JP2010090176A - Protein kinase c peptide for use in withdrawal symptom - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a drug decreasing or overcoming addiction, and if possible, alleviating or removing the symptoms related to the withdrawal of habit-forming and addictive drugs. <P>SOLUTION: A composition for use in managing withdrawal from an addictive substance is described. The composition includes one or more peptides having specific activity on εisozyme and/or γisozyme of protein kinase C (PKC). The peptide can be administered prior to, concurrent with or subsequent to administration of the addictive substance. Also there is described a kit having at least one container containing a peptide having isozyme-specific activity on εPKC or γPKC and instructions for use. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

(Field of Invention)
The present invention relates to treatment compositions and methods for managing withdrawal symptoms and / or reducing patient dependence on addictive and addictive drugs (eg, alcohol, anesthetics, and antidepressants). is connected with.

(Background of the Invention)
Repeated consumption of addictive drugs (eg, alcohol, tranquilizers, stimulants, opiates, hallucinogens and nicotine) often results in some addiction. Typically, such addiction is characterized by requiring or requiring continued use of the drug and, in some cases, a tendency to increase its dosage. Addiction results in psychological and physiological dependence on the effects of such drugs, and ultimately has a detrimental effect on addictive individuals. The spread of drug addiction is well accepted as it imposes great costs on society. The two main categories of motivation in addiction are the desire for experience of the effects of drug abuse pleasure (eg, valuable), and the desire to eliminate the pleasure of drug withdrawal symptoms or unpleasant consequences.

  Withdrawal symptoms resulting from the use of addictive drugs are difficult and presents serious problems, in part due to undesirable physical and / or psychological symptoms with moderation. Both the valuable aspects of addiction and adverse withdrawal symptoms have been studied, especially the mechanism of opiate addiction. What is clear from the literature is that a single brain structure is not a complete cause of addiction and addictive behavior. Repeated use of opiates include, but are not limited to, the nucleus pathways, ventral tegmental area, basolateral amygdala, blue spots, and the segmental striatal nucleus, and nerve pathways in many brain regions and Induces permanent changes in neural processes. The area of the brain above the spine is also susceptible to modulation by ascending inputs from the spinal cord. There is a profound effect of opiates on spinal nerve transmission. Regardless of the area of the brain or spinal cord examined, these permanent changes include adaptation to the neurotransmitter system, which includes glutamatergic, dopaminergic, and adrenergic signaling. These include but are not limited to transmission. These adaptations may be involved in enhancing the pleasure aspects of addiction as well as the harmful aspects of addiction. Opiates act on three classes of receptors (μ, κ, δ) with μ-opioid receptor subtypes that are important for the valuable and harmful effects of opiates. One particular example of how opiates can mediate permanent neural adaptation of neural pathways and processes is described below.

  One mechanism that reveals physical dependence on opiates involves the nodrenergic cells of blue spots. Opiates act as agonists at the inhibitory μ receptors on these cells, thereby reducing presynaptic norepinephrine release by the cells. Over time, this results in up-regulation of post-synaptic norepinephrine receptor expression. At the same time, morphine down-regulates the synthesis of β-endorphin, a normal endogenous agonist at the inhibitory μ receptor. When the opiate is removed, the cells are no longer inhibited and release norepinephrine before synapses. At the same time, post-synaptic hypersensitivity resulting from an increase in norepinephrine receptors results in an amplified response, followed by an adrenergic acute onset. This adrenergic acute onset manifests as a desire for more opiates, and opiate intake resumes the cycle and makes it worse.

  Understanding the central role of μ-opiate receptors in the mechanism of opiate addiction leads to several forbidden-oriented strategies for treating opiate addiction. One such withdrawal-directed strategy relates to regular, typically twice weekly administration of naltrexone, a potent, orally effective, permanent μ-receptor blocker. In another withdrawal-directed treatment, opiate-dependent individuals are maintained on buprenorphine. Because it is a partial μ-receptor agonist, buprenorphine has somewhat enhanced properties and its acceptability by opiate-dependent individuals is high when it is compliant. At the same time, buprenorphine has a high affinity for the μ-receptor, thus blocking the effect of opiates, which causes opiate-dependent individuals to stop requesting opiates.

  Alcohol is another common abuse drug and is a major public health problem worldwide. There are a few drugs that modulate the strong impulse of alcohol consumption, and the molecular causes of alcohol addiction remain largely uncharacterized. Disulfuram (ANTABUSE®) was introduced in 1951 for the treatment of alcohol addiction by inhibition of aldehyde dehydrogenase enzymes (related to the metabolism of alcohol to acetic acid); the drug is the presence of alcohol Underly, it causes headaches, dizziness and vomiting and negatively increases the urge to drink alcohol. Furthermore, administration of naltrexone, an opiate receptor antagonist, reduces alcohol self-administration in experimental animals and relapses in human alcoholism.

  There is a continuing need for compounds that can change consumption behavior by managing withdrawal symptoms. Like opiates, neuroadaptation and neurotransmitter systems in many brain regions underlie the valuable and harmful aspects of alcohol addiction. Similarly, the area of the brain above the spinal column is also susceptible to modulation by ascending inputs from the spinal cord, where alcohol has a profound effect on spinal nerve transmission.

  Protein kinase C (PKC) is a family of isozymes that are heavily involved in signal transduction cascades. Since various PKC isozymes are located throughout the nerve axis (eg, brain, spinal cord, and primary afferent neurons) and regulate the downstream actions of neurotransmitters, PKC acts as a drug abuser and withdrawal symptoms Seems to play a role in the occurrence of. The PKC family of isozymes is an important enzyme in signal transduction involving various cell functions, including cell proliferation, regulation of gene expression, and ion channel activity.

The PKC family of isozymes includes at least 11 different protein kinases that can be divided into at least three subfamilies based on their homology and sensitivity to activators. A member of the classic subfamily, cPKC subfamily (αPKC, β _I PKC, β _II PKC and γPKC) has four homologous domains (C1, C2, C3) with an isozyme specific (variable or V) region between them. And C4), calcium and diacylglycerol are required for activation. Classical PKC family members are found in the spinal cord as well as in the dorsal surface thin films of many brain regions. New subfamilies or members of the nPKC subfamily (δPKC, εPKC, ηPKC and θPKC) lack the C2 homology domain and do not require calcium for activation. εPKC is found in the primary afferent neuron terminals that stimulate the spinal cord as well as in many brain regions. Finally, the atypical subfamily or members of the αPKC subfamily (ζPKC and λ / _I PKC) lack both the C2 homology domain and half of the C1 homology domain and are insensitive to diacylglycerol and calcium.

Studies on the subcellular distribution of PKC isozymes have shown that activation of PKC results in its redistribution (also referred to as translocation) within the cell, so that activated PKC isozymes can be expressed in the plasma membrane, cytoskeletal elements, It demonstrates that it associates with the nucleus and other subcellular compartments (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3). The intrinsic cellular function of different PKC isozymes is determined by their subcellular location. For example, activated β _I PKC is found inside the nucleus, whereas activated β _II PKC is found around the nucleus and the periphery of cardiomyocytes (Non-Patent Document 4). Localization of different PKC isozymes to different regions of the cell is likely due to binding of activated isozymes to specific anchored pups called receptors for activated C-kinase (RACK) . RACKs are thought to function by selectively anchoring activated PKC isozymes to their respective subcellular sites. RACK only binds fully activated PKC and is not necessarily an enzyme substrate. Binding to RACK is not mediated by the catalytic domain of the kinase (Non-Patent Document 5)). PKC translocation reflects the binding of the activating enzyme to RACK anchored to the cellular particulate fraction, and binding to RACK is necessary for PKC to produce its cellular response (Non-Patent Document 6). ). Inhibition of PKC binding to RACK in vivo inhibits PKC translocation and PKC-mediated functions (Non-Patent Document 7; Non-Patent Document 8; Non-Patent Document 9).

  In general, PKC translocation is required for proper functioning of PKC isozymes. PKC binding site on RACK (Non-Patent Document 10; Mochly-Rosen, D. et al., Supra, 1995) or RACK binding site on PKC (Ron et al., Supra, 1995; Johnson, JA et al., Supra. A peptide that mimics any of 1996a) is an isozyme-specific translocation inhibitor of PKC that selectively inhibits the function of the enzyme in vivo.

  Drugs that can reduce or overcome such addiction and, if possible, drugs that can reduce or eliminate symptoms associated with withdrawal of addictive and addictive drugs suffer from addiction Desired by both people and the general public. Inhibitors of PKC can be such drug types.

Saito, N .; Et al., Proc. Natl. Acad. Sci. USA, 86: 3409-3413 (1989). Papadopoulos, V.M. And Hall, P .; F. J. et al. Cell Biol. 108: 553-567 (1989). Mochly-Rosen, D.M. Et al., Molec. Biol. Cell (formerly Cell Reg.), 1: 693-706, (1990) Diasnik, M .; H. Et al., Exp. Cell Res. 210: 287-297 (1994). Mochly-Rosen, D.M. Et al., Proc. Natl. Acad. Sci. USA, 88: 3997-4000 (1991). Mochly-Rosen, D.M. Science, 268: 247-251 (1995). Johnson, J.M. A. Et al. Biol. Chem, 271: 24962-24966 (1996a). Ron, D.C. Et al., Proc. Natl. Acad. Sci. USA, 92: 492-496 (1995). Smith, B.M. L. And Mochly-Rosen, D .; Biochem. Biophys. Res. Commun. 188: 1235-2240 (1992). Mochly-Rosen, D.M. Et al. Biol. Chem. , 226: 1466-1468 (1991a).

(Summary of the Invention)
In one aspect, the invention includes a method for reducing symptoms associated with symptoms of withdrawal from an addictive drug, the method administering a peptide having isozyme-specific inhibitory activity for γPKC or εPKC Process.

  In certain embodiments, the peptide is administered before, during or after delivery of the addictive drug.

  In another embodiment, the peptide has a sequence identified herein as SEQ ID NO: 1 or SEQ ID NO: 2. In other embodiments, the peptide has a sequence selected from the group consisting of the group of sequences identified herein as SEQ ID NO: 4 to SEQ ID NO: 14.

  In other embodiments, the peptide is formulated for intracellular delivery. For example, the peptide is bound to a carrier or mixed with a formulation capable of intracellular delivery.

  In other embodiments of the invention, the addictive drug is an opiate, alcohol, or nicotine.

  In another aspect, the invention includes a method for reducing symptoms associated with symptoms of withdrawal from an addictive drug, the method for εPKC prior to or simultaneously with delivery of an anesthetic. Administering a peptide having isozyme-specific activity; and administering a peptide having isozyme-specific activity for γPKC after delivery of the addictive drug.

  In certain embodiments, a peptide having isozyme-specific activity for εPKC has the sequence identified herein as SEQ ID NO: 1. In another embodiment, a peptide having isozyme specific activity against γPKC has the sequence identified herein as SEQ ID NO: 2.

  In certain embodiments, administration of the peptide is by injection.

  In yet another aspect, the present invention includes a kit for reducing symptoms associated with withdrawal symptoms derived from an addictive drug, the kit comprising (i) isozyme-specific inhibitory activity against γPKC or εPCK. At least one container containing a peptide having: and (ii) instructions for use.

  In one embodiment, the kit consists of a first container containing a peptide having isozyme-specific inhibitory activity against εPKC.

  In another embodiment, the kit includes a second container containing a peptide having isozyme-specific inhibitory activity against γPKC.

  In certain embodiments, the peptide of the kit has a sequence identified herein as SEQ ID NO: 1 or SEQ ID NO: 2.

  In certain embodiments, the kit instructions direct the user to administer a peptide having isozyme-specific inhibitory activity against εPKC prior to or concurrently with administration of the addictive drug. To do.

  In another embodiment, the kit instructions direct the user to administer a peptide having isozyme-specific inhibitory activity against γPKC following administration of the addictive drug.

  In another embodiment, the kit further includes a syringe suitable for injecting at least one peptide.

  Further aspects include the use of peptide inhibitors in the preparation of a medicament for use in the management of withdrawal symptoms from addictive drugs and / or in reducing addictive drug dependence.

  These and other objects and features of the invention will be more fully understood when the following detailed description of the invention is read in conjunction with the accompanying drawings.

(Short description of the sequence)
SEQ ID NO: 1 is an εPKC antagonist peptide.

  SEQ ID NO: 2 is a peptide inhibitor of the PKC gamma isozyme.

  SEQ ID NO: 3 is a carrier peptide derived from Tat (Tat 47-57).

  SEQ ID NO: 4 is a modification of SEQ ID NO: 2.

  SEQ ID NO: 5 is a modification of SEQ ID NO: 2.

  SEQ ID NO: 6 is a modification of SEQ ID NO: 2.

  SEQ ID NO: 7 is a modification of SEQ ID NO: 2.

  SEQ ID NO: 8 is a modification of SEQ ID NO: 2.

  SEQ ID NO: 9 is a modification of SEQ ID NO: 2.

  SEQ ID NO: 10 is a modification of SEQ ID NO: 2.

  SEQ ID NO: 11 is a modification of SEQ ID NO: 2.

  SEQ ID NO: 12 is a modification of SEQ ID NO: 2.

  SEQ ID NO: 13 is a modification of SEQ ID NO: 2.

  SEQ ID NO: 14 is a modification of SEQ ID NO: 2.

SEQ ID NO: 15 is a carrier peptide derived from Drosophila Antennapedia homeodomain.
The present invention also provides the following items.
(Item 1)
A composition for use in the preparation of a medicament for alleviating symptoms associated with withdrawal from an addictive drug, said composition comprising at least one peptide having isozyme-specific inhibitory activity against γPKC or εPCK and A composition comprising a pharmaceutical excipient.
(Item 2)
2. The composition of claim 1, wherein the peptide is provided to the subject before, during or after delivery of the addictive drug.
(Item 3)
3. A composition according to item 1 or 2, wherein the peptide has the sequence identified herein as SEQ ID NO: 1 or SEQ ID NO: 2.
(Item 4)
3. The composition according to item 1 or 2, wherein the peptide has a sequence selected from the group of sequences identified herein as SEQ ID NO: 4 to SEQ ID NO: 14.
(Item 5)
5. A composition according to item 3 or 4, wherein the peptide is formulated for intracellular delivery.
(Item 6)
6. The composition of item 5, wherein the peptide is formulated for intracellular delivery by being coupled to a carrier peptide.
(Item 7)
Item 2. The composition according to Item 1, wherein the addictive drug is an opioid, alcohol, or nicotine.
(Item 8)
A composition according to item 1, wherein a first peptide having isozyme specific activity against εPKC is provided to a patient prior to or simultaneously with delivery of an addictive drug; and has an isozyme specific activity against γPKC A composition wherein the second peptide having is provided to the patient after delivery of the addictive drug.
(Item 9)
9. The item 8 wherein the first peptide has a sequence identified herein as SEQ ID NO: 1 and the second peptide has a sequence identified herein as SEQ ID NO: 2. Composition.
(Item 10)
Item 10. The composition according to Item 8 or 9, wherein the addictive drug is an opioid, alcohol, or nicotine.
(Item 11)
A kit for alleviating symptoms associated with withdrawal of an addictive factor, the kit comprising:
i) at least one container containing a peptide having isozyme-specific inhibitory activity against γPKC or εPCK; and
ii) Instructions for use
Including a kit.
(Item 12)
Item 12. The kit according to Item 11, wherein the kit is composed of a first container containing a peptide having isozyme-specific inhibitory activity against εPKC.
(Item 13)
Item 12. The kit according to Item 11, wherein the kit comprises a second container containing a peptide having isozyme-specific inhibitory activity against γPKC.
(Item 14)
Item 12. The kit according to Item 11, wherein the peptide is selected from the group consisting of SEQ ID NO: 1 or SEQ ID NO: 2.
(Item 15)
In item 11 or item 12, the instructions for use instruct the user to administer the peptide having isozyme-specific inhibitory activity against εPKC before or simultaneously with administration of the addictive factor The described kit.
(Item 16)
14. A kit according to item 11 or item 13, wherein the instructions for use instruct the user to administer the peptide having isozyme-specific inhibitory activity against γPKC after administration of an addictive factor.
(Item 17)
14. The kit according to item 13, wherein the peptide having an isozyme-specific inhibitory activity against εPKC has the sequence identified herein as SEQ ID NO: 1 and has an isozyme-specific inhibitory activity against γPKC The kit, wherein the peptide has the sequence identified herein as SEQ ID NO: 2.
(Item 18)
18. A kit according to item 11 or 17, further comprising at least one syringe suitable for injecting the peptide.

FIGS. 1A-1C show in vitro late progenitors of isolated spinal cord before and after opioid exposure (FIG. 1A) and after exposure (FIG. 1B) and after treatment with naloxone, an opioid antagonist (FIG. 1C). It shows enhanced responsiveness due to naloxone precipitation of potential (sVRP). FIG. 1D is a plot of the area under the curve of sVRP as a function of time (minutes) to show the time course of enhanced responsiveness due to morphine suppression and naloxone precipitation. The horizontal bar indicates the time of application of morphine and naloxone to the isolated spinal cord. Figures 2A-2D show various non-specific PKC inhibitors: broad spectrum inhibitor GF109203X in the presence (Figure 2A) and absence (Figure 2C) of morphine; and in the presence (Figure 2B) and absence of morphine Shows the area under the curve of late root potential (sVRP) as a function of time (minutes) after application of an inhibitor specific for the Ca ^++- dependent PKC isoform, Go6976 in (FIG. 2D). It is a plot. FIGS. 3A-3B show the application of morphine and naloxone in the presence of Tat-binding PKCε-specific peptide inhibitor εV1-2 (FIG. 3A, black square) or Tat alone (FIG. 3A, open circle); and in the absence of morphine FIG. 3 is a plot showing the area under the curve of delayed onset root potential (sVRP) as a function of time (minutes) after application of a Tat-bound PKCε inhibitor alone at (FIG. 3B). FIGS. 3C-3D show morphine and naloxone in the presence of Tat-binding PKCγ isozyme specific antagonist γV5-3 (FIG. 3C, black squares) or Tat carrier alone (FIG. 3C, open circles); FIG. 3 is a plot showing the area under the curve of late root potential (sVRP) as a function of time (minutes) after application of the Tat binding γPKC antagonist γV5-3 in the presence (FIG. 3D). 4A-4B are plots of the average mechanical threshold (grams) (FIG. 4A) and the average thermal response latency (seconds) (FIG. 4B) as a function of time after application of naloxone or saline. Naloxone or saline (vertical striped bars and parallel lined bars, respectively) was administered to 7-day-old rats 30 minutes after delivery of morphine. As a control, naloxone or saline (dotted and white bars) was administered to animals not treated with morphine. The dotted line represents the average baseline mechanical threshold. FIG. 5A shows an experimental protocol for the data reported in FIGS. 5B-5C, where the animals are treated with a PKC inhibitor prior to delivery of morphine; naloxone is administered 30 minutes after delivery of morphine; And the test of the mechanical threshold value and the thermal response latency was performed at 10 minute intervals after naloxone administration. FIGS. 5B-5C show animals treated according to the protocol shown in FIG. 5A (Tat-bound PKCε (vertical striped bar), Tat-bound γPKC (parallel line-patterned) administered intrathecally just prior to morphine). Bars), Tat carrier alone (dotted bars), or mean mechanical threshold (grams) as a function of time after application of naloxone or saline for saline (white bars) (Figure) 5B) and mean thermal response latency (seconds) (FIG. 5C). The dotted line represents the average baseline threshold. FIG. 6A shows the experimental protocol for the data reported in FIGS. 6B-6C, where the animals are treated with a PKC inhibitor 2.5 hours after delivery of morphine; and mechanical threshold and thermal response Latency testing was performed once every hour after peptide administration. 6B-6C show animals treated according to the protocol shown in FIG. 5A (Tat-bound PKCε (vertical striped bars), Tat-bound γPKC (parallel line-patterned) administered intrathecally just prior to morphine). Bars), average mechanical threshold (grams) as a function of time (Figure 6B) and average after application of morphine for Tat carrier alone (dotted bars) or saline (animals treated with white bars) FIG. 6 is a plot of thermal response latency (seconds) (FIG. 6C). The dotted line represents the average baseline threshold. FIG. 7A is a schematic diagram of a lumbar segment for testing alcohol-induced withdrawal responsiveness enhancement in N-methyl-D-aspartate (NMDA) receptor current. FIG. 7B shows individual traces elicited from lumbar spinal cord motoneurons before (control) and after application of the NMDA antagonist 2-amino-5-phosphonovaleric acid (APV). 8A-8C are traces from NMDA-induced currents in motor neurons before (control, FIG. 8A), between (FIG. 8B), and after (FIG. 8C) alcohol-induced withdrawal symptoms. 8D-8E show normalized NMDA current (I _NMDA ) region as a function of time (FIG. 8D) and after application of alcohol (EtOH) to lumbar segment motor neurons and after washing with artificial cerebrospinal fluid FIG. 8 is a plot showing the NMDA current region (as a percentage of control) (FIG. 8E). FIGS. 9A-9C show before application of alcohol (EtOH) in the presence of the calcium chelator BAPTA (1,2-bis (2-aminophenoxy) ethane-N, N, N ′, N′-tetraacetic acid). Traces from NMDA-induced currents in motor neurons (control, FIG. 8A), between (FIG. 8B), and after (FIG. 8C). 9D-9E show normalized NMDA current (I _NMDA ) region as a function of time (FIG. 9D) as well as after application of alcohol (EtOH) in the presence of BAPTA to lumbar spinal cord motor neurons, and artificial FIG. 9E is a plot showing the NMDA current area (as a percentage of control) after lavage with cerebrospinal fluid (FIG. 9E). FIG. 10 shows cells with γPKC localized to the nucleus before application of ethanol (control), after ethanol application, after washing with artificial cerebrospinal fluid, and in the presence of γPKC V5-3 peptide inhibitor. It is a bar graph which shows a number. FIGS. 11A-11F show the results of NMDA-evoked currents in the spinal cord segment in the presence of alcohol and γV5-3PKC peptide inhibitor (SEQ ID NO: 2), where FIGS. 11A, 11C, and 11F are 2 nM γV5-3 Standards as a function of time before, during and after application of ethanol in the presence of peptide (FIG. 11A), 5-10 nM γV5-3 peptide (FIG. 11C), and 10 nM Tat carrier peptide alone (FIG. 11E). Is a plot of the _normalized NMDA current (I _NMDA ) region; FIGS. 11B, 11D, and 11F are histograms corresponding to FIGS. 11A, 11C, and 11F, respectively. FIGS. 12A-12B show the pups of 7 day old (FIG. 12A) and 21 day old (FIG. 12B) rats after injection of 0.5 g / kg (filled circles) and 4 g / kg (stars) of ethanol or physiological FIG. 5 is a plot showing withdrawal force threshold (grams) as a function of time (hours) after saline (white circle) injection. Figures 13A-13B show 7 day old rat pups (Figure 13A) and 21 day old rat pups (Figure 13A) exposed to a single dose of ethanol (dotted bar) or saline (control, white bar). It is a bar graph which shows the incubation time which retracts the leg | foot by the heat | fever in FIG. 13B). FIGS. 14A-14B show the ethanol levels (μg ethanol / ml blood; FIG. 14A) in the blood of 7 day old (black circle) and 21 day old (white circle) rat pups as a function of time after ethanol injection. FIG. 5 is a plot of spinal ethanol concentration (μg ethanol / mg tissue). 15A-15D are pups of 7 day old rats treated with εV1-2 (dotted bars), γV5-3 (vertical striped bars), or Tat alone (white bars) (FIGS. 15A, 15B). , And similarly treated 21 day old rat pups (FIGS. 15C, 15D) at various times after ethanol injection (FIGS. 15A, 15C) or after saline injection (control, FIG. 15B, FIG. 15D) It is a bar graph which shows retraction force threshold value (gram). Figures 16A-16B are bar graphs showing the latency (in seconds) to retract the paw for 7 day old rat pups as a function of time after ethanol injection (Figure 16A) or after saline injection (Figure 16B). It is. At 4 hours, PKC inhibitor peptide εPKC V1-2 (SEQ ID NO: 1; dotted bar) or γPKC V5-3 (SEQ ID NO: 2, vertical striped bar), or Tat carrier peptide alone (SEQ ID NO: 3, white bar) was administered intrathecally.

(Detailed description of the invention)
(I. Definition)
Abbreviations for amino acid residues are standard 3 letter and / or 1 letter codes used in the art to refer to one of the 20 common L-amino acids.

  A “conservative amino acid substitution” is one that does not cause a significant change in the activity or tertiary structure of the selected polypeptide or protein. Such substitution typically involves converting a selected amino acid residue to a different residue having similar physicochemical properties. For example, a Glu substitution for Asp is considered a conservative substitution because both are negatively charged amino acids of similar size. The classification of amino acids by physicochemical properties is known to those skilled in the art.

  “Peptide” and “polypeptide” are used interchangeably herein and refer to a compound composed of a chain of amino acid residues linked by peptide bonds. Unless indicated otherwise, sequences for peptides are given in the order from the amino terminus to the carboxy terminus.

As used herein, the term “anesthetic agent” refers to standard pharmaceutical reference materials (eg, Stetman's Medical Dictionary, 26th edition (Williams & Wilkins Public, Baltimore, 1995) and American Medical Association). (The “more recent” definition used in the “Analogetics” chapter in “Drug Evaluations” of the subscription service published by (Chicago)). That is, as used in any definition (either classical or recent), "anesthetic" includes: (1) opium, (including morphine, codeine, noscapine, papaverine, thebaine, and heroin; Opiates defined as any preparation or derivative of a number of medically important drugs and / or natural mixtures derived from poppy plants containing addictive or addictive drugs; and (2) opiates And opioid drugs, including various synthetic anesthetics with similar or related chemical structures and effects. Exemplary synthetic anesthetics include (DEMEROL ^™ ), hydrocodone (VICODIN ^™ ), hydromorphone (DIALUDID ^™ ), propoxyphene (DARVON ^™ ), oxycodone (PERCODAN ^™ when mixed with aspirin, or acetaminophen When mixed, PERCOCET ^™ ), levorphanol, fentanyl, and methadone are included.

  Drugs typically have as their potency the following: (1) the ability to cause “significant changes in mood and behavior”; (2) the ability to cause the state of “deprived analgesia”; and (3) dependence , Tolerance, and / or sufficient risk of addiction, is classified as “anesthetic”.

  More generally, as used herein, “addictive drugs” refers to various drugs (eg, alcohol); a few tranquilizers (eg, barbiturates (eg, pentobarbital)), and benzodiazepines ( For example, ribulium, barium); stimulants (eg, cocaine, amphetamine and nicotine); anesthetics including opiates (eg, fentanyl, alfentanil and heroin); and hallucinogens (eg, LSD); pure forms or mixtures Or tobacco; cannabis; marijuana;

  Individuals who are addicted are identified, for example, by the presence of any one or more of a number of undesirable symptoms for drug withdrawal. Typical symptoms associated with withdrawal or withdrawal of addictive drugs include general unpleasant feelings, headache, tremor, anxiety, hallucinations, nausea, vomiting, and especially addictive drugs that cause addiction The continuous desire and longing of can be mentioned.

  “Managing, attenuating or reducing withdrawal symptoms” means allodynia (a nociceptive response to stimuli that normally do not cause discomfort), hyperalgesia (an enhanced response to noxious stimuli), headache, One associated with withdrawal from a particular addictive drug, including but not limited to trembling, anxiety, hallucinations, nausea, vomiting, and symptoms of continual desire and craving for addictive drugs that cause addiction Intended for a decrease perceived by the subject in these symptoms.

(II. Compositions and methods for managing withdrawal symptoms)
The present invention shows that ε protein kinase C and γ protein kinase C are related to the mechanism of withdrawal from addictive and addictive drugs, and their selective inhibition can alleviate symptoms associated with withdrawal, or Based on the discovery that it can be removed.

(1. Morphine withdrawal)
Delayed progenitor root potential (sVRP) is an electrophysiological measurement of nociceptive-related responses in the spinal cord lasting approximately 40 seconds (Yanagisawa et al., Eur. J. Pharmacol., 106: 231-239 (1984); Akagi H. et al., British J. Pharmacol., 84: 663-673 (1985); Otsuka M. et al., J. Physiol., 395: 255-270 (1988)). It is strong enough to activate small diameter afferent nerves that transmit nociceptive (painful) stimuli from the periphery to the spinal cord and is triggered by stimulation of the dorsal root of the lumbar region (Lozier) AP et al., J. Neurophysiol., 74: 1001-1009 (1995)). Isolated spinal cord can be used to characterize in vitro nociceptive hyperresponsiveness of opiate withdrawal. Because in this model, withdrawal to opioids is, for example, the administration of the mu opioid receptor agonist morphine followed by the administration of the opioid antagonist naloxone to nociceptive associated late progenitor potential ( This is because it is disclosed as an increase in sVRP). Thus, sVRP is associated with pain and its exacerbation after naloxone is a manifestation of withdrawal symptoms that translate into increased pain sensation in vivo.

  As described in Example 1, neonatal rat spinal cords were isolated from 5-7 day old rats and prepared for measurement of sVRP. 1A-1C show early sVRP of isolated spinal cord (FIG. 1A), sVRP exposed to morphine (FIG. 1B), and then sVRP exposed to naloxone (FIG. 1C). sVRP is reduced by morphine and returns to a level that is significantly above control for naloxone administration. Thirty minutes after naloxone application, the area of sVRP was 140.9% ± 10.12 (mean ± SEM) of controls (P less than 0.001). The hyperresponsiveness lasted for at least 1 hour.

  FIG. 1D shows the time course of morphine reduction and naloxone-induced hyperresponsiveness by plotting the area under the curve of each individual sVRP trace as a function of time (minutes). The horizontal bar indicates the time of application of morphine and naloxone to the isolated spinal cord. The reduction of sVRP and increased responsiveness to naloxone application with morphine administration is evident.

Having established the use and effectiveness of this model to characterize naloxone-induced nociceptive responses, the effects of various PKC antagonists / inhibitors on hyperresponsiveness were evaluated. Figures 2A-2D show various non-specific PKC inhibitors: broad spectrum inhibitor GF109203X in the presence (Figure 2A) and absence (Figure 2C) of morphine; in the presence (Figure 2B) and absence of morphine (Figure 2B). FIG. 2D) is a plot showing late onset root potential (sVRP) as a function of time (minutes) after application of inhibitor Ca6976 specific for Ca ⁺⁺ dependent PKC isoforms in FIG. 2D). PKC antagonist GF109203X inhibited withdrawal hyperresponsiveness, as evidenced by the result that the sVRP region was 87% (± 9.8%) of the control 30 minutes after naloxone (FIG. 2A). . This is not significantly different from the control, but significantly different from the enhanced response after naloxone without antagonist. The PKC antagonist Go6976 did not inhibit withdrawal symptoms because the mean sVRP region was 127.4% (± 5.5%) after 30 minutes of naloxone and the value was not significantly different from naloxone alone. (FIG. 2B). None of the inhibitors alone changed sVRP (FIGS. 2C-2D).

  In another study conducted according to the procedure set forth in Example 1, PKC isozyme-specific antagonists εPKC V1-2 (SEQ ID NO: 1) and γPKC V5-3 (SEQ ID NO: 2) were applied to isolated spinal cord. . The results for the εPKC antagonist are shown in FIGS. FIG. 3A shows pre-late as a function of time after application of morphine and naloxone in the presence of Tat-bound PKCε-specific peptide inhibitors εV1-2 (black squares) or Tat alone (open circles; SEQ ID NO: 3). The root potential (sVRP) region is shown. Tat-bound εPKC V1-2 peptide inhibited withdrawal responsive enhancement, as seen in the result that the sVRP region was 102% of control 30 minutes after naloxone administration. In contrast, the Tat peptide alone (open circles) did not inhibit the withdrawal response. FIG. 6B shows that in the absence of morphine, εPKC V1-2 did not change sVRP.

  The results for the administration of the γPKC antagonist γV5-3 are shown in FIGS. γPKC V5-3 (black squares) reduced enhanced withdrawal responsiveness, but Tat carrier vehicle alone (white circle; FIG. 6C), indicating that the clear decrease in withdrawal symptoms associated with PKCγ inhibitors is due to Tat carrier peptide. ) Not significantly larger. This finding is based on the nonspecific inhibitor finding that the overall PKC antagonist GF109293X (FIG. 2A), but not the Ca 2+ -dependent antagonist Go6976 (FIG. 2B), inhibited enhanced withdrawal responsiveness 30 minutes after morphine exposure. Matches. FIG. 3D shows that γPKC V5-3 does not significantly change sVRP in the absence of morphine and naloxone.

  In another group of animals, the effect of the non-specific PKC antagonist chelerythrine was studied. Administration of chelerythrine to the spinal cord exposed to morphine did not alter the response to morphine (results not shown). However, chelerythrine inhibited morphine withdrawal symptoms as evidenced by the naloxone sVRP response combined with 100.1% cheleryton of control (data not shown).

  These studies demonstrate that naloxone-induced hyperresponsiveness of withdrawal in the spinal cord requires the activity of εPCK. In isolated spinal cords subjected to short-term morphine exposure in vitro, a PKC inhibitor specific for εPKC prevented an increase in sVRP following naloxone treatment. As shown from the following in vivo studies, morphine withdrawal symptoms are also associated with PKC in the gamma isoform, although at a different time than use in in vitro studies.

  In vivo studies of opiate withdrawal allodynia and hyperalgesia were performed using 7 day old rats. As described in Example 2, 7-day-old rats were given morphine subcutaneously 30 minutes after naloxone or saline. To measure foot withdrawal symptom latency, mechanical thresholds were measured as described in Example 2, using a von Frey filament (filament) that causes a hind paw flexion and withdrawal response and a thermal stimulus. . FIG. 4A shows the result of the mechanical threshold test, and FIG. 4B shows the result of the latency test in which the foot is retracted due to heat of the foot. Neonatal rats fed morphine have a deep analgesia that manifests as an increased paw withdrawal threshold for increasing mechanical pressure using von Frey filaments and an increased latency to retract the paw for thermal stimulation. (Parallel line-shaped bars, FIGS. 4A-4B). Thirty minutes after exposure to morphine (vertical bars), administration of the opioid antagonist naloxone promotes mechanical allodynia and thermal hyperalgesia, and baseline pre-morphine measurements (eg, harmful and non-harmful stimuli upon withdrawal from morphine) Increased susceptibility to both), respectively, as a reduced mechanical threshold or latency to retract the shorter paw. Naloxone alone (dotted bar) did not change the paw withdrawal threshold for mechanical stimulation or the latency of paw withdrawal for thermal stimulation.

  FIG. 5A shows a treatment protocol in which rat pups are given a PKC antagonist intrathecal (eg, directly into the cerebrospinal fluid surrounding the spinal cord) prior to subcutaneous morphine delivery. It was. Thirty minutes after morphine delivery, naloxone was administered followed by mechanical allodynia and thermal hyperalgesia (as described in Example 2). The results are shown in FIGS.

  FIG. 5B shows εPKC V1-2 (SEQ ID NO: 1 with vertical stripes), γPKC V5-3 (SEQ ID NO: 2 with parallel lines), Tat carrier alone (SEQ ID NO: 3 with dotted bars) or saline. (White bar) is used to show the mean mechanical threshold (grams) as a function of time after application of naloxone saline for animals treated according to the protocol shown in FIG. 5A. FIG. 5C is a similar plot for the average thermal response latency test. The data show that Tat-bound PKCεV1-2 (vertical bar) administered prior to morphine delivery is effective in inhibiting naloxone-induced morphine withdrawal symptoms allodynia and hyperalgesia (eg, PKCε Mediates withdrawal-induced sensitization of pain pathways). Pretreatment with Tat-bound γPKC V5-3 (parallel lined bars) had a smaller inhibitory effect. Tat carrier alone (dotted bar) and saline (white bar) did not prevent allodynia and hyperalgesia.

  Another study was conducted to investigate the role of PKC isozymes in normal withdrawal symptoms, more closely resembling the clinical dependence sequelae situation. In this study, 7-day-old rats were given a single injection of morphine (1 mg / kg) and received normal withdrawal symptoms (ie, naloxone was not administered to promote withdrawal symptoms). The treatment protocol is shown in FIG. 6A, where εV1-2 or γV5-3 peptide inhibitors were administered intrathecally 2.5 hours after morphine delivery. Mechanical allodynia and thermal hyperalgesia were evaluated at 1 hour intervals according to the procedure described in Example 2. The results are shown in FIGS.

  FIG. 6B shows the results for the mechanical threshold test, and FIG. 6C shows the results for the thermal response latency. Animals were either Tat-bound PKCε (SEQ ID NO: 1; bars with vertical stripes), Tat-bound γPKC (SEQ ID NO: 2, parallel lined bars), Tat carrier alone (SEQ ID NO: 3, dotted bars), or saline (white) ) And each was administered intrathecally just prior to morphine delivery. Rats developed mechanical allodynia and thermal hyperalgesia as a result of withdrawal (see saline-treated animals, white bars). Administration of γV5-3 (parallel bars) attenuated mechanical allodynia and thermal hyperalgesia during normal withdrawal from morphine (γV5-3 vs. Tat carrier alone (dotted bars) or physiological P for saline solution (white bar) is less than 0.05).

  The data in FIGS. 5-6 show the time relationship of PKC isozymes ε and γ in inhibiting the withdrawal response in vivo. εPKC peptide antagonists were effective in preventing naloxone-induced mechanical allodynia and thermal hypersensitivity in vivo and exacerbated nociceptive spinal responses in vitro due to morphine exposure. The εV1-2 peptide was most effective when administered prior to morphine exposure. The γPKC peptide antagonist was effective in alleviating withdrawal symptoms when administered after morphine exposure and was most effective after a longer period of morphine exposure (FIGS. 6B-6C). From this temporal relationship, morphine exposure appears to produce an early phase of PKCε-dependent primary afferent sensitization that causes an increase in transmitter release followed by a late phase associated with PKCγ-dependent spinal cord sensitization.

  Thus, the present invention contemplates administration of an εPKC antagonist or γPKC antagonist for the management of opioid withdrawal symptoms hyperalgesia in a subject. In particular, the εPKC antagonist may be administered prior to, contemporaneously with, or immediately after delivery of the opioid to reduce allodynia and hyperalgesia associated with withdrawal. The γPKC antagonist is preferably administered after opioid delivery, most preferably 1 hour or more after opioid delivery, in order to reduce withdrawal-related allodynia and hyperalgesia. Combination therapy is also contemplated where the εPKC antagonist is administered before, simultaneously with, or immediately after opioid delivery, followed by delivery of the γPKC antagonist after delivery of the opioid. That is, if the εPKC antagonist is administered immediately after opioid delivery, the γPKC antagonist can be administered simultaneously with the εPKC antagonist or after administration of the εPKC antagonist.

(2. Alcohol withdrawal)
Alcohol withdrawal symptoms contribute to alcohol addiction (alcoholism) and pose serious clinical problems. A study was conducted to demonstrate the ability of peptides specific for the PKC and γ isozymes of PKC to alleviate symptoms of withdrawal from alcohol. The study shows the ability of γPKC peptides to mediate enhanced alcohol withdrawal responsiveness of N-methyl-D-aspartate (NMDA) receptor currents mediated by glutamatergic neurotransmission in spinal motor neurons of neonatal rats, as well as young Characterization of the ability of εPKC peptide inhibitors and γPKC peptide inhibitors to attenuate withdrawal symptoms in rats. These studies will now be described.

(A. In vitro study)
To obtain spinal cords from young rats, prepare sections of these spinal cords, and characterize the ability of γPKC peptides to mediate increased alcohol withdrawal responsiveness of N-methyl-D-aspartate (NMDA) receptor currents. Placed in artificial cerebrospinal fluid for electrophysiological analysis. FIG. 7A is a schematic diagram of the technique and shows a lumbar spinal segment 10. The arrangement of the leading electrode 12 and the peptide 14 for the application of NMDA to the motor neuron 16 is shown. FIG. 7B shows individual tracking elicited from spinal motor neurons before (control) and after application of the NMDA antagonist 2-amino-5-phosphonovaleric acid (APV).

8A-8C are follow-ups from NMDA-induced currents in motor neurons before (control, FIG. 8A), during (FIG. 8B), and after (FIG. 8C) alcohol-induced withdrawal symptoms. The time course of the effect of alcohol (EtOH) on the region of NMDA-induced current is shown in FIG. 8D, where the horizontal bar in the figure displays the time of alcohol application. The normalized NMDA current (I _NMDA ) region increases after application of alcohol (EtOH) to lumbar spinal motor neurons (n = 11).

  FIG. 8E is a histogram showing increased withdrawal responsiveness of the same lumbar segment motor neurons (n = 11) of FIG. 8D. NMDA-induced current was measured 18 minutes after application of alcohol (100 mM) and a lavage fluid (artificial cerebrospinal fluid) was applied. After application of ethanol, an increase in NMDA-induced current greater than the control level indicates increased ethanol-induced withdrawal responsiveness.

  Increased ethanol withdrawal responsiveness is calcium-dependent, as shown by the data represented in FIGS. FIGS. 9A-9C show before application of alcohol (EtOH) in the presence of the calcium chelator BAPTA (1,2-bis (2-aminophenoxy) ethane-N, N, N ′, N′-tetraacetic acid). NMDA-induced current tracking in motor neurons (control, FIG. 8A), between (FIG. 8B), and after (FIG. 8C). Individual tracking from motor neurons does not show enhanced ethanol withdrawal responsiveness when the recording pipette contains 30 mM calcium chelator BAPTA. FIG. 9D shows the time course of the mean effect of ethanol on the region of NMDA-induced current (n = 8) in the presence of intracellular BAPTA. The horizontal bar indicates the time of ethanol application, and as can be seen, after stopping ethanol application, the current returned to the control level. FIG. 9E is a histogram showing that enhanced responsiveness after ethanol administration is not observed in the presence of a calcium chelator.

  Fluorescence immunocytochemistry studies were performed on spinal cord sections as described in Example 3. The sections were incubated with anti-γPKC antibody and anti-neuronal antibody to identify neurons. After washing, the sections were labeled with a secondary antibody labeled with fluorescein and observed by laser confocal microscopy. Although not shown herein, visualization of spinal cord sections showed abundant γPKC in anterior horn neurons, indicating that ethanol reversibly induces γPKC transfer from the nucleus to the cytoplasm. . γPKC metastasis was quantified by counting the number of cells with γPKC localized in the nucleus before (control) and after application of ethanol and after washing with artificial cerebrospinal fluid. Metastasis in the presence of γPKC isozyme-specific peptide V5-3 (SEQ ID NO: 2) was also assessed by administering γPKC V5-3 peptide to sections and 20 minutes later by administering ethanol. Two to three sections were counted per animal. The result is shown in FIG.

  FIG. 10 is a bar graph showing the number of cells with γPKC localized in the nucleus. As can be seen, approximately 14 cells were counted as having γPKC localized to the nucleus prior to ethanol application. Following the application of ethanol to spinal cord sections, the number of cells with γPKC localized to the nucleus decreases to approximately 6, consistent with the visible observation that ethanol induces γPKC transfer from the nucleus to the cytoplasm. ing. Metastasis was reversible, as evidenced by an increase in cells with γPKC localized to the nucleus after washing with artificial cerebrospinal fluid. The presence of the γPKC isozyme-specific peptide V5-3 (SEQ ID NO: 2) applied prior to ethanol exposure is evidenced by the number of cells with γPKC in the nucleus remaining at about 14 of the control value. Thus, it was effective in inhibiting γPKC transfer to the cytoplasm.

  FIGS. 11A-11F show the results of NMDA-induced currents in spinal cord sections in the presence of alcohol and γV5-3PKC peptide inhibitor (SEQ ID NO: 2). FIGS. 11A-11B show the effect of ethanol on NMDA-induced currents in the presence of 2 nm γPKC V5-3 (SEQ ID NO: 2). FIG. 11A is a plot of the normalized NMDA-induced current area as a function of time, where the horizontal bar indicates the application of ethanol (100 mM) for 15 minutes. After application of ethanol, spinal cord sections were washed with artificial cerebrospinal fluid. FIG. 11B is a corresponding histogram. At a dose of 2 nM, γPKC peptide inhibitor V5-3 did not completely inhibit enhanced withdrawal responsiveness. However, at doses of 5-10 nM, the peptide effectively inhibited ethanol withdrawal responsiveness enhancement as seen in the data presented in FIGS. 11C-11D. 11E-11F show the effect of 10 nMTat carrier alone on enhanced ethanol withdrawal response, indicating that the carrier peptide does not prevent the symptoms.

  In summary, the data in FIGS. 7-11 show that enhanced ethanol withdrawal responsiveness of NMDA-induced currents is a calcium-dependent phenomenon and γPKC is a calcium-dependent isozyme. The anterior horn of the neonatal rat represents a sufficient amount of γPKC isozyme, which is activated by ethanol to transfer from the nucleus to the cytoplasm. A γPKC isozyme-specific peptide (eg, SEQ ID NO: 2) is effective to inhibit metastasis, thereby reducing or inhibiting enhanced ethanol withdrawal responsiveness.

(B. In vivo studies)
To determine the effect of ε and γ PKC isozyme specific peptides on ethanol withdrawal-induced allodynia and hyperalgesia, an in vivo study was performed using rat rats. As detailed in Example 4, 7 day and 21 day old rats were given ethanol by intraperitoneal injection. Rats were subjected to mechanical allodynia (von Frey hair stimulation) and thermal hyperalgesia (thermal paw withdrawal latency for heat) at predetermined intervals after ethanol administration. To establish a value at 0 hours, a baseline test was performed prior to ethanol treatment.

  The results for the mechanical allodynia test are shown in FIGS. 12A-12B for 7 day old pups (FIG. 12A) and 21 day old pups (FIG. 12B). The threshold withdrawal force (grams) for the y-axis is defined as the logarithm of 10 times the force (milligrams) required to bend the von Frey fiber. Postnatal day 7 rats treated with 4 g / kg 15% EtOH (star symbol) show a slight but significant increase in threshold withdrawal force (eg, analgesia) at the 2 hour test interval. It was. At 6 and 8 hours after ethanol injection, the 7-day-old rats showed mechanical allodynia as evidenced by a decrease in paw threshold withdrawal force compared to saline controls (FIG. 12A). . Administration of 1 g / kg 15% EtOH (black squares) did not significantly change the threshold withdrawal force. When established using a pup injected with saline (open circles), the baseline threshold withdrawal force remained steady throughout the study.

  In contrast to 7-day-old pups, 21-day-old rats did not show an increase in threshold withdrawal force (eg, analgesia) after EtOH administration, as shown in FIG. 12B. The threshold withdrawal force decreased as early as 4 hours after ethanol injection and remained below baseline until 72 hours after the injection. Administration of 15% EtOH at both high (4 g / kg; asterisk) and low (0.5 g / kg; filled circles) doses resulted in the greatest reduction in mechanical threshold at 6 hours after EtOH injection. At both concentrations, the withdrawal threshold remained consistently below baseline for up to 72 hours after injection. When established using rats injected with saline (open circles), baseline threshold withdrawal force in P21 rats remained steady throughout the study.

  FIGS. 13A-13B show 7 day postnatal rats (FIG. 13A) and 21 days postnatally treated with saline (open bars), 4 g / kg 15% EtOH (dashed bars) (FIG. 13B). ) Shows the results of a latent period test that draws a foot against the above heat. Latency to pull to baseline heat is similar in 7-day-old and 21-day-old rats with values of 12.86 ± 0.70 seconds and 12.26 ± 0.79 seconds, respectively. Was. Thermal hyperalgesia, as shown by a decrease in the latency to pull the paw, was observed 6 hours after administration of 4 g / kg 15% EtOH in 7-day-old rats (FIG. 13A). No change in latency to pull the paw was observed with the saline control. In contrast to 7-day-old rats, 21-day-old rats dosed with 4 g / kg 15% EtOH did not show a decrease in the latency to pull the paw when compared to saline controls over the entire duration of the study. (FIG. 13B). No change in latency to pull the paw was observed in 21-day-old pups of saline control.

  Differences observed in determining whether mechanical allodynia and thermal hyperalgesia are temporally correlated with decreasing EtOH concentrations and in the response between 7-day and 21-day-old rats In order to determine whether or not due to different EtOH pharmacokinetics, blood and spinal EtOH concentrations were measured after a single 4 g / kg 15% EtOH administration. Blood and spinal cord ethanol levels were determined according to the procedure described in Example 4. The results are shown in FIGS.

  FIG. 14A shows blood ethanol levels for 7 day old rats (black circles) and 21 day old rats (white circles) as a function of time after ethanol injection. FIG. 14B is a similar plot for ethanol concentration in the spinal cord (reported as ethanol μg / mg tissue). For both 7-day and 21-day-old rats, blood EtOH concentrations reached similar peak levels 30 minutes after ethanol injection. At this point, the maximum blood EtOH concentration was 5.91 ± 0.46 μg / mL for 7-day-old pups and 5.67 ± 0.60 μg / mL for 21-day-old rats (FIG. 14A).

  Loss pharmacokinetics are summarized in Table 1. The disappearance of EtOH from the blood initially showed a fast rate (initial phase) and then a slower rate (lag phase). Both age groups showed a dramatic decrease in EtOH levels with similar initial phase half-lives (1.9 hours and 1.7 hours, respectively) 3 hours after ethanol injection. The 21-day-old rats showed a delayed phase half-life 4 hours longer than the 7-day-old rats, and at 24 hours after injection, low concentrations of EtOH were still detectable.

侵害求心性線維は、脊髄の後角ニューロンで終端している。従って、また、脊髄組織におけるＥｔＯＨの濃度および動力学を検査した。図１４Ｂおよび表１に示されるように、血中とは対照的に、７日齢ラットと２１日齢ラットとの間に脊髄のＥｔＯＨ濃度におけるＥｔＯＨ濃度の明らかな差異が存在した。注射後３０分で最高点のＥｔＯＨレベルが生じ、注射後３時間で、同様の初期相の半減期で最小レベルまで減少した；Ｐ７ラットについて１．６時間およびＰ２１ラットについて１．５時間（図１４Ｂ、表１）。７日齢ラットについての最大ＥｔＯＨ濃度（１４．８μｇ／組織ｍｇ）は、２１日齢のラットについての最大ＥｔＯＨ濃度（１１．４μｇ／組織ｍｇ）よりも高く、初期相の間ずっと高いままであった。脊髄のＥｔＯＨは、血中ＥｔＯＨレベルについての半減期の５０％半減期である遅延相半減期により証明されるように、血中ＥｔＯＨレベルよりも速い速度で検出不可能なレベル付近まで減少した。

Nociceptive afferent fibers terminate in dorsal horn neurons of the spinal cord. Therefore, the concentration and kinetics of EtOH in spinal cord tissue were also examined. As shown in FIG. 14B and Table 1, in contrast to blood, there was a clear difference in EtOH concentration in the spinal cord EtOH concentration between 7-day and 21-day-old rats. The highest EtOH level occurred 30 minutes after injection and decreased to a minimum level with a similar early phase half-life 3 hours after injection; 1.6 hours for P7 rats and 1.5 hours for P21 rats (Figure 14B, Table 1). The maximum EtOH concentration (14.8 μg / tissue mg) for 7 day old rats was higher than the maximum EtOH concentration (11.4 μg / mg tissue) for 21 day old rats and remained much higher during the initial phase. It was. Spinal EtOH decreased to near undetectable levels at a faster rate than blood EtOH levels, as evidenced by a delayed phase half-life, which is 50% of the half-life for blood EtOH levels.

  To study the role of PKCε and PKCγ in mediating ethanol withdrawal-induced pain, a PKCε peptide inhibitor (εV1-2; SEQ ID NO: 1) or PKCγ peptide inhibitor (ε1) 1 hour prior to the expected onset of withdrawal hyperalgesia. γV5-3; SEQ ID NO: 2) was administered intrathecally to 7 and 21 day old rats. Control rats were injected with a vehicle containing Tat carrier protein (SEQ ID NO: 3) (to which the inhibitor was conjugated).

  The results of the mechanical allodynia test are shown in FIGS. FIGS. 15A-15B show ethanol post-injection (FIG. 15A) or saline for 7 day old rats treated with εV1-2 (dashed bar), γV5-3 (vertical bar), or Tat alone (open bar). 15 is a bar graph showing threshold withdrawal force for various times after water injection (control, FIG. 15B). 15C-15D are similar bar graphs for 21 day old rats. For 7 day old rats, administration of εPKC (dashed bar) peptide inhibitor and γPKC (vertical stripes) peptide inhibitor compared to Tat carrier treated rats (open bars, FIG. 15A) at 6 and 7 hours after EtOH injection. Mechanical allodynia was attenuated at both time points. At 8 hours, all rats showed a similar threshold withdrawal response, which was significantly lower than the pre-EtOH baseline level regardless of treatment group. PKC isozyme specific inhibitors had no effect on threshold paw pulling in the absence of EtOH (FIG. 15B).

  For 21 day old rats, FIG. 15C shows that the PKCε inhibitor (dashed bar) slightly attenuated the response at 6 and 8 hours after ethanol injection. PKCγ inhibitor (vertical stripes) prevents a decrease in threshold withdrawal response and is the same withdrawal as the previous EtOH baseline level (FIG. 15C, baseline) and the level at which rats received saline rather than EtOH (FIG. 15D). The response was kept valid.

  16A-16B show the results for thermal hyperalgesia assessment. FIG. 16A is a bar graph showing the latency (seconds) of pulling the paw before ethanol injection (baseline) and at 5, 6, and 8 hours after ethanol injection for 7 day old rat pups. 4 hours after ethanol injection, PKC inhibitor peptide (εPKC V1-2 (SEQ ID NO: 1; dashed bar) or γPKC V5-3 (SEQ ID NO: 2; vertical stripe)), or Tat carrier peptide alone (SEQ ID NO: 3, white) Bar) was administered intrathecally (Example 4). FIG. 16B is a similar graph for animals treated with saline rather than ethanol.

  FIG. 16A shows that in 7-day-old rats, both εPKC and γPKC peptide inhibitors completely prevented thermal hyperalgesia. Rats that received PKC isozyme-specific inhibitors had a similar paw-latency period as saline-treated animals (FIG. 16B).

  To determine whether EtOH alters the expression and cellular localization of εPKC and γPKC, as described in Example 4, L4 / 5 dorsal root ganglia (DRG) and L4 / 5 hips Immunohistochemistry was performed on the spinal cord nodes. Postnatal day 7 and 21 rats treated with saline or 4 g / kg 15% EtOH were euthanized at 2 hours, 4 hours, or 6 hours after injection for lumbar spinal cord isolation. It was. In 7 day old rats injected with saline, a small percentage of cells stained positive for εPKC (data not shown). After 2 hours of exposure to EtOH, the percentage of cells that stained positive for εPKC increased and the staining density in individual cells also increased (data not shown). At 4 and 6 hours, the number of cells staining positive for εPKC was restored to a level comparable to animals injected with saline. In 21-day-old rats injected with saline, it was observed that more cells stained positive for εPKC compared to 7-day-old pups treated with saline. Treatment with EtOH did not cause an increase in the number of positive staining cells and staining intensity within positive staining cells (data not shown). εPKC staining was predominantly cytoplasmic.

  In 7-day-old rats, few γPKC-positive neuronal cell bodies were observed in lamina II of the lumbar spinal cord of control animals. The γPKC staining of the EtOH-injected animals was the same as that of the saline-injected animals at 2 and 4 hours after the injection, but the number of cell bodies positively stained for γPKC was 6 hours after the injection. There was a 4-fold increase over saline control levels (data not shown). In 21 day old rats, there was sufficient γPKC staining in the cell body. The number of γPKC-positive cell bodies did not change after EtOH injection, but the staining intensity of γPKC increased 2 hours after EtOH injection and remained enhanced at 6 hours after injection (data shown). ) In addition to the increase in staining intensity, the tissue expansion rate was found in γPKC in saline-treated pups mainly in the cytoplasm; 2 hours after EtOH injection, γPKC translocated to the plasma membrane; , ΓPKC again proved mainly in the cytoplasm (data not shown).

(III. Usefulness)
Accordingly, administration of peptides or other compounds that antagonize the activity of εPKC or γPKC is contemplated to handle withdrawal from addictive or addictive drugs. In the above studies, exemplary peptides specific for the PKC ε and γ isozymes have been shown to prevent or attenuate symptoms associated with withdrawal from addictive drugs.

  The peptides identified herein as SEQ ID NO: 1 and SEQ ID NO: 2 are understood to be merely exemplary and are modifications derived from εPKC sequences having activities similar to those demonstrated herein; Fragments and derivatives and other peptides are contemplated. Appropriate modifications (eg, conservative amino acid substitutions) are readily determined by those skilled in the art. Exemplary modifications for SEQ ID NO: 2 (RLVLAS) include the following changes shown in lower case: kLVLAS (SEQ ID NO: 4); RLVLgS (SEQ ID NO: 5); RLVLpS (SEQ ID NO: 6); RLVLnS (sequence) Number 7), and any combination of the above. Other modifications include one or two L to I or V changes (eg, RiVLAS (SEQ ID NO: 8); RLViAS (SEQ ID NO: 9); or RiViAS (SEQ ID NO: 10)). Also, L and V can be V, L, I, R, and / or RRLAS (SEQ ID NO: 11), RLdLAS (SEQ ID NO: 12), and RidLAS (SEQ ID NO: 13) or RridAS (SEQ ID NO: 14). Can be changed to D. Any modification that retains the desired activity is suitable. Accordingly, all exemplary fragments listed above, conservative modifications, and other modifications that do not appreciably alter the activity can be made and are included within the scope of the contemplated peptides.

  It will be appreciated that the peptides may be used in their native form or modified by binding to a carrier. In the natural form, the peptide can be formulated to facilitate its delivery into the cell, if desired. Formulations suitable for cell penetration are known in the art and include, for example, micelles, liposomes (charged and uncharged), and lipophilic media. When linked to a carrier, one skilled in the art can select from a variety of peptide carriers known in the art. In addition to the Tat carrier used in the above studies, the Drosophila antennapedia homeodomain (SEQ ID NO: 15; Theodore, L. et al. J. Neurosci. 15: 7158 (1995); Johnson, JA et al., Circ. Res. 79: 1086 (1996b)), where the PKC peptide is cross-linked to the antennapedia carrier via an N-terminal Cys-Cys bond. Polyarginine is another exemplary carrier peptide (Mitchell et al., J. Peptide Res., 56: 318-325 (2000); Rolhbard et al., Nature Med., 6: 1253-1257 (2000)).

  All peptides described herein can be prepared by chemical synthesis using either automated or manual solid phase synthesis techniques known in the art. The peptides can also be prepared recombinantly using techniques known in the art.

  The peptides are prepared for administration in combination with a pharmaceutically acceptable carrier or diluent. Accordingly, a further aspect of the invention provides a pharmaceutical composition containing a γPKC peptide or εPKC peptide in a dosage form suitable for administration to a subject in need of pain treatment. Exemplary dosage forms include, but are not limited to, peptides formulated in a pharmaceutical carrier (eg, starch, lactose, talc, magnesium stearate, aqueous solution, oil-water emulsion, etc.). Dosage forms suitable for injection by any route (including but not limited to intrathecal route, intravenous route, intraperitoneal route, intramuscular route, subcutaneous route) include pharmaceutical carriers (eg, buffered Aqueous solution or non-aqueous medium). The peptides can be administered locally near the site of inflammation or peripheral nerve injury, for example, by topical application, dermal or transdermal administration, or intradermal injection. Mucosal delivery is also contemplated, wherein the peptide is formulated for sublingual delivery, vaginal delivery, intranasal delivery, or ocular delivery. It will be appreciated that certain forms of administration may reach an initial localized site of delivery that becomes more widespread over time. For example, a buccal patch or vaginal suppository provides initially localized delivery at the site of application. Over time, the peptide migrates from the delivery site into body fluids (lymph and blood) to provide a wider area of action. The extent of delivery can be controlled by the choice of formulation and route of administration, as is known to those skilled in the art of pharmaceutical formulations.

  The amount of peptide in the composition can be varied to obtain an appropriate dose and to achieve an effective analgesic effect. The dosage depends on many factors, such as the route of administration, duration of treatment, patient size and physical condition, peptide efficacy and patient response. An effective amount of peptide can be estimated by testing the peptide in one or more pain models described herein.

  The peptide can be administered as needed, hourly, several times a day, every day, or whenever a human experiences pain or as deemed appropriate by the human physician. The peptide can be administered prophylactically in anticipation of pain or, if necessary, before or during an acute episode of pain. The peptides can be administered on a continuous basis while dealing with chronic pain, or can be administered on a short-term basis before and after a pain episode (eg, before and / or after surgery).

  The present invention further contemplates a kit comprising components for use by a user in treating symptoms of withdrawal from an addictive drug. The user may be a health care provider (eg, a nurse or doctor) that cares for a patient treated with an addictive drug or a patient treated for abuse of an addictive drug. The user may also be a person handling addictive drugs, regardless of whether clinical addiction has occurred.

  The kit comprises (i) at least one container containing a peptide having isozyme-specific inhibitory activity against γPKC and / or εPKC; and (ii) instructions for use. In one embodiment, the kit comprises a peptide having isozyme-specific inhibitory activity for εPKC (eg, a peptide identified herein by SEQ ID NO: 1 or any modification discussed above). It consists of a first container.

  The kit also includes a second peptide comprising isozyme-specific inhibitory activity against γPKC (eg, the peptide identified herein by SEQ ID NO: 2 or any modification discussed above). A container may be included. A kit comprising two vials (one containing an ε-specific PKC peptide and the other containing a γ-specific isozyme peptide) isozyme specific for εPKC before or simultaneously with the administration of the addictive agent Manual for instructing the user to administer a peptide having specific inhibitory activity. The user is further instructed to administer a peptide having isozyme-specific inhibitory activity against γPKC after administration of the addictive agent.

  The peptides in the kit can be provided ready for use or in a form that requires the addition of a sterile fluid (eg, saline). Inevitably, if sterile fluid is required, the kit may include the amount of fluid required and a syringe (if injection is required). Peptides formulated for ready use are intended for single use or multiple use dosage forms in any of the forms discussed above for any route of administration.

  The present invention also contemplates a therapeutic regimen for treating withdrawal symptoms in newborns. The studies described here correspond to rats 7 days after birth (developmentally equivalent to newborn human infants) and rats 21 days after birth (developmentally equivalent to human children of preschool age). (Fitzgerald and Anand, Pain Management in Infants, Children and Adolescents (Schechter et al., (Ed.)), Pages 11-32, Baltimore, MD, Williams and Williams, 1993). Human infants are conventionally treated with opioids for pain relief and sedation during mechanical ventilation. Many of these infants show symptoms of neonatal withdrawal syndrome (developmentally specific opiate equivalent withdrawal) (Norton, S., Neonatal Netw., 7: 25-28 (1988)). From the data described herein in postnatal day 7 rats, mediated by γPKC-containing neurons in the spinal cord after the initial phase of major afferent sensitization dependent on εPKC translocation occurring on morphine exposure Followed by a delayed phase involving spinal sensitization. This data shows that in a human neonatal population by administering a peptide having εPKC-specific activity during early phase sensitization, and if desired, a peptide having γPKC-specific activity during delayed phase sensitization. Suggest temporary treatment for opiate tolerance and withdrawal treatment.

  From the above, it can be seen how the various objects and features of the invention fit. Methods are provided for attenuating withdrawal symptoms associated with withdrawal of addictive drugs. Most commonly, the method includes administering to a subject (typically a mammal (particularly a human)) a peptide having isozyme-specific activity against εPKC or γPKC. In one embodiment of this method, both peptides are administered in a transient manner, wherein a peptide having specific activity for the εPKC isozyme is administered prior to or concurrently with the administration of the addictive agent. The The peptide having specific activity for the γPKC isozyme is administered after the addictive agent is given to the subject. “After” means administration of a γPKC isozyme-specific peptide immediately after administration of the addictive agent, or minutes or hours after administration of the addictive agent.

(IV. Examples)
The following examples further illustrate the invention described herein and are in no way intended to limit the scope of the invention.

(Materials and methods)
All peptides were synthesized at Stanford's Protein and Nuclei Acid facility and conjugated to Tat (amino acids 47-57 (SEQ ID NO: 3)) via a cysteine-cysteine bond at the N-terminus. εPKC antagonist (εV1-2 (SEQ ID NO: 1)) and γPKC antagonist (γV5-3 (SEQ ID NO: 2)) were used at a purity of> 90%.

Example 1
(In vitro measurement of sVRP)
The spinal cord from 4-7 day-old rats (Charles River Laboratories) was removed and it was subjected to electrophysiological recording of slow anterior root potential (sVRP) (Woodley SJ et al., Brain Res., 559: 17-21 ( 1991)). Briefly, rat pups were decapitated under halothane anesthesia and the spinal cord was removed into an artificial cerebrospinal fluid containing oxygen. An aspiration stimulating electrode was placed on the posterior root of the hip, and an aspiration recording electrode was placed on the corresponding ipsilateral anterior root. During the experiment, a square wave stimulation duration of 0.2 ms was given at a constant frequency of 1/50 second. The test agent was applied, its response was recorded, digitized, and the area under the curve was measured.

  After reading the baseline of sVRP, morphine (200 nM) was applied to the isolated spinal cord. The sVRP was recorded again to characterize the response to morphine. Naloxone (200 nM) was then applied and sVRP was recorded. The results are shown in FIGS.

  FIGS. 2A-2D show the results after application of a non-specific PKC antagonist (GF109203X (1.2 μM)) and an inhibitor specific for a Ca ++ dependent PKC isoform (Go 6976 (1 μM)).

  Figures 3A-3B show the results after application of an εPKC antagonist (εV1-2; SEQ ID NO: 1, 2 nM) conjugated to a peptide carrier (Tat; SEQ ID NO: 3) and the Tat peptide alone (2 nM; SEQ ID NO: 3). The result after applying is shown. Figures 3C-3D show the results after application of γPKC (γV5-3; SEQ ID NO: 2, 4 nM) conjugated to a peptide carrier (Tat; SEQ ID NO: 3) and the Tat peptide alone (4 nm; SEQ ID NO: 3). The result after application is shown.

(Example 2)
In Vivo Mechanical Threshold and Thermal Paw Withdrawal Studies Male and female 7 day old Sprague-Dawley rats (Charles River Laboratories) were used. For all behavioral experiments, rats were maintained at nest temperature with an overhead heating lamp.

  To measure the mechanical threshold, rats were placed in an elevated wire mesh (2 mm open). Using Von Frey hair (Storing Co., WoodDale, Illinois, USA), Fitzgerald et al. (Fitzgerald M et al. , Md) (1993)), skin flexion withdrawal was pronounced. Von Frey hairs increasing in strength were applied three times to the plantar surface of the left hind paw until paw withdrawal was induced. The lowest intensity Von Frey hair required to produce a withdrawal reflex was recorded as the response threshold to a low intensity mechanical stimulus.

  The latent period of hot paw withdrawal was measured using a Ugo Basile Plant Testing apparatus (Stoeling Co., WoodDale, Illinois, USA). Briefly, 7-day-old rats were placed in an inverted Plexiglas enclosure and the plantar surface of their left hind paw was heated from below with the infrared intensity of the lamp set at 30. Two baseline thermal response latency measurements were collected prior to morphine administration using a 20 second cut-off made to prevent tissue damage.

  Seven days old rats were subcutaneously administered with 1 mg / kg morphine sulfate (Sigma) in 50 μl physiological saline. The mechanical threshold was measured 15 minutes after establishing analgesia. Rats were returned to their dams. At 30 or 120 minutes after morphine administration, rats were administered 0.25 mg / kg naloxone (Sigma) subcutaneously in 50 μl saline. Machine threshold and thermal paw withdrawal latency were measured at 10 minute intervals. Tat-binding peptide inhibitors, Tat carriers (10 μM) or saline specific for ε (εV1-2; SEQ ID NO: 1, 10 μM) and γ (γV3-5; SEQ ID NO: 2, 10 μM) isoforms of PKC were added to halothane. Rats were slightly anesthetized with a sterilized 29-scale 3/10 cc insulin syringe (29 gauge 3/10 cc insulin syringe) and administered intrathecally in a 5 μl volume directly via lumbar puncture. To fully recover from anesthesia, the animals were administered peptide or control solution 30 minutes prior to naloxone. The results are shown in Figures 4A-4B and 5B-5C.

  For natural withdrawal studies, rats receive morphine (1 mg / kg) and 2.5 hours later εV1-2 (10 μM), γV3-5 (10 μM), Tat carrier (10 μM), or saline did. Mechanical threshold and thermal paw withdrawal latency were tested after 3 hours, 4 hours, 5 hours, and 6 hours after morphine. The results are shown in FIGS. 6B-6C.

  An unpaired T test was used to determine the significant difference of the post-naloxone measurement from the baseline measurement. Significant differences between treatment groups for both in vivo and in vitro PKC inhibitor studies were determined by one-way analysis of variance (ANOVA) followed by post hoc Bonferroni analysis. A P value less than 0.05 was considered significant. All statistical analyzes were performed using GraphPad Prism version 3.02.

(Example 3)
(Hyperresponsivity in vitro alcohol withdrawal)
Effect of γPKC peptide on
Seven to ten day old Sprague-Dawley rats were anesthetized with halothane, decapitated, and the spinal cord was quickly removed. A 350 mm thick slice was separated from the lumbar region. Whole cell voltage clamp recordings were recorded from visually identified motor neurons using an infrared video microscope, a 60 × water immersion lens, and a MultiClamp 700A patch clamp amplifier. Cells were maintained at a -60 mV maintenance voltage in artificial cerebrospinal fluid (ACSF) containing bicuculline methiodide (10 mM), strychinin (5 mM), and tetrodoxine (0.5 mM). As shown in FIG. 7A, a post-synaptic current was directly applied to 2 mM N-methyl-D-aspartic acid (NMDA) from a pipette placed in the vicinity of the recording cells at 1-2 minute intervals. Induced by pressure application. The area of evoked current during and after application of 100 mM ethanol (EtOH) was measured and normalized to the average basal current area during 10 minutes of pre-EtOH application. Data are expressed as mean +/- SEM. Statistical significance was determined by one-dimensional configuration ANOVA followed by Dunn's multiple comparison test or Tukey's multiple comparison test with significance set to p less than 0.05.

  Fluorescent immunocytochemistry studies were performed using spinal cord sections from control slices (30 mm) (perfused with ACSF for 70 min) and spinal cord sections from EtOH-treated slices (perfused with ACSF for 30 min, then perfused with 100 mM EtOH for 20 min and ACSF). For 20 minutes). This portion was incubated overnight at 4 ° C. with rabbit anti-PKCg polyclonal antibody (1: 500). In some spinal cords, mouse anti-neuronal nuclear monoclonal antibody (NeuN, 1: 750) was added to identify neurons. After washing several times with ACSF, this part was labeled with a fluorescently labeled secondary antibody (1: 500) for 2 hours at room temperature. Double immunofluorescence was evaluated with a laser confocal microscope.

  The results are shown in FIGS.

Example 4
(Effect of γPKC peptide and εPKC peptide on hyperalgesia to alcohol withdrawal-induced allodynia in vivo)
(1. animals)
Sprague-Dawley rats (Charles River, Mass.) Were housed in nests and exposed to a 12/12 hour light / light cycle with free access to food and water. Unless otherwise specified, experiments were performed on 7 day old male and female pups (14-17 g) or 21 day old male animals (45-55 g). Seven-day-old pups were maintained at the nest temperature with an overhead heating lamp for both mechanical and thermal testing.

  Infants of 7 day old rats were injected intraperitoneally (ip) with a single dose of 1 g / kg or 4 g / kg 15% EtOH or saline. Infants of 21-day-old rats were given 0.5 g / kg or 4 g / kg of 15% EtOH or saline i. p. Injected.

(2. Allodynia test and hyperalgesia test)
A mechanical allodynia test was performed as follows. Under unrestrained conditions, each rat was placed alone in a plexiglass cage with an elevated aluminum shielding surface equipped with a 1 mm empty net. 21-day-old animals were previously habituated to the environment and the experimenter. 7-day-old and 21-day-old juveniles were placed on their hind legs for three contact stimuli using 0.04g-1.4g or 0.16g-6g Von Frey filament (Stoelting Co, Wood Dale, IL), respectively. Exposed. The threshold for pain was determined as described above (Fitzgerald M et al., PAIN MANAGEMENT IN INFANTS, CHILDREN AND ADOLESCENTS; Schechter and Yaster, pages 11-32, Williams and Williams 19 )) Prescribed the lifting of legs. Baseline tests on 7-day-old pups were performed prior to EtOH treatment, followed by measurements every hour up to 6 hours after injection and at 8, 10, 12, and 24 hours. Baseline tests for 21 day old pups were performed as above and measured every 2 hours up to 8 hours after ethanol injection and at 24, 48 and 72 hours. Allodynia was defined as the threshold force below the threshold force required to elicit a saline control response.

  A thermal hyperalgesia test was performed as follows. Thermal paw withdrawal latency was measured using a Ugo Basle Plant Testing apparatus (Storing Co, WoodDale, Illinois, USA). Mainly, 7 or 21-day-old rats were placed in inverted Plexiglas enclosures, and the plantar surface of their hind legs was heated from below, setting the infrared intensity of the lamp to 30 or 40, respectively. Three baseline thermoresponsive latency measurements were collected prior to EtOH administration using a 20 second cut-off attached to prevent tissue damage. Two records separated at 10 minutes after EtOH administration were taken for each juvenile every 2 hours up to 8 hours and then at 24 hours. The recorded foot withdrawal latency is expressed as the average of the individual measurements.

  The results are shown in FIGS. 12A-12B and FIGS. 13A-13B.

  To determine the involvement of εPKC and γPKC in withdrawal-induced mechanical allodynia and thermal hyperalgesia, 7-day and 21-day-old rats were treated with inhibitor peptide 5 hours (7-day-old pups) or 4 hours later ( 21-day-old pups) followed by a single dose of 4 g / kg 15% EtOH or saline (ip). For the thermal test, 7 day old juveniles received the inhibitor peptide 4 hours after EtOH administration. Subarachnoid delivery of peptide drugs was achieved by 5 μL (7 of 20 μM PKCε (εV1-2; SEQ ID NO: 1) inhibitor peptide or PKCγ (γV5-3; SEQ ID NO: 2) inhibitor peptide bound to a Tat protein carrier (SEQ ID NO: 3). Lumbar puncture was performed directly with halothane anesthesia (day-old pups) or 10 μL (21-day-old pups). The PKC isozyme specific inhibitor was administered one hour prior to the predetermined time for the onset of hyperalgesia / allodynia. Mechanical allodynia testing and thermal hyperalgesia testing were performed as described above, leaving the investigator unaware of the peptide drug treatment. The results are shown in Figures 15A-15D and 16A-16B.

(3. Determination of blood and spinal cord ethanol levels)
7-day-old and 21-day-old rat pups were administered 4 g / kg of 15% EtOH (ip) according to the acute procedure described above. The animals were deeply anesthetized at different times after injection and blood was collected into a heparin-containing syringe via a direct cardiac puncture. The spinal cord and blood were removed and stored at −80 ° C. in a microfuge tube until analysis. At the time of analysis, tissues were homogenized for 5 seconds with 1 μL of 6.25% (w / v) trichloroacetic acid (TCA) per mg of tissue using a Polytron® tissue homogenizer. An aliquot of whole blood (100 μL) was added to 900 μL of TCA and vortexed. Tissue and blood samples are centrifuged at 10,000 rpm for 5 minutes at room temperature, and EtOH volume is determined using a colorimetric assay (Sigma Diagnostics, St. Louis) corresponding to a 96-well format according to the manufacturer's instructions. did.

(4. Immunohistochemistry)
Animals receiving a single dose of 4 g / kg 15% EtOH or saline (ip) were deeply anesthetized with halothane and transcardiac perfusion (phosphate buffered saline wash) And then euthanized 2, 4 or 6 hours after injection with 4% formaldehyde wash in PBS, pH 7.4). After perfusion, the lumbar spinal cord and L4 / L5 spinal ganglia (DRG) were isolated, fixed with 4% formaldehyde solution for 3 hours, and cryoprotected in 30% sucrose / PBS at 4 ° C. The cuts were frozen in OCT embedding media, placed on cork blocks and stored at −80 ° C. until analysis. For spinal cord sections, free floating immunohistochemistry was performed on 30 μm L4-L5 spinal cord sections. Spinal ganglia were cut to 10 μm in a cryostat and placed on a glass slide. The glass slide was heated at 32 ° C. overnight. Immunohistochemistry was performed using an avidin-biotin complex (ABC) as described above (Sweitzer et al., Brain Res., 829 (1-2): 209-221 (1999)). Rabbit polyclonal antibodies were used against γPKC (1: 500 from Santa Cruz) or εPKC (1: 1000 from Santa Cruz). Immunohistochemistry was scored while hiding experimental conditions.

  For both spinal cord tissue and DRG, densitometry was performed on at least three parts from each of three individual animals using ImageJ software.

(5. Statistics)
Significant differences between treatment groups and saline controls were performed by one-way analysis of variance. Group mean values were compared by the Newman-Keuls post-hoc test. Significant differences between treatment groups and criteria were tested using a nonparametric t test. All statistics were tested using GraphPad Prizm, model 3.0 (GraphPad Software, San Diego, Calif.).

  While the invention will be described with respect to particular embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the invention.

Claims (1)

Invention described in the specification.

Images