Proc. Nat. Acad. Sci. USA
`Vol. 70, No. 8, pp. 2243-2247, August 1973
`
`Properties of Opiate-Receptor Binding in Rat Brain
`(naloxone/opiate antagonist)
`
`CANDACE B. PERT AND SOLOMON H. SNYDER*
`Departments of Pharmacology and Experimental Therapeutics, and Psychiatry and the Behavioral Sciences, Johns Hopkins University
`School of Medicine, Baltimore, Maryland 21205
`Communicated by Julius Axelrod, May 2, 1973
`
`3HJNaloxone, a potent opiate antagonist,
`ABSTRACT
`binds stereospecifically to opiate-receptor sites in rat-
`brain tissue. The binding is time, temperature, and pH
`dependent and saturable with respect to [3Hinaloxone and
`tissue concentration. The ['Hinaloxone-receptor complex
`formation is bimolecular with a dissociation constant of
`20 nM. 15 Opiate agonists and antagonists compete for the
`same receptors, whose density is 30 pmol/g. Potencies of
`opiates and their antagonists in displacing [3H1naloxone
`binding parallel their pharmacological potencies.
`The elegant conceptualization of Goldstein et al. (1) that
`opiate-receptor binding should be stereospecific enabled us
`to demonstrate and quantify opiate-receptor binding (2).
`Specific-receptor binding of opiates and their antagonists
`closely parallels their pharmacological potency and is con-
`fined to nervous tissue. The present report describes the
`kinetics of specific opiate-receptor binding and the influences
`of temperature, pH, ionic concentrations, and several opiates
`and their antagonists.
`MATERIALS AND METHODS
`Male Sprague-Dawley rats, 7-12 weeks old, were killed by
`cervical dislocation and decapitated, and their brains were
`rapidly removed. After the cerebellum, which is devoid of re-
`ceptor activity (2), was excised, each brain was homogenized
`in 10 ml of 0.05 M Tris HCl buffer, pH 7.4 at 350, by .10
`strokes of a motor-driven ground-glass pestle, and the ho-
`mogenate was diluted to 110 volumes of tissue with cold Tris
`buffer.
`In the standard binding assay, 1.9-ml aliquots of this freshly
`prepared homogenate were incubated for 5 min in triplicate
`with either levorphanol or dextrorphan (0.1 gM). After cool-
`ing to 40, [3H]naloxone was added to a final concentration
`of 8 nM (65,000 cpm) and incubation at 350 in a final volume
`of 2 ml was resumed for 15 min. Samples were cooled to 40
`in an ice bath and then filtered under reduced pressure
`through Whatman glass-fiber circles (GF-B) which were
`chosen because of their ability to hold relatively large quan-
`tities of tissue while maintaining an adequate flow rate. Filters
`were washed twice with 8 ml of cold Tris buffer. The entire
`filtration cycle consumed only 20 sec for each sample. The
`filters were shaken with 1 ml of 10% sodium dodecyl sulfate
`in counting vials for 30 min. After the addition of 12 ml of
`PCS (Amersham-Searle; Phase Combining System), radio-
`activity was determined by liquid-scintillation spectrometry,
`
`* To whom reprint requests should be sent at the Department of
`Pharmacology.
`
`at a counting efficiency of 28%. Protein was measured by
`the method of Lowry et al. (3), with bovine-serum albumin
`as a standard.
`(-)-Naloxone was labeled by tritium exchange at the New
`England Nuclear Corp. 50 mg of naloxone were dissolved
`in 0.3 ml of trifluoroacetic acid with 50 mg of 5% Rh/A1203
`to which were added 25 Ci of 3H20, and the mixture was in-
`cubated 18 hr at 800. In our laboratory, a 70-mCi portion of
`[3H]naloxone was evaporated twice to dryness, and purified by
`thin-layer chromatography on Silica gel G plates (MN-
`Kieselgel G/uv 254) of 0.25-mm thickness (n-butanol-glacial
`acetic acid-H20; 4:1:2). When the purified [3H]naloxone was
`chromatographed in three additional solvent systems, the
`resulting single peak of radioactivity coincided with authentic
`naloxone, which was chromatographed beside it. The specific
`activity of [3H]naloxone was 6.1 Ci/mmol of standard, as
`determined by comparison with the ultraviolet absorption
`of standard solutions of naloxone at 260 nm. Based upon this
`specific activity determination and a counting efficiency of
`28%, 378 cpm is equivalent to 0.1 pmol of naloxone.
`Drugs were generously donated by the following companies:
`Endo (naloxone, oxycodone); Roche [levorphanol, dextror-
`phan, levallorphan, (+)-3-hydroxy-N-allyl-morphinan]; Lilly
`[(-)- and (+)-methadone, (+)-propoxyphene]; Winthrop
`(pentazocine cyclazocine, meperidine); Reckitt and Colman,
`American Cyanamid Agricultural Division and Dr. William
`Martin, Lexington, Kentucky (etorphine); Knoll (hydro-
`morphone); Ciba-Geigy (etonitazene).
`
`RESULTS
`The time-course of [3H naloxone binding to whole rat-brain
`homogenates was studied at 350 in the presence of 0.1 uM
`levorphanol or 0.1 uM dextrorphan (Fig. 1). For most opiates,
`analgesic activity is highly stereospecific with almost all
`activity residing in isomers with a configuration analogous
`to that of D-(-)-morphine. Levorphanol is a potent opiate
`with the D-configuration, while dextrophan is its enantiomer
`and is essentially devoid of analgesic activity.
`Binding of [3H]naloxone occurred rapidly, was linear for
`no more than 1 min, and plateaued between 10 and 30 min
`so that equilibrium was reached by 15 min (Fig. 1). After 30
`min, [3H]naloxone binding gradually decreased by about
`25% and then remained constant up to 70 min. 0.1 ,uM dex-
`trorphan did not reduce [3H]naloxone binding at any time.
`By contrast, 0.1 uM levorphanol greatly reduced the binding
`of [3H]naloxone at all time intervals examined. Increasing
`
`2243
`
`Page 1 of 5
`
`YEDA EXHIBIT NO. 2056
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`2244
`
`Physiology:
`
`Pert and Snyder
`
`Proc. Nat. Acad. Sci. USA 70 (1973)
`
`binding" will refer to the binding of [3H]naloxone in the
`presence of 0.1 MAM dextrorphan minus its binding in the
`presence of 0.1 ,M levorphanol.
`Specific [3Hjnaloxone binding was linear with brain-tissue
`protein over the range 0.2-2.0 mg of protein (Fig. 2). Specific
`binding displayed a sharp pH optimum at 7.4 (Fig. 2), with
`a steep decline at more acid pH values and a gradual decline
`at more alkaline pH values. In some experiments, the fall in
`binding at slightly acid pH was less sharp, presumably be-
`cause of variable tissue aggregation.
`The thermal stability of the opiate receptor was examined
`by prior incubation of the tissue for 10 min at various tem-'
`peratures and then conducting the standard binding assay
`at 35°. Binding was not affected by prior incubation at tem-
`peratures from 20 to 400, but was reduced by heating at
`higher temperatures. Heating for 10 min at temperatures in
`excess of 500 reduced specific naloxone binding by 90% or
`more (Fig. 2).
`Specific binding was temperature dependent with maximal
`binding at 35° and a Qio value of 1.5 when measured between
`25 and 350 and 1.3 when measured between 150 and 25° after
`15 min of incubation. At 4° binding was reduced to 25%
`of values at 35°.
`Specific [3H ]naloxone binding could be altered by various
`ionic manipulations (Fig. 3). The divalent cations calcium
`and magnesium lowered binding by about 40% at concen-
`trations of 3 mM and further lowered binding to 30% of con-
`trol values at 10 mM. In the absence of added sodium and
`potassium, specific binding was the same as in. the presence
`of physiological concentrations of these ions. At concentra-
`tions in excess of 500 mM both lithium and sodium produced
`a gradual decrease of binding. Potassium, which at physio-
`logical concentrations of 5 mM did not affect binding, re-
`duced specific binding 20% at 10 mM and 50% at 30 mM.
`Sodium, lithium, magnesium, and calcium did not alter the
`nonspecific binding of [3HInaloxone in the presence of non-
`radioactive levorphanol.
`Naloxone binding was demonstrated to be saturable by
`two techniques (Fig. 4). In one approach, we measured the
`binding of increasing amounts of [3H]naloxone, while in other
`
`00
`
`800
`
`0 C
`
`z°
`
`z0
`
`0
`
`zF
`0l
`< 200
`0)
`a,
`
`_ Ovvu
`
`EC
`
`L
`
`1600
`
`800
`
`0 c
`
`owzo
`
`0 zI
`
`30
`70
`50
`10
`INCUBATION TIME (MINUTES)
`Time-course of stereospecific [3Hlnaloxone binding to
`FIG. 1.
`rat-brain homogenate. After 5 min preincubation in the presence
`of 0.1 M levorphanol (c), 0.1MuM dextrorphan (0), or no drug (0),
`homogenates were incubated at 350 in the standard binding assay
`from 1 to 75 min with [3H]naloxone. After samples were cooled to
`40 they were filtered rapidly and the radioactive content of the
`tissue-laden filter was determined by liquid scintillation spectro-
`metry. In some experiments binding declined continuously be-
`tween 30 and 70 min.
`
`the levorphanol concentration to 0.1 mM did not further
`diminish naloxone binding.
`In all subsequent experiments, incubations with 0.1 ,M
`concentrations each of levorphanol and dextrorphan were
`included and the radioactivity in the presence of levorphanol
`was subtracted as a "blank" value representing nonspecific
`binding, while the failure of dextrorphan to reduce binding
`ensured that the effect of levophanol was related to its affinity
`for the specific opiate receptor. In a typical experiment in
`which 65,000 cpm of [3H]naloxone (8 nM) were incubated
`in 2 ml with brain homogenate (18 mg wet weight equal to
`1.8 mg of protein) 2000 cpm and 800 cpm were bound, re-
`spectively, in the absence and presence of 0.1 MM levorphanol.
`In the absence of tissue about 300 cpm were bound to the
`filters. Accordingly, only about 500 cpm were bound non-
`specifically to brain tissue in the presence of levorphanol so
`that the ratio of specific to nonspecific binding in typical
`experiments would be about 3. Henceforth, "specific receptor
`
`0
`
`o
`
`>]a0~~~~~~~~~~~~~~
`0100
`
`0.0
`
`w
`
`ED~~~~~~~~~~~~
`
`06
`5
`45
`6
`3
`26
`20
`_j ItIs_
`<<
`
`0~~~~~~~~~~~~~~~~~~~~~~~~u
`
`pH
`
`3
`2
`3
`2
`BRAIN HOMOGENATE PROTEIN (mg)
`Thermal sensitivity, pH dependence, and tissue linearity of stereospecific [3H]naloxone binding to rat-brain homogenate.
`FIG. 2.
`Middle: Rat brains without cerebellum were divided longitudinally and each half was homogenized in 5 ml of 0.05 M citrate-phosphate
`buffer, pH 4, 5, or 5.4; 0.05 M sodium phosphate buffer, pH 6, 6.8, or 7.4; or 0.05 M glycine NaOH buffer, pH 8, 9, or 10. After dilution
`to 75 ml with the appropriate buffers, 1.9-ml aliquots of each homogenate were incubated with 0. 1 MM levorphanol or 0.1MM dextrorphan
`followed by ['H]naloxone in the standard binding assay. Left: Aliquots (1.9 ml) of rat-brain homogenate were maintained for 10 min at
`various temperatures before incubation with 0.1MuM levorphanol or 0.1MM dextrorphan followed by [3H]naloxone in the standard-binding
`assay. Stereospecific [3H]naloxone binding is expressed as percent of the control samples, which were maintained at 40 for 10 min before
`assay. Right: One rat brain without cerebellum was homogenized in 10 ml of Tris buffer and diluted to a total volume of 120 ml with the
`same buffer. Various dilutions of this homogenate were incubated with 0.1 M1 levorphanol or 0.1 MM dextrorphan followed by [3H]-
`naloxone in the standard-binding assay. All experiments were done twice.
`
`Page 2 of 5
`
`YEDA EXHIBIT NO. 2056
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`o~~~~~~~~N
`
`4
`
`16
`
`12
`08
`
`0
`
`30
`20
`NALOXONE (M
`
`10)
`
`40
`
`50
`
`a
`(3H)NALOXONE (Ml -
`Saturation of stereospecific [3H]naloxone binding to
`FIG. 4.
`rat-brain homogenate (left) and the ability of nonradioactive
`naloxone to diminish stereospecific [3H]naloxone binding (right).
`Left: Standard aliquots of homogenate were incubated for 15
`min at 350 with increasing concentrations of [3H]naloxone in the
`presence of 0.1 ,uM levorphanol or 0.1 ,uM dextrorphan. Right:
`Increasing concentrations of naloxone (plotted on the abscissa)
`were used to decrease the stereospecific binding of 8 nM [3H]-
`naloxone in the standard assay. Nonspecific binding in the pres-
`ence of 0.1 MuM levorphanol was subtracted from all samples.
`
`of 0.3 nM, about 1/20th of the corresponding value for mor-
`phine. Etonitazene, an opiate whose potency in vivo is similar
`to that of etorphine (4), was almost as potent as etorphine
`in displacing [3H ]naloxone binding.
`Levorphanol, a potent opiate, had 4000-times the affinity
`of dextrorphan, its analgesically inactive enantiomer. Sim-
`ilarly, levallorphan, the opiate antagonist derived from levor-
`phanol, was 7000-times as potent as its enantiomer. (-)-
`Methadone, the more analgesically active form of methadone,
`was only about 10-times as potent as (+)-methadone, per-
`haps because it has greater conformational mobility than
`levorphanol (5).
`The opiates, morphine, and levorphanol, and their cor-
`responding antagonists, nalorphine and levallorphan, had
`similar affinities for the opiate-receptor binding sites, al-
`
`\
`
`0
`
`a:
`
`0
`w
`W
`
`Rate of stereospecific binding of ['H]naloxone at 250
`FIG. 5.
`(left) and semilog plot of dissociation of bound ['Hinaloxone
`from rat-brain homogenate at 50, 150, and 250
`(right). Left:
`Standard homogenates were incubated for intervals varying
`from 15 sec to 60 min with ['H]naloxone in the presence of 0.1
`MuM dextrorphan or 0.1 ,uM levorphanol at 250 before cooling
`to 4°. Samples were filtered immediately after cooling. Right:
`Brain homogenates were incubated for 15 min at 350 with ['H]-
`naloxone in the presence of 0.1 ,uM levorphanol or 0.1 ,uM dex-
`trorphan in the standard binding assay. After cooling in an ice
`bath, nonradioactive naloxone (10,uM final concentration) was
`rapidly added and samples were filtered immediately (time 0) or
`allowed to incubate for various times at 50, 150, or 250 before
`rapid cooling and filtration.
`
`t EDso is the concentration of drug that reduces specific [3H]-
`naloxone binding by 50%.
`
`Proc. Nat. Acad. Sci. USA 70 (1973)
`
`Opiate Receptor
`
`2245
`
`E 0
`
`oc
`
`z--
`
`Z q
`
`LW 200
`
`w
`
`0
`
`Ka
`v12200
`
`low,
`
`)14M
`
`1000
`!Soo
`i200.
`
`1
`
`100
`
`wz
`oc
`
`-J
`
`0
`
`o za:W co
`
`W
`Cn
`
`400
`
`1000
`
`3
`2200
`1600
`1
`ION CONCENTRATION (mM)
`
`5
`
`7
`
`9
`
`Effect of increasing ionic concentrations on stereo-
`FIG. 3.
`specific ['Hinaloxone binding to rat-brain homogenate. Various
`amounts of ions were included in the standard-binding assay.
`Data are presented from a typical experiment which was done
`twice.
`experiments we studied the extent to which increasing amounts
`of nonradioactive naloxone decreased specific ['H]naloxone
`binding. Half saturation of tissue binding occurred at about
`16 nM with 1.8 mg of brain-homogenate protein.
`To determine the rate constant of opiate-receptor associa-
`tion, we examined the time-course of stereospecific
`[3H]-
`naloxone binding in detail (Fig. 5). Specific binding is a time-
`dependent process, whereas nonspecific binding in the pres-
`ence of nonradioactive levorphanol was maximal even at
`incubation periods of 1 min (Fig. 1). From the data on the
`rate of binding, it was possible to calculate the bimolecular
`rate constant of opiate-receptor association, Ki, which is
`equal to [2.303/t(a - b)] log [b(a - x)/a(b - x)], where a
`= [['H]naloxone], b = [receptors] and x = amount of triti-
`ated naloxone reacting in time (t). The concentration of re-
`ceptors in the standard incubation system was calculated
`to be 0.3 nM based upon the amount of ['H]naloxone spe-
`cifically bound at saturation. The rate of association was 1.15
`+ 0.34 X 106 M-l sec-' at 250.
`The rate of dissociation was examined at four temperatures
`(Fig. 5). At 350, ['Hjnaloxone specifically bound to the re-
`ceptor had completely dissociated by 30 sec. At 250, 150,
`and 50, dissociation appeared to be strictly a first-order pro-
`(3~~~~~~~~~~~~~~~~~~U
`cess. The half lives for dissociation at 250, 150, and 50, re-
`90
`a
`80
`z 1600|
`spectively, were 43 4- 7 sec, 113 ±- 16 sec, and 4.7 i 0.2 min.
`~~~~~~~~~70-
`M
`w 1400
`60
`z
`0
`The rate constant at 250 for [3H]naloxone dissociation from
`0 1200
`z 50
`the receptor (K2) was calculated to be 1.61 4 .24 X 10-2
`040J1000
`.
`4
`sec -'. The calculated value of K2/K, based upon these direct
`0 30-
`_z800
`~~~C_
`~~~~0
`kinetic data was 14.0 4- 4.1 nM, which corresponds to the
`600
`_20
`U.400
`experimentally determined concentration of 16 nM naloxone
`~200C)
`required for half-maximal saturation of receptor binding
`(Fig. 4).
`_lo-__
`10
`20
`30
`6°
`2
`1
`40
`50
`a.
`We compared the affinity of various drugs for opiate-re-
`MINUTES
`INCUBATION TIME AT 25° (MINUTES)
`~~~~~~~~~~~~~~~~~O
`cn
`ceptor binding by incubating several concentrations of non-
`radioactive drugs with tissue homogenate before the addi-
`tion of [3H]naloxone. The concentration of drug that lowered
`specific binding of 8 nM [3H]naloxone by 50% was estimated
`(Table 1) by log-probit analysis (Fig. 6). For the 15 opiate-
`related drugs examined, the slopes of lines describing percent
`inhibition against drug concentration were all parallel. The
`pharmacologic potencies of opiates and opiate antagonists
`were related proportionally to their ability to compete with
`[3H]naloxone for binding to the opiate receptor. Etorphine,
`one of the most potent known analgesics, displayed the great-
`est affinity of any of the drugs examined with an ED5ot value
`
`Page 3 of 5
`
`YEDA EXHIBIT NO. 2056
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`2246
`
`Physiology:
`
`Pert and Snyder
`
`Relative potencies of drugs in reducing stereospecific
`TABLE 1.
`[3H]naloxone binding to rat-brain homogenate
`
`Drug
`(-)-Etorphine
`(-).Etonitazene
`Levallorphan
`Levorphanol
`(-)-Nalorphine
`(-)-Morphine
`(- )-Cyclazocine
`(-)-Naloxone
`(-)-Hydromorphone
`(-)-Methadone
`( i)-Pentazocine
`(+ )-Methadone
`Meperidine
`( i)-Propoxyphene
`(+)-3-Hydroxy-N-
`allyl-morphinan
`Dextrorphan
`
`(-)-Codeine
`(-)-Oxycodone
`
`EDNo(nM)
`0.3
`0.5
`1
`2
`3
`7
`10
`10
`20
`30
`50
`300
`1,000
`1, 000
`7,000
`
`8,000
`
`20,000
`30,000
`
`No effect at 0.1 mM
`Phenobarbital
`Norepinephrine
`Atropine
`Pilocaprine
`Arecholine
`Colchicine
`-y-Aminobutyric acid
`Bicuculline
`Serotonin
`Carbamylcholine
`Neostigmine
`Hemicholinium
`Histamine
`Glycine
`Glutamic acid
`
`A9-Tetrahydrocanna-
`binol
`Acetylsalicylic acid
`Caffeine
`
`Values represent means from 3 log-probit determinations each
`using five concentrations of drug.
`
`though in both cases the antagonist had twice the affinity
`of the agonist.
`Codeine, which is analgesically about 1/10 as potent as
`morphine, displayed less than 1/3000 the receptor affinity
`of morphine. Because codeine is O-demethylated by liver
`
`Proc. Nat. Acad. Sci. USA 70 (1973)
`
`microsomal enzymes to morphine, it may exert analgesic
`activity only after metabolism to morphine (6-8).
`Propoxyphene, which is a weak analgesic (9), had only 1/200
`the affinity of morphine for receptor sites. (=I)-Pentazocine,
`estimated to be from 1/6 as. potent to equipotent to morphine
`in humans (10), had 1/8 the affinity of morphine for receptor
`sites. A wide variety of drugs that are not opiates had no
`significant affinity for the opiate-receptor binding sites (Ta-
`ble 1).
`
`DISCUSSION
`The pharmacological activity of opiates and their antagonists
`parallels their affinity for [3H]naloxone-binding sites. Ap-
`parent discrepancies between potency in vivo and receptor
`affinity in vitro appear related to penetration of the blood-
`brain barrier or drug metabolism. Though etorphine exceeds
`morphine 1,000- to 10,000-times in analgesic potency but only
`20-times in receptor potency, it enters the brain 200-times as
`readily as morphine, rationalizing a 4000-fold difference in
`potency in vivo (11). The moderate analgesic activity of co-
`deine despite its negligible affinity for the opiate receptor
`may be explained by its conversion in vivo to morphine.
`Our data support suggestions that opiates and their antag-
`onists compete for the same receptor sites (12, 13). Thekinetics
`of receptor binding indicate a strictly bimolecular process.
`Log-probit profiles for receptor affinity of a series of 15 opiates
`and their antagonists are parallel. [3H]Levorphanol, [3H]-
`levallorphan, [3H]oxymorphone, [3H]nalorphine, and [3H]di-
`hydromorphine bind stereospecifically to brain homogenates
`(manuscript in preparation). Because agonists and antago-
`nists have similar affinities, their pharmacologic differences
`appear related to differences in "intrinsic activity" (14).
`
`95
`
`80
`50 ED
`
`Iz
`
`15 0
`
`X10-'°
`I'
`
`XIO-9
`9
`
`IxIO-'
`
`IxO-'loIx10-
`DRUG CONCENTRATION (M)
`
`IXI0-5
`
`10-4
`
`IXI
`
`z0
`
`5O~
`
`z50z
`
`' IX-10
`
`IXIOC
`DRUG OONCENTRATION (M)
`Effects of opiates and opiate antagonists on stereospecific [3H]naloxone binding. Five concentrations of each opiate were pre-
`FIG. 6.
`incubated for 5 min with standard aliquots of rat-brain homogenate, and the incubation was continued for 15 min at 350 in the presence of
`[3H]naloxone (8 nM). Percent inhibition of control stereospecific [3H]naloxone binding was computed for each concentration of opiate,
`after subtracton of nonspecific binding from all experimental points, and plotted as a function of drug concentration on log-probit paper.
`Above: Three pairs of enantiomers are presented. Below: 11 Additional opiates and opiate antagonists that exhibit a wide range of in-
`hibitory potencies have been plotted.
`
`1X10?
`
`15
`
`Page 4 of 5
`
`YEDA EXHIBIT NO. 2056
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`Proc. Nat. Acad. Sci. USA 70 (1973)
`
`In our previous study (2), and recent experiments (un-
`published observations) we demonstrated the presence of the
`opiate receptor in mouse, bovine, guinea pig, cat, chick,
`monkey, toad, and fish brain as well as rat brain, indicating
`a broad phylogenetic distribution.
`Physiological concentrations of calcium significantly in-
`hibit and the chelating agents EDTA, EGTA, and citrate
`enhance receptor binding (manuscript in preparation), sug-
`gesting that endogenous calcium plays a role in the action
`of opiates (15). The opiate receptor is extremely sensitive
`to digestion by trypsin and chymotrypsin as well as detergents
`such as Triton X-100, sodium dodecylsulfate, and deoxy-
`cholate (manuscript in preparation).
`The number of opiate receptors increases in the brains of
`mice as early as 2 hr after implantation of a morphine pellet,
`and declines when pellets are removed in parallel with the fall
`in physical dependence (manuscript in preparation).
`Other workers have studied the binding of opiates to
`brain tissue but failed to show specificity and pharmacologic
`relevance (16). Goldstein et al. (1) reported stereospecificity
`in 2% of [3H]levorphanol binding to mouse-brain homog-
`enates. However, unlike the saturable opiate receptor de-
`scribed here, the stereospecificity of binding reported by
`Goldstein et al. (1) increased with increasing concentration
`and accordingly seems to represent a different binding site.
`It can be calculated from the direct measurement of [3H]-
`naloxone bound at saturation that 1 g of rat brain without
`cerebellum has sufficient receptors to bind 30 pmol of naloxone.
`Assuming that each naloxone molecule interacts with one
`receptor unit, we calculate the number of receptor units in
`one rat brain (1.6 g) to be 2 X 1013.
`We gratefully acknowledge the excellent technical assistance
`of Adele Snowman. Supported by USPHS "Johns Hopkins Drug
`
`Opiate Receptor
`
`2247
`
`Abuse Research Center" Grant DA-00266, and by USPHS
`Grants MH-18501 and NS-07275, S.H.S. is a recipient of a Re-
`search Scientist Development Award, MH-33128. C.B.P. is a
`predoctoral student; fellowship from the Scottish Rite Founda-
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`YEDA EXHIBIT NO. 2056
`MYLAN PHARM. v YEDA
`IPR2015-00644