation of the C-6 hydroxyl substituent are different in GPI and in MVD. Since all opioid peptides with known struc ture contain two benzene rings (i.e. ty r1 and phe4) which are substantial for opiate activity, we tested whether presence of the two benzene rings in an opioid structure (6) accounts for (a) the higher activity of an opioid compound in MVD than in GPI, and (b) the harder antagonizability of the bulk of opioid peptides by naloxone or naltrexone in MVD (17, 23) as compared to normorphine. The results obtained for fentanyl suggest that, provided the analogy is relevant, none of the properties described above could be ex plained by the presence of two benzene rings per se.
Acknowledgement Wc are indebted to Professor Dr. K. /•'. Sewing for the valuable help in the preparation of manuscript.
References 1 Austen, B.M. and Smyth, D.G.: The NH,-terminus of ('-fragment is resistant to the action of aminopeptidases. Biochem. biophys. Res. Commun. 76: 477-482 (1977). 2 Bajusz, S.: Ronai, A.Z.; Szckely. J.I.; DunaiKovacs, Zs.: Berzetei, l„ and Graf, L.: Enkephalin analogs with enhanced opiate activity. Acta biochim. biophys. hung. II: 305 309 (1976). 3 Bajusz, S.; Ronai, A.Z.; Szckely, J.I.; Graf, L.; Dunai-Kovacs. Zs.. and Berzetei. I.: A superactive antinociceptive pentapeptidc, (D-met!, pro')enkephalinamidc. EEBS Lett. 76: 91 -92 (19 7 7).
23
4 Baxter, M.G.; Goff, D.; Miller, A.A., and Saunders, I.A.: Effect of a potent synthetic opioid pentapeptide in some antinociceptive and behavioural tests in mice and rats. Br. J. Pharmacol. 59: 455 (1977). 5 Coy. D.H.; Kastin, A.J.; Schally. A.V.; Morin. O.; Caron, N.G.; Labrie, I*'.; Walker, J.M.; Fertcl, R.: Bcrtson, G.G., and Sandman, C.A.: Synthesis and opioid activities of stereoisomers and other /7-amino acid analogs of methionine enkephalin. Biochem. biophys. Res. Commun. 73: 6 3 2 638 (1976). 6 Ecinberg, A.P.; Creese, I., and Snyder, S.H.: The opiate receptor: a model explaining structureactivity relationships of opiate agonists and antago nists. Proc. natn. Acad. Sci. USA 73: 4215 - 4219 (1976). 7 Eeldbcrg, W. and Smyth, D.G.: The C-fragment of lipotropin a potent analgesic. J. Physiol.. Loud. 260: 30 31 (1976). 8 lujimoto, J.M.; Roerig, S.; Wang. R.I.H.; Chattcijie, N., and Inturrisi, C.: Narcotic agonist activ ity of several metabolites of naloxone and naltrex one tested in morphine dependent mice. Proc. Soc. exp. B:ol. Med. 148: 443 448 (1975). 9 Gilbert, P.E. and Martin, W.R.: The effects of morphine- and nalorphine-like drugs in the nondependent. morphine-dependent and cyclazocinedependent chronic spinal dog. J. Pharmac. exp. Thcr. 198: 66-83 (1976). 10 Graf, L. and Li, C.H.: Action of plasmin on ovine-lipotropin: revision of the carboxyl terminal sequence. Biochem. biophys. Res. Commun. 53: 1304-1309 (1973). 11 Graf, I..: Ronai, A.Z.: Bajusz, S.; Csch, G„ and Szckely, J.I.: Opioid agonist activity of d-lipotropin fragments: a possible biological source of morphine-like substances in the pituitary. EEBS Lett. 64: 181 184 (1976). 12 Graf, I..; Szckely, J.I.; Ronai, A.Z.; Dunai-Kovacs, Zs., and Bajusz, S.: Comparative study of analgesic effect of MeO-enkcphalin and related lipotropin fragments. Nature, Lond. 263: 240 242 (1976). 13 Horn, A.S. and Rodgers, J.R.: The enkephalins and opiates: structure-activity relations. J. Pharm. Pharmac. 29: 257 265 (1977). 14 Hughes, J.; Kostcrlitz, H.W., and Leslie, E.M.: Assessment of the agonist and antagonist activities of narcotic analgesic drugs by means of the mouse vas deferens. Br. J. Pharmacol. 51: 139 140 (1974).
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 8/15/2018 7:40:40 PM
Design of Analgesic Peptides
15 Kosterlitz, M.W. and Watt, A.J.: Kinetic parameters of narcotic agonists and antagonists with particular reference to N-allyl noroxymorphonc (naloxone). Br. J. Pharmacol. 33: 266 276 (1968). 16 Lord, J.A.H.: Waterfield, A.A.; Hughes, J., and Kosterlitz, H.W.: Multiple opiate receptors; in Kosterlitz, Opiates and endogenous opioid pep tides, pp. 275 - 280 (North-Holland, Amsterdam 1976). 17 Lord, J.A.H.; Waterfield, A.A.; Hughes. J.. and Kosterlitz, H.W.: Endogenous opioid peptides: multiple agonists and receptors. Nature, Lond. 267: 495 -499 (1977). 18 Martin, W.R.; Eades, C.G.; Thompson, J.G.; llupplcr, R.E., and Gilbert, P.E.: The effects of morphine-and nalorphine-like drugs in the nondependent and morphine-dependent chronic spinal dog. J. Pharmac. exp. Ther. / 97: 517 532 (1976). 19 Paton, W.D.M. and Vizi, E.S.: The inhibitory action of noradrenaline and adrenaline on acetyl choline output on guinea-pig longitudinal muscle strip. Br. J. Pharmacol. 35: 10-28 (1969). 20 Pert. C.B.; Pert, A.; Chang, J.-K., and Long. B.T.: (£>-AlaJ)-met-enkephalinamide: a potent, longlasting synthetic pentapeptide analgesic. Science, N.Y. 194: 330-332 (1976). 21 Roemcr, D.: Bucscher, H.H.; Hill. R.C.; Plcss, J.; Bauer, W.; Cardinaux, E.; Closse, A.; Hauser, D., and Hugucnin, R.: A synthetic enkephalin ana logue with prolonged parenteral and oral analgesic activity. Nature. Lond. 268: 547 549 (1977).
Ronai/Berzetei/Szekely/Graf/Bajusz
22 Ronai, A.Z.; Graf, L.; Szekcly, J.L; Dunai-Kovacs, Zs., and Bajusz, S.: Differential behaviour of LPH(61—91 (-peptide in different model systems: com parison of the opioid activities of LPH-(61-9Dpeptidc and its fragments. EEBS Lett. 74: 182 184 (1977). 23 Ronai, A.Z.; Bcrzetei, L, and Bajusz, S.: Differenti ation between opioid peptides by naltrexone. Eur. J. Pharmacol. 45: 393-394 (1977). 24 Ronai, A.Z.; Foldes, F.F.; Hahn, E.F., and Fish man, J.: Orientation of the oxygen atom atC-6 as a determinant of agonistic activity in the oxymorphone series. J. Pharmac. exp. Ther. 200: 496 500 0977). 25 Szckely, J.L: Ronai, A.Z.; Dunai-Kovacs, Zs.; Miglecz. E.; Berzetei, L; Bajusz, S., and Graf, L.: (£>-met! , pro5)-Enkephalinamide: a potent morphinc-like analgesic. Eur. J. Pharmacol. 43: 293294 (1977). 26 Tseng, L.-F.; Loh, H.H., and Li, C.H.: (5-Endorphin as a potent analgesic by intravenous injection. Nature. Lond. 263: 239-240 (1976).
Received: December 12, 1977 Accepted: March 18. 1978 A.Z. Ronai, Research Institute for Pharmaceutical Chemistry, H 1325 Budapest, PO Box 82 (Hungary)
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 8/15/2018 7:40:40 PM
24
