Application of pUDK-HGF injection

Abstract

The invention discloses application of a pUDK-HGF injection in preparation of a medicine. The medicine is used for treating limb resting pain of an object. The pUDK-HGF injection provided by the invention can be used for effectively treating the limb resting pain, reducing the complete pain disappearance time of a patient, improving the complete pain disappearance proportion of the patient and increasing the pain NRS score decline value of the patient.

Description

[0001] Priority information

[0002] This application claims priority and benefits to patent applications filed with the China National Intellectual Property Administration on August 19, 2024 and August 6, 2025, with patent application numbers 2024111395465 and 2025111006796, which are incorporated herein by reference in their entirety. Technical Field

[0003] This application belongs to the field of biopharmaceuticals, specifically relating to the use of a pUDK-HGF injection. Background Technology

[0004] The main component of recombinant plasmid-hepatocyte growth factor injection (pUDK-HGF injection) is a recombinant plasmid carrying the hepatocyte growth factor gene. This makes it a novel gene therapy product that uses an expression plasmid as a vector and hepatocyte growth factor complementary deoxyribonucleic acid (cDNA) as the therapeutic gene. Hepatocyte growth factor (HGF) is a widely expressed, multifunctional growth factor and angiogenesis factor with multiple functions, including promoting angiogenesis, regulating inflammation, inhibiting fibrosis, and activating tissue regeneration.

[0005] pUDK-HGF injection uses plasmid DNA as a vector. By transfecting the HGF gene into ischemic limb sites, it is expected to promote local angiogenesis and the establishment of collateral circulation, thereby improving blood supply to the limbs. Summary of the Invention

[0006] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a use of pUDK-HGF injection.

[0007] In a first aspect of this application, the use of pUDK-HGF injection in the preparation of a medicament for treating limb resting pain is disclosed. The pUDK-HGF injection of this application can effectively treat limb resting pain, reducing the time to complete pain relief, increasing the proportion of patients experiencing complete pain relief, and increasing the decrease in patients' NRS pain scores.

[0008] According to embodiments of this application, the drug is used for at least one of the following:

[0009] 1) The pain completely disappeared on the 180th day of treatment for limb resting pain;

[0010] 2) The NRS score dropped to 0 on the 180th day of treatment for limb rest pain;

[0011] 3) The NRS score decreased by 50% on the 180th day of treatment for limb resting pain;

[0012] 4) The NRS score decreased by more than 3 points on the 180th day of treatment for limb rest pain;

[0013] 5) The cumulative rate of major amputation or mortality on day 180 of limb rest pain treatment is less than 0.90%.

[0014] It should be noted that "day 180" refers to having the above-mentioned characteristics (e.g., complete disappearance of pain, NRS score reduced to 0, NRS score reduced by more than 3 points, NRS score reduced by 50%, cumulative major amputation or mortality rate less than 0.90%, etc.) within 180 days (including day 180) of limb resting pain treatment. For example, "complete disappearance of pain on day 180 of limb resting pain treatment" means that the pain can be completely eliminated within 180 days (including day 180) of administration.

[0015] In a second aspect, this application discloses the use of pUDK-HGF injection in treating limb resting pain. The pUDK-HGF injection of this application can effectively treat limb resting pain, reduce the time to complete pain relief, increase the proportion of patients experiencing complete pain relief, and increase the decrease in patients' NRS pain scores.

[0016] According to embodiments of this application, the use is at least one of the following:

[0017] 1) The pain completely disappeared on the 180th day of treatment for limb resting pain;

[0018] 2) The NRS score dropped to 0 on the 180th day of treatment for limb rest pain;

[0019] 3) The NRS score decreased by 50% on the 180th day of treatment for limb resting pain;

[0020] 4) The NRS score decreased by more than 3 points on the 180th day of treatment for limb rest pain;

[0021] 5) The cumulative rate of major amputation or mortality on day 180 of limb rest pain treatment is less than 0.90%.

[0022] In a third aspect, this application discloses a pUDK-HGF injection for treating limb resting pain. The pUDK-HGF injection of this application can effectively treat limb resting pain, reduce the time to complete pain relief, increase the proportion of patients experiencing complete pain relief, and increase the decrease in patients' NRS pain scores.

[0023] According to embodiments of this application, the pUDK-HGF injection solution is used for at least one of the following:

[0024] 1) The pain completely disappeared on the 180th day of treatment for limb resting pain;

[0025] 2) The NRS score dropped to 0 on the 180th day of treatment for limb rest pain;

[0026] 3) The NRS score decreased by 50% on the 180th day of treatment for limb resting pain;

[0027] 4) The NRS score decreased by more than 3 points on the 180th day of treatment for limb rest pain;

[0028] 5) The cumulative rate of major amputation or mortality on day 180 of limb rest pain treatment is less than 0.90%.

[0029] According to embodiments of this application, the uses described in the first, second, and third aspects above may further include at least one of the following technical features:

[0030] According to embodiments of this application, the NRS score of the object is 4 to 7.

[0031] According to an embodiment of this application, the object is experiencing moderate to severe pain.

[0032] According to an embodiment of this application, the subject did not receive analgesic drugs.

[0033] According to an embodiment of this application, the subject suffers from severe lower limb ischemia (CLI).

[0034] According to an embodiment of this application, the subject suffers from lower extremity arteriosclerosis obliterans (ASO) or thromboangiitis obliterans (TAO).

[0035] In this article, "the subject did not receive analgesics" should meet the following conditions:

[0036] 1) Analgesics are not allowed to be used at baseline; that is, the score is 4 to 7 when analgesics are not used.

[0037] 2) The assessment of complete pain relief must be performed without the use of analgesics.

[0038] According to an embodiment of this application, the treatment is performed by injecting the subject at 24 injection points.

[0039] According to an embodiment of this application, the injection is an intramuscular injection.

[0040] According to an embodiment of this application, the 24 injection points are located near the following muscle areas:

[0041] 1) Superficial femoral artery; or

[0042] 2) Anterior and posterior tibial arteries; or

[0043] 3) Anterior tibial artery; or

[0044] 4) Posterior tibial artery; or

[0045] 5) Along the superficial femoral artery, anterior tibial artery, and posterior tibial artery; or

[0046] 6) Along the superficial femoral artery and the anterior tibial artery; or

[0047] 7) Along the superficial femoral artery and posterior tibial artery.

[0048] According to embodiments of this application, the 24 injection points are arranged in a single vertical row or in two vertical rows.

[0049] In some optional embodiments of this application, the injection occurs in the following ways:

[0050] 1) Simple superficial femoral artery stenosis or occlusion: 24 injection points are arranged in 2 longitudinal rows and injected into the muscles near the superficial femoral artery;

[0051] 2) Simple stenosis or occlusion of the anterior and posterior tibial arteries: 24 injection points are arranged in two longitudinal rows and injected into the muscles near the anterior and posterior tibial arteries respectively;

[0052] 3) Simple anterior or posterior tibial artery stenosis or occlusion: 24 injection points are arranged in two longitudinal rows and injected intramuscularly along the vicinity of the occluded artery;

[0053] 4) Simultaneous stenosis or occlusion of the superficial femoral artery and the anterior and posterior tibial arteries: 24 points were divided into 2 groups. In the first group, 8 points were injected intramuscularly along the course of the superficial femoral artery in a single longitudinal row. In the second group, 8 points were injected along the course of the anterior tibial artery in a single longitudinal row and 8 points were injected along the course of the posterior tibial artery in a single longitudinal row.

[0054] 5) Stenosis or occlusion of either the superficial femoral artery or the anterior or posterior tibial artery: 24 points are divided into 2 groups. The 8 points in the first group are injected along one side of the superficial femoral artery, and the 16 points in the second group are injected in two longitudinal rows along the course of the anterior or posterior tibial artery.

[0055] According to an embodiment of this application, the highest injection site is above the highest occlusion / stenosis plane of the artery, which is the superficial femoral artery, the anterior tibial artery, and / or the posterior tibial artery.

[0056] According to an embodiment of this application, the pUDK-HGF injection solution comprises pUDK-HGF, disodium hydrogen phosphate, sodium chloride, disodium edetate, hydrochloric acid, and water.

[0057] According to an embodiment of this application, the weight ratio of pUDK-HGF, disodium hydrogen phosphate, sodium chloride, and disodium edetate is 2.0:(1.0-2.0):(8.0-9.0):(0.03-0.04).

[0058] According to an embodiment of this application, the pUDK-HGF injection solution contains pUDK-HGF at a weight-to-volume ratio (g / ml) of 0.2%.

[0059] According to an embodiment of this application, the total dose of the pUDK-HGF injection is 6 mg.

[0060] According to an embodiment of this application, the dosage of pUDK-HGF injection at a single injection site is 0.25 mg.

[0061] According to an embodiment of this application, the pUDK-HGF has a nucleotide sequence as shown in SEQ ID NO:1.

[0062] According to an embodiment of this application, the pUDK-HGF injection solution is available in a specification of 2.0 mg / 1.0 ml / bottle.

[0063] In a fourth aspect, this application provides a method for treating resting pain in limbs. According to an embodiment of this application, the method includes administering a pharmaceutically acceptable dose of pUDK-HGF injection to a subject. The method of this application is effective in treating resting pain in limbs.

[0064] According to an embodiment of this application, the administration is performed via intramuscular injection.

[0065] According to an embodiment of this application, the drug administration is performed via 24 injection sites on the subject.

[0066] According to an embodiment of this application, the 24 injection points are located near the following muscle areas:

[0067] 1) Superficial femoral artery; or

[0068] 2) Anterior and posterior tibial arteries; or

[0069] 3) Anterior tibial artery or

[0070] 4) Posterior tibial artery; or

[0071] 5) Along the superficial femoral artery, anterior tibial artery, and posterior tibial artery; or

[0072] 6) Along the superficial femoral artery and the anterior tibial artery; or

[0073] 7) Along the superficial femoral artery and posterior tibial artery.

[0074] According to an embodiment of this application, the 24 injection points are arranged in two vertical rows.

[0075] According to embodiments of this application, the NRS score of the object is 4 to 7.

[0076] According to an embodiment of this application, the object is experiencing moderate to severe pain.

[0077] According to embodiments of this application, no analgesic drugs are used during the treatment process.

[0078] According to an embodiment of this application, the subject suffers from severe lower limb ischemia (CLI).

[0079] According to an embodiment of this application, the subject suffers from lower extremity arteriosclerosis obliterans (ASO) or thromboangiitis obliterans (TAO).

[0080] According to embodiments of this application, the treatment is used for at least one of the following:

[0081] 1) The pain completely disappeared on the 180th day of treatment for limb resting pain;

[0082] 2) The NRS score dropped to 0 on the 180th day of treatment for limb rest pain;

[0083] 3) The NRS score decreased by 50% on the 180th day of treatment for limb resting pain;

[0084] 4) The NRS score decreased by more than 3 points on the 180th day of treatment for limb rest pain;

[0085] 5) The cumulative rate of major amputation or mortality on day 180 of limb rest pain treatment is less than 0.90%.

[0086] According to an embodiment of this application, the pUDK-HGF injection solution comprises pUDK-HGF, disodium hydrogen phosphate, sodium chloride, disodium edetate, hydrochloric acid, and water.

[0087] According to an embodiment of this application, the weight ratio of pUDK-HGF, disodium hydrogen phosphate, sodium chloride, and disodium edetate is 2.0:(1.0-2.0):(8.0-9.0):(0.03-0.04).

[0088] According to an embodiment of this application, the pUDK-HGF injection solution contains pUDK-HGF at a weight-to-volume ratio (g / ml) of 0.2%.

[0089] According to an embodiment of this application, the total dose of the pUDK-HGF injection is 6 mg.

[0090] According to an embodiment of this application, the dosage of pUDK-HGF injection at a single injection site is 0.25 mg.

[0091] According to an embodiment of this application, the pUDK-HGF has a nucleotide sequence as shown in SEQ ID NO:1.

[0092] According to an embodiment of this application, the pUDK-HGF injection solution is available in a specification of 2.0 mg / 1.0 ml / bottle.

[0093] Beneficial effects:

[0094] 1) After treating patients with resting limb pain with the pUDK-HGF injection of this application, the proportion of patients whose pain completely disappeared was significantly increased. Specifically, the proportion of patients whose pain completely disappeared was 39.55% in the experimental group and 21.37% in the control group. The disappearance rate in the experimental group was higher than that in the control group, and the difference between the two groups was statistically significant (P = 0.0004).

[0095] 2) After treating patients with limb resting pain with the pUDK-HGF injection of this application, the time for complete pain relief was significantly reduced. Specifically, the average time for complete pain relief in the experimental group was 160.80 days, while that in the control group was 186.79 days. Pain relief in the experimental group was earlier than that in the control group, and the difference between the two groups was statistically significant (P = 0.0005).

[0096] 3) After treatment with the pUDK-HGF injection of this application, patients with limb resting pain showed a significant increase in the decrease in their pain NRS scores. Specifically, on days 60, 90, and 180 after treatment, the decrease in pain NRS scores from baseline in the experimental group was 2.20, 2.57, and 3.36, respectively, while the decrease in pain NRS scores from baseline in the control group was 2.12, 2.45, and 2.82, respectively. The improvement in pain NRS scores from baseline in the experimental group was greater than that in the control group, and the difference was greatest on day 180 after treatment, which was statistically significant (P = 0.0028).

[0097] 4) After treatment with the pUDK-HGF injection of this application for patients with limb resting pain, the proportion of patients with a pain NRS score reduction of more than 50% increased significantly. Specifically, on days 60, 90, and 180 after treatment, the proportions of patients with a pain NRS score reduction of more than 50% in the experimental group were 37.85%, 48.02%, and 67.80%, respectively, while the proportions of patients with a pain NRS score reduction of more than 50% in the control group were 35.90%, 44.44%, and 58.12%, respectively. The proportion of patients with a pain NRS score reduction of more than 50% in the experimental group was higher than that in the control group, with the largest difference on day 180. However, there was no statistically significant difference between the groups at each visit point (P>0.05).

[0098] 5) When patients with limb resting pain were treated with the pUDK-HGF injection of this application, the cumulative rate of major amputation or mortality on day 180 was less than 0.90%.

[0099] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0100] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0101] Figure 1 The pUDK-HGF plasmid pattern in Example 1 of this application is shown.

[0102] Figure 2 This is a KM curve showing the time for complete pain relief in Embodiment 1 of this application. Detailed Implementation

[0103] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0104] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0105] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0106] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0107] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0108] In this document, the term "treatment" refers to the administration of a drug or compound to an individual to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in an individual who is susceptible but has not yet been diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0109] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0110] Preparation Example 1: Preparation of pUDK-HGF

[0111] 1. According to Figure 1 pUDK-HGF (nucleotide sequence as shown in SEQ ID NO:1) was constructed, and the positions of the major elements in the plasmid are shown in Table 1.

[0112] Table 1:

[0113]

[0114]

[0115] 2. Using conventional methods in this field, *E. coli* containing the above-mentioned pUDK-HGF were subjected to strain resuscitation, secondary seed culture, tertiary seed culture, and fermentation culture (fermentation culture conditions: pH = 7.00 ± 0.10, temperature 37.0℃ ± 1.0℃). After cell collection and lysis, the supernatant containing the target plasmid was collected, clarified, and concentrated. Then, the target plasmid was purified by Sepharose 6FF chromatography, PlasmidSelect Xtra chromatography, and Source 30Q chromatography. After concentration, displacement, and filtration of the original solution, pUDK-HGF was obtained.

[0116] Preparation Example 2: Preparation of pUDK-HGF Injection

[0117] The pUDK-HGF obtained in Preparation Example 1 was mixed with disodium hydrogen phosphate, sodium chloride, disodium edetate, hydrochloric acid, and water for injection to obtain pUDK-HGF injection solution. Then, the pUDK-HGF injection solution was filtered, filled and capped (theoretical volume is 1.0 ml, actual volume is 1.1 ml), crimped, visually inspected, labeled and boxed.

[0118] The formulation is as follows: Taking the preparation of 1000ml pUDK-HGF injection as an example, the content of each component is shown in Table 1 below:

[0119] Table 1:

[0120]

[0121] Example 1: The therapeutic effect of pUDK-HGF injection on rest pain

[0122] 1. Test Methods

[0123] 1.1 This embodiment recruited patients with moderate to severe pain (NRS score ≤ 4 points ≤ 7 points) caused by severe lower limb ischemic disease. Subjects meeting the inclusion criteria were randomly assigned to the experimental group and the control group at a ratio of 3:1, with 360 patients in the experimental group and 120 patients in the control group, totaling 480 patients (patients currently using analgesics during the screening period were washed out after 2 weeks, and NRS scores were assessed; analgesics were generally not used during the washout period). The experimental group received the recombinant plasmid-hepatocyte growth factor injection obtained in Example 2, while the control group received placebo PBS buffer. The pivotal study lasted 180 days. Subjects received the medication in three injections on day 1 (D1), day 15 (D15), and day 29 (D29). Four visits were conducted on days 1, 60, 90, and 180. The proportion of subjects with complete pain relief by day 180, the time to complete pain relief, the decrease in pain NRS score, the proportion of subjects with a decrease in pain NRS score greater than 50%, the proportion of subjects who discontinued analgesics during the visits, and the cumulative major amputation or mortality rate by day 180 were analyzed.

[0124] The administration method and dosage are shown in Table 2 below:

[0125] Table 2:

[0126]

[0127]

[0128] *Use a 5ml syringe to draw 1ml (1 vial) of recombinant plasmid-hepatocyte growth factor injection solution and 3ml of physiological saline each time, for a total volume of 4ml. Mix well. Use 3 5ml syringes of the same preparation method to draw 3 times of the test drug each time. Each syringe is used for 8 injections, for a total of 24 injections. Inject 0.5ml at each injection point.

[0129] The injection sites are as follows:

[0130] (1) Simple superficial femoral artery stenosis or occlusion: 24 injection points are arranged in two longitudinal rows and injected into the muscles near the superficial femoral artery.

[0131] (2) Simple anterior and posterior tibial artery stenosis or occlusion: 24 injection points are arranged in two longitudinal rows and injected into the muscles near the anterior and posterior tibial arteries respectively.

[0132] (3) Simple anterior or posterior tibial artery stenosis or occlusion: 24 injection points are arranged in two longitudinal rows and injected into the muscle near the occluded artery;

[0133] (4) Stenosis or occlusion of the superficial femoral artery and the anterior and posterior tibial arteries: 24 points were divided into 2 groups. Group 1 (8 points) was injected intramuscularly along the course of the superficial femoral artery, and Group 2 (16 points) was injected along the course of the anterior and posterior tibial arteries.

[0134] (5) Stenosis or occlusion of the superficial femoral artery and one of the anterior or posterior tibial arteries: 24 points are divided into 2 groups. Group 1 (8 points) is injected along one side of the superficial femoral artery, and Group 2 (16 points) is injected intramuscularly in 2 longitudinal rows along the course of the anterior or posterior tibial artery.

[0135] 1.2 Statistical Methods:

[0136] Unless otherwise specified, statistical tests are based on a two-tailed test at a significance level of α = 0.05, and a two-tailed 95% confidence interval (CI) is calculated.

[0137] Data will be summarized by group and visits (if applicable). Quantitative summaries will calculate the number of cases (N, n), mean, standard deviation (SD), median, 25th percentile (Q1), 75th percentile (Q3), minimum (Min), and maximum (Max). Qualitative summaries will calculate the number of cases and percentages (%) for each category. Time-event indicators will be analyzed using the Kaplan-Meier method, describing median time, interquartile range, and their 95% confidence intervals (95% CI), and survival curves will be plotted.

[0138] 2. Results Analysis

[0139] 2.1 Percentage of patients whose pain completely disappeared by day 180

[0140] Pain was assessed using the Numerical Rating Scale (NRS), and the percentage of cases with complete pain relief was calculated as (number of cases with an NRS score of 0 on day 180 / total number of cases) × 100%.

[0141] Table 3 shows the complete disappearance of pain at each visit point. The sensitivity analysis results for the primary endpoint of complete pain disappearance at visit 180, after considering the influence of factors such as baseline NRS score, lower extremity ischemic disease classification (ASO / TAO), and history of surgical or interventional treatment for peripheral vascular disease, are shown in Table 4.

[0142] Based on the FAS (Features, Scale, and Analysis) data, the complete pain resolution rates on day 180 after treatment were 39.55% (140 / 354) in the experimental group and 21.37% (25 / 117) in the control group. The complete pain resolution rate in the experimental group was significantly higher than that in the control group (P = 0.0004). The 95% CI (95% difference) between the experimental and control groups was 18.18 (8.54, 26.46), with a lower limit of 95% CI greater than 0, indicating that the experimental group was superior to the control group. Sensitivity analysis showed that even after considering confounding factors such as baseline NRS score, disease classification, and history of surgery or interventional treatment for peripheral vascular disease, the 95% CI between the experimental and control groups was still 16.76 (7.16, 26.37), with a lower limit of 95% CI still greater than 0, indicating that the experimental group was still superior to the control group.

[0143] Furthermore, based on the proportion of patients whose pain completely disappeared at each visit, the proportion of patients whose pain completely disappeared in the experimental group was higher than that in the control group on day 60 after treatment (15.54% vs 8.55%) and continued until day 180 after treatment, indicating that the experimental drug had a significant effect on limb resting pain relief.

[0144] Table 3: Percentage of patients whose pain completely disappeared on days 60, 90, and 180

[0145]

[0146] Note: Percentages (%) are calculated with the number of people (n) in each group as the denominator.

[0147] Table 4: Multivariate logistic regression analysis (FAS) of the proportion of patients whose pain completely disappeared on day 180.

[0148]

[0149] 2.2 Subgroup analysis of thromboangiitis obliterans (TAO) population

[0150] For patients with thromboangiitis obliterans (TAO) whose treatment options are more limited, considering the influence of baseline NRS score, peripheral vascular disease and history of surgery or interventional treatment, and smoking status on efficacy, it was found that the proportion of pain relief in the experimental group on day 180 was still higher than that in the control group, and the difference was statistically significant, thus validating the superiority test. The results are shown in Table 5.

[0151] Table 5: Multivariate logistic regression analysis of the proportion of patients whose pain completely disappeared on day 180 (FAS, TAO population)

[0152]

[0153]

[0154] 2.3 Time for complete disappearance of pain

[0155] The time to complete pain relief was defined as the time from a NRS score of 0 with no subsequent rebound. An NRS score of 0 must be calculated without the subject taking any analgesics. Detailed data on the time to complete pain relief are shown in Table 6, and the KM curve for the time to complete pain relief is shown in [reference needed]. Figure 2 .

[0156] FAS results: The median time for complete pain disappearance in the experimental group was 204.00 days, and the mean time was 160.80 days; no median time was observed for complete pain disappearance in the control group, and the mean time was 186.79 days. Pain disappeared earlier in the experimental group than in the control group, and the difference was statistically significant (P = 0.0005).

[0157] The FAS results showed that the pain completely disappeared earlier in the experimental group than in the control group, and the difference was statistically significant, suggesting that the experimental drug could relieve patients' pain and shorten the duration of pain.

[0158] Table 6: Time for complete disappearance of pain

[0159]

[0160] 2.4 Decrease in NRS pain score

[0161] The covariance analysis of the decrease in NRS scores from baseline at each visit point is shown in Table 7.

[0162] FAS results showed that pain NRS scores in both the experimental and control groups significantly decreased from baseline at each visit, with statistically significant differences. It is speculated that the improvement within both groups may be related to the consistent use of basic medications after starting the trial. Comparing data between the experimental and control groups, from day 60 post-treatment, the NRS score decrease in the experimental group showed a higher trend than that in the control group, and this trend increased over time. By day 180, the NRS score decrease in the experimental group was significantly higher than that in the control group, with a statistically significant difference (P = 0.0028). This result is consistent with the trend of the primary endpoint, where the treatment effect was observed from day 60 post-treatment, with a significant difference appearing by day 180.

[0163] Table 7: Covariance analysis of the decrease in NRS scores from baseline on days 60, 90, and 180 after treatment.

[0164]

[0165] Note: An ANCOVA model was used, with group as a fixed effect and baseline NRS scores as a covariate, to compare differences between groups. The least squares mean of the change in NRS scores from baseline after treatment and its 95% CI were calculated for each group, as well as the least squares mean difference in the change in NRS scores from baseline after treatment between the two groups and its 95% CI.

[0166] 2.5 Proportion of patients whose pain NRS score decreased by more than 50%

[0167] Table 8 details the proportion of patients whose pain NRS scores decreased by more than 50% at each visit point.

[0168] FAS results showed that, starting from day 60 of treatment, the proportion of patients in the experimental group with a pain NRS score reduction of more than 50% was higher than that in the control group, and this proportion tended to increase over time, but the difference between the two groups was not statistically significant. The trend of this indicator was consistent with the improvement trends of the complete disappearance of pain indicator and the change in pain NRS score from baseline indicator.

[0169] Table 8: Percentage of cases with a pain NRS score reduction of more than 50% on days 60, 90, and 180 after treatment

[0170]

[0171]

[0172] 2.6 Proportion of subjects who discontinued analgesics during the visit period

[0173] FAS results showed that in the experimental group, 82 patients used analgesics at baseline, and 34 patients discontinued them after entering the trial (41.46%). In the control group, 25 patients used analgesics at baseline, and 8 patients discontinued them after entering the trial (32.00%). After starting medication, the proportion of patients discontinuing analgesics in the experimental group was higher than that in the control group.

[0174] 2.7 Cumulative major amputation rate or mortality rate on day 180

[0175] To investigate the incidence of above-knee amputations in lateral limbs, the cumulative major amputation rate or mortality rate on day 180 for each dataset is shown in Table 6.

[0176] The following are the cases of participants who experienced major amputations or death during the study:

[0177] S13019, experimental group: The subject was first administered the drug on October 14, 2021. During the second visit on November 3, 2021, the investigator assessed that the condition had worsened and the subject was no longer suitable to continue participating in the study. The subject withdrew early and underwent femoral artery thrombectomy, endarterectomy, catheter-directed thrombolysis, and balloon dilation on the same day. The subject was discharged on November 14, 2021, in improved condition. However, the condition worsened again within half a month, and the subject was admitted to the hospital on December 2, 2021, with "necrosis and infection of the left lower leg". On December 6, 2021, the subject underwent amputation of the middle and lower segment of the left thigh.

[0178] S15016, the experimental group, the subject was first administered the drug on October 14, 2021, and withdrew from the study early on October 31, 2021, due to worsening of the condition, without receiving subsequent medication. On November 3, 2021, the subject underwent left femoral artery thrombectomy + endarterectomy + left lower extremity arteriography + catheter-directed thrombolysis + balloon dilation. On December 6, 2021, the subject underwent left mid-lower thigh amputation + vascular and nerve exploration. Due to disease progression and postoperative condition, the subject died on December 13, 2021.

[0179] The results showed that the incidence of major amputations and death was low in the study.

[0180] Table 9: Cumulative Major Amputation Rate or Mortality on Day 180

[0181] Viewpoint Statistical description / category FAS: Experimental group (N=354) 180 days after treatment Major amputation or death occurred, n (%) 2(0.56)

[0182] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0183] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. Use of the pUDK-HGF injection in the preparation of a medicament for treating rest pain of a limb in a subject.

2. Use according to claim 1, characterized in that, The medicament is for at least one of: 1) complete pain relief at day 180 of rest pain treatment; 2) NRS score of 0 at day 180 of rest pain treatment; 3) 50% reduction in NRS score at day 180 of rest pain treatment; 4) more than 3 points reduction in NRS score at day 180 of rest pain treatment; 5) cumulative major amputation or death rate of less than 0.90% at day 180 of rest pain treatment.

3. Use of the pUDK-HGF injection in the treatment of rest pain of a limb in a subject.

4. Use according to claim 3, characterized in that, The use is for at least one of: 1) complete pain relief at day 180 of rest pain treatment; 2) NRS score of 0 at day 180 of rest pain treatment; 3) 50% reduction in NRS score at day 180 of rest pain treatment; 4) more than 3 points reduction in NRS score at day 180 of rest pain treatment; 5) cumulative major amputation or death rate of less than 0.90% at day 180 of rest pain treatment.

5. The pUDK-HGF injection for use in the treatment of rest pain of a limb in a subject.

6. Use according to claim 5, characterized in that, The pUDK-HGF injection is for at least one of: 1) complete pain relief at day 180 of rest pain treatment; 2) NRS score of 0 at day 180 of rest pain treatment; 3) 50% reduction in NRS score at day 180 of rest pain treatment; 4) more than 3 points reduction in NRS score at day 180 of rest pain treatment; 5) cumulative major amputation or death rate of less than 0.90% at day 180 of rest pain treatment.

7. Use according to any one of claims 1 to 6, characterized in that, The subject has NRS score of 4-7; and / or, the subject has moderate to severe pain; and / or, the subject has not received analgesic drugs; and / or, the subject has critical lower limb ischemia; and / or, the subject has lower limb arteriosclerosis obliterans or thromboangiitis obliterans.

8. Use according to any one of claims 1 to 6, characterized in that, The treatment is by injection at 24 injection sites of the subject.

9. Use according to claim 8, characterized in that, The injection is intramuscular injection; and / or, the 24 injection sites are disposed along the muscle near: 1) the superficial femoral artery; or 2) the anterior tibial artery and the posterior tibial artery; or 3) the anterior tibial artery; or 4) the posterior tibial artery; or 5) along the superficial femoral artery, the anterior tibial artery and the posterior tibial artery; or 6) along the superficial femoral artery and the anterior tibial artery; or 7) along the superficial femoral artery and the posterior tibial artery; and / or, the 24 injection sites are disposed in a single longitudinal row or in 2 longitudinal rows; and / or, the highest injection site is above the highest occlusion / stenosis plane of the artery, which is the superficial femoral artery, the anterior tibial artery and / or the posterior tibial artery.

10. Use according to any one of claims 1 to 6, characterized in that, The pUDK-HGF injection comprises pUDK-HGF, disodium hydrogen phosphate, sodium chloride, disodium edetate, hydrochloric acid, water; Optionally, the total administration dose of the pUDK-HGF injection is 6 mg; Optionally, the administration dose of the pUDK-HGF injection at a single injection site is 0.25 mg; Optionally, the pUDK-HGF has a nucleotide sequence as shown in SEQ ID NO:

1. Optionally, the pUDK-HGF injection has a specification of 2.0 mg / 1.0 ml / bottle.

11. A method of treating rest pain in a limb, characterized in that, Comprising: administering to a subject a pharmaceutically acceptable dose of pUDK-HGF injection.

12. The method of claim 11, wherein, The administration is intramuscular injection; And / or, the administration is by 24 injection points on the subject.

13. The method of claim 12, wherein, The 24 injection points are arranged along the muscles near: 1) the superficial femoral artery; or 2) the anterior tibial artery and the posterior tibial artery; or 3) the anterior tibial artery; or 4) the posterior tibial artery; or 5) along the superficial femoral artery, the anterior tibial artery and the posterior tibial artery; or 6) along the superficial femoral artery and the anterior tibial artery; or 7) along the superficial femoral artery and the posterior tibial artery; And / or, the 24 injection points are arranged in a single longitudinal row or 2 longitudinal rows; And / or, the highest point of the injection is higher than the highest occlusion / stenosis plane of the artery, which is the superficial femoral artery, the anterior tibial artery and / or the posterior tibial artery.

14. The method according to any one of claims 11 to 13, characterized in that, The subject has an NRS score of 4-7; And / or, the subject has moderate to severe pain; And / or, no analgesic drug is used during the treatment; And / or, the subject has severe lower extremity ischemia; And / or, the subject has lower extremity arteriosclerosis obliterans or thromboangiitis obliterans.

15. The method of any one of claims 11-13, wherein the treatment is for: 1) complete disappearance of pain at day 180 of treatment of rest pain in the limb; 2) reduction of NRS score to 0 at day 180 of treatment of rest pain in the limb; 3) reduction of NRS score by 50% at day 180 of treatment of rest pain in the limb; 4) reduction of NRS score by more than 3 points at day 180 of treatment of rest pain in the limb; 5) cumulative major amputation or mortality rate of less than 0.90% at day 180 of treatment of rest pain in the limb.

16. The method according to any one of claims 11 to 13, characterized in that, The pUDK-HGF injection comprises pUDK-HGF, disodium hydrogen phosphate, sodium chloride, disodium edetate, hydrochloric acid, water; Optionally, the total administration dose of the pUDK-HGF injection is 6 mg; Optionally, the administration dose of pUDK-HGF injection at a single injection point is 0.25 mg; Optionally, the pUDK-HGF has a nucleotide sequence as shown in SEQ ID NO: 1; and optionally, the pUDK-HGF injection has a specification of 2.0 mg / 1.0 ml / bottle.

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