Analgesic activity of intracerebroventricular administration of morphiceptin and β-casomorphins: Correlation with the morphine (μ) receptor binding affinity

Life Sciences, Vol. 30, pp. 1547-1551 Printed in the U.S.A.

Pergamon Press

ANALGESIC ACTIVITY OF INTRACEREBROVENTRICULAR ADMINISTRATION OF MORPHICEPTIN AND ~-CASOMORPHINS: CORRELATION WITH THE MORPHINE (~) RECEPTOR BINDING AFFINITY K.-J. Chang*, P. Cuatrecasas*, E. T. Wei ° and J.-K, Chang

A

*Department of Molecular Biology, Wellcome Research Labs., Research Triangle Park, North Carolina 27709 °Department of Biomedical and Environmental HealthAService , University of California, Berkeley, California 94720 and -Peninsula Labs. Belmont, California 94002 (Received in final form February 19, 1982)

Summar~v Analgesic a c t i v i t i e s

of morphiceptin,

~-casomorphins,

[D-AIa 2,

D-Le~S]enkephalin and Sandoz peptide, FK 33-824, were examined by intracerebroventricular administration in rats. Their relative potencies in vivo were compared with their receptor binding activities. The receptor binding affinities were determined from the competition curves against [3H]naloxone binding in the absence and presence of sodium ions for morphine (~) receptors and against 125I-[D-AIa2, D-LeuS]enkephalin binding for enkephalin (6) receptors. A good correlation between analgesic activity and morphine (~) receptor but not enkephin (6) receptor binding affinity was obtained. These data extend the hypothesis that morphine (p) receptors mediate the major portion of the analgesic activity of opioids. Recently, a new type of opioid peptide (morphiceptin) was identified to be a tetrapeptide amide with the sequence of tyrosylprolylphenylalanylprolinamide (1,2), which is an amide derivative of ~-casomorphin-4 (3-6). Its chemical composition and its selectivity for the opiate receptor subtype are different from those of enkephalins (I). Morphiceptin is a highly selective opioid agonist for both p receptors in guinea pig ileum and morphine binding sites in rat brain (I). This property is contrasted to that of enkephalins (7), which are selective for 6-receptors in the mouse vas deferens and enkephalin binding sites in rat brain (8-10). The affinity of morphiceptin for morphine (~) receptors is at least 1000-times greater than that for enkephalin (6) receptors (I). This peptide, due to its specificity, can serve as a useful tool in studying the physiological roles of the opiate receptor subtype, morphine or ~ receptor, in vivo. Now we report that morphiceptin (I) and ~-casomorphins (3-6) when administered intracerebroventricularly elicit analgesia. Their analgesic activities are correlated with their receptor binding potencies for morphine (p) receptors. Previously, morphiceptin administered intravenously produced transient bradycardia and a fall in blood pressure (2). Methods and Materials Morphiceptin, ~-casomorphins, their related peptides, and the enkephalin analog, FK 33-824, and [D-Ala2,D-LeuS]enkephalin were tested for analgesic activity in male Sprague-Dawley rats weighing 330-400 g. The peptides were 0024-3205/82/181547-05503.00/0 Copyright (c) 1982 Pergamon Press Ltd.

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administered centrally. 22 gauge steel guide cannulae and 28 gauge internal cannulae, obtained from Plastic Products, Roanoke, Virginia, were implanted mid-sagitally into the caudal region of the Sylvian aqueduct (stereotaxic coordinates: 0.5 mm posterior to lambda, 5.5 to 6.0 mm vertical from the dura, with the upper incisor bar at 2.4 mm below the interaural line). Three to five days after surgery, the internal cannula was removed and the guide cannula cleared. Peptides, dissolved in isotonic saline, were injected into the brain in a volume of 5 N1 with a microsyringe, according to the method of Swanson et al. (11). The assay for analgesic activity was based on the inhibition of the tail-withdrawal response of rats to warm water (54 to 56°C) applied to the caudal 5 cm segment of the tail (12,13). Animals which did not receive injections or which received injections of 5 ~i of saline responded within 2.6 ± 0.9 (S.D.) sec, N = 20, after application of the thermal stimulus to the tail. If the tail-withdrawal latency was altered to > 6 sec, within 5, I0 or 15 min after drug injection, the result was considered to be a positive response. The median-effective dose (EDso) for each peptide was estimated according to the up-down method of Dixon (14) with N = 6 and equal log-spacing between doses (3/2 x for a higher dose and 2/3 x for a lower dose). The guide cannula was considered to be correctly placed if clear cerebrospinal fluid could be observed at the external tip of the guide cannula. Injection of 5 pl of I% methylene blue into the guide cannula showed that solutions administered in this manner were distributed in the third ventricle, the aqueduct, the fourth ventricle and, to a lesser extent, in the subarachnoid space. Opiate receptor binding affinity is determined by the concentration which inhibits the specific binding of labeled opioid to rat brain membrane preparations by 50% (IC5o value). [3H]Naloxone (0.2 nM) and 1251-[D-Ala2,DLeuS]enkephalin (0.I riM) were used. Under these conditions, these labels are selectively bound to morphine and enkephalin binding sites, respectively. Rat (Sprague Dawley) brain membranes were prepared as described previously (9,10). Binding assays were performed at 24°C for 60 minutes essentially as described previously, with a filtration (GF/C) method. Nonspecific binding was determined in the presence of 1 N M of the respective unlabeled ligand. 125I-[D-Ala2,D-LeuS]enkephalin was prepared as described previously (4). [3H]Naloxone (50 Ci/ mmole) was purchased from New England Nuclear. Enkephalin and its analogs were available from previous studies (9,10). Morphiceptin and ~-casomorphins were synthesized by a solid-phase method. After hydrogen fluoride cleavage of protected peptide from the resin, the peptides were purified by counter-current distribution with the use of n-butyl alcohol, acetic acid and water (4:1:5 by volume) as partition solvents. Peptides were characterized by thin-layer chromatography, electrophoresis, amino acid analyses and high-pressure liquid chromatography. The purity of the peptide was 98 percent or better (I). Result a n d Discussion Table 1 shows the analgesic activities of morphiceptin, ~-casomorphins and [D-Ala2,D-LeuS]enkephalin and Sandoz peptide FK 33824, [D-AIa2,NMePhe 4, Met(O)Sol]enkephalin, when they are administered intracerebroventricularly. Morphiceptin (No. 3) produces analgesic effect with an median-effective dose (EDso) of about 1.7 nmole/animal. The corresponding tetrapeptide acid (No. 5, casomorphin-4), is about 8-times less active. The pentapeptide (No. 4) is also about 8-times less active. ~-Casomorphin-7 (heptapeptide, No. 7) is about 20-times less active. The amino-terminal tripeptide fragment (No. 6)

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TABLE I Analgesic and Receptor Binding Activities of Peptides Related to Morphiceptin, ~-Casomorphin and Enkephalins

Peptide

Analgesia (EDso) nmole/animal

Receptor Binding Affinity ICso nM p-receptor 6-receptor +

I. Tyr-D-Ala-Gly-NMe-Phe-Met(0)ol (FK 33-824) 2. Tyr-D-Ala-Gly-Phe-D-Leu 3. Tyr-Pro-Phe-Pro-N-H 2 (morphiceptin) 4. Tyr-Pro-Phe-Pro-Gly 5. Tyr-Pro-Phe-Pro 6. Tyr-Pro-Phe 7. Tyr-Pro-Phe-Pro-Gly-Pro-lle (~-Casomorphin) 8. Tyr-D-Pro-Phe-Pro-NH 2

0.03

-Na 0.8

÷ +Na 20

1.4 1.7

I0 50

I00 200

14.2 11.2 >25 35.5

1,300 5,000 >I0,000 6,500

5,500 30,000 14,000

>50

>i0,000 >I0,000

14 1.6 30,000 25,000 70,000 70,000 15,000

50,000

Analgesic potency of intracerebroventricular administration is expressed by median effect dose (EDso). The p-receptor binding affinity was determined from competition studies against [3H]naloxone (0.2 nM) binding in the absence and presence of 0.I M NaCI. The 6-receptor binding affinity was determined from competition curves against 12Sl-[D-Ala2,D-LeuS]enekphalin (0.05 ruM) binding to rat brain membrane preparations. The concentrations which cause 50~ reduction in label binding (IC5o) are thus obtained. shows virtually no activity. The 2-D-proline analog (No. 8) of morphiceptin also shows no activity. Morphiceptin is slightly less active in eliciting analgesia than [D-AIa 2, D-LeuS]enkephalin, a metabolically stable enkephalin analog, and about 80-times less active than Sandoz FK 33-824. Their ranking order of potency in producing analgesic activities in vivo is the same as for their order of potency in guinea pig ileum and binding affinity for morphine (p) receptors (I). At analgesic doses, morphiceptin produces catalepsy and exophthalmus. All these effects were reversed by naloxone hydrochloride, 4 mg/kg, i.p., and thus suggest that these effect of opioids are mediated by morphine (p) receptors. Recently, Brantl et al., also showed that ~-casomorphin-7, (-6), (-5) and (-4) produced naloxone-reversible analgesia after intracerebroventricular injection into rats with a similar ranking order of potency (15). Previously, the activities of morphiceptin and ~-casomorphins for opiate receptors were examined by using labeled compounds as selective p- and 6-agonists (binding) and isolated tissues such as guinea pig ileum and mouse and rat vas deferens (I). In the present studies, the labeled selective p-antagonist, [SH]naloxone, was used under conditions where p-receptors were selectively labeled (9,10). The potency of opioid peptides in competing with the [3H]naloxone binding to morphine binding sites in rat brain membrane preparations was studied in the absence and presence of 0.I M NaCI. The ICso values (the concentration which decreases the [3H]naloxone binding by 50%) are listed in Table 1. Morphiceptin and all other ~-casomorphins show selectivity

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for morphine (~) binding sites. Morphiceptin has the highest affinity and selectivity for p-binding sites among all the ~-casomorphin peptides. The selectivity is reduced proportionally to the decrease in the affinity for morphine (p) binding sites (~able I). The relative binding affinities in the absence and presence of Na are plotted against their in vivo analgesic potency (Fig. I).

1000

>"

100

I---

O

u-6 N E

Io,

.5

...i

-1

-8

-7

-6

-5

-4

log IC50 VALUES MORPHINE (/z) RECEPTOR BINDING AFFINITY

Fig.

I

Correlation between log potency in the in vivo analgesic assay (y) and in the morphine (~) receptor binding assay (x) was studied by using data for Table I. The regression analysis gives a correlation coefficient (r) +0.90 and +0.95 with or without sodium ions. The regression lines are y = 0.65 x -l.00 in the absence of sodium ions and y = 0.77 x -1.85 in the presence of sodium ions. The regression analysis reveals an excellent correlation between analgesic activity and morphine (~) receptor binding affinity. Correlation coefficients of (r) 0.95 and 0.90 were observed for binding affinities in the absence and presence of sodium ions, respectively. Some deviation from the regression lines may be attributed to the difference in the metabolism (i.e. the enzymatic degradation by various peptidases in the tissues or plasma). Except for Sandoz FK 33-824 and [D-AlaZ,D-LeuS]enkephalin, all the other peptides in Table I show very low affinity to enkephalin (6) receptors. No correlation between analgesic activity and enkephalin (6) receptor binding affinity can be obtained. The excellent correlation between analgesic activities and morphine (~) receptors binding affinities strongly suggests and confirms the previously expressed conclusion that morphine (p) receptors mediate the major analgesic

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activity of opioids (16,17,18). However, it does not rule out the possible participitation of other opiate receptor subtypes in mediating analgesic effects. References 1. 2. 3. 4. 5.

6.

7. 8. 9. lO. ll. 12. 13. 14. 15. 16. 17.

18.

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