Life Sciences Vol . 18, pp . 231-244 Printed in the U .S .A .

Pergamon Press

A MODEL SYSTEM FOR OPIATE-RECEPTOR INTERACTIONS : MECHANISM OF OPIATE-CEREBROSIDE SULFATE INTERACTION . Tae Mook Cho, Jung Sook Cho and Horace H . Loh Langley Porter Neuropsychiatric Institute and Departments of Psychiatry and Pharmacology, University of California, San Francisco, Ca .

94143

(Received in final form December 31, 1975) SUMMARY Narcotic analgetics were shown to bind cerebroside sulfate (CS) with high affinity . The binding correlated well with their pharmacological potency . In order to understand opiate receptor interaction at the molecular level, we have proposed the use of CS as a model opiate receptor . In these studies, our data indicate that the binding of opiates is determined by the heptane solubility of the drugs and their affinity to CS . The affinity of the agonist to CS is higher than that of its corresponding antagonist . The difference in affinity between an agonist and its corresponding antagonist is mainly due to the strength of electrostatic bond formed between the protonated nitrogen of the drug and the sulfate group of CS . Furthermore, we have concluded that narcotic agonist-CS complexes are more hydrophobic (intimate ion pairs formation) while the antagonist-CS complexes are more hydrophilic (hydrated ion pairs) in nature .

In recent studies (1), we have shown that cerebroside sulfate exhibited the highest affinity for narcotic agonists among the acidic lipids : cerebroside sulfate, phosphatidylinositol, phosphatidylserine, phosphatidic acid, and triphosphoinositide . Our data also showed that the concentration of various agonists required to inhibit radioactive narcotic binding to cerebroside sulfate correlated with their reported analgetic activity . The chromatographic behavior of the complex formed by cerebroside sulfate (2) with the agonist, levorphanol, was shown to be virtually identical to that of the complex formed with a purified opiate receptor which was isolated from mouse brain and reported to be a proteolipid (3) . Further chemical analysis has confirmed that the purified receptor fraction contains mainly, if not solely, cerebroside sulfates . Based on these observations, the possibility that cerebroside sulfate and opiate receptor may be related is, therefore, indicated . In in vivo, animals with hypothyroidism are known to This investigation was supported by the U .S . Army Medical Research and Development Command under Contract No . DADA17-73-C-3006 . HHL is a recipient of NIDA Research Scientist Development Award K2-DA-70554 . 231

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be deficient in cerebroside sulfate (4) . Sung and Way have also reported that rats rendered hypothyroid become more tolerant and resistant to morphine (5) . Moreover, a genetic mutant, Jimpy mice, which are known to have a low content of cerebroside sulfate (6), were found to be less sensitive to morphine when compared with their littermate controls (7) . Even though the exact role of CS in opiate receptor cannot be established, based on this direct and indirect evidence, we have proposed that cerebroside sulfate can be a good opiate receptor model to study opiate-receptor interaction at the molecular level . The mechanism of opiate interaction with its receptor is not understood ; however, several lines of evidence indicate that the prime mode of opiate receptor interaction is the formation of an electrostatic bond between the protonated nitrogen of opiate and the anionic site of the receptor molecule . Beckett et al . (B) have shown that the anal etic activity of various opiates decreasedwitF the increase in the "effective width' of the basic group . Recently, from the analysis of the structure-activity relationships, Portoghese (9) has concluded that the nitrogen atom plays a pivotal role in the association of the analgetics with their receptors . Moreover, Harris et al . (10) have shown that lanthanum, an inorganic cation, exhibits analgetic activity and the activity was antagonized by naloxone, a pure narcotic antagonist, when administered intraventricularly . This interaction should be dependent on the physicochemical properties of the cationic center of the opiate such as size, hydration and on the polarizability of the anionic center of the receptor and the nature of this interaction in turn determines the physicochemical properties of the drugreceptor complex . In this regard, we have reported that levorphanol (agonist)-CS complex is more hydrophobic than either the complex formed with naloxone (antagonist) or free cerebroside sulfate (11) . In order to understand the molecular mechanisms involved in the interaction of opiates with their receptors, we have proposed to use cerebroside sulfate as a model receptor . In this paper, we report some of our findings, indicating that the lipid solubility of the drug, the affinity of the drug to cerebroside sulfate, and the formation of ion pairs between cationic nitrogen of the opiates and anionic sulfate group are the prime factors determining the degree of binding of opiate to its receptor . Materials and Methods H3- levorphanol (2 .5 Ci/mmole) was custom made by ICN . Cerebrosj ide sulfate was purchased from Analab ., Sephadex LH-20 from Sigma and H -morphine (12 .5 Ci/mmole) from New England Nuclear . The following drugs were generously donated : levorphanol and levallorphan tartrate from Hoffmann-La Roche, Inc ., morphine from Mallinckrodt, nalorphine from Merck Sharp and Dohme, oxymorphone and naloxone from Endo Labs ., GPA-1657 and GPA-2163 from CIBA Pharmaceutical Co ., methadone from Eli Lilly, and ketobemidone from Dr . E .L . May of NIMH . Bindi ng Studies . H 3 -levorphanol binding to cerebroside sulfate was determined in the absence and presence of various unlabelled narcotics aid the degree of inhibition is expressed as a percentage of the total H -levorphanol binding in the absence of the drugs . The organic solvent-water partition method as escgibed below was used . An aqueous solution (1 ml) containing 5 x H 10-levorphanol and varying concentrations of each unlabelled narcotic was adjusted to pH6 .0 and vortexed with 1 ml of organic solvent in the absence and presence of cerebroside sulfate (4 iug) . The organic solvent

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consists of heptane, chloroform, and methanol in the proportion of 1500 :2 :1 . Each mixture was centrifuged at 1500 x g for 10 minutes and the distribution of H3 -levorphanol between the organic phase and aqueous phase was determined by liquid scintillation spectrophotometry . The radioactivity of 0 .5 ml aliquots of the aqueous phase and organic phase was counted in 10 ml of toluene-triton X-100 cocktail solution with an efficiency of 29-33% . The radioactivity at the interface was obtained by subtracting the radioactivity found in water and organic phase from the total âmount added . The amount of drug bound to cerebroside sulfate in the organic phase and at the interface was calculated, using the radioactivity in water and the partition coefficients between heptane and water, as described by Weber et al . (12) . Results H 3-levorphanol binding to cerebroside sulfate (CS) (4 Ng) was determined by organic solvent-water partition with increasing concentration of levorphanol . The results indicated that most of the binding was observed at the interface between organic solvent and w ter . For example, 80% of levorphanol binding was obtained at 5 x 10levorphanol, based on total amount of radioactivity added . The binding was distributed 96% at its interface and 4% in heptane . The radioactivity of H3-levorphanol in the absence and presence of cerebroside sulfate was shown in Fig . 1 . Fig . 2 showed the binding curve of levorphanol to C .S . It is clear from these data that the binding is saturable (Table 1) . Scatchard analysis was applied to the data obtained in Fig . 2 to determine the dissociation constants (K 0 ) and the capacity of these binding sites .

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FIG . 2 The bindin0 curve of levorphanol to cerebroside sulfate . The data were taken from Fig . 1, and the binding of levorphanol was calculated by the method of Weber et al . (12) . As sown in Fig . 3, three components - Lrith dissociation constants of 9 .1 x 10 - M (capacity 0 .45 nmole , 1 x 10 - 'M (capacity 1 .4 nmoles), and 6 .0 x 10 -6 M (capacity 2 .5 nmoles ;were evident . It should be noted that these K 0 's were estimated based on the drug concentration added to this heptane-water partition system . Since the dissociation of the drug-cerebroside sulfate complex will be influenced by the solubilities of the complex and the drug in heptane .(11), a more precise dissociation constant for levorphanol-CS complex may be obtained using the concentration of the drug in heptane (see the discussion for details) . These were estimated by multiplying the dissociation constants based on the drug concentration added + P H/W ), in water by a factor being the partition coefficient PH/W (PH/W/1 of levorphanol between heptane and water (PH/W = 1 .1x10-2 ) . The data are summarized in Table 1 . It is interesting to note that the KD 's in water are similar to the figures reported by Lowney et al, (3) for Purified opiate receptor bindings while the K 0 's in heptane are in the same range of those reported by Pert and Snyder (13) who studied the bindings of opiate to its receptor in brain homogenate .

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Model for Opiate Receptor 4-0

FIG . 3 Scatchard plots of levorphanol binding to cerebroside sulfate : the binding data of levorphanol to cerebroside sulfate were taken from Fig . 2, and plotted by the method of Scatchard . TABLE I Dissociation Constants of Levorphanol-CS Complex Based on Drug Concentration in Water and in Heptane

Water a (M)

Heptane b (M)

Capacity (nmoles/ 4 .4 nmoles of CS)

K1

9 .1 x 10 -8

9 .1 x 10 -10

0 .45

K2

1 .0 x 10 -6

1 .0 x 10 -8

1 .40

K3

6 .0 x 10 -6

6 .0 x 10-8

2 .50

Thé data were taken from Fig . 3 an converted into the dissociation constantsb by multiplying a factor (PH/W/l + PH~W) : P being the partition coefficie2t of levorphanol beiween heptane a~dW water (P H/W = 1 .1 x 10 - ) .

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The concentration of arious unlabelled narcotics required to inhibit the binding of 5 x 10-8M HJ-levorphanol to 4,ug of cerebroside sulfate by 50% (ID50) was estimated from log concentrations - % inhibition curves . The results clearly showed that the ID50's of various narcotics, based on the total concen"E[VW -of the drugs added irrwater (column 1 in- Table - IF), parallel their reported pharmacological potency when the drus were administered by subcutaneous (mice) or by parenteral (man) routes ~14) . The correlation coefficients between the ID50's and their in vivo potency Since obtained by least square analysis were 0 .99 for mice anI Ô?3$ for man . it is possible that the drugs may be first absorbed into heptane phase before their interaction with CS, the ID50's of the drugs based on the concentration in heptane were also calculated by multiplying the ID50's based on the concentration added in water by the factor (P / /(1 + PH/ )) (column 2 in Table II) . When both ID50's were compared wit4Wthe bioTbgical activities by intravenous or intraventricular administration as reported by Kutter et al . (15), the data seem to indicate that the ID50's in water paralleTe-dtheir in vivo potency by intravenous administration, while those ID50's in heptane reacted more their potency by intraventricular administration (Table II) . However, there is a discrepancy between ID59 in heptane and intraventricular dose ; ketobemidone (ID50 = 2 .6 x 10 - ) was more potent than levorphanol (ID50 = 16 .3 x 10 - ) whereas the latter was slightly more potent than the former when they were given by the intraventricular route (Column 6, Table II) . This may be due to the fact that, when the injection site is closer to the action site, i .e . CNS membrane, the potency is more dependent upon the affinity of the drug to the receptor than the lipid solubility of the drug because levorphanol has a higher heptane solubility than ketobemidone . It is interesting to point out that in the drugs with similar rigid structure and same pharmacodynamic groups such as phenol, piperidine and N-methyl (e .g . morphine, metazocine, levorphanol and GPA-1657), the potencies (ID50's) in water and the I .V . potency increased with increasing the partition coefficient of the drug between heptane and water, whereas the reverse was true with the potencies (ID50's) in heptane and the potency when the drugs were given by i .vt . route . When the ID50's and the heptane solubilities of the narcotic agonists were compared with their corresponding antagonists, it was noted, as shown in Table III, that narcotic agonists were more potent in ID50's than their respective antagonists based on the drug concentration added in water but the heptane solubilities of the agoniet were lower than their respective antagonist . Therefore, the differences in ID50's between agonists and their respective antagonists were much bigger when the ID50's were estimated based on the drug concentration in the heptane phase . The binding of opiate to cerebroside sulfate (CS) in the two-phase system, e .g ., liposome in water (2) and heptane-water interface system, depends on at least two factors, i .e ., the lipid solubility and the affinity of the drug for CS . In order to determine the binding merely due to the affinity of the drugs to CS and to eliminate the lipid solubility factor, we have estimated the binding capacity of equimolar concentrations of four pairs of opiate agonists and antagonists to cerebroside sulfate in a pure organic solvent system using sephadex LH-20 column . The data in Table IV show that agonists bound more CS than their corresponding antagonists . Discussion In agreement with previous published results (2), our present findings show that levorphanol binds to CS with high affinity (Table I) and that the ID50's of various opiates correlates well with their reported analgetic

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TABLE III Comparison of ID50 of Narcotic Agonist To Its Respective Antagonist

Drugs

ater

ID50a

Heptane

P H/Wc

pKa d

'UM

nM

Morphine

5 .0

2 .5

5 x 10-4

8 .02

Nalorphine

6 .0

20 .9

3 .5 x 10 -3

7 .93

Oxymorphone

5 .0

10 .0

14 .0

112 .0

8 x 10-3

7 .94

Levorphanol

1 .5

16 .3

1 .1 x 10 -2

8 .18

Levallorphan

1 .8

69 .0

4 x 10-2

8 .30

GPA-1657

0 .75

190 .0

3 .4 x 10

GPA-2163

2 .2

2016 .6

1 .1 x 10

Naloxone

2 x

10-3

8 .25

-1

a.

ID50 is defined as the concentration of th drug required to inhibit the binding of 5 x 10- AM, H -Levorphanol to 4,ug of cerebroside sulfate by 50% .

b.

The ID50's of the drugs in heptane were obtained by multiplying the ID50's in water by the factor partition coefficient of drU6WbetweeP H hëptane aN6Wwater9

c.

Partition coefficients of the drugs between heptane and water (pH 7 .4) were determined in our laboratory .

d.

Negative logarithm of dissociation constants of drugs (17) .

potency (Table II) . Since opiate binding sites are located at the nerve membrane (18) and the interaction between opiate and receptor molecules in the membrane would occur at the interface between the membrane and biological fluid, we feel that the binding of opiate to CS which occurs at the interface between the heptane and water (monolayer) phase would be a good model to study the mechanism of opiate-receptor interaction . Our data indicate that the binding of opiates is determined both by the heptane solubility of the drugs and their affinities to CS . The dissociation constant of the drug-CS complexes is mostly correlated with

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TABLE IV Binding of Opiate Agonists and Their Corrsponding Antagonists To Cerebroside Sulfate

Drugs

Drug bound (P moles)

Morphine

0 .90 ± 0 .05

45

Nalorphine

0 .32 + 0 .04

16

Oxymorphone

0 .98 + 0 .06

49

Naloxone

0 .28 + 0 .03

14

Levorphanol

0 .86 + 0 .04

43

Levallorphan

0 .36 + 0 .02

18

GPA-1657

0 .70 + 0 .03

35

GPA-2163

0 .10 + 0 .01

5 .5

Yields Cx)

The drug-cerebroside sulfate complexes were prepared by mixing 2jumoles of the drugs and 2 mg of cerebroside sulfate in 1 .5 ml of chloroform-methanol mixture (2 :1) . The complexes and free cerebroside sulfate on sephadex LH-20 column were eluted with 100 ml of chloroformmethanol (10 :1) and 100 ml of chloroform-methanol (1 :1), respectively . The yield of the complexes is based on the amounts of cerebroside sulfite added . The detailed procedure has been described before (2) . the concentration in heptane . The reasons are as follows : a) When equimolar concentration of the drugs was used to dejermine binding in a one-phase organic system, morphine (P = 5 x 10- ) bound more CS than GPA-1657 (PH~W = 0 .34) (see Table whereas the reverse was true when the binding ivas determined in a two-phase system (see Table II) . This indicates that morphine has a higher-affinity for CS than GPA-1657, while GPA-1657 has a higher heptane solubility so that it binds more CS in the two-phase system . b) If the drugs in water directly bind with CS, regardless of the heptane-water partition coefficient, the affinity to CS should be sufficient to predict the in vivo potencies when the drugs are administered parenterally . This is,owTously, not the case (Table II) since the in vivo potency is-determined, at least, if not entirely, by blood brainbaker and the affinity of drug to its receptor . Moreover, it is known that the-blood brain barrier is related to the lipid solubility of the drug (Table V) . Our findings that the ID50's in water parallel the intravenous potency of the drugs while the ID50's in heptane resemble more closely to their intraventricular potency (Table II) are in agreement with

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the above discussion . Furthermore, Wilson et al . (19) have reported that the binding of a series of N-alkylnorketobemidones to brain homogenate correlated well with their in vivo potency by subcutaneous administration . In this case, the drugs shouTd7Trst be absorbed into the membrane before they interact with the receptors . The partitions of the free drugs between the membrane and water (in vitro)should linearly correlate with those of the drug from .injectionsile - i:o CNS membrane . Otherwise, the drug binding to brain homogenate should correlate with the in vivo potency only when administered directly to CNS but not by subcutaneous- a-Einistra= tion . However, this is in contrast to their observations (19) . c) Among drugs with a similar rigid ring structure and with same pharmacodynamic groups such as phenol, piperidine and N-methyl (e .g . morphine, metazocine, levorphanol and GPA-1657), the ID50's based on'the drug concentration added in water decreased with increasing heptane-water partition coefficients . This suggests that the heptane solubility of the drug is also important in the binding . However, it should be mentioned that the drug must be reprotonated at the interface between heptane and water after its absorption to heptane since the drug in heptane is mostly a neutral base and the binding occurs at the interface . Based on the dissociation constant of the drugs (pKa ), the nitrogen atom of the drug at the interface would be mostly ionized under the experimental pH by contacting directly with water, but the rest of the drug molecules may still be in heptane like a neutral molecule . If the heptane solubility of this part of the molecule correlates with the whole molecule, it should be possible to conclude that the relative affinity is determined by the drug concentration in heptane rather than the concentration in water . In comparing an agonist with its corresponding antagonist, the substitution of N-methyl group in the agonist by N-allyl group converts it to an antagonist which results in an increased heptane partition coefficient and a decrease of the relative affinities (ID50 in heptane Table III) . Thus, it is clear that two opposing effects, the heptane partition and the affinity, determine ID50 of the drug in water . The fact that the affinity of the agonists (ID50 in heptane) for cerebroside sulfate was higher than their respective antagonists (Table III and IV) and that the agonist-cerebroside sulfate complex was more hydrophobic (11) indicates that the electrostatic bond between protonated nitrogen of the drug and the anionic sulfate group of cerebroside sulfate is extremely important in the binding of the narcotic drug and in the discrimination of agonist from antagonist . It is well known that there are two types of electrostatic bonds ; intimate ion pairs and solvent separated (dissociated) ion pairs (21) . Polyatomic organic cations, unlike uniatom inorganic cations, can still associate with cerebroside sulfate by hydrogen bonding and hydrophobic bonding even if the electrostatic bonds are partially dissociated . The extent of formation of intimate ion pairs for a given ionic concentration is greater, when the effective size of ions is smaller and the dielectric constants of the solvent is lower according to the following equation (22) : l + E e (r te} + r -) where, E = electrostatic bond between a protonated nitrogen of opiate and an anionic sulfate group of cerebroside sulfate .

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Model for Opiate Receptor

e - dielectric constant r+ and r - are the radius of the effective cation and anion, respectively . q + and q - are the charges of drug and receptor, respectively . TABLE V Comparison of the Drug Partition Coefficient With Their Disposition in the Central Nervous Systema

Dose (mg/kg)

Route

Peak Brain Concentration (ug/g)

Ratio b

PH/W c

Morphine

5 10

s .c . i .p .

0 .179 0 .183

3 .6 x 10 -2 1 .8 x 10-2

5 x 10 -4

Codeine

25 30

S.C. i .p .

8 .5 7 .9

3 .4 x 10 -1 2 .6 x 10 -1

2 .5 x 10 -2

2

s .c .

0 .142

7 .2 x 10 -2

1 .1 x 10-2

10 15

S .C . S .C .

17 .0 22 .0

150

s .c .

7 .0

Levorphanol Methadone Nalorphine

1 .7 1 .5 4 .1 x 10 -2

45 1 .5 x 10 -3

bThe.data were taken from S .J . Mule (20) and converted into

the ratio of peak brain concentration to the dose . cThe partition coefficients of the drugs between heptane and water . The effective size of ions which have bound water may be quite different from the dehydrated ion . Therefore, the hydration of an ion is an important factor in the intimate ion pair (dehydrated ion paid) forma tion . Antagonist can form more hydrated ion pairs with cerebroside sulfate than its agonist because it possesses (a) a larger N-allyl group, as compared to the N-CH - of the agonist (b) an unsaturated double bond, which can bind with wat r . These structural modifications tend to increase the effective size of the cationic drug and hence to form hydrated ion pairs . When a hydroxyl group is attached to the carbon atom near the position of the nitrogen such as in the case of naloxone, a pure antagonist, the degree of hydration is even more remarkable . Moreover, when the distances between ions are small, the effective dielectric constant (e) is drastically different from the value determined for the same medium in bulk . The medium does not separate the ions and it is influenced by the ions . Pressman et al . (23) have suggested that tie effective dielectric constants of water Ts given by the equation e=6(r + r - ) - 11 . Thus, the dielectric constant which decreased the forces between ions is largely dependent upon the size of ions . This provides a possible explanation for our conclusion that opiate agonists had higher affinity to CS than their corresponding antagonists .

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Model for Opiate Receptor

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In a solvent of low dielectric constant such as heptane and octanol, where the force of attraction between ions of opposite sign is large, there is an increased tendency for the formation of intimate ion pairs . On the other hand, in a solvent of high dielectric constant such as water, the formation of solvent separated (hydrated) ion pairs would be increased (11) . Further support of this finding is the work of Gray and Robinson (24), who prepared a number of salts of methadone, cyclazocine, and naloxone with various mono and poly basic organic acids in order to develop a long acting narcotic antagonist reparation . They found that, in general, salts of methadone (agonist~ were more insoluble than those of cyclazocine (partial agonist) which were more insoluble than those of naloxone, a pure antagonist . This is in agreement with our conclusion that an agonist interacts with cerebroside sulfate to form more intimate ion pairs (hydrophobic) while an antagonist yields more hydrated ion pairs (hydrophilic) . Thus, the electrostatic bond formed between the protonated nitrogen of the drug and the anionic sulfate group of cerebroside sulfate not only increases the affinity of the drug for cerebroside sulfate but also discriminates agonist from antagonist . The Van der Waals bonding and hydrogen bonding between narcotic and cerebroside sulfate only enhance the affinity of the drug . The same would be true when opiates interact with their receptors . Therefore, we propose that agonistic action is elicited through the formation of intimate ion pairs and antagonistic action through that of hydrated ion pairs . The ratio of intimate ion pairs to hydrated ion pairs determines the purity of an agonist . The analgetic actions of a partial agonist, a drug at low concentration behaves as an antagonist but at high concentration exhibits an agonistic action . This is probably due to a simultaneous increase in formation of the intimate ion pairs and decrease of hydrated ion pairs when the concentration of the drug is increased (22) . It is not surprising that most general anesthetics and alcohols exhibit analgetic activities like opiate agonists . Since the formation of dehydrated ion pairs increases with decreasing dielectric constant of solvent, general anesthetics like organic solvents would increase the formation of intimate ion pairs between endogenous opposing ions . Assuring that the resting state of the nerve membrane is more hydrophobic while the excited state is more hydrophilic, opiate agonists by interacting with their membrane receptors to form intimate (hydrophobic) ion pairs, could stabilize the resting state of the membrane resulting in the decrease of ion conductance . This is supported by the findings that opiates and general anesthetics stabilize the resting state of the membrane (25) and block the sodium conductance (26) . The term "efficacy" as postulated by Stephenson (27) may be due to the physicochemical properties of the drug-receptor complexes which are determined by the nature of electrostatic bond . If this is the case, the affinity and the efficacy of the drug should be interrelated . Acknowledgements The authors are grateful to Barbara Halperin for assistance in the preparation of this manuscript and to Lynne Rappaport for typing the manuscript . References 1.

H .H . LOH, T .M . CHO,and Y .C . WU, Fed . proc . 34, 815 (1975) .

2.

H .H . LOH, T .M . CHO, Y .C . WU, and E . LEONG WAY, Life Sci . 14, 2231 (1974) .

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Model for Opiate Receptor

3.

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