1111111111111111 IIIIII IIIII 11111 1111111111 111111111111111 IIIII 1111111111 1111111111 11111111
`US 20020162125Al
`
`(19) United States
`(12) Patent Application Publication
`Salmon et al.
`
`(10) Pub. No.: US 2002/0162125 Al
`Oct. 31, 2002
`(43) Pub. Date:
`
`(54) METHODS AND COMPOSITIONS FOR THE
`MODULATION OF NEUROGENIC
`INFLAMMATORY PAIN AND PHYSICAL
`OPIATE WITHDRAWAL
`
`(76)
`
`Inventors: Anne-Marie Salmon, Paris (FR);
`Susumu Sekine, Kanagawa (JP);
`Marina Picciotto, Guilford, CT (US);
`Jean-Pierre Changeux, Paris (FR)
`
`Correspondence Address:
`Finnegan Henderson Farabow Garrett &
`Dunner
`Suite 700
`1300 I Street, N.W.
`Washington, DC 20005 (US)
`
`(21)
`
`Appl. No.:
`
`10/091,127
`
`(22)
`
`Filed:
`
`Mar. 6, 2002
`
`Related U.S. Application Data
`
`(60)
`
`Provisional application No. 60/273,349, filed on Mar.
`6, 2001.
`
`Publication Classification
`
`Int. Cl.7 .................................................. A0lK 67/027
`(51)
`(52) U.S. Cl. ................................................... 800/3; 800/18
`
`(57)
`
`ABSTRACT
`
`A method of screening for a compound that is an antagonist
`of calcitonin gene related peptide ( aCGRP) is provided. The
`method comprises: exposing a mutant mouse to a com(cid:173)
`pound. The mutant mouse has a genome that comprises a
`homozygous disruption of the aCGRP gene, wherein the
`disruption results in the mutant mouse lacking detectable
`levels of endogenous aCGRP as compared to a wild type
`mouse. The response of the mutant mouse to a nociceptive(cid:173)
`inducing stimulus is determined. A difference in response
`compared to a wild type mouse is indicative of the com(cid:173)
`pound functioning to alter aCGRP activity. In a preferred
`embodiment, the disruption comprises the insertion of a
`transgene. A compound identified by the method is also
`provided. The compound is useful for ameliorating neuro(cid:173)
`genic inflammatory pain and/or physical opiate withdrawal.
`
`

`

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`Patent Application Publication Oct. 31, 2002 Sheet 2 of 3
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`Patent Application Publication Oct. 31, 2002 Sheet 3 of 3
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`

`

`US 2002/0162125 Al
`
`Oct. 31, 2002
`
`1
`
`METHODS AND COMPOSITIONS FOR THE
`MODULATION OF NEUROGENIC
`INFLAMMATORY PAIN AND PHYSICAL OPIATE
`WITHDRAWAL
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`[0001] This application is based on and claims the benefit
`of U.S. Provisional Application No. 60/273,349, filed Mar.
`6, 2001 (attorney docket no. 03495.6062) The entire disclo(cid:173)
`sure of this application is relied upon and incorporated by
`reference herein.
`
`BACKGROUND OF THE INVENTION
`
`[0002] The present invention relates to methods and com(cid:173)
`positions for the modulation of neurogenic inflammatory
`pain and/or physical opiate withdrawal. In a particular
`embodiment, the methods and compositions of the invention
`include methods and compositions for the specific inhibition
`of calcitonin gene related peptide (aCGRP).
`
`[0003] Calcitonin gene related peptide ( a CG RP)
`is
`expressed in a variety of cell types in both central and
`peripheral nervous systems and the characteristics of the
`gene encoding aCGRP have been disclosed (1). Among its
`various functions, aCGRP has been suggested to contribute
`to local, neurogenic inflammatory responses (2) and to
`nociception (3). aCGRP is expressed in 40% of the sensory
`neurons of the dorsal ganglia, being present in both the
`peripheral Ao and C fibres and in the primary afferent nerves
`to the spinal cord.
`
`[0004] Noxious thermal or mechanical stimulation evokes
`release of aCGRP in the superficial dorsal horn (4). In turn,
`a CG RP potentiates the local effects of other pain mediators,
`including substance P (3). Similarly injection of aCGRP in
`peripheral tissue can elicit visceral pain (5). In contrast,
`central administration of aCGRP in periacqueductal area
`and nucleus raphe magnus produces antinociceptive effects
`(6).
`
`[0005] The putative role of aCGRP in nociception is
`further complicated by its relationship with opioids. Intrath(cid:173)
`ecal a CG RP is known to decrease the analgesia produced by
`opioid agonists (7), whereas naloxone blocks the antinoci(cid:173)
`ceptive effects of aCGRP ( 6). Experiments with the a CG RP
`antagonist 8-37CGRP suggest that aCGRP contributes to
`the development of tolerance to the antinociceptive effect of
`morphine (8). Finally, aCGRP positive fibers are present in
`telencephalic areas involved in motivation, for instance the
`shell of nucleus accumbens and the central nucleus of the
`amygdala (9).
`
`[0006]
`In summary, a great need exists for the definitive
`identification of compounds for the treatment of neurogenic
`inflammatory pain and/or physical opiate withdrawal.
`
`SUMMARY OF THE INVENTION
`
`[0007] This invention aids in fulfilling these needs in the
`art.
`
`[0008] The present invention relates, first, to methods and
`compositions for the modulation of neurogenic inflamma(cid:173)
`tory pain and/or physical opiate withdrawal.
`
`[0009] The compositions of the invention include, in one
`embodiment, compositions, including pharmaceutical com(cid:173)
`positions, for the specific inhibition of aCGRP activity.
`Such compositions of the present invention can include, but
`are not limited to, inhibitors of aCGRP gene activity, such
`as, for example, aCGRP antisense, triple helix and/or
`ribozyme molecules, and inhibitors of aCGRP activity.
`[0010]
`In one embodiment, this invention provides a
`method of screening for a compound that is an antagonist of
`calcitonin gene related peptide (aCGRP). The method com(cid:173)
`prises: exposing a mutant mouse to a compound. The mutant
`mouse has a genome that comprises a homozygous disrup(cid:173)
`tion of the aCGRP gene, wherein the disruption results in
`the mutant mouse lacking detectable levels of endogenous
`aCGRP as compared to a wild type mouse. The response of
`the mutant mouse to a nociceptive-inducing stimulus is
`determined. A difference in response compared to a wild
`type mouse is indicative of the compound functioning to
`alter aCGRP activity. In a preferred embodiment, the dis(cid:173)
`ruption comprises the insertion of a transgene.
`
`[0011] This invention also provides a compound, which is
`an antagonist of aCGRP, identified by the method of the
`invention.
`
`[0012] Further, this invention provides a method for ame(cid:173)
`liorating neurogenic inflammatory pain comprising: admin(cid:173)
`istering a compound capable of specifically inhibiting
`aCGRP activity to an animal having neurogenic inflamma(cid:173)
`tory pain symptoms in an amount sufficient to inhibit the
`aCGRP activity in the animal so that symptoms of neuro(cid:173)
`genic inflammatory pain are ameliorated.
`
`[0013] Another method of the invention involves modu(cid:173)
`lating physical opiate withdrawal comprising: administering
`a compound capable of specifically inhibiting aCGRP activ(cid:173)
`ity to an animal having physical opiate withdrawal symp(cid:173)
`toms for a time and in an amount sufficient to inhibit the
`a CG RP activity in the animal so that symptoms of physical
`opiate withdrawal are ameliorated.
`
`[0014] Another method
`for modulating neurogenic
`inflammatory pain comprises: administering a compound
`capable of specifically inhibiting expression of a CG RP to an
`animal having neurogenic inflammatory pain symptoms for
`a time and in an amount sufficient to inhibit the expression
`of aCGRP in the animal so that symptoms of neurogenic
`inflammatory pain are ameliorated.
`
`[0015] Another method of the invention involves modu(cid:173)
`lating physical opiate withdrawal comprising: administering
`a compound capable of specifically inhibiting expression of
`aCGRP to an animal having physical opiate withdrawal
`symptoms for a time and in an amount sufficient to inhibit
`the expression of aCGRP in the animal so that symptoms of
`physical opiate withdrawal are ameliorated.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0016] This invention will be described with reference to
`the drawings in which:
`
`[0017] FIG. l(A) shows the effects of capsaicin (20 µg)
`injected into the dorsal skin of the right hindpaw of aCGRP
`-/- mice and aCGRP +/+ mice (n=6/group), over 15 min(cid:173)
`utes. Licking manifestations started immediately after injec(cid:173)
`tion in both experimental groups.
`
`

`

`US 2002/0162125 Al
`
`Oct. 31, 2002
`
`2
`
`[0018] FIG. l(B) shows the effects of subcutaneous injec(cid:173)
`tions of formalin (20 µl of 2% paraformaldehyde in PBS)
`(n=lO/group). The cumulative time spent licking the hind(cid:173)
`paw during 0-5 minutes (acute phase) and 5-20 minutes
`(tonic phase) following injection is shown.
`[0019] FIG. 1.(C) shows the results of induction of local
`edema by 20 µl of carrageenan (2% in PBS) injected into the
`dorsal skin of the right hindpaw measured as increased
`hindpaw thickness at 6 h ( early time) and 72 h ( delayed
`time) after injection (n=6/group). All data were analysed
`using Student's T-tests (* P<0.05; ** P<0.01 vs aCGRP +/+
`mice).
`[0020] FIG. 1.(D) shows the effects of acetic acid (10
`µ1/kg of 0.6% in water) or MgSO 4 (120 mg/kg) injected into
`the abdomen on writhing measured over 20 minutes post
`injection (n=lO/group ).
`[0021] FIG. 2. shows the effects of (A) morphine (1 and
`5 mg/kg, ip; n=lO) and (B) nicotine (0.5 and 2 mg/kg, sc;
`n=8) in the tail flick assay, 30 min after drug injection. The
`results are presented as % MPE (maximum possible effect),
`where MPE=( test-control)/( cutoff-control)xlO0. Basal tail
`flick latencies were 1.73+/-0.13 in aCGRP +/+ mice and
`1.79+/-0.12 in aCGRP -/-mice.Data were analysed using
`ANOVA followed by Tukey test(* P<0.05; ** P<0.01 vs
`aCGRP +/+ mice).
`[0022] FIG. 3.(A) shows tolerance to morphine in the
`tail-flick assay: day 1 and 2, 50 mg/kg, day 3 and 4, 100
`mg/kg; aCGRP -/- and +/+ mice did not differ in their
`development of tolerance to morphine analgesia. Acute
`morphine analgesia remained at the starting levels, respec(cid:173)
`tively in -/- and +/+ mice treated with saline (not shown).
`[0023] FIG. 3.(B) shows the results of heroin self-admin(cid:173)
`istration in aCGRP -/- (n=5) and aCGRP +/+ (n=5) mice
`(mean +/- SEM injections per session at each dose under a
`Fixed-Ratio 2 Time-Out 20 sec schedule of reinforcement,
`injection volume 50 µlover 2 sec). Following acquisition of
`stable heroin self-administration at 15.0 µg/kg/injection dose
`(3 consecutive sessions <+/-20% variation, > 70% active
`lever responding), mice were allowed to self-administer
`heroin at each dose during two daily two-hr sessions in a
`Latin square dose order. After all heroin doses had been
`tested, saline vehicle was substituted for heroin until
`responding stabilized for at least 2 consecutive sessions
`( data points above "saline"). Intake of 30 µg/kg/injection
`dose differed significantly from a 7.5 and 15.0 µg/kg!
`injection doses, *, P<0.05 (means comparisons after appro(cid:173)
`priate two-way analysis of variance on self-administration).
`[0024] FIG. 3.(C) shows the results of analysis of somatic
`signs of withdrawal: morphine dependence was induced by
`repeated ip morphine injections for one week: day 1 and 2,
`50 mg/kg, day 3 and 4, 100 mg/kg; days 5-7, 100 mg/kg
`twice/day; day 8, 100 mg/kg in the morning. Then, with(cid:173)
`drawal signs were precipitated by injection of naloxone (0.1
`n=4,0.2 n=5 and 2.0 n=8 mg/kg, ip, 2 h after final morphine
`injection). aCGRP -/- mice exhibited significantly fewer
`total withdrawal signs than aCGRP +/+mice(*, P<0.05, **
`P<0.01). Naloxone (2 mg/kg, ip) in saline-treated control
`mice (mutant and wild type) did not induce any withdrawal
`signs (data not shown).
`[0025] FIG. 3.(D) also shows the difference between
`aCGRP -/- mice and aCGRP +/+ mice when the opiate
`
`dependence syndrome was precipitated by naloxone in mice
`repeatedly injected with morphine. FIG. 3(D) shows that the
`frequency of each somatic sign of morphine withdrawal
`measured was reduced in the aCGRP (-/-) mice Gumping
`is presented as an example in FIG. 3D).
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`[0026] The present invention relates to methods and com(cid:173)
`positions for the modulation of neurogenic inflammatory
`pain and/or physical opiate withdrawal in mammals, includ(cid:173)
`ing humans. More particularly, to establish the role of
`aCGRP, the behavioral responses of aCGRP null-mutant
`mice to chemical pain stimuli, mainly inflammatory, and to
`opiates was examined.
`[0027] As used herein, "modulation" of neurogenic
`inflammatory pain and/or physical opiate withdrawal in
`mammals means increase or decrease of neurogenic inflam(cid:173)
`matory pain and/or physical opiate withdrawal in said mam(cid:173)
`mals compared to such a pain and/or withdrawal in mam(cid:173)
`mals to which the methods/compositions of the invention
`have not been applied/administered.
`[0028] As used herein, "ameliorating" neurogenic inflam(cid:173)
`matory pain and/or physical opiate withdrawal means reduc(cid:173)
`ing symptoms of neurogenic inflammatory pain and/or
`physical opiate withdrawal.
`[0029]
`It was previously shown that homozygous aCGRP
`mutant (-/-) mice, obtained by targeted disruption of exon
`5 of the CT/CG RP gene, displayed a reduced antinociceptive
`tail-flick response to morphine (10). aCGRP (-/-) mice,
`backcrossed on the C57Bl/6 strain to the 7th generation,
`were indistinguishable from wild type aCGRP ( +/+) mice in
`several spontaneous behaviors, including escape response
`latencies to acute thermal pain stimuli using both the hot(cid:173)
`plate (55° C.) and tail-flick tests (10). It was concluded that
`aCGRP is not involved in acute pain sensation.
`[0030] When chemical inflammatory pain was produced
`by capsaicin injection in the hindpaw, a procedure known to
`massively release aCGRP and substance P from nerve
`terminals, a significant attenuation of hindpaw licking
`response was found in aCGRP (-/-) mice (FIG. IA). These
`mice also showed reduced hindpaw-licking time during the
`early (acute) phase of the formalin test, which provides a
`measure of direct chemical stimulation of the primary affer(cid:173)
`ents. Hindpaw licking began immediately following capsai(cid:173)
`cin injections in both aCGRP -/- and aCGRP +/+ mice.
`Moreover a significant decrease in hindpaw-licking time
`was observed in aCGRP mutant mice during the second
`(tonic) phase of the formalin test (FIG. 1B). This second
`phase is thought to involve peripheral inflammatory events
`and ongoing tonic activation of nociceptors.
`[0031] The contribution of the peripheral release of
`aCGRP to neurogenic inflammatory response was further
`confirmed by the profound reduction of edema produced by
`carrageenan injections in the hindpaw of aCGRP (-/-) mice
`when compared with aCGRP ( +/+) (FIG. lC).
`[0032] Two models of visceral pain were also used: acetic
`acid that produces a delayed inflammatory response, and
`MgSO that causes a non-inflammatory response. After intra(cid:173)
`peritodeal injections of acetic acid, significantly fewer epi(cid:173)
`sodes of writhing, a marker of intestinal discomfort, were
`
`

`

`US 2002/0162125 Al
`
`Oct. 31, 2002
`
`3
`
`observed in aCGRP (-/-) than in aCGRP (+/+) mice (FIG.
`1D). In contrast, the episodes of writhing induced by intra(cid:173)
`peritoneal injections of MgSO 4 did not differ in the aCGRP
`(-/-) mice and aCGRP ( +/+) mice.
`
`[0033] Overall, these results indicate that aCGRP is criti(cid:173)
`cal for the production and, possibly, the transmission of
`somatic and visceral pain signals associated with neurogenic
`inflammation.
`
`[0034] Next examined was the analgesic response to mor(cid:173)
`phine and nicotine, two drugs known to act independently on
`the
`tail-flick
`nociception (11). In aCGRP -/- mice,
`responses were attenuated by 1 mg/kg and to a lesser extent
`by 5 mg/kg morphine (FIG. 2A), indicating that opiate
`effects on tail flick spinal reflex are only partially mediated
`by a CG RP. In contrast, the analgesic effects of nicotine were
`significantly increased at both 0.5 and 2.0 mg/kg dose in
`both tail-flick (FIG. 2B) and hot-plate test (data not shown),
`suggesting a possible decrease in aCGRP induced nicotinic
`acetylcholine receptor desensitization. These experiments
`also indicate that the descending pain inhibitory system of
`aCGRP (-/-) mice is not defective, but subject to graded
`activation.
`
`[0035] Tolerance to the antinociceptive effect of opiates
`was studied using a protocol of repeated morphine injec(cid:173)
`tions. The progressive decrease in the amplitude of mor(cid:173)
`phine-elicited antinociceptive response in the tail-flick test
`did not differ between aCGRP (-/-) mice and aCGRP (+/+)
`mice (FIG. 3A), indicating that aCGRP is not involved in
`morphine tolerance.
`
`[0036] To investigate whether the reinforcing properties of
`opiates were affected by the aCGRP mutation, aCGRP
`(-/-) and aCGRP (+/+)mice were trained to self-administer
`heroin in a discriminated lever press operant task. As shown
`in FIG. 3B, aCGRP (-/-) mice and aCGRP (+/+)mice did
`not differ in acquisition or maintenance of heroin self(cid:173)
`administration, showing overlapping dose response curves,
`nor did they differ in acquisition of a food-reinforced lever
`press operant (data not shown). These data indicate that
`aCGRP does not contribute significantly to heroin reinforce(cid:173)
`ment.
`
`[0037] However, when the opiate dependence syndrome
`was precipitated by naloxone (0.1, 0.2 and 2.0 mg/kg) in
`mice repeatedly injected with morphine, a major difference
`between aCGRP (-/-) mice and aCGRP mice was observed
`(FIGS. 3C and D). Total withdrawal scores were signifi(cid:173)
`cantly lower in aCGRP -/- mice across a range of naloxone
`dose (FIG. 3C). The frequency of each somatic sign of
`morphine withdrawal measured was also reduced in the
`aCGRP -/- mice Qumping is presented as an example in
`FIG. 3D). The opiate withdrawal syndrome is known to
`produce a general malaise and intense aversive emotional
`state (12). It is hypothesized to be generated by adaptive
`mechanisms to the prolonged exposure to exogenous opi(cid:173)
`ates. Recent evidence indicates that attenuation of both
`neurogenic inflammatory responses and physical opiate
`withdrawal syndrome is also observed in mice with targeted
`deletion of the substance P NKl receptor (13).
`
`[0038] The present results indicate that an important com(cid:173)
`ponent of the withdrawal malaise is the peripheral nervous
`system that mediates neurogenic inflammatory responses.
`This system can be modulated by increased autonomic
`
`output observed during opiate withdrawal resulting in ampli(cid:173)
`fication of peripheral neurogenic pain signals. In turn,
`increased pain signals from the periphery can augment the
`withdrawal malaise by enhancing both the affective and
`somatic components of opiate withdrawal. The contribution
`of peripheral signals to emotional processing is a well
`established phenomenon (14). This phenomenon can extend
`to the interpretation of the opiate dependence syndrome,
`initially supported by data from targeted deletion of both
`NKl and CT/CGRP genes in mice. Independently from this
`view, the present results indicate that aCGRP antagonists
`can serve as a treatment of both neurogenic inflammatory
`pain and physical opiate withdrawal.
`
`[0039] Described herein are compounds, including phar(cid:173)
`maceutical compositions, which can be utilized for the
`amelioration of neurogenic inflammatory pain and/or physi(cid:173)
`cal opiate withdrawal. More specifically, said compounds
`are antagonists of calcitonin gene related peptide (aCGRP).
`Such compounds can include, but are not limited to, small
`peptides, small organic molecules, antisense, and triple helix
`molecules. Compositions can include polyclonal and/or
`monoclonal antibodies for the modulation of such pain
`and/or withdrawal symptoms.
`
`[0040] A variety of methods can be utilized for the iden(cid:173)
`tification of the compounds of the invention. The identifi(cid:173)
`cation methods comprise isolated protein-based assays, cell(cid:173)
`based assays, and whole animal assays.
`
`[0041] Assays that serve to test inhibition capacity in an in
`vivo situation are preferred. Typically, such assays include
`administering to an animal a test compound and measuring
`its effect. With respect to inhibitors of neurogenic inflam(cid:173)
`matory pain and/or physical opiate withdrawal, animal
`assays using rodents are preferred. As described herein, a
`variety of assays can be employed, including the hot-plate
`assay, the tail-flicks assay, the hindpaw capsaisin injection
`assay, the carrageenan rat paw edema assay, the acetic acid
`assay for visceral pain, and/or the MgSO 4 assay for visceral
`pain. Literature citations describing how these assays are
`carried out are as follows:
`
`[0042]
`"Calcitonin Gene Related Peptide (aCGRP)
`in capsaicin-Sensitive Substance P-Immunoreactive
`Sensory Neurons in Animals and Man: Distribution
`and Release by Capsaicin."
`
`[0043] Franco-Cereda A, Henke H., Lundberg J. M.,
`Peterman J. B., Hokfelt T., and Fischer J. A, Peptides, 8,
`399-410, 1987.
`
`[0044]
`"Differential contribution of the two phases of
`the formalin test to the pattern of C-fos expression in
`the rat spinal cord: studies with remifentanil and
`lidocaine." Abbadie C., Taylor B. K., Peterson M. A,
`and Basbaum A I., Pain, 69, 101-110, 1997.
`
`[0045] "A method for determining loss of pain sen(cid:173)
`sation." D' Amour F. E., and Smith D. L., J. Phar(cid:173)
`macol. Exp. Ther., 72, 74-79, 1941.
`
`[0046]
`"Induction of cyclooxygenase-2 causes an
`enhancement of writhing response in mice." Mat(cid:173)
`sumo H., Naraba H., Ueno A., Fujiyoshi T.,
`Murakami M., Kudo I., and Oh-ishi S., Eur. J.
`Pharmacol., 352 (1), 47-52,1998.
`
`

`

`US 2002/0162125 Al
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`4
`
`[0047] Compounds that ameliorate neurogenic inflamma(cid:173)
`tory pain and/or physical opiate withdrawal can also be
`tested in cell-based assays to test inhibitory capacity within
`the cell. Such cell-based assays can be utilized to test a
`number of features of a potential inhibitor, including, for
`example, the compound's ability to enter the cell, its cyto(cid:173)
`toxicity, as well as its ability to act as an inhibitor once inside
`the cell. Further, cell-based assays can function to identify
`compounds that act more indirectly to inhibit aCGRP activ(cid:173)
`ity.
`
`[0048] A typical cell-based assay can involve contacting a
`cell expressing the activity of interest with a test compound
`for a time and measuring the inhibition of such an activity.
`For measurements, for example, whole cells can be lysed
`according to standard techniques and tested for the presence
`of aCGRP activity.
`
`[0049] Among the inhibitors of the invention are nucleic
`acid antisense and/or triple helix molecules that act to inhibit
`expression of the aCGRP gene involved in one or more of
`the activities relating to neurogenic inflammatory pain and/
`or physical opiate withdrawal processes. Such inhibitors can
`be utilized in methods for the amelioration of neurogenic
`inflammatory pain and/or physical opiate withdrawal.
`
`[0050] Antisense approaches can be utilized to inhibit or
`triple helix
`prevent translation of mRNA transcripts;
`approaches to inhibit transcription of the gene of interest
`itself. Antisense approaches involve the design of oligo(cid:173)
`nucleotides (either DNA or RNA) that are complementary to
`aCGRP mRNA. The antisense oligonucleotides bind to the
`complementary mRNA transcripts and prevent translation.
`Absolute complementarity, although preferred,
`is not
`required. A sequence "complementary" to a portion of an
`RNA, as referred to herein, means a sequence having
`sufficient complementarity to be able to hybridize with the
`RNA, forming a stable duplex. In the case of double(cid:173)
`stranded antisense nucleic acids, a single-strand of the
`duplex DNA may thus be tested, or triplex formation may be
`assayed. The ability to hybridize will depend on both the
`degree of complementarity and the length of the antisense
`nucleic acid. Generally, the longer the hybridizing nucleic
`acid, the more base mismatches with an RNA it may contain
`and still form a stable duplex ( or triplex, as the case may be).
`One skilled in the art can ascertain a tolerable degree of
`mismatch by use of standard procedures to determine the
`melting point of the hybridized complex. Antisense nucleic
`acids should be at least six nucleotides in length, and are
`preferably oligonucleotides ranging from 6 to about 50
`nucleotides in length.
`
`[0051] Regardless of the choice of target sequence, it is
`preferred that in vitro studies are first performed to quanti(cid:173)
`tate the ability of the antisense oligonucleotide to inhibit
`gene expression. It is preferred that these studies utilize
`controls that distinguish between antisense gene inhibition
`and nonspecific biological effects of oligonucleotides. It is
`also preferred that these studies compare levels of the target
`RNA or protein with that of an internal control RNA or
`protein. Additionally, it is envisioned that results obtained
`using the antisense oligonucleotide are compared with those
`obtained using a control oligonucleotide. It is preferred that
`the control oligonucleotide is of approximately the same
`length as the test oligonucleotide and that the nucleotide
`sequence of the oligonucleotide differs from the antisense
`
`sequence no more than is necessary to prevent specific
`hybridization to the target sequence.
`
`[0052] The oligonucleotides can be DNA or RNA or
`chimeric mixtures or derivatives or modified versions
`thereof, single-stranded or double-stranded. The oligonucle(cid:173)
`otide can be modified at the base moiety, sugar moiety, or
`phosphate backbone, for example, to improve stability of the
`molecule, hybridization, etc. The oligonucleotide may
`include other appended groups, such as peptides (e.g., for
`targeting host cell receptors in vivo), or agents facilitating
`transport across the cell membrane. To this end, the oligo(cid:173)
`nucleotide may be conjugated to another molecule, e.g., a
`peptide, hybridization triggered cross-linking agent, trans(cid:173)
`port agent, hybridization-triggered cleavage agent, etc. Oli(cid:173)
`gonucleotides of the invention can be synthesized by stan(cid:173)
`dard methods known in the art, e.g. by use of an automated
`DNA synthesizer (such as are commercially available from
`Biosearch, Applied Biosystems, etc.).
`
`[0053] Any of the compounds identified via the techniques
`described herein can be formulated into pharmaceutical
`compositions and utilized as part of the amelioration meth(cid:173)
`ods of the invention. Such pharmaceutical compositions for
`use in accordance with the present invention may be for(cid:173)
`mulated in conventional manner using one or more physi(cid:173)
`ologically acceptable carriers or excipients.
`
`[0054] Thus, the compounds and their physiologically
`acceptable salts and solvates may be formulated for admin(cid:173)
`istration by inhalation or insufflation ( either through the
`mouth or the nose) or topical, oral, buccal, parenteral or
`rectal administration.
`
`[0055] For oral administration, the pharmaceutical com(cid:173)
`positions can take the form of, for example, tablets or
`capsules prepared by conventional means with pharmaceu(cid:173)
`tically acceptable excipients, such as binding agents ( e.g.,
`pregelatinised maize
`starch, polyvinylpyrrolidone or
`hydroxypropyl methylcellulose ), fillers ( e.g., lactose, micro(cid:173)
`crystalline cellulose or calcium, hydrogen phosphate); lubri(cid:173)
`cants ( e.g., magnesium stearate, talc or silica); disintegrants
`(e.g., potato starch or sodium starch glycolate); or wetting
`agents (e.g., sodium lauryl sulphate). The tablets can be
`coated by methods well known in the art. Liquid prepara(cid:173)
`tions for oral administration can take the form of, for
`example, solutions, syrups, or suspensions, or they can be
`presented as a dry product for constitution with water or
`other suitable vehicle before use. Such liquid preparations
`can be prepared by conventional means with pharmaceuti(cid:173)
`cally acceptable additives such as suspending agents (e.g.,
`sorbitol syrup, cellulose derivatives or hydrogenated edible
`fats); emulsifying agents (e.g., lecithin or acacia); non(cid:173)
`aqueous vehicles ( e.g., almond oil, oily esters, ethyl alcohol
`or fractionated vegetable oils); and preservatives (e.g.,
`methyl or propyl-p-hydroxybenzoates or sorbic acid). The
`preparations can also contain buffer salts, flavoring, color(cid:173)
`ing, and sweetening agents as appropriate.
`
`[0056] For buccal administration the compositions can
`take the form of tablets or lozenges formulated in conven(cid:173)
`tional manner.
`
`[0057] For administration by inhalation, the compounds
`for use according to the present invention are conveniently
`delivered in the form of an aerosol spray presentation from
`pressurized packs or a nebulizer, with the use of a suitable
`
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`5
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`trichlorofluo(cid:173)
`propellant, e.g., dichlorodifluoromethane,
`romethane, dichlorotetrafluoroethane, carbon dioxide, or
`other suitable gas. In the case of a pressurized aerosol, the
`dosage unit can be determined by providing a valve to
`deliver a metered amount. Capsules and cartridges of, e.g.,
`gelatin for use in an inhaler or insufllator can be formulated
`containing a powder mix of the compound and a suitable
`powder base, such as lactose or starch.
`
`[0058] The compounds can be formulated for parenteral
`administration by injection, e.g., by bolus injection or con(cid:173)
`tinuous infusion. Formulations for injection can be pre(cid:173)
`sented in unit dosage form, e.g., in ampoules or in multi(cid:173)
`dose containers, with an added preservative. The
`compositions can take such forms as suspensions, solutions,
`or emulsions in oily or aqueous vehicles, and can contain
`formulatory agents, such as suspending, stabilizing, and/or
`dispersing agents. Alternatively, the active ingredient can be
`in a powder form for constitution with a suitable vehicle,
`e.g., sterile pyrogen-free water, before use.
`
`[0059] The compounds can also be formulated in rectal
`compositions, such as suppositories or retention enemas,
`e.g., containing conventional suppository bases, such as
`cocoa butter or other glycerides.
`
`[0060] All preparations can be suitably formulated to give
`controlled release of the active compound. For example, in
`addition to the formulations described previously, the com(cid:173)
`pounds can also be formulated as a depot preparation. Such
`long acting formulations can be administered by implanta(cid:173)
`tion (for example subcutaneously or intramuscularly) or by
`intramuscular injection. Thus, for example, the inhibitors
`can be formulated with suitable polymeric or hydrophobic
`materials (for example as an emulsion in an acceptable oil)
`or ion exchange resins, or as sparingly soluble derivatives,
`for example, as a sparingly soluble salt.
`
`[0061] The compositions can, if desired, be pr