WO2010018918A1 - Agent comprising g-csf for treatment or prevention of neuropathic pain and method for treating neuropathic pain with the same - Google Patents

Agent comprising g-csf for treatment or prevention of neuropathic pain and method for treating neuropathic pain with the same Download PDF

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WO2010018918A1
WO2010018918A1 PCT/KR2009/002627 KR2009002627W WO2010018918A1 WO 2010018918 A1 WO2010018918 A1 WO 2010018918A1 KR 2009002627 W KR2009002627 W KR 2009002627W WO 2010018918 A1 WO2010018918 A1 WO 2010018918A1
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pain
csf
neuropathic pain
nerve
administration
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PCT/KR2009/002627
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French (fr)
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Kyung-Soo Kim
Jun-Ho Joe
Young-Suck Ro
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Industry-University Cooperation Foundation, Hanyang University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the present invention relates to a preventive or therapeutic agent for neuropathic pain comprising a granulocyte colony stimulating factor (G-CSF) as an active ingredient and a method for treating neuropathic pain with the same.
  • G-CSF granulocyte colony stimulating factor
  • Pain is defined as an unpleasant sensory and emotional experience originating from a specific site in the body.
  • chronic pain is defined as extremely intolerable pain that persists longer than the temporal course of natural healing. Patients who suffer from chronic pain experience a great deal of difficulties in leading a normal life. Pain is generally sensed in accordance with a mechanism wherein a noxious stimulus applied to the body's pain acceptor is converted into a pain signal which is transmitted to the central nervous system.
  • damage to nervous systems may cause continuous pain not associated with any external harmful stimulus. The one is referred to as “nociceptive pain” and the other is referred to as "neuropathic pain”.
  • Nociceptive pain is normally caused by tissue damage or disease. Such pain serves to warn tissue damage and prevent the same. Meanwhile, neuropathic pain or neuralgia is chronic pain caused by damage to peripheral nerves or tissues (J Pain. 2008 Jan;9(1 Suppl 1):S10-8. Mechanisms of pain and itch caused by herpes zoster (shingles)).
  • neuropathic pain symptoms observed in those who suffer from neuropathic pain include hyperalgesia, allodynia, spontaneous pain, etc.
  • Hyperalgesia is defined as an increased sensitivity to a pain stimulus which is normally painful.
  • Allodynia is defined as an extreme response to a usually non-painful stimulus that has no potential to normally induce pain.
  • Spontaneous pain is defined as pain sensed without any stimulus. Since neuropathic pain was first disclosed in 1872 by Mitchell (Mitchell, S.W. Injuries of Nerves and Their Consequences, J.B.
  • neuropathic pain include postherpetic neuralgia, post-thoracotomic pain, trigeminal neuralgia, disseminated sclerosis-associated pain, thalamic pain, phantom limb pain, anesthesia dolorosa, HIV-associated neuropathic pain, spinal cord disorder-associated paraplegic pain, complex regional pain syndromes , etc.
  • CNS Drugs. 2008;22(5):417-42. An update on the pharmacological management of post-herpetic neuralgia and painful diabetic neuropathy).
  • herpes zoster (or simply zoster), commonly known as shingles, is a disease wherein the varicella zoster virus invade spinal sensory nerve roots, sensory ganglia, spinal dorsal-roots, and skin sensory or cerebral nerve peripheral branches corresponding thereto, causing symptoms such as vesicular eruption, pain and oversensitivity in the skin.
  • Postherpetic neuralgia is the most common zoster complication, which is defined as pain persisted for three months or longer after crusts are formed in skin lesions (Young L. Postherpetic neuralgia: a review of advances in treatment and prevention. J Drugs Dermatol 2006; 5:938-41; Wareham D. Postherpetic neuralgia. Clin Evid 2005; 14 :1017-25).
  • Postherpetic neuralgia is one of neuropathic pains such as post-thoracotomic pain, trigeminal neuralgia, diabetic neuropathy, disseminated sclerosis-associated pain, thalamic pain and phantom limb pain.
  • the main symptom of postherpetic neuralgia is unilateral pain arising along nerve ganglia. The pain variably ranges from mild to severe.
  • Postherpetic neuralgia involves burning pain, aching pain, itching, continuous pain and intense shooting pain, followed by hyperalgesia and allodynia.
  • Postherpetic neuralgia is difficult to treat and there are premonitory symptoms associated with postherpetic neuralgia, such as old age, serious initial pain, fever and rash, immune conditions and underlying diseases.
  • Postherpetic neuralgia arises in 10 to 15 % of acute zoster patients.
  • the disease rate rapidly increases with increase in patient's age, rising to 50% for acute zoster patients with an age 60 or over (Rice AS, Maton S. Gabapentin in postherpetic neuralgia: a randomized, double blind, placebo controlled study. Pain 2001; 94 :215-24; Seventer VR, Sadosky A, Lucero M et al. A cross-sectional survey of health state impairment and treatment patterns in patients with postherpetic neuralgia. Age Ageing 2006; 35 :132-7; Volmink J, Lancaster T, Gray S et al . Treatments for postherpetic neuralgia a systematic review of randomized controlled trials.
  • Postherpetic neuralgia is caused by a different mechanism from the pain of acute zoster.
  • Acute zoster is mainly caused by inflammation and necrosis, while postherpetic neuralgia is induced by neuropathic factor caused by nervous system injury or dysfunction.
  • varicella zoster viruses are reactivated, causing injury to adjacent nervous tissues or non-nervous neighboring tissues, while being transferred via blood flow or directly. In particular, elderly people take a long time to recover from nervous injury, thus lengthening neuralgia duration.
  • viruses are diffused into the dermal or hypodermic tissues, inducing inflammation, which causes tissue injury and pain.
  • This injury repeatedly stimulates afferent C-fibers and induces continuous excitation of axons in spinal dorsal-roots.
  • This noxious stimulus induces central sensitization, allowing the pain to be sensed without any stimulus.
  • the central sensitization induces secretion of various neuropeptides from peripheral nerve tissue branches through efferent nerve transmission, which has an important role in neurogenous inflammation.
  • Some neuropeptides stimulate dermal mast cells to secrete histamine and eicosanoid, thus inducing local inflammation. This inflammation stimulates sensory nerves, thus secreting a series of neuropeptides which induce axonal reflex and deepen inflammation.
  • inflammation of dorsal-root ganglia by viruses induces stimulation of sympathetic nerves and reduces blood flow of microvessels in the nerves.
  • Continuous ischemia causes intraneural edema and induces nerve damage and irreversible nerve damage when lasting long (An Seong Gu, update in dermatology for dermatologists, Vol. 2 No.2, 2004).
  • Drugs such as non-steroidal anti-inflammatory drugs (NSAIDs), tricyclic antidepressants, antiepileptic drugs, opioids and local anaesthetics are used for treatment of postherpetic neuralgia.
  • surgical section such as nerve block therapy to block sensory nerves, cordotomy and rhizotomy are used.
  • nerve block therapy to block sensory nerves, cordotomy and rhizotomy are used.
  • chronic postherpetic neuralgias which are difficult to treat with these general treatment methods.
  • the continuous pain affects patient quality of life, impairing physical functions and social activity, causing mental pain and thus increasing health management system costs (Niv D, Maltsman-Tseikhin A, Lang E.
  • Postherpetic neuralgia what do we know and where are we heading Pain Physician 2004; 7:239-47; Oster G, Harding G, Dukes E et al . Pain, medication use, and health-related quality of life in older persons with postherpetic neuralgia: results from a population-based survey. J Pain 2005; 6 :356-63; Schmader KE, Sloane R, Pieper C et al. The impact of acute herpes zoster pain and discomfort on functional status and quality of life in older adults. Clin J Pain 2007; 23 :490-6).
  • G-CSF granulocyte-colony stimulating factor
  • neutrophil progenitor cells specifically acts on neutrophil progenitor cells to promote the proliferation and differentiation of neutrophils and increase antibody-dependent cell-mediated cytotoxicity.
  • G-CSF promotes IgA-mediated phagocytosis and increases superoxide production performance. Accordingly, G-CSF is known to improve reactivity to chemotactic peptides, inhibit occurrence of infectious diseases, and reduce the frequency of pyrexia.
  • G-CSF is believed to have little effect upon leukemic stem cells in the body, since it acts on more differentiated bone marrow cells, as compared to other CSFs such as granulocyte-macrophage CSFs (GM-CSFs). Accordingly, G-CSF is widely used for anti-cancer chemotherapy, administration of a great amount of anti-cancer agent, combination therapy with radiotherapy, and a drug for promoting rehabilitation of neutrophils after bone marrow implantation (Julie M. Vores et al. , Clinical Applications of Hematopoietic Growth Factors, Journal of Clinical Oncology , 13 , 1023-1035, (1995)).
  • G-CSF acts as a hematopoietic agent that primarily acts on the proliferation and differentiation of neutrophils, which is primarily used for the treatment of neutropenia caused by bone marrow transplantation and anti-cancer administration and is responsible for increasing neutrophils in myelodysplastic syndromes, aplastic anemia, serious chronic neutropenia (such as congenital, cyclic or idiopathic neutropenia) , HIV-infected patients and preventing infectious diseases caused by decreased neutrophils.
  • Korean Patent Application No. 10-2005-7019543 discloses a diabetes treatment comprising one or more stem cell-recruiting factors such as G-CSFs as active ingredients.
  • Korean Patent Application No. 10-2006-7008042 discloses a fibroblast-mobilizing agent using G-CSF to simply recruit fibroblasts into wounded tissues and engraft the fibroblasts in the wounded tissues, thereby healing the wounds.
  • the present invention has been made in view of the above problems, and it is an object of the present invention to provide an agent for preventing or treating neuropathic pain comprising a granulocyte colony stimulating factor (G-CSF) as an active ingredient and a method for treating neuropathic pain with the same.
  • G-CSF granulocyte colony stimulating factor
  • an agent for preventing or treating neuropathic pain comprising a granulocyte colony stimulating factor (G-CSF) as an active ingredient and a method for treating neuropathic pain with the same.
  • G-CSF granulocyte colony stimulating factor
  • the G-CSF of the present invention advantageously regenerates blood vessels in the injured peripheral nerve tissues and rehabilitates the injured nerve tissues, thereby improving nerve conduction velocity and alleviating neuropathic pain.
  • FIG. 1 is a graph showing nerve conduction velocity measured before nerve injury, after nerve injury and on 14 th and 50 th days after nerve injury, for a sham group, an experimental group and a control group;
  • FIG. 2(a) is an image showing toluidine blue-stained sciatic nerve tissues of the sham group
  • FIG. 2(b) is an image showing toluidine blue-stained sciatic nerve tissues of the experimental group
  • FIG. 2(c) is an image showing toluidine blue-stained sciatic nerve tissues of the control group
  • FIG. 3 is a graph showing avoidance frequency to cold allodynia for the sham group, the control group and the experimental group;
  • FIG. 4 is a graph showing avoidance frequency to mechanical allodynia for the sham group, the control group and the experimental group;
  • FIG. 5 is a graph showing pain level assessed by BPI scores, before administration of G-CSF and 1, 4, 8, 12 and 48 weeks after administration;
  • FIG. 6 is a graph showing pain severity assessed by CGI scores, before administration of G-CSF and on 1, 4, 8, 12 and 48 weeks after administration thereof.
  • G-CSF granulocyte colony stimulating factor
  • G-CSF relieves pain in neuropathic pain animal models and induces regeneration of nerve cells in the nerve tissues. Furthermore, the inventors found through experiments targeting chronic postherpetic neuralgia patients that after administration of G-CSF, brief pain inventory (BPI) as a pain index and clinical global impression (CGI) as a clinical improvement index are significantly decreased, which indicates that G-CSF is useful as a therapeutic agent for postherpetic neuralgia. Based on such a discovery, the present invention has been completed.
  • BPI brief pain inventory
  • CGI clinical global impression
  • the therapeutic agent of the present invention contains G-CSF as an active ingredient.
  • the method for treating neuropathic pain according to the present invention comprises administering a therapeutically effective amount of G-CSF to a subject in need thereof.
  • G-CSF may be used without particular limitation so long as it exhibits biological activity substantially identical to human G-CSF.
  • useful G-CSF include natural G-CSF and recombinant G-CSF. Preferred is the use of those having the same amino acid sequence as natural G-CSF. Most preferred is the use of recombinant human granulocyte colony stimulating factor (rhG-CSF).
  • the origin of G-CSF is not particularly limited in the present invention.
  • the G-CSF may be prepared by separating from a mammal, synthesizing chemically, or genetically expressing an exogenous DNA sequence obtained by genome or cDNA cloning or DNA synthesis in a prokaryotic or eukaryotic host cell.
  • the prokaryotic host useful for the genetic expression includes various bacteria (e.g., E. coli) and suitable eukaryotic hosts include yeast ( e.g. , S. Cerevisiae) and mammalian cells ( e.g., Chinese hamster ovary cells or monkey cells).
  • the G-CSF obtained by gene recombination includes G-CSF having the same amino acid sequence as natural G-CSF, or G-CSF having an amino acid sequence wherein one or more amino acids are deleted, substituted, or added. These G-CSFs and analogs thereof may be obtained from a variety of suppliers and used after purifying the same.
  • the present inventors confirmed the treatment efficacy of the therapeutic agent of the present invention from experiments for neuropathic pain animal models and chronic experiments for chronic postherpetic neuralgia patients.
  • Rats with partially-injured sciatic nerves are generally used as the neuropathic pain animal models.
  • the neuropathic pain animal model often licks its nerve-injured paws even without any external stimuli. This behavior becomes more severe when slight touch stimulus is sensed.
  • the animal shows active symptoms similar to those observed from postherpetic neuralgia patients. For example, the animal pulls in its legs and assumes a defensive posture.
  • the experimental group wherein the therapeutic agent of the present invention was administered to a neuropathic pain animal model, has a great deal of normal tissues similar to the sham group (saline-administered group), which indicates regeneration of nerve tissues (See FIG. 2).
  • G-CSF releases functional stem cells in the bone marrow to peripheral blood and induces differentiation of the released cells, thereby regenerating nerve cells and blood vessels in the peripheral nerve tissues, restoring the injured nerve tissues, promoting blood supply to nerves and regenerating peripheral nerves.
  • the G-CSF exhibits relief activity to neuropathic pain.
  • peripheral nerves and dorsal-root ganglia show behaviors such as infections, hemorrhagic necrosis and nerve damage. Infections to peripheral nerves are continued for several weeks to several months, inducing demyelination, wallerian degeneration and sclerosis. As a result, peripheral nerves and dorsal-root ganglia are injured (Straus SE, Oxman MN, Schmader KE. Varicella and herpes zoster. In: Fitzpatrick's dermatology in general medicine (Wolff K, Goldsmith LA, Katz SI, Gilchrest BA, Paller AS, Leffell DJ, eds), 7th edn.
  • Postherpetic neuralgia-pathogenesis, treatment, and prevention N Engl J Med 1996; 335:32-42).
  • the mechanism of postherpetic neuralgia has not been clearly determined, but continuous injury to sensory nervous systems is one cause of postherpetic neuralgia.
  • G-CSF The mechanism of G-CSF which has an effect on the postherpetic neuralgia mechanism is not completely found, but is thought to be affected by G-CSF activities including (1) anti-inflammatory activity associated with reduction of inflammatory cytokines such as TNF- ⁇ or INF- ⁇ (Solaroglu I, Cahill J, Jadhav V et al . A novel neuroprotectant granulocyte-colony stimulating factor. Stroke 2006; 37 :1123-8), and (2) anti-apoptotic activity modulated by G-CSF receptor and JAK-STAT3 pathway.
  • G-CSF has an important role in nerve regeneration and neuroblast movement.
  • the therapeutic agent of the present invention comprising G-CSF as an active ingredient may contain the active ingredient in an amount of 0.0001 to 50% by weight, based on the total weight of the therapeutic agent composition.
  • the therapeutic agent of the present invention in addition to the active ingredient, may further comprise one or more active ingredients exhibiting the same or similar functions to the active ingredient.
  • the therapeutic agent of the present invention comprising G-CSF as an active ingredient may further comprise at least one pharmaceutically acceptable carrier in addition to the above-mentioned active ingredient to preferably prepare a pharmaceutical composition.
  • the pharmaceutically acceptable carrier which is suitable for sterilization and use in vivo, may be selected from the group consisting of saline, sterilized water, Ringer's solution, buffered saline, an albumin injection solution, a dextrose solution, a maltodextrin solution, glycerol, ethanol, or a mixture thereof.
  • the composition may further comprise other typical additives such as an antioxidant, a buffer, or a bacteriostatic agent.
  • the composition may be prepared as an injection such as a solution, a suspension or an emulsion, pills, capsules, granules or tablets, by adding a diluent, a dispersant, a surfactant, a binder, or a lubricant thereto.
  • the composition may be used by bonding an antibody specific for a target organ or other ligands to the carrier such that the composition functions specifically upon the target organ.
  • a pharmaceutical form of the therapeutic agent of the present invention comprising the G-CSF as an active ingredient may be granules, powders, coated tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops, injectable solutions, and also preparations enabling sustained release of active compounds.
  • the therapeutic agent of the present invention comprising the G-CSF as an active ingredient may be administered in a typical method through an intravenous, intra-arterial, intraperitoneal, intrasternal, intradermal, nasal, inhalant, topical, rectal, oral, intraocular or subcutaneous route.
  • the administration method is not particularly limited, but non-oral administration is preferable, and subcutaneous administration is more preferable.
  • Dosages of the therapeutic agent of the present invention may be adjusted depending on various factors such as type of homoiothermal animal comprising human in need of administration, a type of disease, degree of illness, type and content of active ingredient and other components contained in a composition, type of formulation, patient's age, weight, general health status, gender and diet, administration time, administration route, flow rate of composition, treatment duration, and other drugs used simultaneously.
  • the G-CSF is administered once daily at a dose of 0.01 ⁇ g /kg/day to 100 ⁇ g/kg/day, and preferably 0.01 ⁇ g/kg/day to 10 ⁇ g/kg/day.
  • the administration may be performed once daily or divisionally several times.
  • the therapeutic agent of the present invention may be used alone or in combination with other methods such as surgical operations.
  • the therapeutic agent of the present invention regenerates nerve cells and blood vessels in peripheral nerve tissues to rehabilitate injured nerve tissues and thus improves nerve conduction velocity and relieves pain due to peripheral nerve injury. Accordingly, the therapeutic agent of the present invention is useful for the prevention and treatment of neuropathic pain.
  • Animals used herein were ⁇ 300g adult Sprague-Dawley (SD) rats which had been acclimated to the laboratory surroundings for 5 days, while being sufficiently fed with a solid feed (Samyang Co., Ltd., for cattle animal application) and water.
  • SD Sprague-Dawley
  • the lab temperature was maintained in the range of 24 to 26 °C and the animals were slept on a 12h/12h cycles.
  • test groups were divided into three groups, i.e. , a sham group (normal group) whose nerve was not injured after operation, an experimental group wherein nerves were injured and G-CSF was administered after operation, and a control group wherein nerves were injured and saline was administered after operation.
  • a sham group normal group
  • an experimental group wherein nerves were injured and G-CSF was administered after operation
  • a control group wherein nerves were injured and saline was administered after operation.
  • the sham group normal group
  • the experimental group is 10
  • control group 10 for a total of 28.
  • Neuropathic pain animal models were established by compressing sciatic nerves with forceps.
  • SD rats were anesthetized by injecting 50 mg/kg of ketamine and 9.6 mg/kg of xylazine into the abdomen thereof, were depilated on the gluteal and femoral regions of both lower limbs, and were then placed in the prone position.
  • the femoral region was sterilized with potadine and 70% alcohol, 2 cm of epidermal cells based on the center thereof were longitudinally incised to turn over the musculus biceps femoris and to thus expose the sciatic nerve.
  • the nerve injury was made by incising 2 cm of epidermal cells interposed between the greater trochanter and the knee joint, stripping gluteal muscles and the knee joint muscles to expose sciatic nerves and applying crushing injury on the region where nerves appear at the sciatic notch with the forceps for 30 seconds.
  • the nerve was injured by marking a black line within 5 mm of the end of the forceps and compressing nerves in the constant region, such that a constant strength is applied to a predetermined region.
  • the sham group normal group
  • G-CSF Leucostim available from Dong-a pharm. Co., Ltd.
  • the nerve conduction test was performed after SD rats were anesthetized with a mixed solution of ketamine and xylazine.
  • the sciatic notch was selected as the stimulated site, an active-recording electrode was placed on the leg muscles, a counter electrode was placed on the foot, and a ground electrode was placed between a stimulating electrode and a recording electrode.
  • An adhesive electrode was used as the recording electrode and a pin electrode as the ground electrode was placed on the subcutis.
  • the nerve conduction test was performed with KeyPoint (Dantec, Denmark). The frequency, sweep velocity, and sensitivity were 2 to 10,000 Hz, 2 msec/division, and 5 mV/division, respectively.
  • the nerve conduction test was performed before operation, on the 14 th day after the operation (injury D14) and on the 50 th day after the operation (injury D50). For the test, the latency and width were determined by measuring onset latency, and the width from a base line to a cathode peak, respectively.
  • the laboratory temperature was maintained at 25 °C or higher and the skin temperature of SD rats was maintained at 30 °C or higher. The results thus obtained are shown in Table 1 below and FIG. 1.
  • the experimental group and the control group showed decreased nerve conduction velocity due to nerve injury.
  • the G-CSF-administered experimental group showed increased nerve conduction velocity, as compared to the control group and the control group (saline-administered group).
  • the experimental group As can be seen from FIG. 2, as compared to the control group (saline-administered group), the experimental group (G-CSF-administered group) had a great deal of normal tissue similar to the sham group, which indicates that nerve tissues were regenerated.
  • the sham group normal group
  • the experimental group G-CSF-administered group
  • the avoidance frequency to cold allodynia which indicates that neuropathic pain induced by nerve injury was alleviated.
  • the sham group normal group
  • the experimental group G-CSF-administered group
  • the avoidance frequency to mechanical allodynia which indicates that neuropathic pain induced by nerve injury was relieved.
  • the patients were not affected with serious diseases such as cancers, blood diseases, infectious or immune diseases. Average age and average disease duration of the patients were 69 and 26.3 months, respectively.
  • recombinant human G-CSF (Dona-A Pharmaceutical Co., Ltd., Leucostim ® ) was administered to target patients in a dose of 10 ⁇ g/kg once daily for five days by subcutaneous injection. During the administration, physical examination and experimental tests were monitored daily for patients and all side effects were recorded. After the administration duration of 5 days, experimental and medical details of patients were reviewed and then discharged from the hospital. 4, 8, 12 and 48 weeks after discharge, the patients were invited to the department of dermatology at Hanyang University and data associated with symptoms and side effects of the patients were recorded.
  • Treatment efficacies were evaluated from self-reported brief pain inventory (BPI) and clinical global impression (CGI) scales.
  • BPI Brief pain inventory
  • Pain severity includes four items including "present pain”, “severe pain”, “mild pain” and “moderate pain” for the past one week, and each item is ranked on an 11-point numeric scale from 0 (no pain) to 10 (pain as bad as one imagine).
  • Pain interference on daily function includes seven items. Specifically, pain interference is assessed by measuring intensity 11-point numeric scale on seven items of general activity, mood, walking, normal work, relationships with others, sleep and leisure, wherein 0 represents “does not interfere”and 10 represents "completely interferes"
  • CGI rating scales require the clinician to assess patient's general conditions. Severity assessment is ranked on a 7 point scale from 1 (not ill at all) to 7 (extremely ill). In total clinical experience, all items were assessed by the same researcher to prevent bias. The results thus obtained are shown in FIGs. 5 and 6.
  • FIG. 5 is a graph showing the pain level assessed by BPI scales before administration of G-CSF and 1, 4, 8, 12 and 48 weeks after administration.
  • BPI was initiated to improve within one week after administration of G-CSF.
  • One week after administration BPI was the most significantly decreased, and for the test duration, 48 weeks, BPI score was improved.
  • 48 weeks after administration mean pain severity showed a decrease of 4 from 5.0 to 1.0, which corresponds to 80% improvement in pain level.
  • Mean pain interference showed a decrease of 6.7 (corresponding to 91%) from 7.3 to 0.6, which was significantly improved, as compared to pain severity.
  • FIG. 6 is a graph showing pain severity assessed by CGI scales, before administration of G-CSF and 1, 4, 8, 12 and 48 weeks after administration thereof.
  • CGI was initiated to improve within one week after administration of G-CSF and continuously improved for 12 weeks.
  • a mean CGI score was decreased from 4.3 to 1.0 (corresponding to a top baseline). The value 1.0 was continued for 48 weeks after administration.
  • the therapeutic agent of the present invention is useful for the prevention and treatment of neuropathic pain.

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Abstract

Disclosed herein is an agent for preventing or treating neuropathic pain, comprising a granulocyte colony stimulating factor (G-CSF) as an active ingredient. The therapeutic agent advantageously regenerates nerve cells and blood vessels in peripheral nerve tissues and thus rehabilitates the injured nerve tissues to improve nerve conduction velocity, and relieves neuropathic pain.

Description

AGENT COMPRISING G-CSF FOR TREATMENT OR PREVENTION OF NEUROPATHIC PAIN AND METHOD FOR TREATING NEUROPATHIC PAIN WITH THE SAME
The present invention relates to a preventive or therapeutic agent for neuropathic pain comprising a granulocyte colony stimulating factor (G-CSF) as an active ingredient and a method for treating neuropathic pain with the same.
Pain is defined as an unpleasant sensory and emotional experience originating from a specific site in the body. In particular, chronic pain is defined as extremely intolerable pain that persists longer than the temporal course of natural healing. Patients who suffer from chronic pain experience a great deal of difficulties in leading a normal life. Pain is generally sensed in accordance with a mechanism wherein a noxious stimulus applied to the body's pain acceptor is converted into a pain signal which is transmitted to the central nervous system. On the other hand, damage to nervous systems may cause continuous pain not associated with any external harmful stimulus. The one is referred to as "nociceptive pain" and the other is referred to as "neuropathic pain".
Nociceptive pain is normally caused by tissue damage or disease. Such pain serves to warn tissue damage and prevent the same. Meanwhile, neuropathic pain or neuralgia is chronic pain caused by damage to peripheral nerves or tissues (J Pain. 2008 Jan;9(1 Suppl 1):S10-8. Mechanisms of pain and itch caused by herpes zoster (shingles)).
Clinically, symptoms observed in those who suffer from neuropathic pain include hyperalgesia, allodynia, spontaneous pain, etc. Hyperalgesia is defined as an increased sensitivity to a pain stimulus which is normally painful. Allodynia is defined as an extreme response to a usually non-painful stimulus that has no potential to normally induce pain. Spontaneous pain is defined as pain sensed without any stimulus. Since neuropathic pain was first disclosed in 1872 by Mitchell (Mitchell, S.W. Injuries of Nerves and Their Consequences, J.B. Lippincott, Philadelphia, PA, 1872; p.252), it has been found in a number of patients and individuals suffering from neuropathic-like pain are common in Korea (Bae Hwan Lee, et al., Korean Journal of Brain Science and Technology, Vol.1, No.1, pp.53-64, June 2001).
Representative examples of neuropathic pain include postherpetic neuralgia, post-thoracotomic pain, trigeminal neuralgia, disseminated sclerosis-associated pain, thalamic pain, phantom limb pain, anesthesia dolorosa, HIV-associated neuropathic pain, spinal cord disorder-associated paraplegic pain, complex regional pain syndromes , etc. (CNS Drugs. 2008;22(5):417-42. An update on the pharmacological management of post-herpetic neuralgia and painful diabetic neuropathy).
Meanwhile, herpes zoster (or simply zoster), commonly known as shingles, is a disease wherein the varicella zoster virus invade spinal sensory nerve roots, sensory ganglia, spinal dorsal-roots, and skin sensory or cerebral nerve peripheral branches corresponding thereto, causing symptoms such as vesicular eruption, pain and oversensitivity in the skin. Postherpetic neuralgia is the most common zoster complication, which is defined as pain persisted for three months or longer after crusts are formed in skin lesions (Young L. Postherpetic neuralgia: a review of advances in treatment and prevention. J Drugs Dermatol 2006; 5:938-41; Wareham D. Postherpetic neuralgia. Clin Evid 2005; 14:1017-25).
Postherpetic neuralgia is one of neuropathic pains such as post-thoracotomic pain, trigeminal neuralgia, diabetic neuropathy, disseminated sclerosis-associated pain, thalamic pain and phantom limb pain. The main symptom of postherpetic neuralgia is unilateral pain arising along nerve ganglia. The pain variably ranges from mild to severe. Postherpetic neuralgia involves burning pain, aching pain, itching, continuous pain and intense shooting pain, followed by hyperalgesia and allodynia. Postherpetic neuralgia is difficult to treat and there are premonitory symptoms associated with postherpetic neuralgia, such as old age, serious initial pain, fever and rash, immune conditions and underlying diseases.
Postherpetic neuralgia arises in 10 to 15 % of acute zoster patients. The disease rate rapidly increases with increase in patient's age, rising to 50% for acute zoster patients with an age 60 or over (Rice AS, Maton S. Gabapentin in postherpetic neuralgia: a randomized, double blind, placebo controlled study. Pain 2001; 94:215-24; Seventer VR, Sadosky A, Lucero M et al. A cross-sectional survey of health state impairment and treatment patterns in patients with postherpetic neuralgia. Age Ageing 2006; 35:132-7; Volmink J, Lancaster T, Gray S et al. Treatments for postherpetic neuralgia a systematic review of randomized controlled trials. Fam Pract 1996; 13:84-91). In addition, pain duration increases with patient age and approaches one year or longer for 48% of patients (≥ 70 years of age) (Kost RG, Straus SE. Postherpetic neuralgia-pathogenesis, treatment, and prevention. N Engl J Med 1996; 335:32-42; Niv D, Maltsman-Tseikhin A, Lang E. Postherpetic neuralgia: what do we know and where are we heading Pain Physician 2004; 7:239-47). The recent increase in the elderly population has brought about increases in zoster and thus postherpetic neuralgia.
Postherpetic neuralgia is caused by a different mechanism from the pain of acute zoster. Acute zoster is mainly caused by inflammation and necrosis, while postherpetic neuralgia is induced by neuropathic factor caused by nervous system injury or dysfunction. There are hypotheses known up to date. First, varicella zoster viruses are reactivated, causing injury to adjacent nervous tissues or non-nervous neighboring tissues, while being transferred via blood flow or directly. In particular, elderly people take a long time to recover from nervous injury, thus lengthening neuralgia duration. Second, viruses are diffused into the dermal or hypodermic tissues, inducing inflammation, which causes tissue injury and pain. This injury repeatedly stimulates afferent C-fibers and induces continuous excitation of axons in spinal dorsal-roots. This noxious stimulus induces central sensitization, allowing the pain to be sensed without any stimulus. The central sensitization induces secretion of various neuropeptides from peripheral nerve tissue branches through efferent nerve transmission, which has an important role in neurogenous inflammation. Some neuropeptides stimulate dermal mast cells to secrete histamine and eicosanoid, thus inducing local inflammation. This inflammation stimulates sensory nerves, thus secreting a series of neuropeptides which induce axonal reflex and deepen inflammation. Third, inflammation of dorsal-root ganglia by viruses induces stimulation of sympathetic nerves and reduces blood flow of microvessels in the nerves. Continuous ischemia causes intraneural edema and induces nerve damage and irreversible nerve damage when lasting long (An Seong Gu, update in dermatology for dermatologists, Vol. 2 No.2, 2004).
Drugs such as non-steroidal anti-inflammatory drugs (NSAIDs), tricyclic antidepressants, antiepileptic drugs, opioids and local anaesthetics are used for treatment of postherpetic neuralgia. In addition, surgical section such as nerve block therapy to block sensory nerves, cordotomy and rhizotomy are used. However, there are chronic postherpetic neuralgias which are difficult to treat with these general treatment methods. The continuous pain affects patient quality of life, impairing physical functions and social activity, causing mental pain and thus increasing health management system costs (Niv D, Maltsman-Tseikhin A, Lang E. Postherpetic neuralgia: what do we know and where are we heading Pain Physician 2004; 7:239-47; Oster G, Harding G, Dukes E et al. Pain, medication use, and health-related quality of life in older persons with postherpetic neuralgia: results from a population-based survey. J Pain 2005; 6:356-63; Schmader KE, Sloane R, Pieper C et al. The impact of acute herpes zoster pain and discomfort on functional status and quality of life in older adults. Clin J Pain 2007; 23:490-6).
Accordingly, there is an increasing need for development of a method for efficiently treating neuropathic pain including postherpetic neuralgia.
Meanwhile, granulocyte-colony stimulating factor (G-CSF) specifically acts on neutrophil progenitor cells to promote the proliferation and differentiation of neutrophils and increase antibody-dependent cell-mediated cytotoxicity. In addition, G-CSF promotes IgA-mediated phagocytosis and increases superoxide production performance. Accordingly, G-CSF is known to improve reactivity to chemotactic peptides, inhibit occurrence of infectious diseases, and reduce the frequency of pyrexia.
In addition, G-CSF is believed to have little effect upon leukemic stem cells in the body, since it acts on more differentiated bone marrow cells, as compared to other CSFs such as granulocyte-macrophage CSFs (GM-CSFs). Accordingly, G-CSF is widely used for anti-cancer chemotherapy, administration of a great amount of anti-cancer agent, combination therapy with radiotherapy, and a drug for promoting rehabilitation of neutrophils after bone marrow implantation (Julie M. Vores et al., Clinical Applications of Hematopoietic Growth Factors, Journal of Clinical Oncology, 13, 1023-1035, (1995)).
Such G-CSF acts as a hematopoietic agent that primarily acts on the proliferation and differentiation of neutrophils, which is primarily used for the treatment of neutropenia caused by bone marrow transplantation and anti-cancer administration and is responsible for increasing neutrophils in myelodysplastic syndromes, aplastic anemia, serious chronic neutropenia (such as congenital, cyclic or idiopathic neutropenia) , HIV-infected patients and preventing infectious diseases caused by decreased neutrophils.
In recent years, a great deal of research has been conducted on, in addition to clinical use of G-CSF for neutropenia, administration of G-CSF alone or in combination with an antibiotic for the treatment of infectious diseases, based on the expectation that G-CSF promotes neutrophil production and reinforces neutrophil performance, thus being potent for preventing and treating various infectious diseases such as pneumonia or septicemia.
Several therapeutic agents utilizing G-CSF, based on various physiological activities, have been suggested. For example, Korean Patent Application No. 10-2005-7019543 discloses a diabetes treatment comprising one or more stem cell-recruiting factors such as G-CSFs as active ingredients. In addition, Korean Patent Application No. 10-2006-7008042 discloses a fibroblast-mobilizing agent using G-CSF to simply recruit fibroblasts into wounded tissues and engraft the fibroblasts in the wounded tissues, thereby healing the wounds.
However, there is no research that recognizes the treatment of neuropathic pain as a novel application of G-CSF.
Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide an agent for preventing or treating neuropathic pain comprising a granulocyte colony stimulating factor (G-CSF) as an active ingredient and a method for treating neuropathic pain with the same.
In accordance with one aspect of the present invention, the above and other objects can be accomplished by the provision of an agent for preventing or treating neuropathic pain comprising a granulocyte colony stimulating factor (G-CSF) as an active ingredient and a method for treating neuropathic pain with the same.
The G-CSF of the present invention advantageously regenerates blood vessels in the injured peripheral nerve tissues and rehabilitates the injured nerve tissues, thereby improving nerve conduction velocity and alleviating neuropathic pain.
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a graph showing nerve conduction velocity measured before nerve injury, after nerve injury and on 14th and 50th days after nerve injury, for a sham group, an experimental group and a control group;
FIG. 2(a) is an image showing toluidine blue-stained sciatic nerve tissues of the sham group, FIG. 2(b) is an image showing toluidine blue-stained sciatic nerve tissues of the experimental group, and FIG. 2(c) is an image showing toluidine blue-stained sciatic nerve tissues of the control group;
FIG. 3 is a graph showing avoidance frequency to cold allodynia for the sham group, the control group and the experimental group;
FIG. 4 is a graph showing avoidance frequency to mechanical allodynia for the sham group, the control group and the experimental group;
FIG. 5 is a graph showing pain level assessed by BPI scores, before administration of G-CSF and 1, 4, 8, 12 and 48 weeks after administration; and
FIG. 6 is a graph showing pain severity assessed by CGI scores, before administration of G-CSF and on 1, 4, 8, 12 and 48 weeks after administration thereof.
Hereinafter, the present invention will be illustrated in more detail.
During research on various physiological activities of granulocyte colony stimulating factor (hereinafter, referred to as 'G-CSF'), the inventors of the present invention discovered that G-CSF regenerates blood vessels in peripheral nerve tissues and rehabilitates the injured nerve tissues, thus improving nerve conduction velocity and pain sensitivity, thereby being useful as a preventive and therapeutic agent for diabetic peripheral neuropathies. Korean Patent No. 0812274 was granted to the present inventors, based on this discovery.
In the process of a great deal of research to confirm whether G-CSF is efficacious for novel indications, in addition to diabetic peripheral neuropathies, the present inventors discovered that G-CSF relieves pain in neuropathic pain animal models and induces regeneration of nerve cells in the nerve tissues. Furthermore, the inventors found through experiments targeting chronic postherpetic neuralgia patients that after administration of G-CSF, brief pain inventory (BPI) as a pain index and clinical global impression (CGI) as a clinical improvement index are significantly decreased, which indicates that G-CSF is useful as a therapeutic agent for postherpetic neuralgia. Based on such a discovery, the present invention has been completed.
The therapeutic agent of the present invention contains G-CSF as an active ingredient. In addition, the method for treating neuropathic pain according to the present invention comprises administering a therapeutically effective amount of G-CSF to a subject in need thereof.
Any G-CSF may be used without particular limitation so long as it exhibits biological activity substantially identical to human G-CSF. Representative examples of useful G-CSF include natural G-CSF and recombinant G-CSF. Preferred is the use of those having the same amino acid sequence as natural G-CSF. Most preferred is the use of recombinant human granulocyte colony stimulating factor (rhG-CSF).
The origin of G-CSF is not particularly limited in the present invention. The G-CSF may be prepared by separating from a mammal, synthesizing chemically, or genetically expressing an exogenous DNA sequence obtained by genome or cDNA cloning or DNA synthesis in a prokaryotic or eukaryotic host cell. The prokaryotic host useful for the genetic expression includes various bacteria (e.g., E. coli) and suitable eukaryotic hosts include yeast (e.g., S. Cerevisiae) and mammalian cells (e.g., Chinese hamster ovary cells or monkey cells).
The G-CSF obtained by gene recombination includes G-CSF having the same amino acid sequence as natural G-CSF, or G-CSF having an amino acid sequence wherein one or more amino acids are deleted, substituted, or added. These G-CSFs and analogs thereof may be obtained from a variety of suppliers and used after purifying the same.
The present inventors confirmed the treatment efficacy of the therapeutic agent of the present invention from experiments for neuropathic pain animal models and chronic experiments for chronic postherpetic neuralgia patients.
Rats with partially-injured sciatic nerves are generally used as the neuropathic pain animal models. Clinically, the neuropathic pain animal model often licks its nerve-injured paws even without any external stimuli. This behavior becomes more severe when slight touch stimulus is sensed. As a result, the animal shows active symptoms similar to those observed from postherpetic neuralgia patients. For example, the animal pulls in its legs and assumes a defensive posture.
As can be seen from Experimental Example 2, the experimental group, wherein the therapeutic agent of the present invention was administered to a neuropathic pain animal model, exhibited decreased nerve conduction velocity due to nerve injury, as compared to the control group (See FIG. 1).
In addition, as can be seen from Experimental Example 3, the experimental group, wherein the therapeutic agent of the present invention was administered to a neuropathic pain animal model, has a great deal of normal tissues similar to the sham group (saline-administered group), which indicates regeneration of nerve tissues (See FIG. 2).
In addition, as can be seen from Experimental Examples 4 and 5, when the therapeutic agent of the present invention was administered to a neuropathic pain animal model, the pain of nerve injury was alleviated owing to the administration of G-CSF (See FIGs. 3 and 4).
These behaviors occur because G-CSF releases functional stem cells in the bone marrow to peripheral blood and induces differentiation of the released cells, thereby regenerating nerve cells and blood vessels in the peripheral nerve tissues, restoring the injured nerve tissues, promoting blood supply to nerves and regenerating peripheral nerves. In addition, the G-CSF exhibits relief activity to neuropathic pain.
Referring to Experimental Example 6, when G-CSF was administered to postherpetic neuralgia patients that had taken chronic pain drugs for one year or longer, pain intensity and general quality of life were improved. Clinical improvement in pain had already been initiated within one week and BPI was improved for 48 weeks after administration. In particular, of BPI scales, pain interference on general activity was improved 90% from a base scale, which means that self-reported functional prognosis is significantly improved. Furthermore, after clinical tests, all patients sensed no or almost no pain, thus eliminating the necessity of pain drug administration.
In an acute stage of zoster, peripheral nerves and dorsal-root ganglia show behaviors such as infections, hemorrhagic necrosis and nerve damage. Infections to peripheral nerves are continued for several weeks to several months, inducing demyelination, wallerian degeneration and sclerosis. As a result, peripheral nerves and dorsal-root ganglia are injured (Straus SE, Oxman MN, Schmader KE. Varicella and herpes zoster. In: Fitzpatrick's dermatology in general medicine (Wolff K, Goldsmith LA, Katz SI, Gilchrest BA, Paller AS, Leffell DJ, eds), 7th edn. New York: McGraw-Hill, 2007:1885-98; Watson CP, Deck JH, Morshead C. Post-herpetic neuralgia: further post-mortem studies of cases with and without pain. Pain 1991; 44:105-17). The central nervous system is significantly pathologically varied. Such a variation may cause acute degeneration of dorsal horn, unilateral segmental myelitis and leptomeningitis in spinal cords adjacent to the infected skin. As a result of autopsy of patients who suffered from zoster, patients affected with postherpetic neuralgia showed atrophy of spinal dorsal horn, unlike patients not affected with postherpetic neuralgia (Kost RG, Straus SE. Postherpetic neuralgia-pathogenesis, treatment, and prevention. N Engl J Med 1996; 335:32-42). The mechanism of postherpetic neuralgia has not been clearly determined, but continuous injury to sensory nervous systems is one cause of postherpetic neuralgia.
The mechanism of G-CSF which has an effect on the postherpetic neuralgia mechanism is not completely found, but is thought to be affected by G-CSF activities including (1) anti-inflammatory activity associated with reduction of inflammatory cytokines such as TNF-α or INF-γ (Solaroglu I, Cahill J, Jadhav V et al. A novel neuroprotectant granulocyte-colony stimulating factor. Stroke 2006; 37:1123-8), and (2) anti-apoptotic activity modulated by G-CSF receptor and JAK-STAT3 pathway. In addition, for chronic nerve injury such as postherpetic neuralgia, G-CSF has an important role in nerve regeneration and neuroblast movement.
The therapeutic agent of the present invention comprising G-CSF as an active ingredient may contain the active ingredient in an amount of 0.0001 to 50% by weight, based on the total weight of the therapeutic agent composition.
In addition, the therapeutic agent of the present invention, in addition to the active ingredient, may further comprise one or more active ingredients exhibiting the same or similar functions to the active ingredient.
The therapeutic agent of the present invention comprising G-CSF as an active ingredient may further comprise at least one pharmaceutically acceptable carrier in addition to the above-mentioned active ingredient to preferably prepare a pharmaceutical composition. In preparing the composition in a liquid solution, as the pharmaceutically acceptable carrier, which is suitable for sterilization and use in vivo, may be selected from the group consisting of saline, sterilized water, Ringer's solution, buffered saline, an albumin injection solution, a dextrose solution, a maltodextrin solution, glycerol, ethanol, or a mixture thereof. If necessary, the composition may further comprise other typical additives such as an antioxidant, a buffer, or a bacteriostatic agent. Further, the composition may be prepared as an injection such as a solution, a suspension or an emulsion, pills, capsules, granules or tablets, by adding a diluent, a dispersant, a surfactant, a binder, or a lubricant thereto. The composition may be used by bonding an antibody specific for a target organ or other ligands to the carrier such that the composition functions specifically upon the target organ.
A pharmaceutical form of the therapeutic agent of the present invention comprising the G-CSF as an active ingredient may be granules, powders, coated tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops, injectable solutions, and also preparations enabling sustained release of active compounds.
The therapeutic agent of the present invention comprising the G-CSF as an active ingredient may be administered in a typical method through an intravenous, intra-arterial, intraperitoneal, intrasternal, intradermal, nasal, inhalant, topical, rectal, oral, intraocular or subcutaneous route. The administration method is not particularly limited, but non-oral administration is preferable, and subcutaneous administration is more preferable.
Dosages of the therapeutic agent of the present invention may be adjusted depending on various factors such as type of homoiothermal animal comprising human in need of administration, a type of disease, degree of illness, type and content of active ingredient and other components contained in a composition, type of formulation, patient's age, weight, general health status, gender and diet, administration time, administration route, flow rate of composition, treatment duration, and other drugs used simultaneously. In case of an adult, the G-CSF is administered once daily at a dose of 0.01 ㎍ /kg/day to 100 ㎍/kg/day, and preferably 0.01 ㎍/kg/day to 10 ㎍/kg/day. The administration may be performed once daily or divisionally several times.
The therapeutic agent of the present invention may be used alone or in combination with other methods such as surgical operations.
The therapeutic agent of the present invention regenerates nerve cells and blood vessels in peripheral nerve tissues to rehabilitate injured nerve tissues and thus improves nerve conduction velocity and relieves pain due to peripheral nerve injury. Accordingly, the therapeutic agent of the present invention is useful for the prevention and treatment of neuropathic pain.
Hereinafter, examples will be provided for a further understanding of the invention. The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Experimental Example 1: Therapeutic activity of G-CSF on Neuropathic Pain
Animals used herein were ~300g adult Sprague-Dawley (SD) rats which had been acclimated to the laboratory surroundings for 5 days, while being sufficiently fed with a solid feed (Samyang Co., Ltd., for cattle animal application) and water. The lab temperature was maintained in the range of 24 to 26 ℃ and the animals were slept on a 12h/12h cycles.
The test groups were divided into three groups, i.e., a sham group (normal group) whose nerve was not injured after operation, an experimental group wherein nerves were injured and G-CSF was administered after operation, and a control group wherein nerves were injured and saline was administered after operation. For the number of respective groups, the sham group (normal group) is 8, the experimental group is 10 and the control group 10, for a total of 28.
Neuropathic pain animal models were established by compressing sciatic nerves with forceps.
Specifically, SD rats were anesthetized by injecting 50 mg/kg of ketamine and 9.6 mg/kg of xylazine into the abdomen thereof, were depilated on the gluteal and femoral regions of both lower limbs, and were then placed in the prone position. The femoral region was sterilized with potadine and 70% alcohol, 2 cm of epidermal cells based on the center thereof were longitudinally incised to turn over the musculus biceps femoris and to thus expose the sciatic nerve. The nerve injury was made by incising 2 cm of epidermal cells interposed between the greater trochanter and the knee joint, stripping gluteal muscles and the knee joint muscles to expose sciatic nerves and applying crushing injury on the region where nerves appear at the sciatic notch with the forceps for 30 seconds. The nerve was injured by marking a black line within 5 mm of the end of the forceps and compressing nerves in the constant region, such that a constant strength is applied to a predetermined region. The sham group (normal group) was subjected to operation in the same manner as in the experimental and control groups except that nerves were not injured with forceps. After operation, the wound was sealed and sterilized.
14 days after operation (injury D14), G-CSF (Leucostim available from Dong-a pharm. Co., Ltd.) was administered once daily at 100 ㎍/kg/day for 5 days to the abdominal subcutis of the experimental group, and 0.2 mL of saline was intraperitoneally administered once daily for 5 days to the control group and the sham group (normal group). Then, the test groups were observed for about 4 weeks.
Experimental Example 2: Nerve conduction test on Neuropathic Pain Animal Model
The nerve conduction test was performed after SD rats were anesthetized with a mixed solution of ketamine and xylazine. The sciatic notch was selected as the stimulated site, an active-recording electrode was placed on the leg muscles, a counter electrode was placed on the foot, and a ground electrode was placed between a stimulating electrode and a recording electrode. An adhesive electrode was used as the recording electrode and a pin electrode as the ground electrode was placed on the subcutis.
The nerve conduction test was performed with KeyPoint (Dantec, Denmark). The frequency, sweep velocity, and sensitivity were 2 to 10,000 Hz, 2 msec/division, and 5 mV/division, respectively. The nerve conduction test was performed before operation, on the 14th day after the operation (injury D14) and on the 50th day after the operation (injury D50). For the test, the latency and width were determined by measuring onset latency, and the width from a base line to a cathode peak, respectively.
The nerve conduction test was carried out by obtaining three respective values from the both sides, for the sham group (normal group, n=8), the experimental group (n=10) and the control group (n=10). The laboratory temperature was maintained at 25 ℃ or higher and the skin temperature of SD rats was maintained at 30 ℃ or higher. The results thus obtained are shown in Table 1 below and FIG. 1.
Table 1 Nerve conduction velocity (m/s)
Test Groups Before operation D 14 D 50
Sham group 83.4 82.1 86.9
Control group (saline-administration) 82.8 39.2 40.8
Experimental Group(G-CSF administration) 85.8 47.8 69.7
As can be seen from FIG. 1, the experimental group and the control group showed decreased nerve conduction velocity due to nerve injury. On the other hand, the G-CSF-administered experimental group showed increased nerve conduction velocity, as compared to the control group and the control group (saline-administered group).
Experimental Example 3: H istopathological examination on Neuropathic Pain Animal Model
On the 50th day after operation (injury D 50), sciatic nerve tissues of each group were extracted in order to identify the tissue variation and neurohistological examination was performed using toluidine blue staining. The results thus obtained are shown in FIG. 2.
As can be seen from FIG. 2, as compared to the control group (saline-administered group), the experimental group (G-CSF-administered group) had a great deal of normal tissue similar to the sham group, which indicates that nerve tissues were regenerated.
Experimental Example 4: Cold Allodynia Test on Neuropathic Pain Animal Model
Whether or not cold allodynia was observed on 1, 2, 6, 8, 10, 14, 20, 27, 31, 38, 42, 45 and 50th days after operation, was confirmed for respective groups.
Specifically, pain by cold stimuli was measured by dropping acetone on the plantar of the injured leg to perform avoidance response (Tal et al., Onset of ectopic firing in the Chung model of neuropathic pain coincides with the onset of tactile allodynia, Proceedings of the 11th World Congress on Pain). The acetone was dropped on the plantar of the injured leg five times at five-minute intervals using a polyethylene tube connected to a syringe. The response frequency (%) was determined by dividing the leg avoidance frequency by the total number of tests and calculating as a percentage. The results thus obtained are shown in FIG. 3.
As can be seen from FIG. 3, the sham group (normal group) was not injured by operation, but sensed pain only thereby. As compared to the control group (saline-administered group), the experimental group (G-CSF-administered group) exhibited decreased avoidance frequency to cold allodynia, which indicates that neuropathic pain induced by nerve injury was alleviated.
Experimental Example 5: Mechanical Allodynia Test on Neuropathic Pain Animal Model
Whether or not mechanical allodynia was observed on 1, 2, 6, 8, 10, 14, 20, 27, 31, 38, 42, 45 and 50th days after operation, was confirmed for respective groups.
Specifically, harmless mechanical stimuli not normally inducing pain were applied to the plantar of the injured leg using von Frey hair (15 gm; 147 mN). The plantar was slightly stimulated with the von Frey hair, to find and mark the site sensitive to the mechanical stimuli. The leg skin was stimulated ten times at 10-20-minute intervals on the marked point. As the response frequency increases, the pain response increases. 0.5 cm or higher of rough sudden avoidance response was counted as a frequency. The response frequency (%) was represented by the frequency of avoidance responses/10(trial frequency)×100, and the results thus obtained are shown in FIG. 4.
As can be seen from FIG. 4, the sham group (normal group) was not injured by operation, but sensed pain by the operation. As compared to the control group (saline-administered group), the experimental group (G-CSF-administered group) exhibited decreased avoidance frequency to mechanical allodynia, which indicates that neuropathic pain induced by nerve injury was relieved.
Experimental Example 6: Clinical Test to confirm treatment efficacy of G-CSF for postherpetic neuralgia
6-1. Test subjects
Three patients suffering from chronic postherpetic neuralgia that had been invited to the department of dermatology at Hanyang University from February to July in 2007 were targeted for tests. The patients that participated in the test had failed to alleviate pain in spite of administration of conventional therapeutic agents including nonsteroidal anti-inflammatory drugs, antidepressants and antiepileptic drugs and had taken these drugs for one year or longer. Basic information, medical history and therapeutic history of the patients are shown in Table 2 below.
Table 2
Age Gender Disease duration (month) Therapeutic agent, Maximum dosage, Administration duration Pain site Systemic disease
62 Male 22 Amitryptiline, 75 mg/day, 12 monthsGabapentin, 1200 mg/day, 12 monthsAceclofenac, 200 mg/day, 12 monthsAmitryptiline 100 mg/day, 38 monthsGabapentin, 600 mg/day, 28 monthsNonsteroidal agent, if necessary Left face scalp DiabetesHypertension
66 Female 38 Gabapentin, 600 mg/day, 19 monthsAceclofenac, 200 mg/day, 19 monthsAntihistamine agent, if necessary Left face scalp None
79 Female 19 Amitryptiline, 75 mg/day, 19 monthsGabapentin, 1,200 mg/day, 12 monthsAceclofenac, 200 mg/day, 12 monthsAntihistamine agent, if necessary Left arm Front breast AsthmaHypertension
The patients were not affected with serious diseases such as cancers, blood diseases, infectious or immune diseases. Average age and average disease duration of the patients were 69 and 26.3 months, respectively.
6-2. Test method
Patients hospitalized were evaluated for serious medical history including zoster records, present prescriptions and vital sign records depending on individual medical charts. For general tests including overall blood tests, kidney function tests, liver function tests and electrolyte level measurement, blood samples were collected. Prior to screening tests, nonsteroidal anti-inflammatory drugs, local anesthetics (lidocaine, capsaicin), tricyclic antidepressants (amitryptiline, nortriptyline), antiepileptic drugs (gabapentin, carbamazepine), opiates and novel antidepressants (fluoxetine, bupropion) were maintained for a washout period of 14 days.
After all data were recorded, recombinant human G-CSF (Dona-A Pharmaceutical Co., Ltd., Leucostim®) was administered to target patients in a dose of 10 ㎍/kg once daily for five days by subcutaneous injection. During the administration, physical examination and experimental tests were monitored daily for patients and all side effects were recorded. After the administration duration of 5 days, experimental and medical details of patients were reviewed and then discharged from the hospital. 4, 8, 12 and 48 weeks after discharge, the patients were invited to the department of dermatology at Hanyang University and data associated with symptoms and side effects of the patients were recorded.
6-3. Evaluation of treatment efficacy
Treatment efficacies were evaluated from self-reported brief pain inventory (BPI) and clinical global impression (CGI) scales.
Brief pain inventory (BPI) is a simple and readily applicable multi-dimensional pain measurement which provides pain information including pain history, pain intensity, pain location and pain level. Validity and reliability of this measurement were verified in 1993 by Cleeland and Flanery.
Pain severity includes four items including "present pain", "severe pain", "mild pain" and "moderate pain" for the past one week, and each item is ranked on an 11-point numeric scale from 0 (no pain) to 10 (pain as bad as one imagine).
Pain interference on daily function includes seven items. Specifically, pain interference is assessed by measuring intensity 11-point numeric scale on seven items of general activity, mood, walking, normal work, relationships with others, sleep and leisure, wherein 0 represents "does not interfere"and 10 represents "completely interferes"
Clinical global impression (CGI) rating scales require the clinician to assess patient's general conditions. Severity assessment is ranked on a 7 point scale from 1 (not ill at all) to 7 (extremely ill). In total clinical experience, all items were assessed by the same researcher to prevent bias. The results thus obtained are shown in FIGs. 5 and 6.
6-4. Results of Clinical Tests
All patients clinically experienced alleviation of pain, without any serious side effects. Throughout the test duration, all patients participated in tests.
FIG. 5 is a graph showing the pain level assessed by BPI scales before administration of G-CSF and 1, 4, 8, 12 and 48 weeks after administration.
Referring to FIG. 5, BPI was initiated to improve within one week after administration of G-CSF. One week after administration, BPI was the most significantly decreased, and for the test duration, 48 weeks, BPI score was improved. 48 weeks after administration, mean pain severity showed a decrease of 4 from 5.0 to 1.0, which corresponds to 80% improvement in pain level. Mean pain interference showed a decrease of 6.7 (corresponding to 91%) from 7.3 to 0.6, which was significantly improved, as compared to pain severity.
FIG. 6 is a graph showing pain severity assessed by CGI scales, before administration of G-CSF and 1, 4, 8, 12 and 48 weeks after administration thereof.
Referring to FIG. 6, similar to BPI, CGI was initiated to improve within one week after administration of G-CSF and continuously improved for 12 weeks. On 12 weeks after administration, a mean CGI score was decreased from 4.3 to 1.0 (corresponding to a top baseline). The value 1.0 was continued for 48 weeks after administration.
As apparent from the foregoing, the therapeutic agent of the present invention is useful for the prevention and treatment of neuropathic pain.

Claims (6)

  1. An agent for preventing or treating neuropathic pain, comprising a granulocyte colony stimulating factor (G-CSF) as an active ingredient.
  2. The agent according to claim 1, wherein the G-CSF is obtained and separated from natural or recombinant origin.
  3. The agent according to claim 1, wherein the G-CSF is a recombinant human granulocyte colony stimulating factor (rhG-CSF).
  4. The agent according to claim 1, wherein the neuropathic pain is postherpetic neuralgia, post-thoracotomic pain, trigeminal neuralgia, disseminated sclerosis-associated pain, thalamic pain, phantom limb pain, anesthesia dolorosa, HIV-associated neuropathic pain, spinal cord disorder-associated paraplegic pain or complex regional pain syndrome.
  5. The use of granulocyte-colony stimulating factor (G-CSF) for treating neuropathic pain.
  6. A method of treating neuropathic pain, comprising administering a therapeutically effective amount of granulocyte-colony stimulating factor (G-CSF) to a patient in need thereof.
PCT/KR2009/002627 2008-08-12 2009-05-19 Agent comprising g-csf for treatment or prevention of neuropathic pain and method for treating neuropathic pain with the same WO2010018918A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004082703A1 (en) * 2003-03-20 2004-09-30 Keiichi Fukuda Combined use of g-csf with angiogenetic factor
US20060153799A1 (en) * 2004-11-05 2006-07-13 Northwestern University Use of SCF and G-CSF in the treatment of cerebral ischemia and neurological disorders
US20070196279A1 (en) * 2006-02-21 2007-08-23 Shen Che-Kun J Methods for treating progressive neurodegenerative disorders
WO2008054098A1 (en) * 2006-10-30 2008-05-08 Dong-A Pharm.Co., Ltd. Agent comprising g-csf for prevention and treatment of diabetic peripheral neuropathy

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004082703A1 (en) * 2003-03-20 2004-09-30 Keiichi Fukuda Combined use of g-csf with angiogenetic factor
US20060153799A1 (en) * 2004-11-05 2006-07-13 Northwestern University Use of SCF and G-CSF in the treatment of cerebral ischemia and neurological disorders
US20070196279A1 (en) * 2006-02-21 2007-08-23 Shen Che-Kun J Methods for treating progressive neurodegenerative disorders
WO2008054098A1 (en) * 2006-10-30 2008-05-08 Dong-A Pharm.Co., Ltd. Agent comprising g-csf for prevention and treatment of diabetic peripheral neuropathy

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