Patent Application
20220105153
The present invention relates to a use of a protein-based long-acting preparation in improving sexual dysfunction.
Erectile dysfunction (hereinafter referred to as ED for abbreviation) is the most common male sexual dysfunction. Its etiology can be divided into: psycho mental ED, organic ed (including vascular and neurological causes), or mixed ED (referring to erectile dysfunction caused by psycho mental factors and organic causes).
Existing chemical drugs for treating ED, such as sildenafil citrate, can only restore an abnormal erectile function to a normal state temporarily but cannot treat the organic lesion that causes ED.
As ED has a high incidence rate and is a chronical disease, it is imperative to develop new, long-acting drugs that can improve ED by reversing the underlying organic lesion.
Because the incidence of ED is high and it is a chronic disease, it is very necessary to develop new long-acting drugs that can provide the treatment to ED by reversing the organic lesions.
One objective of the present invention is to find a long-acting preparation that can improve ED.
The inventor of the present invention has found through experimentation that a long-acting preparation made from a protein having the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2 can improve ED in experimental animals.
The long-acting preparation disclosed herein includes various sustained-release preparations or controlled-release preparations made from the aforesaid protein and a pharmaceutically acceptable auxiliary substance.
The long-acting preparation disclosed herein also includes long-acting preparations made from a modified protein of the aforesaid protein.
The aforesaid modified protein is a protein molecule having the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2 and connected with another protein or with another chemical substance.
Experimental results have proved that the protein-based long-acting preparation provided by the present invention can effectively improve ED, as detailed below with reference to an embodiment of the invention.
Explanation: In
ZX indicates the experimental group using a protein having the amino acid sequence of SEQ ID NO. 1.
In the following embodiment, “ZX1305 protein” indicates a protein having the amino acid sequence of SEQ ID NO. 1.
Embodiment 1: Experimental Study on Rat ED Improvement by ZX1305-Protein-Based Sustained-Release Preparation
40 specific-pathogen-free (SPF) male Sprague Dawley (SD) rats provided by Qing Long Shan Animal Propagation Station, Jiangning District, Nanjing City
The 40 male SD rats received adaptive feeding for three days. After that, ten rats were randomly selected as the normal-animal blank control group and fed with normal feed. The remaining 30 rats were fed with high-fat feed in order to create a diabetes model.
In the fourth week, the rats in the normal-animal blank control group were given a citric acid-sodium citrate buffer solution for comparison purposes.
Meanwhile, the rats fed with high-fat feed were intraperitoneally injected with a streptozotocin (STZ) solution (with a dose of 60 mg/kg, and the solvent being a citric acid-sodium citrate buffer solution), and the drug was administered for three consecutive days.
The blood sugar level was tested on the fourth day after drug administration. The intended diabetes model would be considered established if a random plasma glucose level higher than 16.7 mmol/L was detected.
The effects of the ZX1305 sustained-release preparation on the body weight and blood sugar level of the rats are tabulated as Table 1.
Six weeks after the injection of STZ, a comparison with the normal-animal blank control groups shows that the rats in the diabetes-model blank control group, in the high-dose ZX1305 group, and in the low-dose ZX1305 group all had a significant decrease in body weight (P<0.01) and a significant increase in blood sugar level (P<0.01). This indicates that the diabetic rat model was successfully established.
Streptozotocin (STZ) repackaged by sigma, purchased from ThermoFisher Biochemical Reagent Co., Nanjing
ZX1305 sustained-release preparation provided by XINTRUM Pharmaceuticals, Jiangsu. The preparation is a nanoparticle long-acting preparation prepared by the methods disclosed in PCT/US2019/015, filed on Jan. 29, 2019, and U.S. Provisional Patent Application No. 62/623,018, filed on Jan. 29, 2018.
{circle around (1)} Citric acid-sodium citrate buffer solution: 2.1 g of citric acid (FW: 210.4) was weighed and then dissolved in 100 ml of double distilled water to produce solution A. 2.94 g of sodium citrate (FW: 294.10) was dissolved in 100 ml of double distilled water to produce solution B. Solutions A and B were mixed at a ratio of 1:1, and the pH value of the mixture was adjusted to 4.2-4.5 with a solvent or water. The buffer solution was used immediately after the preparation.
{circle around (2)} Solutions of the ZX1305 sustained-release preparation: The specification of the sustained-release preparation was 0.5 mg/piece. Immediately before use, an entire vial of the sustained-release preparation/drug was directly dissolved in 8 ml of ZX1305 preparation buffer solution to produce the high-dose drug solution (62.5 μg/ml) to be administered to the high-dose group. The high-dose drug solution was then diluted ten times with the ZX1305 preparation buffer solution to produce the low-dose drug solution (6.25 μg/ml) to be administered to the low-dose group.
The rats in the successfully established diabetes model were randomly divided into three groups:
the diabetes-model blank control group, the low-dose (10 μg/kg) ZX1305 sustained-release preparation group, and the high-dose (100 μg/kg) ZX1305 sustained-release preparation group. These three groups and the normal-animal blank control group made up a total of four groups.
The drug-administered groups were intramuscularly injected with their respective ZX1305 sustained-release preparation solutions. The volume of the drugs administered was 1.6 ml/kg, and the drugs were administration only once. After drug administration, the behaviors of the rats were observed on a daily basis, changes in body weight were recorded on a weekly basis, and the blood sugar level was recorded every two weeks. The observation and recording continued for six weeks.
An APO induction experiment was conducted in the tenth week after drug administration. Each rat was subcutaneously injected in the neck with apomorphine (APO) at 100 μg/kg and was observed for 30 minutes after the injection, with the occurrence or non-occurrence of penile erection and the number of times of penile erection recorded. The erection rates were then calculated. Glans engorgement plus exposure of the terminal end of the penis body was counted as one penile erection.
Rat cages were prepared, in each of which a single male experimental rat was placed. The cages were placed in the dark for five minutes, allowing the rats to adapt to the environment. Then, a normal female rat was put into each cage, and the following began to be recorded: {circle around (1)} the latent capture period: the time period starting from the instant a female rat was put into a cage to the instant the male rat in the cage captured the female rat for the first time (the latent capture period was recorded as 20 minutes if the male rat did not capture the female rat at all); {circle around (2)} the number of times of capture: the number of times for which a male rat captured the female rat in its cage during the 20 minutes after the female rat was put into the cage (the number of times of capture was recorded as 0 if the male rat did not capture the female rat at all).
The cavernous pressure of each rat was recorded with the electrophysiological recording system while the cavernous nerves of the rat were stimulated with a 5-V, 15-Hz, 5-ms direct current.
Once the intracavernous pressure of a rat was determined, the abdominal aorta of the rat was exposed, and a PE50 tube connected to the pressure transducer was placed into the aorta in order to monitor the average arterial pressure of the rat directly and continuously.
After the rats in each group were weighed and anesthetized, whole blood was drawn from the abdominal aorta and then centrifuged at 4° C. and 2000 g for ten minutes. Serum was subsequently obtained and kept at −20° C. for later use. The rats were then executed by cervical dislocation. The testicles of the rats were taken and weighed with precision in order to calculate the testicular index as follows: testicular index (%)=testicle weight (g)/body weight (g)×100%.
Once the rats were executed, the heart, liver, spleen, lungs, kidneys, brain, eyeballs, cavernous body, and sciatic nerves of each rat were extracted.
Observation of the ten rats in the normal-animal blank control group reveals that every rat had penile erection; the erection rate was 100%, and the average number of times of erection was 5.3. Observation of the eight rats in the diabetes-model blank control group reveals that none but one of the rats had penile erection, the erection rate being 12.5%. The results indicate that a pathological model for sexual dysfunction of diabetic rats was successfully established.
According to the blood sugar levels of the rats and the results of the APO induction experiment, a sexual dysfunction model for diabetic rats was successfully established.
The latent capture periods and the numbers of times of capture of the experimental rats are shown in Table 2 and
Compared with the normal-animal blank control group, the diabetes-model blank control group showed a significant increase in the latent capture period (P<0.01) and a significant reduction in the number of times of capture (P<0.01).
Compared with the diabetes-model blank control group, the high-dose ZX1305 group showed a significant improvement in both the latent capture time and the number of times of capture (P<0.01), and the low-dose ZX1305 group showed a significant reduction in the latent capture period (P<0.01) but no significant change in the number of times of capture (P>0.05).
The test results are shown in
##P<0.01, compared with the normal-animal blank control group; *P<0.05, compared with the diabetes-model blank control group.
Compared with the normal-animal blank control group, the diabetes-model blank control group showed a highly significant reduction in the ratio of the intracavernous pressure to the average arterial pressure (P<0.01).
Compared with the diabetes-model blank control group, the high-dose ZX1305 group showed a significant increase in the ratio of the intracavernous pressure to the average arterial pressure (P<0.01).
While the low-dose ZX1305 group also showed an increase in the ratio of the intracavernous pressure to the average arterial pressure, the change is not significant (P>0.05).
#P<0.05, compared with the normal-animal blank control group; **P<0.01, compared with the diabetes-model blank control group.
Compared with the normal-animal blank control group, the diabetes-model blank control group showed a significant reduction in the testicular index (P<0.05).
Compared with the diabetes-model blank control group, the high-dose ZX1305 group showed a significant increase in the testicular index (P<0.01), and the low-dose ZX1305 group showed no significant change in this respect (P>0.05). The experiment results are plotted in
The foregoing experiments have shown that:
1) When administered at a dose of 100 μg/kg, the ZX1305 sustained-release preparation produced the following experimental results on the diabetic rats with sexual dysfunction:
{circle around (1)} A highly significant increase in the number of times of capture;
{circle around (2)} A highly significant increase in the testicular index;
{circle around (3)} A significant increase in the ratio of the intracavernous pressure to the average arterial pressure; and
{circle around (4)} A highly significant reduction in the latent capture period.
2) When administered at a dose of 10 μg/kg, the ZX1305 sustained-release preparation caused a highly significant reduction in the latent capture period of the diabetic rats with sexual dysfunction but did not have a significant effect on the other indices.
3) The ZX1305 sustained-release preparation had no effect on the body weights or blood sugar levels of the diabetic rats.
The ZX1305-protein-based sustained-release preparation had an improving effect on the sexual dysfunction of the diabetic rats.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201910809334.6 | Aug 2019 | CN | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2020/108912 | Aug 2020 | US |
| Child | 17555374 | US |
