Gene therapy for diabetic neuropathy using an HGF isoform
10759841 ยท 2020-09-01
Assignee
Inventors
Cpc classification
A61K38/1833
HUMAN NECESSITIES
C07K14/4753
CHEMISTRY; METALLURGY
A61K31/711
HUMAN NECESSITIES
A61K48/005
HUMAN NECESSITIES
International classification
A61K31/711
HUMAN NECESSITIES
Abstract
The present invention relates to a pharmaceutical composition for the prevention or treatment of diabetic neuropathy, wherein the pharmaceutical composition comprises, as active ingredients, different types of isoforms of HGF or a polynucleotide encoding the isoforms. The present invention is the first invention demonstrating that diabetic neuropathy can be prevented and treated using different types of isoforms of HGF. According to the present invention, it is possible to very effectively treat diabetic neuropathy.
Claims
1. A method for treating a human subject having painful diabetic peripheral neuropathy, the method comprising: intramuscularly administering to an affected limb of the human subject in need thereof, a pCK-HGF-X7 DNA construct at a dose of 8 mg per affected limb, equally divided into a plurality of injections to reduce pain in said human subject, wherein the pCK-HGF-X7 DNA construct comprises the nucleotide sequence as set forth in SEQ ID NO: 13, and wherein each of the plurality of injections is performed with 0.25 mg of the pCK-HGF-X7 DNA construct.
2. The method of claim 1, wherein the step of intramuscularly administering to the affected limb is done at a dose of 8 mg, equally divided into 32 injections over two visits.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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MODE FOR CARRYING OUT THE INVENTION
(21) The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.
EXAMPLES
Example 1: Preparation of Plasmid DNA Expressing Isoforms of HGF
(22) In order to carry out the following various experiments, the present inventors used the pCK vector as a vector capable of expressing isoforms of HGF. The pCK vector is constructed such that the expression of a subject to be expressed, e.g., an HGF gene, is regulated under enhancer/promoter of the human cytomegalovirus (HCMV), and is disclosed in detail in Lee et al., Biochem. Biophys. Res. Commun. 272:230 (2000); WO 2000/040737. Currently, the pCk vector is used for clinical trials on human body, and its safety and efficacy were confirmed (Henry et al., Gene Ther. 18:788 (2011)). In order to prepare plasmid DNAs expressing hybrid HGF genes as a therapeutic agent for diabetic neuropathy, the present inventors inserted each of the hybrid HGF genes into the pCK vector according to the method disclosed in U.S. Pat. No. 7,812,146.
Example 2: Verification of Hybrid HGF Genes Co-Expressing Isoforms of HGF
(23) 2-1. Construction of Vector Expressing Isoforms of HGF
(24) In order to verify the expression of isoforms of HGF, gene expression vectors for cHGF (flHGF), dHGF, and a hybrid form thereof were prepared, and the HGF gene expressing vector was compared with the cHGF or dHGF expressing vector. The cHGF obtained by treating the pCK-cHGF disclosed in U.S. Pat. No. 7,812,146 with BamHI was inserted into the BamHI site of the pVAX1 (Invitrogen, USA) to construct pVAX1-cHGF (
(25) The promoter obtained by treating the pVAX1-cHGF with NdeI and BstEII was inserted into the pCK-cHGF and pCK-dHGF without promoters, respectively, which were obtained by treatment with the same enzymes, to construct new plasmids, pCY-cHGF and pCY-dHGF, using the term pCY, respectively (
(26) 2-2. Verification of RNA Expression of Isoforms of HGF
(27) Each of the plasmid DNAs was transfected into 110.sup.6 cells of 293T cells (ATCC CRL 1573) using FuGENE6 (Roche, USA) according to the manufacturer's instructions. At 48 hours after transfection, cells for each of the plasmids were harvested. RNA was extracted from the harvested 293T cells using the Trizol method (Trizol; Invitrogen, USA), and subjected to RT-PCR to obtain cDNA. PCR was conducted using the harvested cDNA as a template DNA and synthetic oligonucleotides of SEQ ID NO: 15 and SEQ ID NO: 16 as a primer pair. The PCR was conducted such that 3 of the template DNA, 1
each of 10 pmol/
primer, 5
of 2.5 mM dNTP, 3.5 units of High fidelity enzyme mix (Roche, USA), and 5
of an enzyme buffer solution were mixed to prepare a total of 50
of a mixture liquid, which was then subjected to PCR amplification under conditions of 40 cycles of 30 seconds at 95, 30 seconds at 60, and 30 seconds at 72. The thus amplified PCR products correspond to the boundary region between exon 4 and exon 5 of the HGF gene. Here, the nucleotide sequence of 142 bp is amplified for cHGF cDNA and the nucleotide sequence of 127 bp is amplified for dHGF cDNA.
(28) As for the HGF-X gene, nucleotide sequences of at least 1 kb are amplified when the splicing does not occur, and both of the nucleotide sequences of 142 bp and 127 bp are amplified when alternative splicing occurs and thus cHGF and dHGF simultaneously are produced. The amplified PCR products were confirmed by electrophoresis on 15% polyacrylamide gels. As a result, the bands of 142 bp and 127 bp were detected for cHGF cDNA and dHGF cDNA, respectively, and both bands of 142 bp and 127 bp were detected for the hybrid HGF (
(29) 2-3. Verification of Protein Expression of Isoforms of HGF
(30) Each of the plasmid DNAs was transfected into 110.sup.6 cells of 293T cells (ATCC CRL 1573) using FuGENE6 (Roche, USA) according to the manufacturer's instructions. At 48 hours after transfection, the supernatant of each of the plasmid DNAs was harvested. The amount of HGF protein in the supernatant was measured using an enzyme-linked immunosorbent assay (ELIS; R&D System, MN, USA). As a result, it was verified that, among the hybrid HGF genes, HGF-X7 showed the highest protein expression level.
Example 3: Effect of Hybrid HGF Expressed in pCK Vector on Growth and Survival of Neuronal Cells
(31) 3-1. Effect of Hybrid HGF on Growth of Neuronal Cells
(32) (1) Cell Line and Cell Culture
(33) Rat-derived P12 pheochromocytoma (CRL-1721; ATCC, MD, USA) was used in this experiment. P12 cells are commonly used in the research of diabetic neuropathy. It has been recently validated that glucose reduces neuritis of PC12 cells (Fan Zhang et al., THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS. 323:508-515 (2007)). In addition, it has been reported that glucose induces the reduction in proliferation of PC12 cells and DNA disruption, resulting in apoptosis of PC12 cells (EFRAT LELKES et al., Neurotoxicity research. 3:189-203 (2000)). PC12 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum and antibiotics under 37 and 5% CO.sub.2. The cell culture medium, reagent, and serum were purchased from Gibco (Gibco BRL life technologies, inc., MD, USA), and plastic products for culture were purchased from BD Falcon (BD Falcon, NJ, USA).
(34) (2) Preparation of Supernatants Containing Hybrid HGF Proteins and Recombinant Human HGF Protein
(35) Supernatants expressing hybrid HGF proteins, that is, HGF-X2, HGF-X3, HGF-X4, HGF-X5, HGF-X6, HGF-X7, and HGF-X8 were produced using DNA transfection. The transfection was conducted by using the Cellphect phosphate calcium transfection system (GE Healthcare BioSciences, NJ, USA) according to the manufacture's protocol. 293T cell lines seeded at 110.sup.6 cells per well one day before were transfected with pCK, pCK-HGF-X2, pCK-HGF-X3, pCK-HGF-X4, pCK-HGF-X5, pCK-HGF-X6, pCK-HGF-X7, and pCK-HGF-X8, and then the cells were incubated for 48 hours. Upon the completion of culturing, the supernatants were all harvested, and then filtered through a 0.22-m filter. The harvested protein supernatants were frozen at 80 before use.
(36) Recombinant human HGF protein was purchased from R&D (R&D Systems, Inc., MSP, USA) for use.
(37) (3) Verification of Protein Expression and Protein Quantification
(38) In order to verify the expression of the respective proteins in the supernatants of 293T cells, the human HGF immunoassay by R&D (R&D Systems, Inc., MSP, USA) was used. The expression levels of the respective proteins were measured, and then the respective supernatants were again diluted to 1 g/ml for the use of experiments.
(39) (4) Comparison of Cell Growth Among Hybrid HGF Proteins in PC12 Cells
(40) In order to compare effects of hybrid HGF proteins on the growth of neuronal cells, the following experiment was conducted using PC12 cells. PC12 cells were seeded in a 6-well plate at 110.sup.5 cells per well, and the next day, the medium was exchanged with a medium containing 1% FBS. The 293T cell supernatant expressing each protein was added thereto at a concentration of 5 ng/ml, followed by culturing for 7 days, and then cell counting was conducted. As control groups, the supernatant of 293T cells transfected with the pCK vector and the recombinant human HGF protein were used. As a result, all the experiment groups added with the supernatants expressing all the hybrid HGF proteins excluding HGF-X4 were observed to exhibit higher cell growth than the control groups. The experiment groups added with the supernatants expressing HGF-X6, HGF-X7, and HGF-X8 showed statistically significant differences as compared with the control group (pCK vector) (P<0.05 or P<0.005;
(41) Since the pCK-HGF-X7 showed the highest gene expression level among the hybrid HGF genes (see,
(42) 3-2. Comparison Between Effects of HGF-X7 and cHGF on Growth of Neuronal Cells
(43) (1) Cell Line and Cell Culture
(44) Cell lines used in the present experiment were a total of two, PC12 cell line and human-derived SH-SY5Y neuroblasts (22266; KCLB, Korea). The SH-SY5Y cell line, like the PC12 cell line, is one of the most used cell lines for research of diabetic neuropathy. According to the study on diabetic neuropathy using SH-SY5Y cells, it has been known that glucose increases the depolarization of mitochondrial membranes of the SH-SY5Y cells and activates inactivated caspase-3, leading to apoptosis of the SH-SY5Y cells (G M Leinninger et al., Cell Death and Differentiation. 11:885-896 (2004)). All the cells were cultured under conditions of 37 and 5% CO.sub.2. The PC12 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum and antibiotics, and the SH-SY5Y cells were cultured in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum and antibiotics. The cell culture medium, reagent, and serum were purchased from Gibco and the ATCC (American Type Culture Collection, MD, USA).
(45) (2) Production and Quantification of Supernatants Expressing HGF Proteins
(46) 293T cells were seeded at 110.sup.6 cells, and the next day, the cells were transfected with pCK, pCK-cHGF, pCK-dHGF, and pCK-HGF-X7. After culturing for 48 hours, the supernatants were all harvested, and then filtered through a 0.22-m filter. The expression levels of the HGF proteins contained in the respective supernatants were measured using human HGF immunoassay. The respective supernatants were again diluted to 1 g/ for the use of experiments.
(47) (3) Comparison Between Growths of PC12 Cells by HGF-X7 and cHGF
(48) In order to compare effects on the growth of neuronal cells, the cell proliferation degrees by the respective proteins were evaluated using PC12 cells. For achieving this, PC12 cells were seeded in a 6-well plate at 110.sup.5 cells per well, and the next day, the medium was exchanged with a medium containing FBS. The respective proteins obtained from 293T cells transfected with pCK, pCK-cHGF, pCK-dHGF, and pCK-HGF-X7 were added thereto at concentrations of 5 ng/. The pCK vector was used for a control group.
(49) As a result of cell counting after culturing for 7 days, the experiment group added with the supernatant of 293T cells containing HGF-X7 was verified to have the highest cell number. The experiment group added with HGF-X7 showed a cell growth effect, which was about 50% higher than that in cHGF and about 70% higher than that in dHGF (
(50) (4) Comparison Between Cell Growths of SH-SY5Y Cells by HGF-X7 and cHGF
(51) In order to compare effects on the growth of neuronal cells, SH-SY5Y cells, the cell proliferation degrees by the respective proteins were measured. For achieving this, SH-SY5Y cell line was seeded in a 6-well plate at 510.sup.4 cells per well. The next day, the medium was exchanged with a medium containing 1% FBS. The respective proteins obtained from 293T cells transfected with pCK, pCK-cHGF, pCK-dHGF, and pCK-HGF-X7 were added thereto at concentrations of 5 ng/. The pCK vector was used for a control group.
(52) As a result of cell counting after culturing for 7 days, the experiment group added with the supernatant of 293T cells containing HGF-X7 was verified to have the highest cell number. The experiment group added with HGF-X7 showed a cell growth effect, which was about 25% higher than that in cHGF and about 80% higher than that in dHGF (
(53) 3-3. Effect of HGF-X7 on Growth of PC12 Cells in Culture Conditions of High-Concentration Glucose
(54) (1) Selection of Glucose Concentration and Culture Time for Inhibition of Growth of PC12 Cells
(55) Prior to the verification of an effect of HGF-X7 on the growth of PC12 cells under the culture conditions of high-concentration glucose, the glucose concentration and the culture time for inhibiting the growth of PC12 cells were selected. PC12 cells were seeded in a 96-well plate at 510.sup.4 cells per well, and the next day, the medium was exchanged with 100 mM and 200 mM glucose media containing 1% FBS, respectively. As a control group, a medium containing 50 mM glucose, which was a culture medium of PC12 cells, was used. At 24, 48, and 72 hours after medium exchange, the cell growth was measured using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Wis., USA). The growth of PC12 cells was verified to be reduced in the high-concentration glucose medium. In particular, the growth of PC12 cells was observed to be reduced by about 50% in the 200 mM glucose medium at 48 hours and 72 hours. Based on these results, the glucose concentration and the culture time for inhibiting the growth of PC12 cells were selected to be 200 mM and 72 hours, respectively.
(56) (2) Verification of Effect of HGF-X7 on Growth of PC12 Cells in Culture Conditions of High-Concentration Glucose
(57) The effect of HGF-X7 on the growth of PC12 cells in the culture conditions of high-concentration glucose was confirmed. PC12 cell line was seeded in a 96-well plate at 510.sup.4 cells per well. The next day, the medium was exchanged with a 200 mM glucose medium, and then 50 ng/ml of the 293T cell supernatant expressing HGF-X7 was added thereto.
(58) As a result of confirming the cell growth after culturing for 72 hours, it was observed that the experiment group added with the supernatant expressing HGF-X7 showed an increase by about 23% or more in cell growth as compared with the control group (pCK vector), and an increase by about 10% or more in cell growth as compared with the experiment group added with the same concentration of the supernatant containing cHGF.
(59) 3-4. Effect of HGF-X7 on Apoptosis Inhibitory Effect of PC12 Cells Under the Culture Conditions of High-Concentration Glucose
(60) (1) Selection of Glucose Concentration and Culturing Time for Inducing Apoptosis of PC12 Cells
(61) Prior to the estimation of an effect of HGF-X7 on apoptosis of PC12 cells under the culture conditions of high-concentration glucose, the glucose concentration and the culture time for inducing apoptosis of PC12 cells were selected. The PC12 cell line was seeded in a 6-well plate at 110.sup.5 cells per well, and the next day, the medium for the PC12 cell line was exchanged with 50 mM, 100, mM, and 200 mM glucose media containing 1% FBS. The cells were cultured for 48 hours or 72 hours, and then all the cells were collected. The supernatants were removed by centrifugation for 3 minutes at 12000 rpm, followed by washing with PBS. This procedure was repeated once more. The degrees of apoptosis for the collected cells were measured using the Annexin V apoptosis assay system (BD Biosciences, NJ, USA). A 1 Annexin V binding buffer was put into the collected cells at a volume of 1 ml per 110.sup.6 cells, so that the cells were suspended in the buffer. 5 of Annexin-V and a propidium iodide buffer were added to 100
of the suspended cells to stain the suspended cells for 20 minutes in the dark. 400
of a 1 Annexin V binding buffer was further added to the stained cells to detect apoptosis by flow cytometry.
(62) As a result, the apoptosis of PC12 cells was not induced when the cells were cultured in the 100 mM glucose medium for 48 hours, as compared with the control group, but about 2.5-fold of apoptosis was induced in the 200 mM glucose medium as compared with the control group. Whereas, it was verified that, under the culture conditions for 72 hours, the apoptosis was induced in both 100 mM and 200 mM glucose media as compared with the control group, and the significant difference between 100 mM and 200 mM glucose media was not shown. Based on these results, the glucose concentration and the culture time for inducing apoptosis of PC12 cells were selected to be 200 mM and 48 hr, respectively.
(63) (2) Effect of HGF-X7 on Apoptosis of PC12 Cells in Culture Conditions of High-Concentration Glucose
(64) The PC12 cell line was seeded in a 6-well plate at seeded in at 110.sup.5 cells per well, and the next day, the medium for the PC12 cell line was exchanged with 200 mM glucose medium containing 1% FBS. 50 ng/ml of the 293T cell supernatant expressing cHGF or HGF-X7 was added thereto. As a control group, the supernatant of 293T cells transfected with the pCK vector was used. After culturing for 48 hours, all the cells were collected. Staining was conducted using the Annexin V apoptosis assay system, and then the degrees of apoptosis were confirmed by flow cytometry.
(65) As a result, the experiment group added with the 293T cell supernatant expressing HGF-X7 was verified to lead to a 2-fold decrease in apoptosis as compared with the control group added with the 293T cell supernatant expressing the pCK vector and show an apoptosis inhibitory effect of about 1.5 times or higher as compared with the experiment group added with the supernatant containing cHGF (
Example 4: Clinical Trial of pCK-HGF-X7 Against Diabetic Neuropathy
(66) 4-1. Subjects and Administration
(67) A phase I clinical trial for safety and efficacy of pCK-HGF-X7 was conducted for 12 patients diagnosed with diabetic neuropathy. The time and dose of administration were different for three trial groups as shown in Table 1.
(68) TABLE-US-00001 TABLE 1 Number of times Trial Dose of of administration Total dose of group administration Day 0 Day 14 administration I 4 mg 8 8 8 ml II 8 mg 16 16 16 ml III 16 mg 32 32 32 ml
(69) 4-2. Methods
(70) (1) Informed Consent Form and Screening Procedure
(71) After receiving informed consent forms from patients, a screening procedure for checking the possibility of participating in the present clinical trial was conducted. The screening procedure was conducted within 30 days before day 0 of primary administration, and the possibility of participating in the present clinical trial was determined for each of the patients based on the following items.
(72) a. complete medical history
(73) b. complete physical exam
(74) c. cancer screening tests
(75) d. retinal fundoscopy
(76) e. viral screening tests
(77) f. hematology and serum chemistry
(78) g. urinalysis
(79) h. urine pregnancy test (for only females)
(80) i. Ulcer screening (if possible)
(81) j. ECG
(82) k. Michigan Neuropathy Screening Instrument
(83) l. Visual Analogue Scale
(84) (2) Administration of Trial Drug
(85) The pCK-HGF-X7 was injected in the right calf muscle of each of the subjects undergoing screening at an interval of two weeks (Day 0 and Day 14). The subjects assigned to trial group I were administered with 2 mg of pCK-HGF-X7 on Day 0, and again administered with 2 mg of pCK-HGF-X7 on Day 14. Therefore, trial group I was administered with a total of 4 mg of pCK-HGF-X7. On Day 0, each of the subjects was administered with 2 mg of pCK-HGF-X7, which was injected in eight sites of the calf muscle at a divided dose of 0.25 mg/0.5 ml/site. On Day 14, the administration was also conducted in the same manner. Trial group II was administered with a total of 8 mg of pCK-HGF-X7 (4 mg on Day 0 and 4 mg on Day 14). The administration was conducted similarly to trial group I. That is, on Day 0, each of the subjects of trial group II was administered with 4 mg of pCK-HGF-X7, which was injected in 16 sites of the calf muscle at a divided dose of 0.25 mg/0.5 ml/site. On Day 14, the administration was conducted in the same manner. Trial group III was administered with a total of 16 mg of pCK-HGF-X7 (8 mg on Day 0 and 8 mg on Day 14). On Day 0, each of the subjects of trial group III was administered with 8 mg of pCK-HGF-X7, which was injected in 32 sites of the calf muscle at a divided dose of 0.25 mg/0.5 ml/site. On Day 14, the injection in 32 sites was conducted in the same manner.
(86) 4-3. Clinical Evaluation Indicator
(87) The primary endpoint of the present clinical trial is to confirm the safety of pCK-HGF-X7 injected in the calf muscle of each of the patients with diabetic neuropathy, and the secondary endpoint of the present clinical trial is to confirm the efficacy of pCK-HGF-X7 on pain, which is a main symptom of diabetic neuropathy.
(88) (1) Safety Analysis
(89) All the subjects administered with the trial drug in the present clinical trial are to be tested for safety analysis. Through follow-up observation of 12 months after administration, adverse event data (including adverse events and adverse events to stop administration of trial drug) were all recorded according to the extents thereof and relations with the trial drug. If possible, safety analysis was conducted through all statistical analysis. In addition, in order to avoid risks associated with cancers, all the subjects were screened by the method specified in the American Cancer Society Cancer Screening Guideline during the screening procedure.
(90) (2) Pharmacokinetic Analysis
(91) The level of HGF protein in serum of the subject and the amount of pCK-HGF-X7 in blood of the subject were measured before and after the administration of the trial drug of Day 0, and before and after the administration of the trial drug of Day 14, on Day 21, on Day 30, on Day 60, and on Day 90.
(92) (3) Efficacy Analysis
(93) A visual analogue scale (VAS) method was used to record the change in pain for all the subjects. According to the VAS method, the individual preference for a health state was directly measured. That is, each of the subjects is allowed to directly score a scale for the severity of pain. A 100 mm-long line was drawn, and No pain at all was marked at one side of the line and Pain as bad as it can be was marked at the other side of the line. Then, the subjects are allowed to determine and record the severity of pain by themselves according to the VAS indicator. This method cannot show the comparison between different subjects, but can show the change in the severity of pain for the same subject (
(94) 4-4. Results
(95) (1) Safety Results (Adverse Event Report)
(96) As for the adverse events due to administration of pCK-HGF-X7 of the present invention, seven adverse events occurred in a total of three subjects of trial group I; two adverse events occurred in two subjects of trial group II; and two adverse events occurred in two subjects of trial group III. The adverse events were reported to be dry eyes, injection site pain, dry mouth, diarrhea, and the like in trial group I; back pain and sinusitis in trial group II; and right rib pain and viral syndrome in trial group III. The number of adverse drug events was five, which were reported in two subjects of trial group I, dry eyes (two events), injection site pain, dry mouth, and diarrhea, but they correspond to mild adverse drug events and thus recovered soon. Whereas, no serious adverse events were reported.
(97) (2) Pharmacodynamics (PD) Results
(98) As a result of confirming the amount of HGF protein produced in serum after administration of pCK-HGF-X7, it was verified that the level of HGF protein in serum after administration of pCK-HGF-X7 was not increased but maintained during the clinical trial (
(99) (3) Pharmacokinetics (PK) Results
(100) As a result of confirming the amount of pCK-HGF-X7 remaining after pCK-HGF-X7 treatment, the pCK-HGF-X7 DNA was not detected in ten subjects during follow-up observation of 60 days, and was detected at under 100 copies/ for all the subjects (Table 2).
(101) TABLE-US-00002 TABLE 2 Day 0 Day 14 Prior Post Prior Post Trial Patient admin- admin- admin- admin- Day Day Day Day group ID istration istration istration istration 21 30 60 90 I 1-01 NEG 45846.3 NEG 62,762.8 10.0 7.1 NEG NEG 2-01 NEG 38401.5 NEG 18,215.9 NEG NEG NEG NEG 2-02 NEG 5871.8 NEG 38,401.5 NEG NEG NEG NEG 2-03 NEG 18215.9 NEG 5,871.8 NEG NEG NEG NEG II 2-04 NEG 562,669.0 NEG 300,852.0 51.0 NEG 38.1 NEG 1-02 NEG 114,319.0 333.0 139,297.0 56,266.9 219.0 91.1 NEG 2-05 NEG 183,514.0 63.0 582,978.0 3,875.0 69.0 NEG 28.9 1-03 5.1 177,131.0 319.0 1,532,729.0 262.8 108.1 NEG NEG III 1-04 NEG 1,920,770.8 148 6,252,606.8 1,637.5 162.2 NEG 42.7 2-07 NEG 368,173.0 NEG 23,198.3 32.9 NEG NEG NEG 2-08 NEG 76,888.4 170.7 101,424.0 157.6 58.6 50.6 NEG 2-09 NEG 491,690.2 77.1 432,454.6 77.6 33.7 NEG NEG
(102) (4) Efficacy Test Results
(103) The severity of pain was measured through the Pain VAS (Visual Analogue Scale). As for a total of twelve subjects, the mean baseline VAS value was 48.0, and the mean VAS value at six months after the pCK-HGF-X7 treatment was 25.4, which showed a 47% reduction in the pain VAS value (
(104) In the case of the first dose group (4 mg), the mean baseline VAS value was 39.5, and the mean VAS value at two months after treatment was 23.8, which showed a 39.7% reduction in the pain VAS value, but the mean VAS value at six months after treatment was 31.3, which merely showed a 20.8% reduction in the pain VAS value as compared with the baseline value. In the first dose group, the pain reduction was observed in three of four subjects and the pain reduction of 50% or higher was observed in two of four subjects (
(105) In the case of the second dose group (8 mg), the mean baseline VAS value was 59.1, and the VAS value from one month after treatment was sharply reduced and the mean VAS value at six months after treatment was 27.5, which showed a 53.5% reduction in the pain VAS value as compared with the baseline value (
(106) In the case of the third dose group (16 mg), the mean baseline VAS value was 45.3. Similarly to the second dose group, the VAS value from one month after treatment was sharply reduced and the mean VAS value at six months after treatment was 17.3, which showed a 61.4% reduction in the pain VAS value as compared with the baseline value. In the third dose group, the pain reduction was observed in all four subjects and the pain reduction of 50% or higher was observed in three of four subjects (
(107) As a result of surveying the efficacy using the pain VAS, the pain, which is the main symptom of diabetic neuropathy, was reduced after the pCK-HGF-X7 injection, and the pain reduction rate and the response rate to pain reduction were more remarkable in the medium-dose group (8 mg) or the high-dose group (16 mg) than in the low-dose group (4 mg). These results supported that the pain reduction observed in the present clinical trial was due to the administration of pCK-HGF-X7 and not the placebo effect (
(108) Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.