Application of melatonin in improving the side effects caused by chemotherapy drugs
Combining melatonin with hydroxycamptothecin in pharmaceutical compositions addresses male reproductive impairment caused by chemotherapy, enhancing sperm quality and fertility by reducing apoptosis and oxidative stress.
Patent Information
- Application Number
- CN202310733804.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-20
- Publication Date
- 2024-12-17
- Estimated Expiration
- 2043-06-20
AI Technical Summary
Current chemotherapy drugs like hydroxycamptothecin cause significant side effects, including male reproductive impairment, such as testicular oxidative stress, sperm abnormalities, and decreased fertility, with no existing methods to mitigate these effects while maintaining anti-cancer efficacy.
Combining melatonin with hydroxycamptothecin in a pharmaceutical composition, such as tablets, capsules, or injections, to alleviate the side effects on male reproductive capacity by reducing apoptosis, oxidative stress, and improving sperm quality and testosterone production.
Melatonin significantly reduces testicular damage, increases sperm-egg binding, and enhances testosterone levels, thereby protecting male fertility during chemotherapy.
Smart Images
Figure CN116687918B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of reproductive biology and relates to a new use of melatonin, and specifically to the use of melatonin in improving the side effects caused by chemotherapy drugs. Background Art
[0002] Malignant tumors are a persistent public health challenge that the world is currently facing. As the second most common cause of death in the world, cancer is expected to become the main obstacle to the increase in life expectancy in the 21st century. Hydroxycamptothecin (HCPT) is an active alkaloid isolated from Camptotheca acuminata with high anticancer effects. It can form a stable complex with type I topoisomerase of DNA, induce single-strand breaks of DNA and unwind its double-stranded structure, thereby preventing the synthesis of cellular RNA. It is often used clinically to assist in the treatment of various malignant tumors such as primary liver cancer, gastric cancer, and esophageal cancer. In addition, the anticancer activity of HCPT exceeds that of Camptothecin, and it has anti-tumor, immunosuppressive, antiviral, anti-early pregnancy and insecticidal effects. Because HCPT has no cross-resistance, a wide anticancer spectrum and high efficacy, it is also used clinically to treat leukemia, psoriasis, warts, etc. Studies have shown that HCPT can also inhibit fibrosis and scar adhesion outside the dura mater. In short, HCPT is widely used clinically. However, hydroxycamptothecin also has some side effects, such as nausea, vomiting, inhibition of bone marrow proliferation and damage to reproductive ability.
[0003] Melatonin (MT) is an indole neuroendocrine hormone secreted by the pineal gland. It regulates the circadian rhythm and seasonal rhythm of the human body through the rhythm of low daytime and high nighttime. It has important physiological and pathological functions. In recent years, melatonin has been widely used and paid attention to. A large number of studies have shown that melatonin has the functions of regulating the body's immune response, body weight, reproduction, adjusting jet lag, and inhibiting tumor growth. It can also act as a reducing agent to scavenge free radicals and anti-oxidation, which helps to delay aging. Melatonin can have anti-proliferative effects on a variety of cancers through receptors MT1 and MT2, such as breast cancer, lung cancer, metastatic renal cell carcinoma, liver cancer, brain metastasis of malignant tumors, ovarian cancer, neuroblastoma, bladder cancer, leukemia, etc. There is currently no precedent for melatonin to be used with chemotherapy drugs to inhibit the growth of cancer while protecting other organs and cells from damage.
[0004] Currently, there is no research on the correlation between chemotherapy drugs and male reproductive ability in the existing technology. If a method can be developed to alleviate the impact of chemotherapy drugs on male reproductive ability, it will provide a reference for clinical cancer patients to preserve their fertility during chemotherapy. Summary of the invention
[0005] In view of the deficiencies in the prior art, the present invention provides a new use of melatonin, specifically the use of melatonin in improving the side effects caused by chemotherapy drugs, providing a chemotherapy regimen or reference for malignant tumor patients who want to preserve their fertility during chemotherapy.
[0006] The present invention is achieved through the following technical solutions:
[0007] Application of melatonin in the preparation of drugs for improving side effects caused by chemotherapy drugs.
[0008] Preferably, the chemotherapy drug is hydroxycamptothecin, and the side effect is male reproductive impairment caused by the use of hydroxycamptothecin.
[0009] Application of melatonin combined with hydroxycamptothecin in the preparation of anti-tumor drugs.
[0010] A combined drug composition for treating tumors, wherein the active ingredients are melatonin and hydroxycamptothecin.
[0011] Preferably, a pharmaceutically acceptable excipient is also included.
[0012] Preferably, the pharmaceutical composition is an oral dosage form or a non-oral dosage form.
[0013] Preferably, the pharmaceutical composition is a tablet, capsule, powder, pill, granule, solution, suspension, syrup, injection, suppository, inhalant or spray.
[0014] The beneficial effects of the present invention are as follows:
[0015] (1) The present invention is aimed at the existing problem that continuous use of the chemotherapy drug hydroxycamptothecin by male cancer patients can cause a decline in testicular reproductive capacity, including a significant increase in the number of apoptotic cells in the seminiferous tubules, a significant increase in the rate of sperm acrosome abnormalities, severe testicular oxidative stress damage, a decrease in the sperm-egg binding rate, and a significant decrease in testicular testosterone content. A new solution is provided: injecting melatonin while injecting the chemotherapy drug hydroxycamptothecin. Melatonin can alleviate the effects of chemotherapy drugs on testicular reproductive capacity, including relieving a significant increase in apoptotic cells in the seminiferous tubules and sperm acrosome abnormalities in the chemotherapy group, protecting the testicles from oxidative stress damage and improving sperm quality, which is beneficial to sperm-egg binding and testosterone production.
[0016] (2) The new use of melatonin of the present invention can be used to provide a new chemotherapy regimen or reference for cancer patients who want to protect their fertility after chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1The effects of hydroxycamptothecin and melatonin, chemotherapy drugs, on the size of male testicles and their underlying mechanisms in Example 1 are explored: A is a representative diagram showing the size of testicles in each group; B is a bar graph showing the testicular weight of each group; C is a statistical diagram showing the number of apoptotic cells in each flexor cavity of each group; D is a representative bar graph showing the protein expression of Bax, Bcl-2, P53, Caspase-3, CleavedCaspase-3 and GAPDH in testicular tissue of each group; E is a relative expression diagram of different proteins in testicular tissue of each group; wherein, * P<0.05, ** P<0.01, *** P<0.005, # P<0.05, ## P<0.01, ns means no significant difference, marked with * The difference between the two groups is significant compared with the NC group, and is marked with # represents the significance compared with the HCPT group;
[0018] Figure 2 The effects of chemotherapeutic drugs hydroxycamptothecin and melatonin on male testicular sperm quality and testosterone production in Example 2: A is a representative graph of sperm concentration in each group; B is a statistical graph of sperm concentration in each group; C is a representative picture of sperm-egg combination in each group; D is a statistical graph of the number of sperm-egg combination in each group; E is a normal morphological graph (left) and an abnormal morphological graph (right) of the sperm acrosome in the tail of the epididymis; F is a statistical graph of the abnormal rate of sperm acrosome in the tail of the epididymis in each group; G is a statistical graph of testosterone content in testicular tissue in each group; wherein, * P<0.05, ** P<0.01, *** P<0.005, # P<0.05, ## P<0.01, ### P<0.005, ns means no significant difference, marked with * The difference between the two groups is significant compared with the NC group, and is marked with # represents the significance compared with the HCPT group;
[0019] Figure 3 The effects of melatonin and chemotherapeutic drug hydroxycamptothecin on male testicular oxidative stress in Example 3: A is a representative graph of protein immunoblotting of Nrf2, Keap1, HO-1 and GAPDH in each group; B is a bar graph of the relative expression levels of Keap1, Nrf2 and HO-1 in each group; C is a statistical graph of glutathione (GSH) content in testicular tissue of each group; D is a statistical graph of malondialdehyde (MDA) content in testicular tissue of each group; wherein, * P<0.05, ** P<0.01, *** P<0.005,# P<0.05, ## P<0.01, ### P<0.005, ns means no significant difference, marked with * The difference between the two groups is significant compared with the NC group, and is marked with # The represents significant difference compared with the HCPT group. DETAILED DESCRIPTION
[0020] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.
[0021] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0022] The animals used in the following examples were 12-week-old CD1 male mice from the Experimental Animal Center of Nantong University. They were fed and watered freely, and the light was controlled (12 h / d). The indoor temperature was 20-22°C.
[0023] Example 1 Effects of hydroxycamptothecin and melatonin on male testicles and their underlying mechanisms
[0024] 1. Male mice were intraperitoneally injected with normal saline (NC group), melatonin 15 mg / kg (MT group), hydroxycamptothecin 5 mg / kg (HCPT group), and simultaneously injected with melatonin 15 mg / kg and hydroxycamptothecin 5 mg / kg (HCPT+MT group) for 14 consecutive days. The drugs were stopped for 2 weeks and the mice were euthanized to collect their corresponding testicular tissues.
[0025] The results of comparing the testicular size of each group are as follows Figure 1 As shown in A and B, the testis size of mice injected with the chemotherapy drug hydroxycamptothecin (HCPT group) was significantly smaller than that of the NC group ( *** P<0.005). However, the injection of melatonin at the same time as HCPT (HCPT+MT group) can significantly alleviate the effect of HCPT on testicular size. Compared with the HCPT group, the testicles of the HCPT+MT group were significantly larger ( # P<0.05).
[0026] 2. To explore the reasons for the above experimental results, half of the testicular tissues from each group were fixed with paraformaldehyde, dehydrated, embedded in paraffin and sectioned in paraffin. Finally, the number of apoptotic cells in the testicles of each group was detected using the TUNELL kit.
[0027] The results are as follows Figure 1 As shown in C, compared with the NC group, the number of apoptotic cells in the testes of mice injected with the chemotherapy drug hydroxycamptothecin (HCPT group) increased significantly ( ***P<0.005). However, the injection of melatonin at the same time as the chemotherapy drug (HCPT+MT group) can significantly alleviate the number of testicular cell apoptosis caused by hydroxycamptothecin. Compared with the HCPT group, the number of testicular apoptotic cells in the HCPT+MT group was significantly reduced ( # P<0.05).
[0028] 3. In order to further explore the intrinsic mechanism of testicular cell apoptosis, the testicular tissues of the other half of the above groups were taken, lysed with protein lysis buffer to extract total protein, and then the protein expression levels of apoptosis-related factors were detected according to the conventional method of protein immunoblotting.
[0029] The results are as follows Figure 1 As shown in D and E, compared with the NC group, the apoptosis factor Bax / Bcl protein expression ratio, P53 and cleaved Caspase 3 protein expression levels of mice injected with the chemotherapy drug hydroxycamptothecin (HCPT group) were significantly increased ( * P<0.05, *** P<0.05). However, compared with the HCPT group, the testicular Bax / Bcl protein expression ratio, P53 and cleaved Caspase 3 protein expression levels in the HCPT+MT group were significantly decreased ( # P<0.05, ## P<0.01).
[0030] Comprehensive analysis of the above experimental results shows that melatonin can significantly improve the damage of the chemotherapy drug hydroxycamptothecin to testicular tissue cells.
[0031] Example 2 Effects of HCPT and Melatonin on Male Testicular Sperm Quality and Testosterone Production
[0032] 1. Male mice were intraperitoneally injected with normal saline (NC group), melatonin 15 mg / kg (MT group), hydroxycamptothecin 5 mg / kg (HCPT group), and melatonin 15 mg / kg and hydroxycamptothecin 5 mg / kg (HCPT+MT group) for 14 consecutive days. After 2 weeks of drug withdrawal, the mice were euthanized and the sperm of their corresponding epididymal tails were collected. The sperm concentration of each group was detected by using a CASA instrument.
[0033] The results are as follows Figure 2 As shown in A and B, compared with the NC group, the sperm concentration in the tail of the epididymis decreased significantly after mice were injected with the chemotherapy drug hydroxycamptothecin (HCPT group) ( *** P<0.005). However, compared with the HCPT group, the sperm concentration in the cauda epididymis of the HCPT+MT group increased significantly ( ## P<0.05).
[0034] 2. In order to determine the quality of sperm obtained from each group, a portion of sperm from each group was taken and incubated with oocytes in the second stage of meiosis at 37°C and 5% CO2 for 1 hour to observe the sperm-oocyte binding ability.
[0035] The results are as follows Figure 2 As shown in C and D, compared with the NC group, the number of sperm in the tail of the epididymis that could combine with the egg decreased significantly after the mice were injected with the chemotherapy drug hydroxycamptothecin (HCPT group) ( *** P<0.005). However, compared with the HCPT group, the number of spermatozoa in the cauda epididymis in the HCPT+MT group increased significantly ( ### P<0.005).
[0036] 3. In order to further explore how the chemotherapy drug hydroxycamptothecin affects sperm-egg binding and the role of melatonin in it, a portion of sperm from each group was stained acrosome (PNA fluorescence staining method) and the complete morphology of the sperm acrosome in each group was counted.
[0037] The results are as follows Figure 2 As shown in E and F, compared with the NC group, the number of abnormal acrosomes in sperm in the cauda epididymis of mice injected with the chemotherapy drug hydroxycamptothecin (HCPT group) increased significantly ( *** P<0.005). However, compared with the HCPT group, the number of abnormal acrosomes in the sperm of the cauda epididymis in the HCPT+MT group decreased ( ## P<0.01).
[0038] 4. Since studies have found that sperm quality is also affected by the level of testosterone production, a testosterone detection kit was used to detect the testosterone levels in the testicular tissues of the above groups.
[0039] The results are as follows Figure 2 As shown in G, compared with the NC group, the testosterone level in the testes of mice injected with the chemotherapy drug hydroxycamptothecin (HCPT group) was significantly decreased ( *** P<0.005). However, compared with the HCPT group, the testosterone level in the HCPT+MT group increased ( ### P<0.005).
[0040] Based on the analysis of the above experimental results, it can be concluded that melatonin can improve the ability of sperm to bind to eggs by increasing the level of testosterone in the testicles and maintaining the integrity of the sperm acrosome, ultimately alleviating the damage to sperm quality caused by the chemotherapy drug hydroxycamptothecin.
[0041] Example 3 Melatonin can alleviate hydroxycamptothecin-induced oxidative stress damage to male testicles
[0042] 1. To explore the reasons why hydroxycamptothecin causes testicular cell apoptosis and decreased sperm quality in the cauda epididymis, and how melatonin alleviates the decreased testicular spermatogenesis caused by hydroxycamptothecin. Male mice were intraperitoneally injected with normal saline (NC group), melatonin 15 mg / kg (MT group), hydroxycamptothecin 5 mg / kg (HCPT group), and melatonin 15 mg / kg and hydroxycamptothecin 5 mg / kg (HCPT+MT group) respectively. After continuous injection for 14 days, the drugs were stopped for 2 weeks, and the mice were euthanized to collect their corresponding testicular tissues. Half of the testicular tissues of the above groups were taken, lysed with protein lysis buffer, and total protein was extracted. Then, the expression levels of oxidative stress and anti-oxidative stress-related proteins were detected according to the conventional method of protein immunoblotting.
[0043] The results are as follows Figure 3 As shown in A and B, compared with the NC group, the expression levels of anti-oxidative stress proteins Nrf2 and HO-1 were significantly decreased, while the oxidative stress protein Keap1 was significantly increased in mice injected with the chemotherapy drug hydroxycamptothecin (HCPT group). *** P<0.005). However, compared with the HCPT group, the expression levels of the anti-oxidative stress proteins Nrf2 and HO-1 in the testis of the HCPT+MT group were significantly increased, while the oxidative stress protein Keap1 was significantly decreased ( ## P<0.005, # P<0.05).
[0044] 2. In order to further verify that the chemotherapy drug hydroxycamptothecin can cause testicular oxidative stress damage, the testicular tissues of the other half of the above groups were ground into slurry, and the contents of GSH and malondialdehyde (MDA) in the testicles of each group were measured according to the instructions of the glutathione (GSH) and malondialdehyde (MDA) kits.
[0045] The results are as follows Figure 3 As shown in C and D, compared with the NC group, the peroxidation index MDA in the testis of mice injected with the chemotherapy drug hydroxycamptothecin (HCPT group) increased significantly, while the antioxidant index GSH content decreased significantly ( *** P<0.005). However, compared with the HCPT group, the antioxidant index GSH content in the testis of the HCPT+MT group increased significantly, while the peroxidation index MDA decreased significantly ( ## P<0.01).
[0046] Comprehensive analysis of the above experimental results shows that melatonin can protect testicular tissue by alleviating the oxidative stress damage of hydroxycamptothecin to testicular tissue.
[0047] It should be understood that the solutions given in the above embodiments are only for the purpose of illustration, and are not intended to limit the present invention. Those skilled in the art may make various modifications and substitutions to the above embodiments without departing from the purpose and spirit of the present invention, and all of these shall fall within the scope of protection of the present invention.
Claims
1. Use of melatonin in the preparation of a drug for improving the side effects caused by chemotherapeutic drugs, characterized in that: The chemotherapy drug is hydroxycamptothecin, and the side effect is a decrease in testicular reproductive ability caused by the use of hydroxycamptothecin.