Use of momordicin IV in preparation of tumor treatment drugs

Abstract

The application discloses application of momordin IV in preparation of tumor treatment drugs. The application firstly proposes and verifies that the momordin IV can inhibit activity of cancer cells by inducing double-sulfur death of the cancer cells, and can further promote gefitinib to overcome drug resistance of the cancer cells and inhibit progress of drug-resistant tumors, so that a new direction and theoretical basis are provided for drug research and treatment of gefitinib-resistant non-small cell lung cancer.

Description

Technical Field

[0001] This invention relates to the field of natural medicinal chemistry, and more particularly to the application of momordicin IV in the preparation of tumor therapeutic drugs. Background Technology

[0002] Lung cancer is one of the leading causes of cancer-related morbidity and mortality, with non-small cell lung cancer (NSCLC) accounting for approximately 85%. NSCLC is characterized by its aggressiveness and poor prognosis, and treatment strategies have transitioned from traditional chemotherapy to targeted therapy. EGFR mutation is one of the most common driver genes in NSCLC, and EGFR tyrosine kinase inhibitors (EGFR-TKIs) targeting this mutation have significantly improved patient prognosis, achieving a response rate of up to 80% and extending progression-free survival (PFS) to 10-14 months. However, long-term use of EGFR-TKIs often leads to acquired resistance, severely limiting its long-term efficacy. For some resistance mutations, there are no effective drugs, limiting treatment options. Studies have found that inhibiting ferroptosis is a novel mechanism promoting gefitinib resistance. Targeting the ELK3-SERPINE1-PCBP2 axis or using novel complexes to enhance ferroptosis may become a new strategy to overcome resistance.

[0003] Momordicin IV is a natural triterpenoid compound extracted from bitter melon. Other active components of bitter melon (such as momordicin-I) have been shown to inhibit glycolysis and lipid metabolism in cancer cells through multiple targets, demonstrating anti-cancer potential without significant toxicity in head and neck cancer models. However, the relevant biological activities of momordicin IV have not yet been clearly reported. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide the application of momordicin IV in the preparation of a treatment for non-small cell lung cancer, especially gefitinib-resistant non-small cell lung cancer, whether used alone or in combination with gefitinib.

[0005] Technical solution: The application of momordicin IV described in this invention in the preparation of tumor therapeutic drugs.

[0006] Preferably, the CAS number of the bitter melon extract IV is 894412-35-2.

[0007] Preferably, the application is in the preparation of drugs for treating drug-resistant tumors.

[0008] Preferably, the drug-resistant tumor is a gefitinib-resistant tumor; more preferably, the tumor is non-small cell lung cancer.

[0009] Preferably, the drug contains momordicin IV or its pharmaceutically acceptable salts, solvates, or hydrates as active ingredients.

[0010] Preferably, the drug further contains gefitinib as an active ingredient; more preferably, the mass ratio of momordicin IV to gefitinib in the drug is 1:2~3.

[0011] Preferably, the drug further contains pharmaceutically acceptable excipients; more preferably, the pharmaceutically acceptable excipients include any one or more of excipients, diluents, lubricants, flow aids, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

[0012] Preferably, the dosage form of the drug includes tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

[0013] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention proposes and verifies for the first time that bitter melon extract IV can significantly inhibit the activity of cancer cells by inducing disulfiram-induced death of cancer cells, and can further promote gefitinib to overcome the drug resistance of cancer cells and inhibit the progression of drug-resistant tumors, providing a new direction and theoretical basis for the drug development and treatment of gefitinib-resistant non-small cell lung cancer. Attached Figure Description

[0014] Figure 1 The IC50 of gefitinib on PC9 and PC9 / GR cells 50 Value measurement results graph; Figure 2 The IC50 of momordicin IV on PC9 / GR cells 50 Value measurement results graph; Figure 3 The image shows the cell viability assay results of PC9 / GR cells after treatment. Figure 4 The image shows the results of the colony-forming ability assay for PC9 / GR cells after treatment. Figure 5 A graph showing the quantitative analysis of the colony-forming ability of PC9 / GR cells after treatment; Figure 6 Figure 1 shows the flow cytometry results of apoptosis levels in PC9 / GR cells after treatment. Figure 7 A flow cytometry graph showing the quantitative analysis of apoptosis levels in PC9 / GR cells after treatment; Figure 8 The graph shows the flow cytometry results of cell cycle determination in PC9 / GR cells after treatment. Figure 9 A flow cytometry graph showing the quantitative analysis of cell cycle determination results in treated PC9 / GR cells; Figure 10 The figure shows the flow cytometry results of mitochondrial membrane potential measurement in PC9 / GR cells after treatment. Figure 11 A flow cytometry graph showing the quantitative analysis of mitochondrial membrane potential measurements in PC9 / GR cells after treatment. Figure 12 The results of transcriptome sequencing analysis of PC9 / GR cells before and after treatment are shown in the figure. Figure 13 NADP for PC9 / GR cells after treatment + / NADPH ratio determination results graph; Figure 14 Figure showing the results of disulfiram-related protein level measurement in PC9 / GR cells after treatment; Figure 15 The graph shows the statistical results of the body weight of mice in different treatment groups; Figure 16 A graph showing the statistical results of lung tissue coefficients in mice from different treatment groups; Figure 17 The statistical results of tumor volume growth curves in mice of different treatment groups are shown in the figure. Figure 18 The graph shows the statistical results of tumor weight in mice from different treatment groups; Figure 19 Representative images of Ki67 protein immunohistochemical staining in tumor tissues of mice in different treatment groups; Figure 20 The figure shows the results of measuring the levels of disulfiram-related proteins in tumor tissues of mice in different treatment groups. Detailed Implementation

[0015] The technical solution of the present invention will be further described below.

[0016] Example 1: In vitro efficacy validation of momordicin IV (MOM) in the treatment of drug-resistant non-small cell lung cancer 1. Gefitinib (GEFI) IC50 in PC9 and PC9 / GR cells 50 Value determination PC9 cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., catalog number CL-0668) or PC9 / GR cells (purchased from Zhejiang Meisen Cell Technology Co., Ltd., catalog number CTCC-ZHYC-0141) were used at a rate of 2×10⁻⁶. 3Cells were seeded at a density of 1 cell / well in 96-well plates and cultured for 24 h. Then, the original medium was replaced with DMEM complete medium containing GEFI (purchased from MedChemExpress LLC., catalog number HY-50895) at final concentrations of 0, 0.5, 1, 2, 4, 8, 16, 32, and 64 µM. After 24 h of treatment, 100 μL of DMEM basal medium containing 10% CCK8 reagent (purchased from Novizan, catalog number A311-01) was added to each well and the plates were incubated at 37 °C for 2 h. The absorbance was measured at 450 nm using a microplate reader.

[0017] The results are as follows Figure 1 As shown, with the continuous increase of GEFI treatment concentration, the viability of both PC9 and PC9 / GR cells showed a decreasing trend. For PC9 cells, the IC50 value after 24 h of GEFI treatment was [not specified]. 50 The value was 1.839 µM, while the IC50 value for PC9 / GR cells was [missing value]. 50 The value was 8.632 µM, which indicates that PC9 / GR cells have developed a certain degree of resistance to GEFI. Therefore, in subsequent experiments, the GEFI treatment concentration was selected as 8 µM.

[0018] 2. MOM's effect on the IC50 of PC9 / GR cells 50 value PC9 / GR cells were loaded at 2×10 3 Cells were seeded at a density of 1 cell / well in 96-well plates and cultured for 24 h. The original medium was replaced with DMEM complete medium containing MOM (purchased from MedChemExpress LLC., catalog number HY-N13579) at final concentrations of 0, 0.5, 1, 2, 4, 8, 16, 32, and 64 µM. After 24 h of treatment, 100 μL of DMEM basal medium containing 10% CCK8 reagent was added to each well and the plates were incubated at 37°C for 2 h. The absorbance was measured at 450 nm using a microplate reader.

[0019] The results are as follows Figure 2 As shown, MOM significantly inhibited the activity of PC9 / GR cells in a dose-dependent manner, with an IC50 value of [value missing] at 24 h. 50 The value was 10.107 µM, therefore, in subsequent experiments, the MOM treatment concentration was chosen to be 10 µM.

[0020] 3. Assay of PC9 / GR cell viability after treatment PC9 / GR cells were loaded at 2×10 3Cells were seeded at a density of 1 cell / well in 96-well plates and cultured for 24 h. The original medium was replaced with DMEM complete medium containing 1‰ DMSO, or MOM with a final concentration of 10 µM, or GEFI with a final concentration of 8 µM and MOM with a final concentration of 10 µM. After 24 h of treatment, 100 μL of DMEM basal medium containing 10% CCK8 reagent was added to each well and the plates were incubated at 37°C for 2 h. The absorbance was measured at 450 nm using a microplate reader.

[0021] The results are as follows Figure 3 As shown, the combined treatment of the two drugs has a more significant effect on inhibiting the activity of PC9 / GR cells. That is, MOM can not only overcome the GEFI resistance problem, but also increase the sensitivity of PC9 / GR cells to GEFI treatment.

[0022] 4. Clonogenic capacity assay of PC9 / GR cells after treatment PC9 / GR cells were seeded at a density of 500 cells / well in 6-well plates and cultured for 24 h. The original medium was then replaced with DMEM complete medium containing 1‰ DMSO, or MOM at a final concentration of 10 µM, or a combination of GEFI at a final concentration of 8 µM and MOM at a final concentration of 10 µM, for another 24 h. After treatment, the medium was replaced with fresh, drug-free DMEM complete medium, and the medium was changed every 2 days for a total of 14 days. Cells were then washed with PBS, fixed with methanol, stained with Beyotime crystal violet reagent (C0121-100ml), and observed under a microscope for quantitative analysis.

[0023] The results are as follows Figure 4 , 5 As shown, compared with DMSO-treated PC9 / GR cells, MOM treatment significantly inhibited the clonogenic ability of PC9 / GR cells, and further significantly enhanced the inhibitory effect of GEFI on the clonogenic ability of PC9 / GR cells, with a significant difference.

[0024] 5. Measurement of apoptosis level in PC9 / GR cells after treatment PC9 / GR cells were used at 2×10 4 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured overnight. After adhesion, the cells were treated for 24 h with DMEM complete medium containing 1‰ DMSO, or MOM at a final concentration of 10 µM, or a combination of GEFI at a final concentration of 8 µM and MOM at a final concentration of 10 µM. Cells were then collected and stained using the Annexin V-PE / 7AAD apoptosis detection kit (catalog number A213-01). The stained cells were analyzed using a Beckman CytoFLEX flow cytometer to quantify the level of apoptosis.

[0025] The results are as follows Figure 6 , 7 As shown, after treatment with 10 µM MOM, the apoptosis level of PC9 / GR cells was significantly increased, and GEFI-induced apoptosis in PC9 / GR cells was also significantly increased. This means that MOM can effectively overcome the GEFI resistance problem that occurs in the treatment of non-small cell lung cancer.

[0026] 6. Cell cycle detection of PC9 / GR after treatment PC9 / GR cells were used at 2×10 4 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured overnight. After adhesion, the original medium was replaced with DMEM complete medium containing 1‰ DMSO, or MOM at a final concentration of 10 µM, or a combination of GEFI at a final concentration of 8 µM and MOM at a final concentration of 10 µM, for 24 h. Cells were then collected and stained using the Novizan Cell Cytometry Kit (catalog number AC101-01). The stained cells were analyzed using a Beckman CytoFLEX flow cytometer to quantify the cell cycle.

[0027] The results are as follows Figure 8 , 9 As shown, MOM treatment significantly increased the proportion of PC9 / GR cells in the G2 / M phase, with a statistically significant difference; at the same time, the proportion of PC9 / GR cells in the G2 / M phase was even higher after MOM and GEFI combined treatment.

[0028] 7. Measurement of mitochondrial membrane potential in PC9 / GR cells after treatment PC9 / GR cells were used at 2×10 4 Cells were seeded at a density of cells / well in 6-well plates and cultured overnight. After adhesion, the original medium was replaced with DMEM complete medium containing 1‰ DMSO, or MOM with a final concentration of 10 µM, or both GEFI with a final concentration of 8 µM and MOM with a final concentration of 10 µM for 24 h. After treatment, cells were collected and incubated in PBS buffer containing 10 µg / mL JC-1 (purchased from MedChemExpress LLC., catalog number: HY-15534) at 37°C in the dark for 30 min. After incubation, the cells were washed twice with pre-cooled PBS, resuspended, and fluorescence signals were detected using a Beckman CytoFLEX flow cytometer. The excitation wavelength for JC-1 monomer (green fluorescence) was set to 488 nm, and the emission wavelengths were detected at 525 / 50 nm. The emission wavelengths for JC-1 multimer (red fluorescence) were detected at 590 / 40 nm.

[0029] The results are as follows Figure 10 , 11As shown, the mitochondrial membrane potential of cells in the DMSO treatment group was higher (89.6 / 8.87=10.1), while MOM treatment significantly reduced the mitochondrial membrane potential of PC9 / GR cells (70.2 / 28.3=2.5). Meanwhile, the mitochondrial membrane potential of PC9 / GR cells was lowest after combined treatment with MOM and GEFI (55.2 / 43.8=1.3). These experimental results preliminarily indicate that MOM can induce a decrease in mitochondrial activity in PC9 / GR cells, thereby inhibiting cell activity and effectively overcoming GEFI resistance in non-small cell lung cancer.

[0030] 8. Transcriptome sequencing analysis of PC9 / GR cells before and after treatment PC9 / GR cells were used at 2×10 4 Cells were seeded at a density of cells / well in 6-well plates and cultured overnight. After adherence, the original medium was replaced with DMEM complete medium containing 1‰ DMSO, or MOM at a final concentration of 10 µM, or both GEFI at a final concentration of 8 µM and MOM at a final concentration of 10 µM for 24 h. After treatment, cells were collected, and RNA was extracted using Absin total RNA extraction reagent (catalog number abs60154). Transcriptome sequencing and analysis were performed by Beijing Novogene Technology Co., Ltd.

[0031] The results are as follows Figure 12 As shown, the KEGG analysis results of transcriptome sequencing showed that the disulfiram-related pathway in PC9 / GR cells was significantly activated after MOM treatment. Therefore, it can be preliminarily inferred that MOM can effectively induce disulfiram-related cell death in PC9 / GR cells, ultimately overcoming GEFI resistance in non-small cell lung cancer.

[0032] 9. NADP in PC9 / GR cells after treatment + / NADPH ratio determination PC9 / GR cells were used at 2×10 4 Inoculate 6-well plates at a density of 10 cells / well. After overnight incubation and adhesion, replace the original medium with DMEM complete medium containing 1‰ DMSO, or MOM at a final concentration of 10 µM, or both GEFI at a final concentration of 8 µM and MOM at a final concentration of 10 µM, and treat for 24 h. Collect the culture supernatant and use Beyotime NADP... + / NADPH Detection Kit (Catalog No. S0179) for detecting NADP in supernatant + The ratio of / NADPH.

[0033] The results are as follows Figure 13 As shown, the ratio of NADP in the MOM-treated group was significantly increased, showing a significant difference compared to the DMSO-treated group. Furthermore, the NADP in cells of the MOM and GEFI combined treatment group was significantly higher. +The highest NADPH ratio indicates a decrease in the antioxidant capacity of PC9 / GR cells, which can induce high levels of disulfiram-like cell death.

[0034] 10. Measurement of ferroptosis-related proteins in PC9 / GR cells after treatment PC9 / GR cells were used at 2×10 4 Cells were seeded at a density of 10 cells / well in 6-well plates and cultured overnight until adherence. Then, the original medium was replaced with DMEM complete medium containing 1‰ DMSO, or MOM at a final concentration of 10 µM, or both GEFI at a final concentration of 8 µM and MOM at a final concentration of 10 µM for 24 h. Cells were collected and lysed in RIPA lysis buffer (WB3100) supplemented with 0.1 volume of cyprotease inhibitor mixture (P001) and 0.1 volume of cyprotease inhibitor mixture (P003). Lysis was performed at 4°C for 30 min, followed by centrifugation at 12000 rpm for 30 min at 4°C. The supernatant was collected and quantified using a cyprotease BCA protein quantification kit (WB6501). Subsequently, cyprotease 5× Loading buffer (WB2001) was added proportionally, and the mixture was boiled in a metal bath for 10 minutes. After a period of time, SDS-PAGE electrophoresis was performed. After transfer and blocking, SLC7A11 primary antibody (purchased from Proteintech, catalog number 26864-1-AP) diluted 1:1000 or GAPDH primary antibody (purchased from Proteintech, catalog number 10494-1-AP) diluted 1:10000 was added and incubated overnight at 4°C. After rinsing, secondary antibody (purchased from Proteintech, catalog number SA00001-2) diluted 1:10000 was added and incubated at room temperature for 2 h. Finally, the images were developed and acquired using the Neo-Synemax Ultrasensitive ECL Chemiluminescence Kit (catalog number P10100).

[0035] The results are as follows Figure 14 As shown, compared with DMSO treatment, MOM significantly increased the SLC7A11 protein level in PC9 / GR cells, and the increase in SLC7A11 protein level was most significant after combined treatment with MOM and GEFI. These experiments preliminarily verify that MOM can effectively induce disulfiram-like cell death in PC9 / GR cells and ultimately inhibit its activity.

[0036] Example 2: Validation of the therapeutic effect of momordicin IV in a mouse model of drug-resistant non-small cell lung cancer. Female Balb / c nude mice aged 6-8 weeks and weighing 18-20 g were purchased from the Experimental Animal Center of Nantong University and housed in an SPF-grade animal room at a temperature of 22±2℃ and a humidity of 50±10%, with a 12-hour day-night cycle. They were acclimatized for one week before the experiment.

[0037] PC9 / GR cells were administered at a rate of 5 × 10⁵ cells per mouse. 5 One cell was inoculated subcutaneously under the left axilla to create a tumor. The tumor was induced when it reached approximately 100 mm in size. 3 At that time, tumor-bearing mice were randomly divided into 3 groups: saline group, MOM treatment group (dose of 20 mg / kg), and MOM+GEFI treatment group (MOM dose of 20 mg / kg and GEFI dose of 50 mg / kg), and drug treatment was started at the same time.

[0038] The drug was administered by gavage every 4 days, while the saline group received the same volume of saline. Treatment continued until day 24. Upon reaching the experimental endpoint, the mice were euthanized, and tumor and lung tissues were collected from each group for subsequent analysis of relevant indicators.

[0039] 1. Weight monitoring Starting from day 0 of the drug administration, the body weight of mice in each experimental group was measured every 4 days.

[0040] The results are as follows Figure 15 As shown, the weight changes of the three groups of nude mice were basically the same, and there was no significant difference between the groups.

[0041] 2. Lung tissue coefficient measurement The collected intact lung tissue was blotted dry with filter paper and weighed. The lung tissue coefficient was calculated using the following formula: Lung tissue coefficient (%) = wet weight of lung tissue / mouse body weight × 100.

[0042] The results are as follows Figure 16 As shown, there was no significant difference in lung tissue coefficients among the three groups of nude mice.

[0043] The above experiments show that the concentrations of MOM and GEFI drugs selected in the experiment did not produce biological toxicity.

[0044] 4. Tumor volume monitoring and weight measurement Starting from day 0 of the drug administration, the tumor volume of mice in each experimental group was measured every 4 days. The length and width of the tumor were recorded, and the tumor volume was then calculated and statistically analyzed according to the following formula: Tumor volume (mm) 3 () = length × width × width / 2. After reaching the end of the experiment, weigh the collected tumor tissue.

[0045] Tumor volume monitoring results in nude mice are as follows Figure 17 As shown, compared with the saline treatment group, the tumor volume after MOM treatment was much smaller than that in the saline group, and the tumor volume in the nude mice in the MOM+GEFI treatment group was the smallest. This indicates that MOM is effective in treating non-small cell lung cancer and can significantly enhance the therapeutic effect of GEFI drugs.

[0046] Tumor weight measurement results in nude mice are as follows Figure 18 As shown, the MOM treatment group significantly inhibited tumor weight, and the MOM+GEFI treatment group had the lowest tumor weight in nude mice, indicating the best tumor inhibition effect.

[0047] 5. Immunohistochemical detection of Ki67 expression in tumor tissue Tumor tissues were fixed overnight with 4% paraformaldehyde, dehydrated in a gradient manner, embedded in paraffin, and cut into 4 μm sections. Antigen retrieval was performed at 95°C for 20 min with citrate buffer (pH 6.0). Endogenous peroxidase activity was inhibited with 3% hydrogen peroxide solution for 10 min, followed by blocking with 5% bovine serum albumin solution for 1 h. The sections were then incubated overnight at 4°C with Ki67 primary antibody (purchased from Proteintech, catalog number 27309-1-AP) diluted 1:10000 or SLC7A11 primary antibody diluted 1:200. After washing, the sections were incubated with horseradish peroxidase (HRP) diluted 1:500 with secondary antibody (Proteintech, catalog number SA00004-2) at room temperature for 1 h. Signal detection was performed using Beyotime DAB chromogenic reagent kit (catalog number P0202), and the cell nuclei were counterstained with hematoxylin. After mounting with neutral resin, the sections were observed and images were acquired under a microscope.

[0048] The results are as follows Figure 19 As shown, after MOM treatment, Ki67 expression in tumor tissue decreased significantly, indicating that MOM has a good in vivo anti-tumor effect. Simultaneously, the SLC7A11 protein expression level in nude mice treated with MOM was significantly increased, consistent with the in vitro experimental results. Furthermore, MOM+GEFI treatment caused even greater changes in Ki67 and SLC7A11 expression, i.e., lower Ki67 and higher SLC7A11, indicating a better effect.

[0049] 6. Western blot detection of ferroptosis-related proteins in tumor tissues 100 mg of tumor tissue was collected, ground in liquid nitrogen using a mortar and pestle, and then lysed thoroughly with 1 mL of Beyotime Western chemiluminescence buffer containing protease inhibitors and phosphatase inhibitors and IP cell lysis buffer. After lysis at 4°C for 30 min, the tissue was centrifuged at 12,000 rpm for 30 min to obtain the supernatant, which was then quantified using the Neo-SyneBCA protein quantification kit. Subsequently, Neo-SyneBCA 5× Loading buffer was added in proportion, and the mixture was heated to boiling in a metal bath for 10 min. SDS-PAGE electrophoresis was then performed, followed by membrane transfer and blocking. Then, SLC7A11 primary antibody diluted 1:1000 or GAPDH primary antibody diluted 1:10000 was added and incubated overnight at 4°C. After rinsing, the membrane was incubated with secondary antibody diluted 1:10000 at room temperature for 2 h. Finally, the membrane was developed and images were acquired using the Neo-SyneBCA ultrasensitive ECL chemiluminescence kit.

[0050] The results are as follows Figure 20 As shown, compared to the saline group, MOM effectively upregulated SLC7A11 expression to promote disulfiram-like cell death in tumors. Furthermore, the highest SLC7A11 protein expression level was observed in tumor tissues after MOM+GEFI combination therapy, indicating that the combination of MOM and GEFI can more significantly induce disulfiram-like cell death in GEFI-resistant tumors, meaning that MOM can significantly increase the effectiveness of GEFI treatment for GEFI-resistant non-small cell lung cancer.

Claims

1. The application of momordicin IV in the preparation of tumor therapeutic drugs, characterized in that, The CAS number of the bitter melon extract IV is 894412-35-2, and the tumor is gefitinib-resistant non-small cell lung cancer.

2. The application according to claim 1, characterized in that, The drug contains momordicin IV or a pharmaceutically acceptable salt thereof as its active ingredient.

3. The application according to claim 2, characterized in that, The drug also contains gefitinib as an active ingredient.

4. The application according to claim 3, characterized in that, The mass ratio of momordicin IV to gefitinib in the drug is 1:2~3.

5. The application according to any one of claims 1 to 4, characterized in that, The drug also contains pharmaceutically acceptable excipients.

6. The application according to claim 1, characterized in that, The dosage forms of the drug include tablets, capsules, granules, powders, injections, suspensions, patches, and suppositories.

7. The application according to claim 6, characterized in that, The tablets include chewable tablets, effervescent tablets, and sustained-release tablets; the injections include infusion solutions; and the patches include transdermal patches.

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