I wo 94/06426 2 1 4 5 2 0 7 PCI/US93/08869
- ME~IODS FOR IDE~NTIFYING AND USING LOW/NON-ADDICTIVE OPIOID
AN~rrJF.~ICS
The present invention relates to a specific group of
opioid agonists for use as low/non-addictive analgesics and
for treatment of opioid addiction. More particularly, the
present invention is directed to etorphine,
dihydroetorphine, ohmefentanyl and other opioid and
analogues thereof that are effective as low/non-addictive
analgesics and for the treatment of opioid addiction. In
addition, this invention provides a bioassay method to
screen and identify such compounds with the ability to
1~ selectively activate inhibitory but not excitatory opioid
receptor-mediated functions.
The present invention also relates to the preparation
of etorphine, dihydroetorphine and analogues thereof using
thebaine as the starting material. More specifically, the
present invention relates to the preparation of
dihydroetorphine hydrochloride (7~-tl-(R)-hydroxy-l-
methylbutyl]-6,14-endo-ethano-tetrahydrooripavine
hydrochloride) and pharmaceutical compositions thereof.
Since the introduction of morphine [Fig. l(I)] to the
clinic as a pain reliever, clinicians have been troubled
with the problem of drug addiction. For more than a
century, chemists, pharmacologists, and clinicians have
strived to find an ideal analgesic with high potency, yet
low addictivity. A series of opioids such as meperidine,
methadone tFig. l(II)], and fentanyl were subsequently
developed.
However, none of these drugs exert sustained analgesic
effects in patients without developing addiction. In
Western countries, methadone substitution has been employed
for the treatment of drug abuse for some time.
Unfortunately, methadone induces significant psychological
and physical dependencies. Consequently, patients
PCT/US93/08869 -
W094/06426
21 4~ 207 2
undergoing such treatment usually convert to methadone
dependence during withdrawal from chronic use of morphine,
heroin or other opioids (Jaffe, 1990). Therefore the need
remains to develop better methods based upon insights into
the molecular and cellular mechanisms underlying opioid
addiction for treating drug abuse and particularly a means
to identify compounds for use as low- or non-addictive
analgesics and for suppression of opioid withdrawal
symptoms.
The present invention is directed to an n vitro
screening method for identifying a low- or non-addictive
opioid analgesic by screening opioids to identify a compound
which is capable of evoking an inhibitory effect but not an
excitatory effect on opioid receptor-mediated functions of
sensory neurons in a dose-dependent manner over the
concentration range of from about femtomolar (fM~ to about
micr~molar (~M). In particular, such opioid compounds are
identified by recording the action potential duration (APD)
of a sensory neuron elicited by the compound in a cell
culture screening assay and selecting those opioid compounds
which shorten the APD but do not prolong the APD relative to
a control APD when the compounds are assayed in the
concentration range of about fM to about ~M. Opioid
compounds with these characteristics are thereby identified
as low- or non-addictive opioid analgesics of the invention.
Preferably, the cell culture screening assay comprises
exposing a dorsal-root ganglion (DRG) neuron to the
candidate compound, typically by bath perfusion, applying a
brief intracellular depolarizing current to said DRG neuron,
and recording opioid-induced alteration in the APD of the
DRG neuron using standard electrophysiological techniques.
Another aspect of the invention, thus, provides low- or
non-addictive analgesics, particularly as identified by the
method of the present invention, which are capable of
evoking the inhibitory but not the excitatory effects of
opioid receptor-mediated functions, particularly on sensory
PCT/US93/08869
~ W094/06426 2 1 4 5 2 0 7
neurons, in a dose-dependent manner in concentrations
ranging from about fM to about ~M. In a preferred
embodiment these opioids include etorphine, dihydroetorphine
or ohmefentanyl. Pharmaceutical compositions containing the
subject low- or non-addictive opioids, or pharmaceutically
acceptable salts thereof, together with pharmaceutically
acceptable carriers are also provided. In addition, the
subject pharmaceutical compositions can also contain a
replacement opioid or naloxone.
Yet another aspect of this invention provides a method
of treating opioid addiction by administering an effective
amount of a non-addictive opioid analgesic, or an analog
thereof, to a patient for a time sufficient to relieve or
suppress withdrawal symptoms that occur when the addictive
opioid is withheld from the addict. After the initial
administration of the non-addictive opioid analgesic for a
period to permit alleviation of the withdrawal symptoms, the
dose of the non-addictive opioid analgesic is gradually
decreased from the original dose to zero over a time
sufficient to fully wean said patient from said analgesic
without untoward side effects. Typically the initial
administration of the non-addictive opioid analgesic lasts
for about 1 to about 5 days and the weaning period lasts
from about 1 to about 7 days, so that a patient can be
withdrawn from opioid addiction within an overall about 2 to
12 day period. In a preferred embodiment the non-addictive
opioid analgesic is etorphine or dihydroetorphine initially
administered at a dose of from about lo ~g to about lOOo ~g
per day. Such dosages are usually administered
sublingually, intramuscularly or intravenously, pre~erably
by intravenous dripping, depending on the severity of the
withdrawal symptoms in the patient. Even more preferably,
opioid addiction is treated by administering about 40 to
about 500 ~g of dihydroetorphine per day to a patient for
about one to about three days, administering a decreasing
amount of dihydroetorphine for the following about four to
PCT/US93/08869
W094/06426
~ 1~5 2Q~ 4
about seven days so that no further dihydroetorphine is
necessary by about lO days after the first administration of
dihydroetorphine.
A further aspect of the invention provides a method of
s treating opioid addiction by administering an effective
amount of a non-addictive opioid analgesic, to~a patient for
a time sufficient for immediate relief or suppreesion of
withdrawal symptoms due to said opioid addiction;
administering an effective amount of a longer-acting
replacement opioid for a time sufficient to maintain the
relief or suppression of withdrawal symptoms, followed by
administering a decreasing dose of the non-addictive opioid
analgesic for a time sufficient to wean said patient from
said opioid analgesic without untoward side effects.
Typically the initial administration of the non-addictive
opioid analgesic lasts for about 1 to about 3 days, the
administration of the replacement opioid lasts for about 1
to about 3 days, and the return to the non-addictive opioid
analgesic with its concomitant weaning period lasts from
about 1 to about 8 days, so that a patient can be withdrawn
from opioid addiction within an overall 3 to 14 day period.
Alternatively, the initial administration of the non-
addictive opioid analgesics and administration of the
replacement opioid can be made simultaneously. Thus, these
two opioids are co-administered until the patient is
relieved of withdrawal symptoms (e.g. the symptoms are
effectively suppressed). Thereafter, administration of the
replacement opioid is discontinued and the non-addictive
opioid analgesic dosage is stepwise or gradually reduced
until the patient is weaned off of the non-addictive opioid
analgesic. The time periods for co-administering these
opioids is about 2 to about 6 days and for weaning is about
1 to about 8 days, so that the patient can be withdrawn from
opioid addiction within an about 3 to about 14 day period.
In a preferred embodiment the non-addictive opioid analgesic
is etorphine or dihydroetorphine initially administered at a
A~ ~ PCT/US93/08869
W094/06426 ~l ~J ~ V (
dose of from about 10 ~g to about 1000 ~g per day. Such
dosages are usually administered sublingually,
intramuscularly or by intravenous dripping depending on the
severity of the withdrawal symptoms in the patient.
Preferably the replacement opioid is methadone administered
per os at a dose of about 5-100 mg/day.
A still further aspect of the invention provides a
method of treating acute or chronic pain with a low- or non-
addictive opioid analgesic. In particular, dihydroetorphine
hydrochloride (DHE) is administered to a patient for a time
and in an amount effective to relieve or suppress pain
without resultant addiction. Treatment for acute pain is
typically accomplished by administration of about 20-60 ~g
DHE sublingually, up to about 180 ~g per day for the
duration of the pain, and typically no longer than 1 week.
Treatment for chronic pain is typically accomplished by
administration of about 20-100 ~g DHE sublingually, up to
400 ~g per day, and such administration can last several
months. In rare instances treatment of chronic pain can
result in mild addiction.
Alternatively, chronic or acute pain can be treated by
co-administering a low- or non-addictive opioid analgesic
and a replacement opioid for a time and in an amount
effective to relieve or suppress pain without resultant
addiction. The potent inhibitory effects exerted by low- or
non-addictive opioid analgesics, such as DHE and etorphine,
block the excitatory effects exerted by replacement opioids
such as morphine and methadone. Typically the amounts of
the non-addictive opioid analgesics are about 10 to about
1000 ~g per day as well as those dosages described above for
treatment of pain with only a low- or non-addictive opioid
analgesic. The amounts of the replacement opioid are about
5 to about 100 mg per day. Dosages of the analgesic can
also be determined as about 0.05% to about 5% of the
replacement opioid on a weight basis. Using a combination
of replacement opioid with a relatively lesser amount of a
PCT/US93/08869 ~
Wo94/06426 2 1 ~ 5 2 ~ ~
low- or non-addictive opioid analgesic permits treatment of
pain without addiction or with a low incidence of addiction.
Preferably the analgesic is DHE, etorphine, ohmefentanyl or
a pharmaceutically acceptable salt thereof, and the
replacement opioid is morphine, methadone or fentanyl.
The present invention also relates to improved methods
for the preparation of etorphine, DHE, and analogues thereof
using thebaine as the starting material. ~For example, the
present invention provides a method for the preparation of
dihydroetorphine hydrochloride (7~-tl-(R)-hydroxy-l-
methylbutyl]-6~14-endo-ethano-tetrahydrooripavine
hydrochloride) and other salts of DHE.
In particular, the method for preparing
dihydroetorphine and its analogues tFigs. 13, 14 and 15]
comprises (1) reacting thebaine with an excess of methyl
vinyl ketone for a time and under conditions sufficient to
produce a first product and recovering that first product;
subjecting the first product to catalytic hydrogenation to
produce a second product and recovering that second product;
reacting the second product with a Grignard reagent of the
formula RMgX for a time and under conditions to produce a
third product and recovering that third product; reacting
the third product with a strong base in an anhydrous
solution for a time and under conditions sufficient to
produce dihydroetorphine or its corresponding analog,
wherein R is a lower alkyl group and X is a halogen. The R
group is preferably n-propyl or i-amyl. The method of
preparing etorphine and its analogues in accordance with
this invention follows the same method for preparation of
DHE except that catalytic hydrogenation step is omitted.
When preparing etorphine or etorphine-related components, R
is preferably n-propyl, n-butyl, n-amyl, i-amyl or
cyclohexyl.
Fig. 1 depicts the structure of opioids: (I) morphine;
(II) methadone; (III) etorphine (a) and analogues (b,c,d,e)
thereof, (IV) dihydroetorphine(a) and an analogue (b)
PCT/US93/08869
~ W094/06426 2 1 4 ~ 2 0 7
-
thereof, and (V) naloxone.
Fig. 2 illustrates positive- and negative-feedback
phosphorylation mechanisms in dorsal root ganglion (DRG)
neurons that may result in opioid excitatory
supersensitivity and opioid inhibitory desensitization
during chronic opioid exposure. Sustained activation of
excitatory Gs-coupled opioid receptors increases adenylate
cyclase activities and PKA, resulting in: (a) cAMP-dependent
elevation of GM1 ganglioside via PKA, and (b) activation of
voltage-sensitive K+ and Ca2+ channels, leading to action
potential duration (APD) prolongation and enhanced
transmitter release (if similar APD modulation occurs in
presynaptic DRG terminals). Elevation of GM1, in turn,
enhances the efficacy of excitatory Gs-coupled opioid
receptor functions, i.e., heterologous sensitization
(resulting in dependence). The upregulated AC/cAMP/PKA
system may concomitantly phosphorylate ligand-bound
inhibitory opioid receptors, thereby attenuating their
coupling to Gi/Go, i.e., heterologous desensitization
(resulting in tolerance to opioid inhibitory effects).
Abbreviations: AC, adenylate cyclase; PKA, cAMP-dependent
protein kinase; gK, membrane K+ conductance; gc" membrane
Ca2+ conductance.
Fig. 3 illustrates that acute application of pM-~M
concentrations of etorphine to a naive DRG neuron elicits
inhibitory shortening of the APD. 1: Action potential (AP)
generated by a DRG neuron in Hank's balanced salt solution
containing 5 mM Ca2+ and 5 mM Ba2+ (BSS). AP response in
this record (and in all records below) was evoked by a brief
(2 msec) intracellular depolarizing current pulse. 2-5: The
APD is progressively shortened by bath perfusion of 1 fM, 1
pM, 1 nM and 1 ~M etorphine, respectively. 6: After washout
of etorphine, the APD recovers.
Fig. 4 shows the dose-response relationship of
etorphine, DHE and dynorphin (1-13) (Dyn 1-13) effects on
the APD of DRG neurons. Etorphine and DHE elicited a dose-
- F
PCT/US93/08869
W094/06426
21~2~7
dependent shortening of the APD (n=ll and 13, respectively).
In contrast, Dyn (1-13) elicited a dose-dependent
prolongation of the APD at fM-nM concentrations and required
much higher concentrations (ca. ~M) to shorten the APD
(n=35)-
Fig. 5 illustrates that chronic exposure of a DRG
neuron to a bimodally acting opioid (DADLE~ causes the DRG
neuron to become supersensitive to the excitatory effects of
dynorphin (1-13) (Dyn), whereas perfusion of etorphine
effectively shortened the APD of the same DRG neuron
(inhibitory response). 1: Action potential generated by a
DRG neuron treated for 3 wks in culture with 1 ~M DADLE and
then tested in BSS with 1 ~M DADLE. 2: APD is prolonged by
bath perfusion of 1 fM Dyn with 1 ~M DADLE (5 min test).
3,4: APD is further prolonged by sequentially raising the
Dyn concentration to 1 nM and 1 ~M (5 min tests). 5:
Control response 5 min after washout of Dyn with BSS
containing 1 ~M DADLE. 6: 1 fM etorphine (Etorp) shortens
the APD of the same DRG neuron in the presence of 1 ~M
DADLE. 7-9: Further increases in the concentration of
etorphine from 1 pM to 1 ~M progressively shorten the APD.
10: APD returns to control value after removal of etorphine.
Fig. 6 shows that chronic exposure to a bimodally
acting opioid (DADLE) followed by acute application of low
concentrations of etorphine can block the excitatory APD-
prolonging effects precipitated by naloxone (NLX) in this
supersensitive DRG neurons. 1: Action potential generated by
a DRG neuron treated for 2 wks in culture with 1 ~M DADLE
and then tested in BSS with 1 ~M DADLE. 2: 1 nM NLX
prolongs the APD of this DRG neuron (5 min test). In
contrast, nM naloxone is ineffective on naive DRG neurons
(Crain & Shen, 1992a,b~ 3: Acute addition of 1 pM etorphine
attenuates the naloxone-induced APD prolongation (5 min
test). 4: Further increase in concentration of etorphine to
1 nM almost completely blocks the naloxone-induced APD
prolongation.
PC~r/US93/08869
~ W O 94/06426 2 1 4 ~ 2 0 7
Fig. 7 illustrates the relief of naloxone-precipitated,
sustained body weight loss by morphine, DHE and methadone
injections in morphine-dependent rats. Daily dose: morphine
100 mg/kg, divided into 2 subdoses; DHE 12 ~g/kg, divided
into 4 subdoses; methadone 24 mg/kg, divided into 4
subdoses. Filled circle: morphine group; open circle: DHE
group; filled triangle: methadone; cross: saline control
group. X ~ SD, ***p~0.01, as compared with saline control
group.
Fig. 8 depicts the effect of DHE and methadone
substitution on naloxone precipitated body weight loss in
morphine-dependent rats. The body weight loss from the
first naloxone precipitation test is provided in Column A.
The second naloxone precipitation test was performed after 4
days of maintaining one group of rats with morphine (100
mg/kg/day, divided into 2 subdoses), a second group with DHE
(12 ~g/kg/day, divided into 4 subdoses) and a third group
with methadone (24 mg/day, divided into 4 subdoses). The
body weight loss after the second naloxone precipitation
test is provided in Column B. Statistical p values between
the first and second naloxone precipitation test are "**",
p<o.o5 and "***", p<o.ol. The p value for the DHE group
relative to the methadone group is p<o.o5.
Fig. 9 depicts the withdrawal symptom scores after
naloxone precipitation for DHE and methadone substitution in
morphine-dependent rats. Rats were treated as described in
Fig. 8. Column A: Withdrawal scores from the first naloxone
precipitation test. Column B: Withdrawal scores from the
second naloxone precipitation test. Statistical p values
between the first and second naloxone precipitation test are
"**", p<o.05 and "***", pco.01. The p value for the DHE
group relative to the methadone group is p<0.01.
Fig. 10 shows the development of withdrawal symptoms
in morphine-dependent monkey after cessation of morphine.
Fig. 11 illustrates the relief of withdrawal symptoms
by DHE in morphine-dependent monkeys. The arrows indicate
PCT/US93/08869 ~
W094/06426 21 4~ 2 Q 7
time of DHE injection (3 ~g/kg). Open circle: control
group; filled circle: DHE group.
Fig. 12 illustrates the therapeutic effect of DHE and
methadone on withdrawal symptoms of morphine-dependent
S monkeys. The arrows indicate the time~o`~ naloxone (NLX)
precipitation (1 mg/kg). open circle: control group; filled
circle: DHE group; filled triangle: methadone group.
Figure 13 illustrates a reaction scheme for the
synthesis of etorphine and analogues thereof from thebaine.
Figure 14 illustrates a reaction scheme for the
synthesis of dihydroetorphine and analogues thereof from
thebaine.
~ igure 15 illustrates the synthesis of dihydroetorphine
hydrochloride from thebaine.
BRIEF DESCRIPTION OF ABBREVIATIONS USED
~ DADLE [D-Ala2,D-Leu5]enkephalin
DAGO tD-Ala~,MePhe~,Gly-ol]enkephalin
DPDPE Tyr-D-Pen-Gly-Phe-D-Pen (Pen = penicillamine)
U-50,488H 3,4 dichloro-N-methyl-N-(2-tl-pyrrolidinyl3-
cyclohexyl)benzene-acetamide
Dynorphin 1-13 dynorphin A, Fragment 1-13
tTyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-
Pro-Lys-Leu-Lys)
Dynorphin 1-17 dynorphin A, Fragment 1-17
(Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-
Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln)
Etor etorphine
DHE dihydroetorphine
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention,
electrophysiologic assay of the effects of opioids on the
action potential duration of sensory neurons in organotypic
cultures provides an extremely sensitive in vitro bioassay
to screen and characterize agonists with the ability to
activate inhibitory, but not excitatory, opioid
receptor-mediated functions. This assay permits
21~5207
W094/06426 PCT/US93/08869
identification of low- or non-addictive opioid analgesics as
well as agents useful for treatment of drug addiction.
As used herein "non-addictive" and "low-addictive" are
used interchangeably to describe the addiction potential of
the opioids of the present invention. In treating opioid
addiction in accordance with the present invention, the
subject opioids are essentially non-addictive at the
prescribed dosages when used for periods of up to several
weeks. For example, DHE administration to a drug addict in
the range of about 10 ~g to about 1000 ~g per day which is
gradually withdrawn over a period of up to 14 days, does not
result in addiction to DHE. In contrast, even well-
controlled treatment of drug addicts by methadone
substitution invariably results in transfer to methadone
addiction. The present invention, thus, greatly improves
the present methods for treating opiate drug abuse without
concomitant addiction to another opioid.
Likewise, treatment of acute pain for several days with
the subject opioids in accordance with the present
invention, does not result in addiction. While treatment of
chronic pain of long duration with the subject opioids
generally does not result in addiction, mild addiction may
result in exceptional cases. Hence use of the subject
opioids in accordance with this invention is non-addictive
for the vast majority of chronic pain patients. The
addiction potential of the subject opioids, as illustrated
with DHE, for chronic pain patients is thus low, typically
less than 1 in lO0 for patients treated longer than 3
months. For example, no addiction has been observed when
treating patients for up to 3 months with DHE. Moreover, in
the rare cases of addiction, such addiction may result from
minor contaminants present in bimodally-acting thebaine, a
starting material for drug synthesis which is carried into
certain preparations of DHE.
As used herein "opioid" refers to any substance that
binds specifically to an opiate receptor (Casy & Parfitt,
PCT/US93/08869
W094/06426
2~4~2o~
12
1986; Pasternak 1988).
As used herein "replacement opioid" is a bimodally-
acting opioid that has both inhibitory and excitatory
effects on opioid receptors. Such opioids, generally, have
a longer duration of action than the non-addictive opioid
analgesics with which it is combined for use. Combination
therapy of replacement opioids with non-addictive opioid
analgesics permit the latter compounds to block or mask the
excitatory effects of the replacement opioids.
lo Activation of opioid receptors has been known to
produce inhibitory effects on neuronal activity which in
turn provides the primary cellular mechanism underlying
opioid analgesia in vivo (e.g. North, 1986). However,
recent electrophysiological studies indicated that specific
mu-, delta- and kappa-opioid receptor agonists elicit both
- excitatory and inhibitory modulation of the action
potentials of sensory DRG neurons isolated in culture in a
concentration dependent manner (Shen & Crain, 1989; Crain &
Shen, 1990).
These opioid agonists were found to elicit excitatory
effects at low (<nM) concentrations and inhibitory effects
at high (~M) concentrations as measured by prolo~gation or
shortening of the calcium-dependent component of the action
potential duration (APD), respectively (Fig. 2; Table 1).
Earlier experiments demonstrated that the excitatory
effects of opioids are mediated by opioid receptors that are
positively coupled via a cholera toxin-sensitive Gs-like
regulatory protein to adenyl cyclase and cyclic
AMP-dependent voltage-sensitive ionic conductances
(resembling, for example, beta-adrenergic receptors)(Fig. 2;
Shen & Crain, 1989, l990a; Crain & Shen, 1990, 1992),
whereas inhibitory effects are mediated by opioid receptors
linked to pertussis toxin-sensitive Gi/Go proteins
(resembling alpha2-adrenergic receptors)(Fig. 2; Shen &
Crain, 1989; Gross et al, 1990).
The ability to differentiate between these bimodal
~ W094/06426 2 1 4 5 2 0 7 PCT/US93/08869
properties of opioids, i.e. excitatory and inhibitory
activities mediated by two distinct groups of opioid
receptors, has led to the present invention, and
particularly to a method for identifying low- or non-
addictive opioid analgesics. Hence, this method provides anin vitro bioassay to identify compounds that can selectively
activate the inhibitory but not excitatory opioid response.
Since sustained activation of excitatory opioid receptor
functions plays a crucial role in development of tolerance
and dependence in chronic opioid-treated neurons in vitro
(Crain & Shen, 1992; Shen & Crain, 1992), compounds with
such properties, i.e. which activate the inhibitory response
but not the excitatory response, are useful as non-addictive
analgesics ia vivo.
In particular, the in vitro bioassay uses a cell
culture system of DRG neurons to screen candidate compounds
by exposing the DRG neurons to the candidate compound and
observing its effect on the APD using standard
electrophysiological recording methods. The detailed
methodology for growing neurons, treating with a candidate
compound and recording the APD are provided in Example 1.
Any opioid compound screened by this bioassay that exhibits
inhibitory effects (e.g., shortening the APD in DRG neurons)
but not excitatory effects (e.g., prolonging the APD in DRG
neurons) in about the fM-pM range to ~M range is a low- or
non-addictive opioid analgesic in vivo. Generally, these
compounds effect the APD in a concentration-dependent manner
and the responses are mediated by specific opioid receptors.
Hence, the method of the present invention provides a
powerful tool to identify low- or non-addictive opioid
analgesics.
Nearly all the opioids tested by this bioassay,
including morphine, enkephalins, dynorphins, endorphins and
synthetic opioid peptides, have dose-related dual modulatory
effects (i.e. both inhibitory and excitatory) on the action
potential of sensory DRG neurons. All such compounds are
W094/06426 PCT/US93/08869 ~
~2~2o7
14
well-known to be addictive. However, in accordance with
this invention etorphine and dihydroetorphine (thebaine
derivatives) (Bentley and Hardy, 1963; Bentley and Hardy,
1967), compounds previously believed and classified as
addictive (WHO Rep 1966), have the selective characteristic
(Table 1) of inhibiting opioid-receptor mediated functions
without exciting such functions. Both e~t,orphine and
dihydroetorphine elicit dose-dependen~inhibitory)
shortening of the APD, starting at about pM levels in some
of the DRG neurons, and reaching a maximum effect at ~M
levels in most of the DRG neurons (Example 1). Furthermore,
no excitatory prolongation of the APD occurs with these two
compounds at <pM concentrations in contrast to the
characteristic excitatory effects elicited at low
concentration by the bimodally-acting opioids.
It is well known that chronic exposure of DRG-spinal
cord explants to bimodally-acting opioids (e.g., morphine or
DADLE) causes sensory DRG neurons to become desensitized to
the inhibitory effects of opioid agonists, resulting in
tolerance (Crain et al, 1988), and supersensitized to the
excitatory effects of opioid agonists as well as
antagonists, resembling significant features of abstinence,
dependence and withdrawal syndrome in vivo (Crain & Shen,
1992a,b; Shen & Crain, 1992).
Sustained activation of excitatory opioid receptors
after chronic treatment with an opioid agonist triggers a
positive-feedback mechanism that results in up-regulation of
a Gs/adenylate cyclase/cyclic AMP/protein kinase A/GM1
glycosyl-transferase system that may account for the
remarkable supersensitivity of chronically opioid-treated
neurons to the excitatory effects of opioid antagonists and
agonists (Fig. 2, Crain & Shen, 1992a,b; Shen & Crain,
1992).
When DRG-cord explants are chronically treated with a
bimodally-acting delta/mu agonist, DADLE (1 ~M) or morphine
(1 ~g/ml) for 3 weeks, acute treatment with etorphine still
~ W094/06426 2 1 4 S 2 0 7 PCT/US93/08869
elicits a marked inhibitory dose-dependent shortening of the
APD of DRG neurons even at concentrations as low as 1 fM
(Example 2), whereas bimodally-acting mu, delta and kappa
opioid agonists show a high degree of opioid excitatory
supersensitivity at concentrations ranging from pM to ~M
(Example 2).
Furthermore, the excitatory APD prolongation of chronic
opioid-treated DRG neurons precipitated by acute application
of nM naloxone tFig. l(V)](Crain & Shen, 1992a,b), which
provides a cellular model of naloxone-induced withdrawal
supersensitivity in opiate addicts in vivo (Crain & Shen,
1992b), can be blocked by acute application of etorphine,
but not by morphine or other bimodally acting opioid
agonists (Example 2).
Tissue culture studies provide strong support that
excitatory opioid receptor functions of sensory neurons play
important roles in vivo, both by attenuating analgesic
effects mediated by inhibitory opioid receptors and by
facilitating the cellular mechanisms underlying addiction.
The use of opioids (e.g. etorphine, dihydroetorphine), that
at low concentrations preferentially activate inhibitory but
not excitatory opioid receptor functions n vitro, as
indicated by the screening model, results in much more
potent analgesia n vivo and far less evidence of
dependence/addiction than occurs during chronic treatment
with morphine and most other bimodally-acting opioids.
The present invention demonstrates that etorphine (and
compounds with similar properties as identified by the
present bioassay (e.g. dihydroetorphine and ohmefentanyl)
elicits potent dose-dependent inhibitory APD-shortening
effects on naive and chronic opioid-treated, "addicted"
sensory DRG neurons, even at low (pM-nM) concentrations
where most bimodally-acting opioids generally elicit
excitatory APD-prolonging effects. Hence etorphine and
similar compounds of this invention selectively activate
inhibitory rather than excitatory opioid receptors on DRG
PCT/US93/08869
W094/06426
,2~4~2~
16
neurons, even when the cells are supersensitive to the
excitatory effects of bimodally-acting opioids following
chronic treatment.
Etorphine has long been known to be >1,000 times more
potent than morphine as an analgesic in animals (Blane et
al, 1967) and humans (Blane & Robbie, 197Q, Jasinski et al,
1975). This invention shows that the high inhibitory
potency of etorphine may be due, in part, to its selective
activation of inhibitory opioid receptors whose effects are
not attenuated by the concomitant activation of
higher-affinity excitatory opioid receptors.
The clinical trial results of the present invention
show that low doses of dihydroetorphine, a specifically
inhibitory opioid-receptor agonist, are remarkably effective
in relieving postoperative pain and chronic pain in terminal
cancer patients, yet tolerance and addiction are far less
evident than observed with morphine and other conventional
bimodally-acting opioids (Example 5). Thousands of patients
have been treated with a >90~ effective rate and no
significant adverse side-effects have been observed.
Furthermore, the potent inhibitory effect exerted by the
low- or non-addictive opioid analgesics of this invention
can block or suppress the excitatory effects of bimodally-
acting opioids, i.e. the replacement opioid as defined
herein, to alleviate tolerance and addiction commonly
observed by sole usage of bimodally-acting opioids (e.g.
morphine or methadone).
In addition, several hundred heroin addicts have been
successfully treated over a two year period. In this group,
withdrawal symptoms were rapidly blocked and
dihydroetorphine substitution therapy was maintained for
about a week with minimal rebound after final opioid
withdrawal (Example 6). Similar results were obtained in
tests on morphine-dependent monkeys and rats (Examples 3 &
4). The successful results obtained with dihydroetorphine
in treating heroin and morphine addiction are in sharp
~ W094/06426 2 1 ~ 5 2 ~ 7 PCT/US93/08869
contrast to the unreliable results obtained in comparative
clinical studies with methadone and other bimodally-acting
or mixed agonist-antagonist opioids.
Hence, another aspect of the present invention provides
a method of treating opioid addiction by administering an
opioid or analog therof, in an amount effective and for a
time sufficient to relieve the withdrawal symptoms of opioid
addiction and subse~uently withdrawing administration of
said opioid or analog thereof.
Another aspect of the present invention provides
improved synthetic methods for the preparation of DHE,
etorphine and analogs of these compounds. In addition, a
method for preparing salts, particularly pharmaceutically
acceptable salts, of the foregoing compounds is also
provided.
The reaction scheme for preparing etorphine (Fig. 1
(III)) and related analogues is shown in Fig. 13. As
depicted, thebaine [1] is reacted with an excess of methyl
vinyl ketone under reflux for about 1 hour. Any remaining
ketone is then distilled off, preferably under pressure.
The thick oil can be dissolved in warm methanol, cooled to
allow crystallization and the crystals recovered.
Conveniently, the crystals can be washed several times with
ice cold methanol and dried to yield 7~-acetyl-6,14-endo-
etheno-tetrahydrothebaine [~]. This compound [2] is then
reacted with a Grignard reagent of the formula RMgX to form
a tertiary alcohol with the R group of the Grignard reagent
at the 7~ position of thebaine as represented by compound
t3] in Fig. 13.
The R group of the Grignard reagent is lower alkyl and
X is a halogen. Analogs of etorphine are thus prepared by
varying the R group. As used herein lower alkyl refers to
alkyl groups containing one to six carbon atoms. These
groups may be straight, branched or cyclic chains and
include such groups as methyl, ethyl, propyl, isopropyl,
butyl, sec-butyl, isobutyl, t-butyl, pentyl (amyl),
PCT/US93/08869
W094/06426
2l~52o7
18
isopentyl (i-amyl), neopentyl (n-amyl), hexyl, cyclopentyl,
cyclohexyl and the like. When R is propyl, the final
product [4] is etorphine. Preferred R groups for etorphine
and its analogues are n-propyl, n-butyl, n-amyl, i-amyl or
cyclohexyl. ;
Compound t3~ is thus prepared by rea-cting an anhydrous
solution of [2] with the desired Grignà`rd reagent for a time
and under conditions for the formation of the tertiary
alcohol. For example, t2] can be dissolved in benzene and
refluxed with the Grignard reagent for several hours, or as
needed, until the reaction goes to completion. Upon
completion, the anhydrous solution can be added to an
immiscible aqueous solution (e.g., saturated ammonium
chloride), the product extracted into that aqueous solution,
followed by separation of the organic and aqueous layers.
The aqueous layer is recovered and extracted with ether or
other suitable solvent several times to yield a neutral
solution containing compound t3]- Compound t3] can be
further purified by recrystallization.
Reaction of compound t3] under strong basic conditions
yields the 7~-tl-hydroxy-1-methylR]-6,14-endo-etheno-tetra-
hydrooripavine compounds t4] Such compounds t4] can be
recovered by extraction, filtration, recrystallization and
the like. When R is n-propyl, then t4] is etorphine.
Any of various salts of etorphine or its analogues t4]
can be prepared by reacting the free base with the desired
free acid and recovering the resultant salt by
crystallization, filtration or the like. In a preferred
embodiment, t4] is dissolved in an alcoholic ether solution
and an ether solution containing the desired acid is added
thereto until the reaction mixture reaches a pH of about 2.
Additional ether is added thereto until a crystalline solid
[5] forms. The solid is collected, washed with ether and
dried. If desired the solid can be recrystallized.
Examples of various acids which can be used to prepare salts
of t4] are provided in Example 9.
~1 A~ ~ PCT/US93/08869
W094/0~26 ~ 0 7
19
A reaction scheme for the preparation of DHE, its
analogues and salts is shown in Fig. 14. Like etorphine,
the starting material is thebaine and the first reaction
with methyl vinyl ketone is identical to form the 6,14-endo-
etheno derivative of the thebaine [2]. However, [2] isfirst subjected to catalytic hydrogenation and recovery to
yield the 6,14-endo-etheno derivative of thebaine [3] before
reaction with a Grignard reagent to produce [4]. The
remaining synthetic steps proceed as described above for
synthesis of etorphine. Hence, for DHE and related
compounds, ~2] is hydrogenated to produce, [3]; t3] is
reacted with a Grignard reagent to produce [4]; and [4] is
reacted with strong base to produce [5], the free base DHE
or a related analog. Finally, [5] is reacted with an acid
as described above to produce [6].
For DHE and related compounds, the R group of the
Grignard reagent is lower alkyl as defined herein before.
When R is n-propyl, then t5] of Fig. 14 is DHE. The
preferred R group for DHE and an analogues thereof are n-
propyl and i-amyl, respectively.
Another aspect of the invention is directed to
pharmaceutical compositions of the opioid compounds of the
present invention including dihydroetorphine and its
analogues, etorphine and its analogues, ohmefentanyl as well
as pharmaceutically acceptable salts of any of the foregoing
compounds.
Dosage forms (compositions) suitable for administration
can contain from about 10 ~g to about 1000 ~g of active
ingredient per unit. In these pharmaceutical compositions
the active ingredient will ordinarily be present in an
amount of about 0.5-95% by weight based on the total weight
of the composition.
The active ingredient can be administered sublingually
in solid dosage forms, such as capsules, tablets, and
powders, or be administered parenterally in sterile liquid
dosage forms.
W094/06426 PCT/US93/08869
21 4~ 20~ 20
Gelatin capsules contain the active ingredient and
powdered carriers, such as lactose, sucrose, mannitol,
starch, cellulose derivatives, magnesium stearate, stearic
acid, and the like. Similar diluents can be used to make
compressed tablets. Both tablets and capsules can be
manufactured as sustained release products to provide for
continuous release of medication over a period of hours.
Compressed tablets can be sugar coated or film coated to
mask any unpleasant taste and protect the tablet from the
atmosphere.
In general, water, a suitable oil, saline, aqueous
dextrose (glucose), and related sugar solutions and glycols
such as propylene glycol or polyethylene glycols are
suitable carriers for parenteral solutions. Solutions for
parenteral administration preferably contain a water soluble
salt of the active ingredient, suitable stabilizing agents,
and if necessary, buffer substances. Antioxidizing agents
such as sodium bisulfite, sodium sulfite, or ascorbic acid,
either alone or combined, are suitable stabilizing agents.
Also used are citric acid and its salts and sodium EDTA. In
addition, parenteral solutions can contain preservatives,
such as benzalkonium chloride, methyl- or propyl-paraben,
and chlorobutanol.
Suitable pharmaceutical carriers are described in
Reminqton's Pharmaceutical Sciences, A. Osol, a standard
reference text in this field.
Yet another aspect of this invention provides a
pharmaceutical composition which comprises a low- or non-
addictive opioid analgesic, or a pharmaceutically acceptable
salt thereof, in admixture with naloxone which is an opioid
antagonist. The low- or non-addictive opioid analgesics are
those compounds as provided herein, e.g. etorphine, DHE,
ohmefentanyl and the like, in amounts as provided herein.
The subject compositions are thus useful to avoid
diversion or abuse of take-home preparations of solid forms,
e.g. tablets of low- or non-addictive opioid analgesics
~ W094/06426 2 1 4 5 2 ~ 7 PCT/US93/~8869
21
administered orally or sublingually to uses other than
detoxification or severe pain relief. Since naloxone has
low oral or sublingual bioavailability, an amount of
naloxone can be introduced into the preparations that has no
effect when taken orally or sublingually but antagonizes the
effect of the low or non-addictive opioid analgesic, e.g.
DHE, when the preparation is dissolved in water and
injected. The amount of naloxone can be readily determined
by one of ordinary skill in the art.
A still further aspect of this invention provides a
pharmaceutical composition which comprises a low- or non-
addictive opioid analgesic, or a pharmaceutically acceptable
salt thereof, in admixture with a "replacement opioid".
These compositions are useful for treating chronic or acute
pain as well as opioid addictions. The dosages appropriate
for each use can be readily determined by one of ordinary
skill in the art. The low or non-addictive opioid
analgesics include the compounds provided herein, e.g.
etorphine, DHE, ohmefentanyl and the like, in amounts as
provided herein. These pharmaceutical compositions are
provided in formulations as described above.
The "replacement opioid" is a bimodally-acting opioid
that has both inhibitory and excitatory effects on opioid
receptors. The replacement opioid is formulated into
compositions in an amount effective to (partially or wholly)
relieve or suppress the withdrawal symptoms of opioid
addiction, or an amount to alleviate pain. The amount of
the low- or non-addictive opioid analgesic in these
compositions is likewise that amount necessary to provide
relief or suppression of withdrawal symptoms or to alleviate
pain when used with a replacement opioid. One of ordinary
skill in the art can readily determine a suitable ratio and
dosage of analgesic and replacement opioid.
For example, suitable dosage forms for administration
can contain from about 10 to about lOOO ~g of the analgesic.
When the analgesic is DHE, a preferred formulation for a
W094/06426 PCT/US93/08869 ~
2l4s2a~
22
sublingual dosage contains about 20 ~g to about 40 ~g DHE,
or the corresponding equivalent of a salt thereof, per
tablet. A preferred formulation for an injectable dosage
form contains about 20 ~g to about 100 ~g of DHE or the
corresponding equivalent of a salt thereof.
Similarly, suitable dosage formsSfor administration of
the replacement opioid can contain~àn amount which provides
from about 5 mg to about 100 mg pe~ day to the patient.
Preferred replacement opioids include morphine, methadone,
fentanyl and buprenorphine.
In a preferred embodiment, these pharmaceutical
compositions contain DHE, or a pharmaceutically acceptable
salt thereof such as DHE hydrochloride, and either methadone
or morphine. Methadone is preferred for treating drug
addiction whereas morphine is preferred for treating pain.
The examples serve to illustrate the present invention
and are not to be used to limit the scope of the invention.
REFERENCES CITED
Bentley, K.W. & Hardy, D.G.:New potent analgesics in the
morphine series. Proc. Chem. Soc. p.220, 1963.
Bentley, K.W. & Hardy, D.G.:Novel analgesics and molecular
rearrangements in the morphine-thebaine group. III.
Alcohols of the 6,14-endo-ethenotetrahydro-oripavine
series and derived analogues of n-allylnormorphine and
norcodeine. J. Amer. Chem. Soc. 89:3281-3286, 1967.
Blane, G.F. & Robbie, D.S.:Trial of etorphine hydrochloride
(M99 Reckitt) in carcinoma pain:preliminary report.
Brit. J. Pharmacol. Chemother. 20:252-253, 1970.
Blane G.F., Boura, A.L.A., Fitzgerald, A.E. and Lister,
R.E.: Actions of etorphine hydrochloride (M99):A potent
morphine- like agent. Brit. J. Pharmac. Chemother.
30:11-22, 1967.
Casy, A.F. & Parfitt, R.T.: Opioid Analgesics: ChemistrY and
RecePtors~ Plenum Press, New York, 1986.
~ W094/06426 2 1 ~ 5 2 0 7 PCTJUS93/08869
23
Crain, S.M. & Shen, K.-F.:Opioids can evoke direct receptor-
mediated excitatory effects on sensory neurons. Trends
Pharmacol. Sci. 11:77-81, 1990.
Crain, S.M. & Shen,K.-F.:After chronic opioid exposure
sensory neurons become supersensitive to the excitatory
effects of opioid agonists and antagonists as occurs
after acute elevation of GM1 ganglioside. Brain Res.
575:13-24, 1992a.
Crain, S.M. & Shen, K.-F.:After GM1 ganglioside treatment of
sensory neurons naloxone paradoxically prolongs the
action potential but still antagonizes opioid
- inhibition. J. EXD. Pharmacol. Ther. 260:182-186,
1992~.
Crain, S.M., Shen, K.-F. & Chalazonitis, A.:Opioids excite
lS rather than inhibit sensory neurons after chronic
opioid exposure of spinal cord-ganglion cultures.
Brain Res. 455:99-109, 1988.
Deneau, G.A. & Seevers, M.H.: Drug dependence. In: Lawrence
D.R., Bacharach, A.L. eds. Evaluation of Druq
Activities: Pharmacometrics. Vol. 1, London: Academic
Press, 1964, pp. 167-179.
Gross, R.A., Moises, H.C., Uhler, M.D. & Macdonald, R.C.:
Dynorphin A and cAMP-dependent protein kinase
independently regulate neuronal calcium currents.
Proc. Natl. Acad. Sci. 87:7025-7029, l99o.
Huang, M & Qin, B.Y.: Acta Pharmacol. Sinica, 3(1):9, 1982.
Jaffe, J.H.:Drug addiction and drug abuse. in The
Pharmacoloqical Basis of Therapeutics, 8thed. (eds.
Gilman,A.G.,Rall, T.W., Nies, A.S. & Taylor, P.)
Pergamon Press, N.Y. pp.522-573, 1990.
~asinski, D.R., Griffith, J.D. & Carr, C.B.:Etorphine in man
1. Subjective effects and suppression of morphine
abstinence. Clin. Pharmacol. Ther. 17:267-272, 1975.
Pasternak, G.W.: The Opiate Receptors, Humana Press, New
Jersey, 1988.
Shen, K.-F. & Crain, S.M.:Dual opioid modulation of the
W094/06426 PCT/US93/08869 ~
2145207
24
action potential duration of mouse dorsal root ganglion
neurons in culture. Brain Res. 491:227-242, 1989.
Shen, K.-F. & Crain, S.M.:Cholera toxin-A subunit blocks
opioid excitatory effects on sensory neuron action
potentials indicating mediation by Gs-linked opioid
receptors. Brain Res. 525:225-231, 1990.
Shen, K.-F. & Crain, S.M.:Chronic~selective activation of
excitatory opioid receptor functions in sensory neurons
results in opioid"dependence" without tolerance. Brain
Res. (in press), l99Z.
Wei, E., Loh, H.H. & Way, E.L.: Quantitative aspects of
precipitated abstinence in morphine-dependent rats. J.
Pharmacol. Ex~. Therap. 184:398-403, 1973.
W.H.O. Expert Committee on Dependence-Producing Drugs, WHO
Tech. Rep. Ser. Vol 343, p.5, 1966.
Winger, G., Skjoldager, P. & Woods, J.H.:Effects of
buprenorphine and other opioid agonists and antagonists
on alfantanil- and cocaine-reinforced responding in
Rhesus monkey. J.Pharmacol. Ex~. TheraP. 261:311-317,
1992.
~mple 1
SELECTIVE INHIBITORY BUT NOT EXCITATORY EFFECT OF ETORPHINE
AND DIHYDROETORPHINE ON THE ACTION POTENTIAL DURATION OF
SENSORY DORSAL ROOT GANGLION NEURONS IN CULTURE
Tissue culture: The experiments were carried out on
dorsal root ganglion (DRG) neurons in organotypic explants
of spinal cord with attached DRGs (from 13-day-old fetal
mice) after 3 to 5 weeks of maturation in culture. The DRG-
cord explants were grown on collagen-coated coverslips in
Maximow depression-slide chambers. The culture medium
consisted of 65% Eagle's minimal essential medium, 25% fetal
bovine serum, 10% chick embryo extract, 2 mM glutamine and
0.6% glucose. During the first week n vitro, the medium
was supplemented with nerve growth factor (NGF-7s) at a
concentration of about 0.5 ~g/ml to enhance survival and
~ W094/06426 2 1 ~ S 2 0 7 PCT/US93/08869
growth of the fetal mouse DRG neurons.
~ lectrophvsioloqical recordinqs: The culture coverslip
was transferred to a recording chamber containing about 1 ml
of Hanks' balanced salt solution supplemented with 5 mM Ca2+
and 5 mM Ba2+ (BSS) to provide a prominent baseline response
for pharmacological tests. Intracellular recordings were
obtained from DRG perikarya selected at random within the
ganglion with micropipette probes. The micropipettes were
filled with 3M KCl lresistance about 60-100 megohms) and
were connected via a chloridized silver wire to a
neutralized input capacity preamplifier (Axoclamp 2A) for
current clamp recording. After impalement of a DRG neuron,
brief (2 msec) depolarizing current pulses were applied via
the recording electrode to evoke action potentials (at a
frequency of 0.1 hz). Recordings of the action potentials
were stored on a floppy disc using the p-clamp program (Axon
Instruments) in a microcomputer (IBM AT-compatible).
Dru~ test: Drugs were applied by bath perfusion with a
manually operated push-pull syringe system at a rate of 2-3
ml/min. Perfusion of test agents was begun after the action
potential and the resting potential of the neuron reached a
stable condition during >4 min pretest periods in control
BSS. Opioid-mediated changes in the APD were considered
significant if the APD alteration was >10% of the control
value for the same cell and was maintained for the entire
test period (about 5 min). The APD was measured as the time
between the peak of the APD and the inflection point on the
repolarizing phase.
OPioid ResPonsiveness: The opioid responsiveness of DRG
neurons was analyzed by measuring opioid-induced alterations
in the APD of DRG perikarya. DRG neurons in DRG-cord
explants were examined for sensitivity to acute application
of etorphine or dihydroetorphine at fM to ~M concentrations.
None of the cells (n=12) showed APD shortening or
prolongation in 1 fM etorphine. However, naloxone-
reversible APD shortening was observed in 25% of the cells
W O 94/06426 PC~r/US93/08869 ~
2l452~
26
(n=8) after application of pM and nM concentrations of
etorphine and in 100% of the cells (n=7) after application
of ~M concentrations of etorphine (Figs. 3 and 4). None of
the DRG neurons tested with different concentrations of
etorphine (n=13) showed APD prolongation.
These results are in sharp contra~t to other mu, delta
or kappa opioids (e.g. morphine, methadone, DAG0, DPDPE,
DADLE, dynorphin (amino acids 1-13) or (amino acids 1-17)
and U-50,488H), each of which show bimodal action such that
low concentrations (<nM) evoked excitatory APD-prolonging
effects and higher concentrations (-~M) evoked inhibitory
APD-shortening effects on many DRG neurons (Fig. 4; Table
1). For Fig. 4, data were obtained from 11 neurons for
etorphine test, half of which were tested with all four
concentrations of etorphine (from fM to ~M).
Like etorphine, electrophysiologic tests with
dihydroetorphine (over fM-~M ranges) on DRG neurons (n=15)
showed concentration-dependent inhibitory APD shortening
effects, with threshold at fM-pM, and no evidence of
excitatory APD prolonging effects (Fig. 4).
~ W094/06426 2 1 4 ~ 2 ~ 7 PCTIUS93/08869
27
TABLE 1
Alteration of action potential duration of
dorsal root ganglion neurons treated
5with high and low concentrations of opioids
-
A~teration of Action Potential Duration
Opioid at low Opioid at high
concentrationconcentration
< nM ~M
Morphine Prolongation Shortening
DAGO Prolongation Shortening
20 DADLE Prolongation Shortening
DPDPE Prolongation Shortening
U-50,488H Prolongation Shortening
Dynorphin 1-13 Prolongation Shortening
Dynorphin 1-17 Prolongation Shortening
30 Met-enkephalin Prolongation Shortening
Leu-enkephalin Prolongation Shortening
~-endorphin Prolongation Shortening
Methadone Prolongation Shortening
Fentanyl Prolongation Shortening
40 Levorphenol Prolongation Shortening
Thebaine Prolongation Shortening
Etorphine* Shortening Shortening
Dihydroetorphine* Shortening Shortening
* Selectively activate inhibitory (APD shortening), but not
excitatory, opioid receptor-mediated functions.
W O 94/06426 P~r/US93/08869
2 l 4 S ~ ~ ~ 28
Exam~le 2
ENHANCED INHIBITORY EFFECT OF ETORPHINE ON CHRONIC OPIOID-
TREATED, ADDICTED SENSORY NEURONS THAT HAD BECOME
SUPERSENSITIVE TO THE EXCITATORY EFFECTS OF BIMODALLY ACTING
OPIOID AGONISTS AND TO NALOXONE
Druq tests: Mouse DRG-cord expla~nts, grown for >3
weeks as described in Example 1, wer~ chronically exposed to
the bimodally acting (excitatory/inhibitory) delta/mu opioid
agonist, DADLE (3 ~M) or morphine (1 ~M) for 1 week or
longer. Electrophysiological recordings were made as in
Example 1.
Results: After such chronic exposure, DRG neurons are
supersensitive to the excitatory effects of opioids (Crain &
Shen 1992a; Shen & Crain, 1992). Whereas pM-nM Dyn (amino
acid 1-13) is generally required to prolong the APD of naive
DRG neurons (Fig. 4), fM levels and lower are effective at
prolonging the APD after chronic opioid treatment (Fig. 5,
traces 1-4). In contrast, acute application of etorphine to
chronic DADLE-treated neurons effectively shortened the APD
of the same DRG neurons that showed supersensitive
excitatory responses to low concentrations of bimodally-
acting opioids (Fig. 5, traces 6-9). Furthermore, the
inhibitory APD-shortening effect of etorphine on DRG neurons
appears to be significantly enhanced. While pM etorphine
was effective in shortening the APD of 25% of the DRG
neurons tested in naive explants (Figs. 3 and 4), this low
opioid concentration was effective in all of the chronic
DADLE-treated DRG neurons tested in the presence of 1 ~M
DADLE (n=4; Fig. 5, traces 5 and 6). This same low
concentration of etorphine (pM) was effective in 71% of the
chronic morphine-treated (1 ~g/ml) DRG neurons tested in the
presence of 1 ~g/ml morphine (n=7). Dose response tests on
chronic DADLE-treated DRG neurons showed, in fact, that the
magnitude of the APD was progressively shortened when the
acute etorphine concentration was increased sequentially
from 1 fM to 1 ~M (Fig. 5, traces 6-9).
The opioid antagonist, naloxone (nM-~M), does not alter
W094/06426 2 1 ~ ~ 2 0 7 PCT/US93/08869
29
the APD of naive DRG neurons (Crain & Shen 1992a, b). In
contrast, after chronic opioid, such as DADLE treatment,
acute application of low concentrations of naloxone prolongs
the APD of sensory neurons (Crain et al, lgs2b; Shen &
Crain, 1992). The naloxone-induced excitatory APD-
prolonging effect on chronic opioid-treated DRG neurons is
shown in Fig. 6, traces 1 and 2. Acute application of low
concentrations of etorphine (pM-nM) effectively blocks the
naloxone-induced APD prolongation of DRG neurons (n=3; Fig.
6, traces 3 and 4) whereas bimodally acting opioids are
ineffective.
Since etorphine and dihydroetorphine elicit potent
inhibitory effects on naive sensory neurons even when
applied at extremely low (pM) concentrations and show no
signs of concomitantly activating excitatory opioid
receptors on these cells, these in vitro electrophysiologic
analyses predict that application of etorphine and
dihydroetorphine in vivo at the relatively low doses
required to produce analgesia (<1,000 times lower than
morphine) are not addictive even after sustained application
for treatment of chronic pain.
Example 3
SUPPRESSION OF WITHDRAWAL SYMPTOMS BY DHE
IN MORPHINE-DEPENDENT RATS
Mor~hine-dependent rat model: Wistar rats of both
sexes, 120-150 g body weight, were administered morphine
subcutaneously (s.c.) twice a day (8:00 a.m., 4:00 p.m.)
starting at a dose of 20 mg/kg/day, with an increment of
20 mg/kg/day for 5 consecutive days until the final dose
reached 100 mg/kg/day.
Naloxone (NLX) precipitation for the scorinq of
withdrawal sYmptoms: 3-4 hrs after administering the last
dose of morphine (or other test drug(s)), withdrawal
symptoms of morphine-dependent rats were precipitated by
intraperitoneal (i.p.) injection of naloxone (4 mg/kg).
Naloxone-induced withdrawal symptoms were monitored for 1 hr
w094/06426 l 45 ~ ~ 7 PCT/US93/08869 -
thereafter and scored according to the method of Wei et al,
1973.
Animal qrouPs: After 5 days of morphine addiction, the
animals were divided into 7 groups according to Table 2.
Each group contained 5-6 rats.
Groups 1, 2, and 3 received 20 mg/kg morphine (4 times
the EDso for analgesia), 9 mg/kg methadone (9 times the ED50
analgesia) or 6 ~g/kg DHE (12 times the ED5~ analgesia) by
i.p. injection, respectively. These opioid agonists were
injected 15-30 min before naloxone precipitation was
initiated. After the naloxone withdrawal test was
completed, groups 1, 2, and 3 were continued on morphine 100
mg/kg (s.c.) for another 4 consecutive days. A second
naloxone precipitation test was given on the 4th day but
- lS only saline was administered (i.p.) prior to naloxone.
The first naloxone precipitation test was performed on
the animals of groups 4, 5, and 6 in the same manner as for
groups 1-3, except the administration of opioid agonists 15-
30 min prior to the naloxone test. For the second naloxone
test, groups 4, 5 and 6 received loo mg/kg morphine (s.c.)
twice a day, 3 ~g/kg DHE 4 times a day, or 6 mg/kg
methadone, 4 times a day, instead of morphine at 100 mg/kg,
for 4 days, respectively. The second naloxone test was
performed as above on the 4th day.
After the first naloxone precipitation test, Group 7
animals were given saline (s.c.) as control for 4 days
before the second naloxone test.
The body weight of the animals was monitored during the
entire period.
Results: One to 2 min after intraperitoneal injection
of naloxone, the morphine-dependent rats began to show
naloxone induced withdrawal symptoms with a peak response
occurring within 15 min. An hour later the body weight of
the animals was greatly reduced. Intraperitoneal injection
of morphine (20 mg/kg), DHE (6 ~g/kg) or methadone (9 mg/kg)
prior to the administration of naloxone suppressed the
W094/06426 2 1 g 5 2 0 7 PCT/US93/08869
naloxone induced withdrawal symptoms of the rats. No
significant differences in suppressing effect were detected
among these three opioid substitutes. For morphine, DHE and
methadone, prevention of body weight loss was 43.5%, 49.8%
and 48.15%, respectively, and suppression of other
withdrawal symptoms was scored as 45.5%, 63.7% and 49.4%,
respectively.
After naloxone precipitation, the body weight of the
dependent rats continued to decrease. The loss of body
weight reached its maximum 24 h after naloxone
precipitation. A gradual weight recovery was achieved by 9O
h.
Subcutaneous injection of morphine, DHE or methadone
was given for several days after the first naloxone
precipitation test. The loss of body weight of morphine-
dependent rats was found to be reduced in all three groups
treated with opioid agonists. Su~cutaneous administration
of morphine ~one hour after naloxone precipitation) reversed
the body weight loss in 3 hours, with occasional weight gain
in some of the rats. A complete recovery of weight loss
was observed 48 h later. The effect of subcutaneous
injection of DHE or methadone on body weight loss was not as
dramatic as with morphine. However, both opioids did
prevent further body weight loss. When compared with the
untreated control group (saline injected), the effect of
both DHE or methadone on body weight loss was highly
significant (Fig. 7).
After the first naloxone precipitation test, some of
the animals continued to be maintained on morphine (s.c.).
Four days later, a second naloxone test was given. The
second naloxone test resulted in more severe withdrawal
symptoms relative to the first test. In contrast, in those
animals that were treated with DHE (s.c., 4 days) instead of
morphine, the second naloxone test failed to precipitate any
withdrawal symptoms except minor loss in body weight. In
the animals maintained with methadone (s.c., 4 days), the
W094/06~26 PCT/US93/08869 -
?~45~1
32
second naloxone injection precipitated less severe
withdrawal symptoms in comparison to the morphine group, yet
more severe when compare with the DHE group (Figs. 8 and 9).
Table 2
Animal Groups Used to Test the Suppression of NLX-induced
Withdrawal Symptoms by Different Opioids
1 0 Development of Continued
Horphine Pretreotment 1st M~inten~nce ~ith Pretre~tment 2nd
Animal D~ e ~15-30' prior ~LX Opioids (15-30~ prior NLX
Groups 5 daysto 1st test) Test4 days to 2nd test) Test
1 Horphine Horphine NLXMorphine 100 mg/k~Saline ULX
20--~100 mg~kg~20 mg/k~)
2 S~me ~s 1Methadone NLX S~me ~s 1 S~line NLX
(9 ~9/k9)
2 0
3 S~me ~s 1 DHE NLX S~me ~s 1 S-line ULX
. (6 ~/kg)
Same ~ 1 NLXHorphine 50 m~/kg -- ~ILX
Same ~s 1 -- ~LXDHE 3 ~g/kg -- NLX
6 S~me ~ 1 -- NLX~ICt'~ _ 6 mg/kg -- NLX
7 SAme ~S 1 -- NLX Saline -- NLX
~Y~m~le 4
ANTI-ADDICTIVE EFFECTS OF DHE TREATMENT
OF MORPHINE-DEPENDENT MONKEYS
Morphine-de~endent monkey model: Seven male rhesus
monkeys (Macaca mulatta, 3.4-5 kg) were injected with
morphine (s.c.) twice a day (8:00 a.m., 4:00 p.m.), starting
at a dose of 10 mg/kg/day and increasing the dose by
increments of 5 mg/kg/day every third day until the dosage
reached 50 mg/kg/day on the 24th day. This dosage was
continued for another 10 days prior to performing drug
tests.
Staqe 1 druq tests: The monkeys were randomly divided
into 2 groups. At 24 h after withdrawal of morphine, Group
A (4 animals) received 3 ~g/kg DHE (s.c.) every 3 h. The
interval between DHE administration was increased gradually
so that by the 3rd day, DHE was only given twice a day, and
then stopped for 2 days of observation. Group B (3 animals)
was treated in the same manner as group A except, this group
received saline instead of DHE. After completion of these
~ W094/06426 2 1 4 5 2 0 7 PCT/US93/08869
33
tests, all the animals were treated with morphine for 12
consecutive days by administration of 50 mg/kg/day morphine
(s.c.) twice a day. The test was repeated except the Group
A monkeys received the saline controls and the Group B
monkeys received the DHE treatment. Withdrawal symptoms of
the animals were observed and scored according to Deneau &
Seevers (1964), during the entire experimental period.
Sixteen hours after withdrawal of morphine, withdrawal
symptoms began to appear in the morphine-dependent monkeys.
Symptoms were moderate at first and included yawning,
salivation, agitation and fear. These signs became more
severe as time went on. Within 20-60 h after withdrawal of
morphine, the animals' withdrawal symptoms included
vomiting, tremor, teeth-gritting on chain, eye closing,
lying on its side and dyspnea. All these symptoms are
indicative of extreme agitation. After 60 h these symptoms
gradually subsided. By 120 h after withdrawal of morphine,
some moderate withdrawal symptoms were still detected (Fig.
10). One week later all the withdrawal symptoms had
disappeared.
In sharp contrast, all of these withdrawal symptoms
were completely suppressed by DHE one minute after its
administration (3 ~g/kg. s.c.). Two and a half to three
hours later, withdrawal symptoms reappeared which were again
suppressed by another dose of DHE (Fig. 11). This
suppressing effect of DHE on morphine withdrawal symptoms
was observed with each monkey. DHE continued to be
effective at suppressing withdrawal symptoms for 3-4 days
with repeated injections at 2.5-3 h intervals.
Discontinuation of DHE injection at 80 h after morphine
withdrawal did not trigger any withdrawal symptoms,
indicating that the animals had not become dependent on DHE
during this substitution treatment.
Staqe 2 druq tests: After the stage 1 experiments, all
7 monkeys were administered morphine (s.c.) at a dose of 50
mg/kg/day for 7 days. The morphine-addicted monkeys were
W094/06426 PCT/US93/08869 ~
34
then randomly divided into 3 groups. Group 1 was maintained
with 8.C. injection of 2s mg/kg morphine twice a day for 9
days. Group 2 was substituted with DHE by s.c. injection of
3 ~g/kg DHE (equi-analgesic dose) four times a day for 4
days, of 1.5 ~g/kg DHE three times a day for 2 days and then
twice a day for 3 days. Group 3 was substituted with
methadone by s.c. injection of 6 mg/kg methadone (equi-
analgesic dose) four times a day for 4 days, of 3 mg/kg
three times a day for 2 days and then twice a day for 3
lo days.
Sixteen hours after the last injection of opioid, each
animal was precipitated with naloxone (1 mg/kg, s.c.) to
evaluate the severity of naloxone withdrawal symptoms for 1
day. Seven days later, another naloxone precipitation test
was performed on these monkeys for 1 day. After completion
of all tests, 3 monkeys were randomly selected for morphine
addiction (25 mg/kg, s.c., twice a day for 7 days).
Naloxone precipitation tests were performed twice on these 3
monkeys, the first trial given after the last injection of
morphine and the second trial given 7 days thereafter.
Since the action period of DHE and methadone is
relatively short, some moderate withdrawal symptoms appeared
during the 6 hr intervals between injections on the first 3
days. After these 3 days, the withdrawal symptoms became
milder and gradually disappeared.
Naloxone precipitation tests were carried o~t after 9
days of substitution treatment with DHE or methadone. For
the monkeys maintained on morphine, naloxone injection
precipitated a series of withdrawal symptoms after 15 sec.
These symptoms included squeaking, coughing, rolling,
tremor, vomiting, agitation, teeth-gritting on chain,
dyspnea, and finally lying down on the ground. The animals
recovered by 7 days later. The monkeys substituted with
methadone showed moderate naloxone withdrawal symptoms
including yawning, placing hands on the belly, tremor of
extremities, frequent teeth-gritting on chain and agitation.
~ W094/06426 2 1 ~ 5 2 0 7 PCT/US93/08869
However, those animals substituted with DHE showed no change
in behavior both before and after naloxone precipitation.
Table 3 shows the scores of naloxone withdrawal symptoms
from the 3 different groups of monkeys. Once morphine was
fully excreted from the body (7 days after withdrawal),
naloxone no longer precipitated any withdrawal symptoms.
The naloxone precipitation test was used to evaluate
whether these animals were dependent on morphine or had
become dependent on the substitution opioid.
Fig. 12 illustrates the variations in the scores of
withdrawal symptoms in monkeys after DHE or methadone
substitution relative to compulsive withdrawal. In the
compulsive withdrawal group (upper trace) that withdrawal
sympto~s reached a maximal score during the first several
days, but returned to zero by 7 days after abrupt morphine
withdrawal. On day 9, naloxone no longer precipitated any
withdrawal symptoms. For the methadone substitution group
(middle trace), the withdrawal symptoms during the first
several days were partially suppressed. On day 9, naloxone
precipitated withdrawal symptoms, suggesting that the
animals have already switched to methadone dependence. For
DHE substitution group (lower trace) only minor withdrawal
symptoms were observed. Naloxone precipitation tests on day
g did not trigger any withdrawal symptoms. These results
indicate that DHE is an ideal low- or non-addictive
substitution drug for treatment of opioid abstinence
problems.
W094/06426 PCT/US93/08869 ~
2,~.4~2~ 36
Table 3
Scores of naloxone withdrawal syndromes
in morphine dependent monkeys with or without
5DHE or methadone substitution treatment
Scores of withdrawal syndromes (X + SD)
Treatment-
(n) 1st naloxone 2nd naloxone
precipitationb precipitationC
morphine (4) 49.0 + 2.2 1.5 + 1.0
DHE (3) 2.0 + 1.0 1.0 + 1.0
methadone (3)17.0 + 4.6 1.3 + 1.2
Daily treatment dosages were 50 mg/kg morphine (divided
into 2 subdoses), 12 ~g/kg DHE (divided into 4 subdoses)
decreased to 3 ~g/kg (divided into 2 subdoses), and 24 mg/kg
methadone (divided into 4 subdoses) decreased to 6 mg/kg
(divided into 2 subdoses).
b The first naloxone precipitation test was performed 16
h after the last injection of opioid.
' The second naloxone precipitation test was performed
7 days after the last injection of opioid.
*** p< 0.01, compared to the morphine group. For the DHE
group compared to the methadone group, then p<O.O1.
~xam~le 5
DHE ELICITS POTENT LOW- OR NON-ADDICTIVE ANALGESIA
IN ACUTE AND CHRONIC PAIN PATIENTS
The results in the first stage clinical trial showed
that none of the 20 volunteers had euphoria feeling after
DHE administration through sublingual route at 60 ~g single
dose. At high dosage (e.g. > 1 mg per day), dizziness,
nausea, vomiting and lethargy appeared. The results from
second stage clinical trial demonstrated that DHE can
effectively relieve postoperative pain and pain caused by
terminal stage of cancer. The effective rate of 730 cases
that have complete medical records was 97.6%. Among them,
the effective rate of acute pain in patients from
W094/06426 2 1 ~ 5 2 0 7 PCT/US93/08869
departments of surgery, obstetric and gynecology approached
nearly 100%. The effective rate for relief of chronic
severe pain and terminal stage of cancerous pain was 90-95%.
The clinical data indicate that the analgesic effect of DHE
is substantial with only mild side effects. DHE treatment
was effective in those terminal stage cancer patents that
were unresponsive to morphine or pethidine (demerol)
treatment. No cross tolerance to DHE was found in these
patients. Long-term use of DHE can result in tolerance;
lo however, the degree of tolerance is less than that observed
with morphine or pethidine.
Clinical treatment with DHE has been conducted in more
than one hundred thousand patients in China. As an
analgesic, the main disadvantage of DHE is its short action
period (about 3-4 hours). Compared with morphine, DHE has
high analgesic effect and low addictivity, whereas morphine
has relatively low analgesic effect and high addictivity.
During many years of trials using DHE as an analgesic, no
cases of drug abuse were ever reported. This phenomena may
be attributed to the strict regulation of DHE treatment.
The medication period for ordinary pain is typically limited
to 1 week; whereas, for patients with terminal cancer pain,
the treatment period is much longer. Although some of the
patients became tolerant to DHE after long-term use, there
is a slight chance that a few patients may become addicted
to the drug after long-term use (e.g., > 6 months).
W O 94/06426 PC~r/US93/08869 -
~4s2a~
38
Examle 6
DHE SUB~ llON TREATMENT SUPPRESSES WITHDRAWAL SYMPTOMS IN
OPIATE ADDICTS WITHOUT CONCOMITANT DHE ADDICTION
~eneral ~rotocol: Institution of DHE therapy as a
substitute drug began with a sufficient dose on days 1-3 to
suppress completely the withdrawal symptoms. On days 4-7,
the dosage was reduced and by days 8-10 the DHE substitution
therapy was terminated. This protocol was followed because
(1) withdrawal symptoms are most severe during the first 3
days after abrupt withdrawal of heroin or other addictive
opioid; (2) withdrawal symptoms gradually decline and
disappear after 7-10 days; and (3) consecutive use of DHE as
a substitution agent for 7-10 days does not produce any
self-dependence.
DHE administration: More than 300 cases of chronic
heroin users were treated for 7-10 days with DHE in 10
hospitals in China. DHE was administered either sublingual
in tablet form (40 ~g) or by intramuscular injection (20 ~g)
or by intravenous dripping (20 ~g). The tablet form was
used more often. At the onset of withdrawal symptoms,
sublingual administration of 1-2 tablets (20-40 ~g) of DHE
effectively suppressed the symptoms. Sustained suppression
of withdrawal symptoms required repeated DHE medication
every 2-4 h. Total dosage was adjusted according to the
severity of the withdrawal symptoms. Typically after 4 days
of DHE medication, the dosage required to suppress
withdrawal symptoms was generally reduced. The entire
course of DHE substitution was generally 7 days. (In one
instance of overdose of DHE, respiratory side effects
occurred.)
For addicts whose withdrawal symptoms were so severe
and violent that sublingual medication was not enough to
subdue them, intramuscular injection of DHE (20 ~g) gave
instant relief. The addict generally became quiet and
cooperative. However, to maintain the therapeutic effect,
it was necessary to administer DHE by intravenous dripping
W 0 94/06426 2~452a7 PC~r/US93/08869
39
(100 ~g in Soo ml glucose saline for 6-10 hr). The
transfusion rate depended upon the severity of symptoms: the
drip rate was increased when the patient showed sign of
restlessness or decreased when the patient was quiet and
= 5 complained of lethargy. In severe cases, intravenous
transfusion of DHE was maintained for 3-4 days with
progressive decrease in dosage. On day 4 or 5, intravenous
dripping of DHE was converted to sublingual DHE
administration and terminated on day 7. Occasionally
treatment was prolonged to 8-9 days, but never more than 10
days to prevent possible occurrence of dependence. Hence,
by using this optimal 7-10 day treatment period DHE is
effectively employed as a substitute for drug addiction
therapy.
The effectiveness of DHE substitution therapy was
evaluated on day 10 by the naloxone precipitation test (0.4-
0.8 mg naloxone, intramuscular injection) and urine analysis
of the residual amount of opioid. The treatment course was
considered successful if both tests were negative.
One of the primary advantages of DHE over methadone
substitution therapy is the early onset of DHE
effectiveness. Withdrawal symptoms were markedly relieved
after 10-20 min of sublingual administration or 5 min of
intramuscular injection of DHE. In contrast, with methadone
substitution the first dose usually begins at 10 mg and is
increased every hour until the therapeutic effect is
achieved. Such treatment can last from one to several hours
before symptomatic relief. This period is intolerable to a
patient with severe withdrawal symptoms. Furthermore,
methadone substitution therapy often results in the rebound
of withdrawal symptoms, suggesting dependence on methadone.
In contrast, cessation of DHE administration generally
proceeded smoothly. As with methadone, the side effects of
DHE were negligible during substitution treatment for drug
addiction. Since DHE is short acting (only 2-4 hr),
frequent administration may be necessary. To avoid this,
W O 94/06426 - PC~r/US93/08869 -
,2~4s2~
intravenous dripping of DHE is recommended. However,
intravenous administration can only be prescribed in the
hospital and is not applicable to the ordinary drug
rehabilitation clinic. Since methadone is effective orally
(once or twice a day), one alternatlve is to combine DHE and
methadone treatments. For example, DHE is used initially
for swift control of the withdrawal symptoms and is then
replaced by methadone to maintain the suppressing effect for
2-3 days. The treatment is then switched back to DHE on a
decreasing dosage regime until DHE is no longer needed
(usually another 5-10 days). This combined therapy is safe,
pragmatic and convenient.
~AmPle 7
According to the following synthetic route (Figure 15),
dihydroetorphine hydrochloride was prepared using thebaine
[Compound 1] as a starting material.
(a) PreDaration of 7a-acetvl-6.14-endo-etheno-di77hYdro-
thebaine r ComPound 2~
A mixture of thebaine (49.8 g, 0.16 mol, Compound
1) and methyl vinyl ketone (150 mL) was refluxed in a 250 mL
round-bottomed flask for 1 hour. The excess ketone was
distilled off under reduced pressure. Warm methanol (60 mL)
was added to dissolve the thickened oil. Under cooling a
crystalline product formed and it was filtered, washed with
ice-cold methanol for 2-3 times and dried. The solid (56.6
g, yield 93%) was obtained, m.p. 120-122C.
(b) PreParation of 7-acetyl-6,14-endo-ethano-tetrahYdro-
thebaine r ComPound 31
A mixture of compound [2] (19.1 g, 0.05 mol), 10% Pd/C
(4 g) and absolute alcohol (200 mL) was catalytically
hydrogenated under the hydrogen pressure of 40-50 kg/cm2 at
55-60C with stirring for 8-12 hours. The catalyst was
filtered off and filtrate was concentrated. After cooling,
the crystalline product was collected and washed with
absolute alcohol. The white solid (15.7 g, yield 82~) was
obtained, m.p. 135-137C.
W094/06426 2 1 g 5 2 ~ 7 PCT/US93/08869
41
(c) Pre~aration of 7~-rl-fR)-hYdroxY-l-methylbutyll-6,14-
endo-ethano-tetrahYdrothebaine r Compound 4l
The Grignard reagent was prepared by the reaction of
bromopropane (127.9 g, 1 mol) and magnesium (24.3 g, 1 mol)
in 1100 mL of absolute ether. The compound [3] (99.7 g,
0.26 mol) in 1100 m~ of benzene was added dropwise with
vigorous stirring and under reflux. The reaction mixture
was stirred and refluxed for another 2 hours. A saturated
ammonium chloride solution was poured into the mixture,
which separated the organic layer. The aqueous layer was
extracted with ether for several times. The combined ether
extract was washed with water until the washings became
neutral. The extract was dried over anhydrous magnesium
sulphate. After evaporating the solvent, the crude product
was recrystallized from absolute alcohol. The white solid
(75-79 g, yield 67-71%) was obtained, m.p. 184-186C.
(d) PreParation of 7~- r 1- (R)-hYdroxy-l-methYlbutvll-6,14-
endo-ethano-tetrahYdrooriPavine r ComPound 5l
A mixture of compound t4] (85.5 g, 0.2 mol), diethylene
glycol (1700 mL) and potassium hydroxide (616 g) was placed
in a four-necked flask. After the low-boiling substance was
distilled off under a nitrogen stream, the reaction mixture
was heated at an internal temperature of 200-210C and
stirred for 14-16 hours. The resultant mixture was poured
into 10 L of water to dissolve it. A suitable amount of
ammonium chloride was added until the solid separated out
completely. The solid was filtered, washed with water until
the washings became neutral, dried and extracted with
absolute ether. After ether was distilled off, the crude
product was recrystallized from methanol. The pure compound
t54-58 g, yield 66-70%) was obtained, m.p. 204-206C.
Chemical and Spectral Analysis: C25H3sNO~
Calc:% C 7-2.63 H 8.53 N 3.38
Found:~ C 72.40 H 8.65 N 3.22
35M+: 413
IR (KBr): r (cm-~) 3528, 3478, 3314, 3186 (OH);
W O 94/06426 PC~r/US93/08869 -
2,~4S2~
42
1213 (Ar-0); 1110 (tert. alc. C-0); 3040
(Ar-H); 1633, 1612, 1498 (Ar C=C); 1152
(C-O-C); 1283,1085, 882 (Ar-O-C); 2790,
2764 (=N--CH3); 820 (Ar--H).
IHNMR: (CDCl3, 400 MHz, ~ ppm)
0.78 (m, lH, 8~H); 0.87 (m, 3H, 25CH3);
1.02, 2.66 (m, 2H, 18CH2); 1.07 (m, lH,
813H); 1.32 (s, 3H, 22CH3); 1.47 (m, 2H,
23CH2); 1.40 (m, 2H, 24CH2); 1.64, 2.00
(m, 2H, 15CH2); 1.76, 1.90 (m, 2H,
l9CH2); 1.78 (m, lH, 7CH); 2.21 (d, lH,
lO~H); 2.26, 2.44 (m, 2H, 16CH2); 2.30
(s, 3H, 17CH3); 2.65 (m, lH, 9CH); 3.09
(d, lH, lOl~H); 3.46 (s, lH, 30H); 3.55,
(s, 3H, 20CH3); 4.37 (s, lH, 5CH); 5.32
(s, lH, 260H~; 6.50 (d, lH, lCH); 6.67
(d, lH, 2CH).
Hl,H2 = 8 Hz, Hlo~Hlo~r = 18.3 Hz,
H2s~H24 = 6 Hz, H~5,H~5 = 11.7 Hz,
H4~HIo~ = 6.5 Hz.
CNMR: (CHCl3, 100 Hz, ~ ppm)
C25=14.65, C24=15.86, C~9=17.87, Clo=21.94,
C22=23.36, C8=29.73, C~8=31.70, C,s=35.38,
C~4=36.00, C23=43.44, C~=43.70, C~6=45.13,
C~45.40,C2~=46.30, C2o=52.62, C9=61.23,
C~3=76.13, C6=30.38, C5=96.95, C~=116.76,
C2=119.35, C~=127.57, C~2=131.92,
C3=137.72, C4=145.67.
(e) PreParation of dihYdroetorPhine hYdrochloride rComPound
61
The free base t5] (14 g, 0.034 mol) was dissolved in a
mixed solvent of absolute alcohol (400 mL) and absolute
ether (640 mL). An amount of ether saturated with dried
hydrogen chloride was added dropwise until the solution
35 became acidic (pH = 2). After adding 200 mL of absolute
ether, a crystalline solid formed. The white solid was
21gS2~7
W094/0~26 ~ PCT/US93/08869
43
collected, washed with absolute ether and dried. The
desired final product (14-14.5 g, yield 92-94%) was
obtained, m.p. 297-298C, [a]20 -6S.
Chemical and Spectral Analysis: C2sH3sNO~.HCl
Calc:% C 66.72 H 8.06 N 3.11
Found:% .C 66.70 H 8.15 N 3.09
Exam~le 8
PREPARATION OF VARIOUS DIHYDROETORPHINE (DHE) SALTS
AND ANALYSIS OF DURATION AND POTENCY
OF THE ANALGESIC EFFECTS THEREOF
A total of 26 dihydroetorphine (DHE) salts were
prepared according to step (e) of Example 7 except the
various acids listed below were substituted for HCl.
I. Structures of 26 acids employed to form derivatives of
dihydroetorphine salts
*1.COOH
I
COOH
*2. TH2COOH
CH2COOH
*3. CH3COOH
*4. HO-CH-COOH
I
CH2COOH
5.CH3CHCOOH
OH
6.HO-TH-COOH
HO-CH-COOH
*7-TH2COOH
HO-C-COOH
CH2COOH
W O 94/06426 PC~r/US93/08869 -
2~4~2~
8. ~OH
s ~L COOH
9. COOH
HO ~OH
OH
* 10 . HCCOOH
Il
HCCOOH
11. CH2CH=CHCH=CHCOOH
12 . CH3 ( CH2 ) loCOOH
2013. CH3(CH2)14COOH
14 . CH3 (CH2) 16COOH
* 15 . C6H5CH (OH) COOH
*16 . (C6Hs) 2C (OH) COOH
17. C76Hs2O46
3 018 . C6H5CHCOOH
CH20H
19. C6HsSO3H
20. H IOH H
HOH2CC-(~-C--(~-COOH
O H OH
Cl~,O~
llo J
o~
~ W094/06426 2 1 ~ ~ 2 0 7 PCT/US93/08869
4S
21. ~ COOE~
N
22. CH3CHCOOH
0}~
23. C6H4(NH2)(S03H)
*24. HBr
*25. HO2CCH(NH2)CH2COOH
*26. HO2CCHNH2(CH2)2COOH
20 27. ~
c~;_C_D ~3
28.
~CU~
c~ c~ ~
* Preliminary screening tests have been done.
A "mouse hot plate" (55C + 0.5C) method as previously
described (Huang and Qin, 1982) was used to score % analgesia
to measure the potency of each DHE salt which was injected to
animals subcutaneously. The ED50 (the dose that gives rise to
50% analgesia as calculated by the following formula) was
measured for the DE~E salts shown in Table 4.
W094/0~6 PCT/US93/08869 ~
~4~
46
Pain thre~hold (~ec) _ Pain threshold (sec)
after admini~trat~on before admini~tration
Analge~ia % =
60 - Pain threshold (~ec) before administration
A dose of 5 EDso was used for~feach salt to measure the
corresponding analgesic duration. "Analgesia %" was
recorded at 90, 120 and 150 min after administration (Table
5).
In summary, an ED50 (~g/kg) in the range of 0.50 to 2.0
was observed with the 12 DHE salts tested, indicating an
equivalent level of analgesic effect conferred by these
salts (see data presented in Table 4). Furthermore, except
for acetyl DHE, DHE maleate, and DHE amygdalate, all DHE
salts demonstrated an equivalent level of analgesia over a
120-150 min duration.
Table 4
Analgesic Effect of Various DHE Salts
Salts of DHE- EDso (~g/Kg)
DHE hydrochloride 1.43
Acetyl DHE (27) 0.47
DHE maleate (10) 0.58
DHE succinate (2) 0.83
DHE oxalate (1) 0.68
DHE acetate (3) 0.51
DHE malate (4) 0.62
DHE asparagate (25) 1.12
DHE glutamate (26) 0.65
DHE amygdalate (15) 0.61
DHE dibenzoylhydroxyl acetate (16) 1.29
DHE citrate (7) 1.73
The numbers following each salt correspond
to the numbered compounds of Example 80
-
~ W094/06426 2 1 4 ~ 2 0 7 PCT/US93/08869
47
Table 5
Ans~gesia X (x ~ SD)
S~lts of DHE
90 min 120 min 150 ~in
DHE hydrochloride 42.39 ~ 31.34 20.05 ~ 10.91 28.49 ~ 14.21
- Ac~tyl DHE 5.96 ~ 12.65 8.85 ~ 9.47 ~D
DHE maleate 15.84 13.82 ~D ~D
DHE succinate 26.16 ~ 14.86 30.77 ~ 42.35 32.26 ~ 35.89
DHE oxalate 14.21 ~ 6.37 19.35 ~ 24.14 ND
0 DHE scetate 21.64 ~ 10.11 29.54 ~ 30.93 UD
DHE malate 26.92 ~ 23.75 22.36 ~ 19.29 ND
DHE ~ te 40.96 ~ 37.28 35.94 ~ 45.06 21.44 ~ 35.68
DHE glutamate 29.33 ~ 36.15 9.27 ~ 12.57 14.09 ~ 17.07
DHE nmygdalate 29.15 ~ 26.n 1.87 ~ Z.97 ND
1 5 DHE dibenzoylhy- 45.01 ~ 49.66 13.85 l 17.49 ND droxyl acetate
DHE citrste 56.12 ~ 46.42 25.95 37.46 14.27 ~ 14.49
.
ExamPle 9
The pharmaceutical preparations of dihydroetorphine
hydrochloride include a parenteral injectable sterile
solution and a sublingual tablet.
(a) Preparation of iniectable dihYdroetorphine
2 5 h Y dr O C hl O r i de
This injectable is a pharmaceutical preparation in a
sterile aqueous solution. Its outward appearance is
transparent and colorless. Each ampule contains 20 ~g of
said compound as the active ingredient in 1 mL of solution.
The prescription is shown as follows:
Dihydroetorphine hydrochloride 20 mg
0.001 N Hydrochloric acid q.s. 1000 mL
(b) PreParation of dihYdroetorPhine hYdrochloride
sublinqual tablet
The outward appearance of the sublingual tablet is
white. Each tablet contains 20 ~g or 40 ~g of said compound
as active ingredient.
For example, the prescription for 10000 tablets at 40
~g per tablet is as follows:
Dihydroetorphine hydrochloride 400 mg
w094/06426 ~ 45 ~ ~ ~ PCT/US93/08869 -
48
Lactose:starch:mannitol:sucrose (3:1:3:3) 600 g
Sodium carboxymethyl cellulose (1% aque sol'n) 18 mL
Ethyl alcohol (50%) 24 mL
Magnesium stearate 6 g
According to the above-mentioned prescription, a
designated amount of dihydroetorphine hydrochloride was
weighed and dissolved in 50% ethyl alcohol. This solution
was added dropwise onto excipients under mechanical stirring
to ensure uniformity. Meanwhile, 1% sodium carboxymethyl
cellulose solution was added dropwise. The soft material
thus formed was screened through a 20-mesh sieve and the
- same operation was repeated for 3 times. The product was
then dried in an oven at 60C. Magnesium stearate was added
as a -lubricating agent for the tablets.
