Neuropsychopharmacology (2003) 28, 1973–1982
`& 2003 Nature Publishing Group All rights reserved 0893-133X/03 $25.00
`
`www.neuropsychopharmacology.org
`
`Vivitrexs, an Injectable, Extended-Release Formulation of
`Naltrexone, Provides Pharmacokinetic and Pharmacodynamic
`Evidence of Efficacy for 1 Month in Rats
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`Raymond T Bartus*,1, Dwaine F Emerich1, Joyce Hotz1, Marc Blaustein1, Reginald L Dean1, Brigido
`Perdomo1 and Anthony S Basile1
`1Alkermes, Inc., Cambridge, MA, USA
`
`While oral naltrexone is effective in treating alcohol and opiate dependencies, poor patient adherence and widely fluctuating plasma
`levels limit its efficacy. To overcome these problems, an extended-release formulation of naltrexone (Vivitrexs) was developed by
`encapsulating naltrexone into injectable, biodegradable polymer microspheres. Pharmacokinetic studies in rats demonstrated that this
`formulation produced stable, pharmacologically relevant plasma levels of naltrexone for approximately 1 month following either
`subcutaneous or intramuscular injections. While rats receiving placebo microspheres demonstrated a pronounced analgesic response to
`morphine in the hot-plate test, morphine analgesia was completely blocked in rats treated with extended-release naltrexone. This
`antagonism began on day 1 following administration and lasted for 28 days. Rats reinjected with extended-release naltrexone 34 days
`after the initial dose and tested for another 35 days showed consistent suppression of morphine analgesia for an additional 28 days. m-
`Opioid receptor density, as measured by [3H]DAMGO autoradiography,
`increased up to two-fold following a single injection of
`extended-release naltrexone. Saturation binding assays using [3H]DAMGO showed changes in the midbrain and striatum at 1 week after
`extended-release naltrexone administration, and after 1 month in the neocortex. These receptor increases persisted for 2–4 weeks after
`dissipation of the morphine antagonist actions of naltrexone. These data suggest that therapeutically relevant plasma levels of naltrexone
`can be maintained using monthly injections of an extended-release microsphere formulation, and that changes in m-opioid receptor
`density do not impact its efficacy in suppressing morphine-induced analgesia in the rat. Clinical trials of extended release naltrexone for
`treating alcohol and opiate dependency are currently ongoing.
`Neuropsychopharmacology (2003) 28, 1973–1982, advance online publication, 20 August 2003; doi:10.1038/sj.npp.1300274
`Keywords: naltrexone; analgesia; extended-release; Vivitrexs; opiate antagonist; alcoholism
`
`INTRODUCTION
`
`Alcohol and opiate dependence represent serious medical
`problems in the United States and throughout the world. In
`the US alone, it is estimated that 10–14 million people are
`alcohol-dependent, while 1 million people suffer from
`opiate addiction (Grant, 1997). Evidence implicating the
`functional involvement of the endogenous opioid system in
`mediating many of
`the reinforcing aspects of drug
`consumption (Spanagel et al, 1992; Herz, 1997) has led to
`the use of opioid receptor antagonists as a pharmacothe-
`rapeutic intervention for treating drug dependency syn-
`dromes (Martin et al, 1973; Litten and Allen, 1998; Garbutt
`et al, 1999). The opiate antagonist naltrexone (Resnick et al,
`
`Inc., 9381 Judicial
`*Correspondence: RT Bartus, now at Ceregene,
`Drive, San Diego, CA 92121, USA, Tel: +1 858 458 8834, Fax: +1
`858 458 8801, E-mail: rtbartus@ceregene.com
`Received 30 January 2003; accepted 13 June 2003
`Online publication: 20 June 2003 at http://www.acnp.org/citations/
`Npp06200303043/default.pdf
`
`1974) is effective and approved by the FDA for treating
`opiate and alcohol dependence (Food and Drug Adminis-
`tration NDA 18-932/S-010, 1994). Despite its utility, the
`efficacy of oral naltrexone in treating drug dependencies is
`limited by at least two deficiencies. The first involves poor
`adherence to the prescribed daily dosing schedule (Volpi-
`celli et al, 1997). Although adherence is important
`in
`achieving efficacy with all drugs, it is particularly important
`in treating alcohol and opiate dependence.
`In these
`syndromes,
`the
`compulsion to self-administer drugs
`directly conflicts with the need to remain abstinent. This
`daily conflict further exacerbates problems of poor adher-
`ence, contributing to the ‘spiral towards relapse’ common to
`both maladies.
`Indeed, a recent
`study of alcoholics
`(Volpicelli et al, 1997) found that patients who were highly
`adherent benefited significantly from naltrexone, while
`nonadherent patients responded no differently than those
`receiving placebo. Moreover, it is not uncommon for less
`than 30% of the opioid-dependent patients to continue with
`antagonist therapy for more than 6 months (Greenstein et al,
`1984; Capone et al, 1986; D’Ippoliti et al, 1998). These data
`
`ALKERMES EXHIBIT 2010
`Amneal Pharmaceuticals LLC v. Alkermes Pharma Ireland Limited
`IPR2018-00943
`
`Page 1 of 10
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`

`1974
`
`Vivitrexs pharmacokinetics and pharmacodynamics
`RT Bartus et al
`
`argue that the efficacy of naltrexone might be significantly
`enhanced if adherence could be improved. A second
`deficiency with oral naltrexone involves the widely fluctuat-
`ing plasma levels that occur with daily oral dosing (Verebey,
`1980). These daily fluctuations may cause side effects during
`peak plasma levels (Croop et al, 1997; King et al, 1997),
`while compromising efficacy during the nadir in plasma
`concentration (Verebey, 1980).
`One approach for improving upon the effectiveness of
`naltrexone would be to develop an injectable formulation
`that would maintain plasma levels within the therapeutic
`range for several weeks, but below levels that induce serious
`side effects. Polymers such as poly-lactide co-glycolide
`(PLG, (Shive and Anderson, 1997)) can be formulated into
`small-diameter (o100 mm),
`injectable microspheres that
`incorporate active moieties to provide extended release for
`several weeks (Lewis, 1990). Such an extended-release
`formulation of naltrexone might provide several advantages
`over oral naltrexone, including a significant reduction in
`daily high plasma peaks and a decrease in the gastro-
`intestinal exposure and first-pass hepatic metabolism
`associated with oral delivery (Kranzler et al, 1998). These
`improvements could reduce the incidence of adverse effects
`associated with oral naltrexone administration. Similarly,
`the ability to maintain therapeutically relevant plasma levels
`of naltrexone continuously should provide for a more
`uniform occupation of opioid receptors, and therefore, a
`more consistent pharmacodynamic response throughout
`the course of
`treatment. Finally, adherence would be
`assured for an entire month after each injection, providing
`yet another important advantage.
`Owing to these potential advantages, we formulated
`naltrexone into PLG-microspheres using Alkermes’ pro-
`prietary Medisorbs technology to provide a product
`intended to deliver therapeutic levels of naltrexone for a
`full month following each injection. The following experi-
`ments describe preclinical pharmacokinetic and pharmaco-
`dynamic results from such an extended-release microsphere
`formulation of naltrexone (ie Vivitrexs), demonstrating
`that it provides plasma concentrations of naltrexone that
`are consistently elevated above therapeutic levels for
`approximately a 1-month period following each injection.
`Moreover, the plasma levels of naltrexone show a close,
`temporal correlation with a sustained pharmacodynamic
`response (morphine-induced analgesia as measured in the
`hot-plate test) that reflects an effective opioid receptor
`blockade.
`
`MATERIALS AND METHODS
`
`Subjects
`Male Sprague–Dawley rats (450 7 50 g; Taconic Farms,
`Germantown, NY) were used in all studies. Rats were
`pair-housed in polypropylene cages with free access to food
`and water. The vivarium was maintained on a 12 h
`light : dark cycle with a room temperature of 22 7 11C and
`relative humidity level of 50 7 5%. All studies were
`approved by Alkermes Institutional Animal Care and Use
`Committee and were conducted in adherence with the NIH
`‘Guide for the Care and Use of Laboratory Animals, 1996’.
`
`Neuropsychopharmacology
`
`Drug Treatments and Experimental Design
`Naltrexone-containing microspheres (Vivitrexs) were fabri-
`cated from the PLG polymer using a proprietary process
`(Lewis, 1990) to provide loading densities of approximately
`35% (w/w) naltrexone base. Placebo (nonloaded) micro-
`spheres were prepared in an identical manner, except that
`naltrexone was omitted.
`Microspheres were suspended in 1 ml of an aqueous
`diluent (0.9% saline, 0.1% Tween-20 and 3.0% low-viscosity
`carboxymethylcellulose) and injected using a 22 G needle to
`provide a total of 50 mg/kg naltrexone, or a comparable
`mass of placebo microspheres.
`In the first series of
`experiments, animals received either a subcutaneous (s.c.)
`or intramuscular (i.m.) injection of placebo or naltrexone-
`loaded microspheres. Animals received an intraperitoneal
`(i.p.) injection of morphine (1 mg/kg) or saline 1, 3, 7, 14,
`21, 28, and 35 days after microsphere administration and
`were retested on the hot plate 30 min after each morphine
`injection.
`Rats receiving i.m. injections were also used in studies
`further exploring the relationships between extended-
`release naltrexone, morphine antagonism, and changes in
`brain m-opioid receptor binding and immunoreactivity. Half
`of the animals receiving extended-release naltrexone i.m.
`were killed 36 days after injection, a time when the
`behavioral effects of naltrexone were diminished. The
`placebo and remaining extended-release naltrexone-treated
`animals received a second, identical microsphere injection
`34 days after the first. These animals were retested on the
`hot plate 1, 7, 14, 21, 28, 30, 32, 34, and 36 days later and
`were sacrificed on day 37, a time when the pharmacody-
`namic effects of naltrexone had completely disappeared.
`Injections of extended-release naltrexone or placebo were
`well tolerated by the rats,
`independent of the route or
`number of injections. This was evidenced by the absence of
`local site reactions (redness, swelling, exudation, or skin
`scratching) upon clinical examination, both in vivo and ex
`vivo. Body weights of rats receiving extended release
`naltrexone and placebo microspheres were measured
`weekly for 35 days. The body weight of extended-release
`naltrexone-treated rats was 2.8 7 0.27% lower than placebo-
`treated rats over this period (Po0.01, two-way ANOVA).
`
`Quantitation of Plasma Levels of Naltrexone
`
`Blood samples were collected from all animals immediately
`after each behavioral test (see below). Animals were briefly
`anesthetized with 1–2% isoflurane and blood samples
`(approximately 500 ml of whole blood) were collected via a
`lateral tail vein into tubes containing EDTA. The tubes were
`centrifuged for 10 min at 1000 g to separate plasma, which
`was subsequently stored at 701C until levels of naltrexone
`were determined by LC-MS (Naidong et al, 2002). The lower
`limit of quantitation (LOQ) for these studies was 1.0 ng/ml,
`and the coefficient of variation for the assay was o4.4%.
`
`Hot-Plate Testing
`
`Morphine-induced (1 mg/kg) analgesia was used to deter-
`mine the ability of extended-release naltrexone to block
`opioid receptors in the central nervous system. Analgesia
`
`Page 2 of 10
`
`

`

`was monitored using a commercially available hot-plate
`apparatus (Columbus Instruments, Columbus, OH). Rats
`were
`individually placed on the hot plate
`(surface
`temperature¼ 481C) and the latency (60 s maximum) to
`lick either hind paw was recorded. Animals received two
`baseline trials on the hot-plate test and were then randomly
`assigned to treatment groups. The effect of placebo or
`extended-release naltrexone formulations on morphine-
`induced analgesia was assessed on the indicated days after
`injection by administering morphine, starting at 1000 h.
`Each rat was tested on the hot plate 20 min after morphine
`injection, and then returned to its cage. The testing order
`was randomized over the course of the investigation.
`
`l-Opioid Receptor Changes Following Administration
`of Extended-Release Naltrexone to Rats
`Increases in m-opioid receptor density are commonly
`observed in response to antagonist administration (Lahti
`and Collins, 1978; Zukin et al, 1982). Therefore, the status of
`m-opioid receptor density following the administration of an
`extended-release naltrexone preparation was investigated as
`a biochemical measure of pharmacodynamic efficacy. The
`time course of the changes in m-opioid receptor density and
`expression following the administration of extended-release
`naltrexone microspheres was investigated using two differ-
`ent radioligand binding assays and immunohistochemical
`techniques. For the saturation radioligand binding assays,
`male Sprague–Dawley rats were injected once i.m. with
`79.2 mg of microspheres (placebo or naltrexone, approxi-
`mately 50 mg/kg naltrexone) suspended in 0.75 ml of
`diluent. The rats were then killed 3, 5, 7, 28, 30, 32, 36, or
`40 days after naltrexone, and 3, 7, 38, or 40 days after
`placebo administration. The brains were rapidly removed,
`placed in isotonic sucrose (0–41C), the cortex, midbrain
`(from approximately bregma 5 to 10 mm), and striatum
`dissected free on ice, rapidly frozen on dry ice, and stored at
`801C until use. For autoradiographic and immunohisto-
`chemical studies, rats were injected twice with extended-
`release naltrexone (see above).
`
`m-Opioid receptor binding.
`[3H]DAMGO (D-ala2, N-
`activity¼
`methyl-phe4,
`glycol5)
`enkephalin;
`specific
`55.0 Ci/mmol; Amersham Pharmacia Biotech, Arlington
`Heights, IL) binding to m-opioid receptors in the midbrain,
`striatum, and cortex was performed using a modification of
`a previously described technique (Goldstein and Naidu,
`1989). At
`the time of
`the assay,
`these regions were
`homogenized using a probe sonicator in 10 vol of 50 mM
`Tris-HCl, 1 mM EDTA, and 280 mM sucrose buffer, pH 7.4,
`then centrifuged at 20 000 g for 20 min. The pellet was
`retained and resuspended in 50 mM Tris-HCl pH 7.4 buffer
`alone and then recentrifuged. The latter step was repeated a
`total of four times.
`The equilibrium binding constants (Kd and Bmax) for
`[3H]DAMGO binding to m receptors in brain homogenates
`were determined using saturation binding assays consisting
`of 0.5–10 nM concentrations of [3H]DAMGO, tissue (0.08–
`0.7 mg protein), 10 mM naltrexone (for determination of
`nonspecific binding), and sufficient 50 mM Tris-HCl buffer
`to yield a final volume of 250 ml. The assays were performed
`in duplicate in polystyrene 96-well plates incubated at 251C
`
`1975
`
`Vivitrexs pharmacokinetics and pharmacodynamics
`RT Bartus et al
`
`for 1 h, and terminated by filtration. The Bmax and Kd values
`were determined by nonlinear regression fitting of satura-
`tion isotherms to the data (Prism, GraphPad Software, San
`Diego, CA).
`
`m-Opioid receptor autoradiography. At the conclusion of
`behavioral testing (see above), animals were killed, their
`brains removed, flash frozen, and stored at 801C. Frozen
`brains were cut into 20 mm thick sections using a cryostat,
`the sections thaw-mounted onto glass slides, and stored at
`801C until they were used for quantitative autoradio-
`graphy of m-opioid receptor binding (Morris et al, 2001).
`Brain sections were prewashed in 120 mM NaCl and 50 mM
`Tris-HCl buffer, then incubated in a solution containing
`5 nM [3H]DAMGO and 120 mM NaCl in Tris-HCl buffer (pH
`7.4, 1 h, 251C). Nonspecific binding was determined using
`1 mM DAMGO. At the end of the incubation period, the
`sections were washed 5 1 min in Tris buffer (pH 7.4,
`0–41C) with a final rinse in distilled H2O (0–41C), then dried
`under a cool stream of air. The sections were apposed to
`film (Hyperfilm-3H; Amersham Pharmacia Biotech), to-
`gether with a tritium standard calibration slide (American
`Radiolabeled Chemicals, St Louis, MO) and nonspecific
`control sections. The films were stored at 801C and
`developed 9 weeks later. The optical density of autoradio-
`graphic exposures was quantified using an MCID 4 image
`analysis system (Imaging Research, St Catherines, Ontario,
`Canada).
`
`m-Opioid receptor immunoreactivity. Brain sections ad-
`jacent to those used for receptor binding were processed for
`m-opioid receptor immunoreactivity (Unterwald et al, 1998).
`Sections were immersion fixed in 6% paraformaldehyde,
`20% sucrose, 20% ethanol, 20% ethylene glycol, and 10%
`glycerol in 0.05 M phosphate buffer (Jones et al, 1992), then
`washed in phosphate-buffered saline. Subsequently,
`the
`sections were treated to suppress endogenous peroxidase
`activity and the nonspecific sites were blocked. The slides
`were then incubated with the primary antibody to m-opioid
`receptors (Ab-1, Oncogene Research Products, Cambridge,
`MA; 1 : 2500) in a humidifying chamber for 24 h. The next
`day, the slides were washed and incubated for 2 h with the
`secondary antibody (I125 anti-rabbit
`IgG; Amersham
`Biosciences, Piscataway, NJ, 1 : 100) in the humidifying
`chamber. The sections were then washed and dried under a
`stream of cool air. Nonspecific immunoreactivity was
`assessed by deleting either the primary antibody or using
`sections treated with antibody preabsorbed to the antigenic
`peptide (Opioid m-Receptor Control Peptide, Oncogene).
`These sections, together with an I125 microscale standard
`(Amersham Biosciences), were apposed to film (Hyperfilm-
`max, Amersham Biosciences) for 7 days. The films were
`developed and the autoradiographs analyzed using the
`MCID 4 image analysis system.
`
`RESULTS
`
`Quantitation of Plasma Levels of Naltrexone in Rats
`
`Plasma naltrexone levels were below the LOQ in all rats
`tested prior to treatment. The route of administration (i.m.
`or s.c.) had no significant effect on either the plasma
`
`Neuropsychopharmacology
`
`Page 3 of 10
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`

`

`Vivitrexs pharmacokinetics and pharmacodynamics
`RT Bartus et al
`
`1976
`
`Rx 1
`
`Rx 2
`
`Naltrexone
`1 mo only
`Naltrexone
`2 mo
`
`LOQ
`
`0
`
`60
`50
`40
`30
`20
`10
`Time after Administration (D)
`
`70
`
`a
`
`**
`
`**
`
`Placebo +
`Morphine
`Naltrexone +
`Morphine, 1 mo
`Naltrexone +
`Morphine, 2 mo
`
`Rx 1
`
`Rx 2
`
`B
`
`0
`
`60
`50
`40
`30
`20
`10
`Time after Administration (D)
`
`70
`
`30
`
`25
`
`20
`
`15
`
`10
`
`05
`
`60
`
`55
`
`50
`
`45
`
`40
`
`35
`
`30
`
`25
`
`20
`
`a
`
`Plasma Naltrexone (ng/ml)
`
`b
`
`Response Latency (Sec)
`
`Figure 2 (a) Plasma levels of naltrexone in rats following either a single
`i.m. injection of extended-release microspheres (open circles) or a second,
`identical
`injection 34 days following the first treatment (closed circles).
`Plasma levels of naltrexone were maintained for approximately 21 days
`following a single injection, an effect that was repeated with a second
`injection after an additional 34 days. Each point represents the mean 7
`SEM naltrexone plasma concentration (ng/ml) from nine rats. LOQ: lower
`limit of quantitation (o1 ng/ml). (b) Analgesic actions of morphine (1 mg/kg
`i.p.) following either one or two i.m.
`injections of extended-release
`naltrexone microspheres in rats as tested on the hot plate. Naltrexone,
`whether administered for 1 or 2 months i.m., was capable of antagonizing
`morphine-induced analgesia, with responses equivalent to those observed
`under baseline conditions. The analgesic actions of morphine in rats treated
`once with naltrexone increased to levels observed in the placebo+mor-
`phine by 41 days.
`In contrast, two naltrexone treatments consistently
`suppressed morphine-induced analgesia for a total of 68 days. Data
`represent the mean 7 SEM of latency to lick a hind paw by nine rats.
`**Performance of the naltrexone+morphine, 1 or 2 months groups, are
`significantly different from the placebo+morphine group, Po0.01, two-
`way ANOVA followed by Tukey’s post hoc analysis. (a) Performance of the
`naltrexone+morphine 1 month group is significantly different from the
`naltrexone+morphine 2 months group, Po0.01,
`two-way ANOVA
`followed by Tukey’s post hoc analysis. B: Baseline conditions. Rx 1, 2:
`Times of first and second naltrexone injections.
`
`plasma naltrexone levels during the plateau phase between
`the first and second injections. Moreover, naltrexone was no
`longer quantifiable in the plasma (o1 ng/ml) 35 days after
`the second injection.
`
`Hot-Plate Testing
`
`The pharmacodynamic effects of extended-release naltre-
`xone corresponded well with the pharmacokinetic profile
`derived from the same animals. Extended-release naltre-
`xone significantly suppressed the analgesia produced by
`morphine,
`independent of
`the route of microsphere
`administration (Treatment Effect, Po0.01, Route Effect,
`P¼ 0.35, Multiway ANOVA, Figures 1b, 2b). Morphine
`
`LOQ
`
`0
`
`30
`25
`20
`15
`10
`5
`Time after Administration (D)
`
`35
`
`**
`
`a
`
`b
`
`Placebo +
`Morphine
`Naltrexone +
`Morphine
`Placebo +
`Saline
`Naltrexone +
`Saline
`
`B
`
`0
`
`5
`10
`15
`20
`25
`30
`Time after Administration (D)
`
`35
`
`30
`
`25
`
`20
`
`15
`
`10
`
`05
`
`60
`
`55
`
`50
`
`45
`
`40
`
`35
`
`30
`
`25
`
`20
`
`a
`
`Plasma Naltrexone (ng/ml)
`
`b
`
`Response Latency (Sec)
`
`Figure 1 (a) Plasma levels of naltrexone in rats following a single, s.c.
`injection of extended-release naltrexone microspheres (50 mg/kg naltrex-
`one). Blood was sampled from the tail vein immediately after the hot-plate
`test. Note that the plasma levels of naltrexone were maintained for 21 days
`and were above the LOQs for at least 28 days. Each point represents the
`mean 7 SEM of plasma naltrexone concentrations (ng/ml) from eight rats.
`(b) Pharmacodynamic effects of a single s.c. injection of extended-release
`naltrexone microspheres. This was assessed by testing naltrexone (circles)
`or placebo formulation (squares)-treated rats on the hot plate 30 min after
`an injection of morphine (1 mg/kg i.p., closed symbols) or saline (open
`symbols). Rats receiving placebo microspheres manifested a pronounced
`analgesic response to morphine that consistently approached the
`maximum latency of 60 s. In contrast, animals pretreated with naltrexone
`microspheres showed substantial block of morphine analgesia, responding
`at or near
`the level of saline-treated animals. Data represent
`the
`mean 7 SEM of the latency to lick a hind paw measured in eight rats.
`**Performance of the naltrexone+morphine group is significantly different
`from placebo+morphine, placebo+saline, and naltrexone+saline groups,
`Po0.01, two-way ANOVA followed by Tukey’s post hoc analysis. (a)
`Performance of the naltrexone+morphine group is significantly different
`from placebo+morphine and placebo+saline groups, Po0.01, two-way
`ANOVA followed by Tukey’s post hoc analysis. (b) Performance of the
`naltrexone+morphine group is significantly different from placebo+saline
`and naltrexone+Saline GROUPS, Po0.01, two-way ANOVA followed by
`Tukey’s post hoc analysis. B: baseline conditions.
`
`naltrexone levels (P¼ 0.72, two-way ANOVA, Figures 1a,
`2a), or the area under the curves (332 vs 360 ng day/ml, i.m.
`vs s.c., respectively). However, plasma naltrexone concen-
`trations changed significantly with time (Po0.01, Two-way
`ANOVA),
`increasing to approximately one-half of
`the
`maximum within 24 h of injection, with maximum levels
`(15 7 1.4 and 19 7 3.6 ng/ml, s.c. and i.m., respectively)
`observed by 3 days. Plasma concentrations of naltrexone
`did not differ significantly from each other between 3 and
`14 days (i.m.) or 21 days (s.c.) postinjection, with detectable
`levels of naltrexone maintained to 35 days. A similar pattern
`was observed in animals that received a second i.m.
`injection of extended-release naltrexone microspheres
`(Figure 2a, Rx 2). There were no significant differences in
`
`Neuropsychopharmacology
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`Page 4 of 10
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`

`

`Vivitrexs pharmacokinetics and pharmacodynamics
`RT Bartus et al
`
`1977
`
`Cerebral Cortex
`
`Placebo
`Naltrexone
`
`a
`
`**
`
`0
`
`30
`20
`10
`Time after Administration (D)
`
`40
`
`Midbrain
`
`Placebo
`Naltrexone
`**
`
`a
`
`*
`
`0
`
`30
`20
`10
`Time after Administration (D)
`
`40
`
`Striatum
`
`Placebo
`Naltrexone
`*
`
`a
`
`*
`
`*
`
`0
`
`30
`20
`10
`Time after Administration (D)
`
`40
`
`300
`
`250
`
`200
`
`150
`
`100
`
`50
`
`350
`
`300
`
`250
`
`200
`
`150
`
`100
`
`50
`
`350
`
`300
`
`250
`
`200
`
`150
`
`100
`
`50
`
`a
`
`(fmol/mg protein)
`[3H]DAMGO Bmax
`
`b
`
`(fmol/mg protein)
`[3H]DAMGO Bmax
`
`c
`
`(fmol/mg protein)
`[3H]DAMGO Bmax
`
`Figure 3 Regional changes in the Bmax of [3H]DAMGO binding to m-
`opioid receptors in the brain following a single i.m. dose of extended-
`release naltrexone. Data represent the mean 7 SEM of data from eight rats
`(panels a, b) or eight sets of striata pooled from 16 rats (panel c). Significant
`increases in the density of m receptors in the cerebral cortex were not
`observed until 32 (relative to t0 control) to 40 (relative to contempora-
`neous placebo control) days after naltrexone administration (panel a). In
`contrast, m-opioid receptor density in the midbrain (panel b) and striatum
`(panel c) was significantly increased by 7 days (relative to contempora-
`neous placebo) after naltrexone administration. (a) Significantly different
`from t0 control, Po0.05; *, **significantly different from contemporaneous
`placebo control, Po0.05, 0.01, two-way ANOVA followed by Tukey’s post
`hoc comparison matrix.
`
`densities continued to increase at 2 months, from 100% in
`the subiculum to 220% in the dorsal raphe.
`
`Immunohistochemistry.
`using
`Immunohistochemistry
`brain sections adjacent to those used in the radioligand
`autoradiography also revealed significantly increased m-
`opioid receptor immunoreactivity (Table 2, Figure 5). After
`
`Neuropsychopharmacology
`
`induced a profound analgesia in rats receiving placebo
`microspheres, as evidenced by hot-plate times approaching
`the maximum duration (57 7 0.60 s).
`In contrast,
`the
`analgesic effects of morphine were suppressed in rats
`previously administered extended-release naltrexone (Fig-
`ures 1b, 2b). Over a 21-day period, the rats receiving
`extended-release naltrexone+morphine showed hot-plate
`response times (40 7 0.54 s) that were approximately 70%
`of
`the level of
`those that
`received extended release
`placebo+saline (34 7 0.80 s). After 28 days,
`the mor-
`phine-associated response latencies of the extended-release
`naltrexone-treated rats increased to the level of those rats
`receiving placebo microspheres (59 7 0.84 vs 57 7 1.8 s,
`placebo+morphine vs naltrexone+morphine). Rats receiv-
`ing extended-release naltrexone i.m. had hot-plate response
`times after morphine treatment (36 7 0.40 s) that were
`statistically indistinguishable from saline-treated animals
`placebo+saline¼ 31 7 5.1 s;
`naltrexone+saline¼
`(eg
`34 7 3.5 s). Animals receiving a second i.m. injection of
`extended-release naltrexone on day 34 continued to exhibit
`complete antagonism of the analgesic effects of morphine
`throughout
`the second month (36 s, Figure 2b). The
`analgesic effects of morphine on naltrexone-treated rats
`reverted to the level of the placebo-treated rats (59 7 0.48 vs
`59 7 0.58 s, placebo+morphine vs naltrexone+morphine)
`by 35 days after treatment (Figure 2b).
`
`Opioid Receptor Changes Following Extended-Release
`Naltrexone in Rats
`
`Radioligand binding assays. Saturation binding assays
`revealed that the Bmax for [3H]DAMGO binding to the
`midbrain and the striatum was significantly increased (110
`and 110% vs placebo treatment, Po0.01, 0.05, respectively,
`ANOVA followed by Tukey’s post hoc comparison test) by 1
`week after administration of extended-release naltrexone
`(Figures 3b, c). Evidence of increased m-opioid receptor
`density was observed as early as 5 days after administration.
`These increases in receptor density were sustained through-
`out the subsequent 33 days, at least 1 week after the
`significant decline in pharmacodynamic effectiveness of the
`extended-release preparation. Interestingly, the density of
`cortical m-opioid receptors did not begin to increase until 30
`days after
`extended-release naltrexone administration
`(Figure 3a), reaching significance at 40 days (120% increase
`vs placebo, Po0.01, ANOVA followed by Tukey’s post hoc
`comparison test). No significant changes in radioligand
`affinity for the receptors were observed in any brain region
`at any time, regardless of treatment (cortex: 7.1 7 0.40;
`midbrain: 4.2 7 0.30; striatum: 3.1 7 0.10 nM).
`Similar results, albeit with higher regional resolution,
`were obtained using radioligand binding autoradiography.
`In this study, rats received either one or two injections of
`extended-release naltrexone spaced 34 days apart, and the
`changes in m-opioid receptor density quantified at 1 month
`and 24 h after the antagonism of morphine’s analgesic
`effects had dissipated (ie 2 months from initial injection).
`Autoradiography revealed that radioligand binding to m-
`opioid receptors was significantly increased above control
`in all brain regions examined, ranging from 90% in the
`habenular nucleus to 160% in the dorsal raphe nucleus
`(Table 1, Figure 4) after 1 month. In most regions, these
`
`Page 5 of 10
`
`

`

`Vivitrexs pharmacokinetics and pharmacodynamics
`RT Bartus et al
`
`1978
`
`Table 1 Regional Changes in m-Opioid Receptor Density Following Extended-Release
`Naltrexone
`
`Brain region
`
`Central grey
`
`Dentate gyrus
`
`Dorsal raphe nucleus
`
`Habenular nucleus
`
`Hippocampus CA1
`
`Inferior colliculus
`
`Lateral orbital cortex
`
`Nucleus accumbens
`
`Perirhinal cortex
`
`Striatum
`
`Subiculum
`
`Substantia nigra
`
`Superior colliculus
`
`Taenia tecta
`
`Thalamus
`
`Control
`
`7.5 7 0.27
`7.1 7 0.39
`7.2 7 0.86
`19 7 1.4
`4.8 7 0.29
`10 7 1.7
`8.8 7 0.72
`13 7 1.1
`6.4 7 0.24
`8.0 7 0.67
`20 7 1.6
`8.7 7 0.77
`8.9 7 1.2
`9.2 7 1.1
`11 7 1.5
`
`Treatment
`
`Naltrexone
`(1 month)
`
`% Increase
`
`Naltrexone
`(2 months)
`
`% Increase
`
`18 7 0.74*
`16 7 0.82*
`19 7 2.3*
`36 7 2.5*
`12 7 0.79*
`23 7 1.9*
`20 7 0.81*
`25 7 2.2*
`13 7 0.51*
`15 7 0.79*
`34 7 2.3*
`18 7 0.68*
`20 7 1.6*
`23 7 1.3*
`22 7 1.4*
`
`140
`
`125
`
`160
`
`90
`
`150
`
`130
`
`120
`
`90
`
`100
`
`80
`
`70
`
`100
`
`120
`
`150
`
`100
`
`20 7 0.62*
`17 7 0.89*
`23 7 1.1*
`41 7 2.4*
`14 7 0.75*
`27 7 1.5*
`24 7 1.3*
`22 7 1.3*
`15 7 0.57*
`14 7 0.38*
`40 7 1.8*
`21 7 1.5*
`20 7 1.4*
`29 7 1.7*
`21 7 1.1*
`
`160
`
`140
`
`220
`
`120
`
`190
`
`170
`
`170
`
`70
`
`130
`
`75
`
`100
`
`140
`
`120
`
`215
`
`90
`
`Brains from animals treated with extended-release naltrexone (50 mg/kg) were processed for [3H]DAMGO
`autoradiography of m-opioid receptors. One group of animals was killed 29 days after administration when the
`behavioral effects of naltrexone were maximal. The remaining animals received a second treatment with extended-
`release naltrexone at 34 days and were killed after an additional 29 days. Significant increases in radioligand binding
`to the m-opioid receptor were observed in all brain regions by 1 month, with only the subiculum and taenia tecta
`showing further changes following 2 months of naltrexone. Data represent the mean 7 SEM mCi/g, n¼ 8 (1 month)
`or 9 (2 months). *Significantly different, naltrexone vs control groups (Po0.05).
`
`1 month of treatment with extended-release naltrexone,
`immunoreactivity was significantly increased above control
`levels in only two of 15 brain regions studied, including the
`nucleus accumbens (20%) and the substantia nigra (20%,
`Table 2). Following 2 months of
`treatment,
`increased
`immunoreactivity over placebo controls was observed in
`14 of 15 brain regions examined. However, the magnitude of
`these increases was
`lower
`than those observed with
`radioligand receptor binding, ranging from 10% in the
`perirhinal cortex to 40% in the substantia nigra (Table 2).
`
`DISCUSSION
`
`Naltrexone has repeatedly been shown to be effective in
`suppressing a number of indices of ethanol consumption in
`animal models, including decreasing ad libitum (Altshuler
`et al, 1980; Davidson and Amit, 1997; Boyle et al, 1998) and
`limited access (Stromberg et al, 1998) ethanol intake, while
`delaying the acquisition of voluntary ethanol consumption
`(Davidson and Amit, 1997). Moreover, naltrexone has been
`shown to be effective in several human studies in reducing
`alcohol consumption and relapse to heavy drinking when
`combined with psychosocial treatment (Garbutt et al, 1999;
`O’Malley et al, 1992; Volpicelli et al, 1992). Nonetheless, the
`efficacy of naltrexone in treating alcohol dependence has
`
`Figure 4 Representative photomicrographs of brain sections incubated
`with [3H]DAMGO from rats receiving i.m. injections of either naltrexone-
`containing or placebo microspheres for approximately 2 months (see text
`for details). Note the marked increase in the density of m-opioid receptor
`binding in the prefrontal cortex, striatum, thalamus, hippocampus, and
`superior colliculus in those animals receiving extended-release naltrexone
`relative to rats receiving placebo microspheres. See Table 1 for quantitative
`data.
`
`Neuropsychopharmacology
`
`Page 6 of 10
`
`

`

`Vivitrexs pharmacokinetics and pharmacodynamics
`RT Bartus et al
`
`1979
`
`Table 2 Regional Changes in m-Opioid Receptor Immunoreactivity Following Extended-Release
`Naltrexone
`
`Brain region
`
`Central grey
`
`Dentate gyrus
`
`Dorsal raphe nucleus
`
`Habenular nucleus
`
`Hippocampus CA1
`
`Inferior colliculus
`
`Lateral orbital cortex
`
`Nucleus accu