Use of a combination of Achyranthes japonica polypeptide and human umbilical cord mesenchymal stem cell exosomes in the preparation of drugs to prevent and / or treat Parkinson's disease.

Abstract

This invention relates to the use of a combination of Achyranthes japonica polypeptide and human umbilical cord mesenchymal stem cell exosomes in the preparation of drugs for the prevention and / or treatment of Parkinson's disease. A Parkinson's disease model (PD) was created in mice using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The mice were then treated with a combination of Achyranthes japonica polypeptide and human umbilical cord mesenchymal stem cell exosomes. The results showed that this treatment significantly alleviated PD-related symptoms, including restoring the number of dopaminergic neurons in the substantia nigra, improving tyrosine hydroxylase expression in the midbrain, mitigating motor and olfactory dysfunction in PD model mice, reducing the activation of microglia and astrocytes in the olfactory bulb and midbrain, and improving neuronal activity in the olfactory bulb.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals and relates to the use of a hinataino kozuchi polypeptide and a human umbilical cord mesenchymal stem cell exosome in the preparation of a drug for preventing and / or treating Parkinson's disease.

Background Art

[0002] Parkinson's disease (PD), also known as paralysis agitans, is the second most common neurodegenerative disease that seriously affects the quality of human life after Alzheimer's disease. According to statistics, 1 in 800 people worldwide suffers from Parkinson's disease. By 2030, due to the accelerating aging population, the prevalence of Parkinson's disease is expected to double, and the number of patients is expected to exceed 9 million. Parkinson's disease is characterized by progressive, multiple, and insidious onset, and the main symptoms include bradykinesia, muscle rigidity, resting tremor, and postural instability. The main pathological feature of Parkinson's disease is the decrease of dopamine neurons in the substantia nigra. Dopamine-producing cells in the brain gradually lose their ability to affect the nervous system, and the patient's muscle control ability is limited. Clinically, the treatment of Parkinson's disease mainly focuses on dopamine replacement therapy. Long-term use of this treatment method may cause various side effects such as anxiety, insomnia, hallucinations, and other mental symptoms. Therefore, the search for new therapeutic drugs for PD will bring great economic and social benefits.

[0003] The dried roots of the plant Achyranthes bidentata BI. of the Amaranthaceae family are widely used as traditional Chinese medicines and have physiological effects such as removing blood stasis, improving the flow of meridians, nourishing the liver and kidneys, strengthening tendons and bones, relieving fluid retention, improving lymph flow, and promoting blood flow. Achyranthes bidentata polypeptide is a substance with biological activity isolated from Achyranthes bidentata. Further purification by high-performance liquid chromatography (HPLC) yielded a purified Achyranthes bidentata polypeptide fraction.

[0004] Human umbilical cord mesenchymal stem cells (hUCMCS) are obtained from human umbilical cord tissue and are characterized by their ease of extraction, strong self-renewal capacity, low immunogenicity, and ability to secrete multiple functional factors. Therefore, human umbilical cord mesenchymal stem cells are considered the first-line mesenchymal stem cell for clinical stem cell therapy and are widely used for the repair of various tissues. Exosomes are cystic vesicles of a lipid bilayer, 50-150 nm in diameter, that are produced and expelled from cells through active secretion. Exosomes contain a variety of substances, including lipid molecules, proteins, and nucleic acids (including DNA, RNA, and various non-coding RNAs). Exosomes play a crucial role in intercellular information exchange and substance transport. Currently, there is a growing research finding that stem cell exosomes possess physiological activity similar to that of stem cells.

[0005] The nasal cavity is rich in lymphatic capillaries, and the surface of respiratory cells has numerous fine villi, making it an ideal mucosal drug delivery route. The nasal mucosa has relatively high permeability and relatively few enzymes, resulting in lower degradation of protein-based drugs compared to the gastrointestinal mucosa. Furthermore, with intranasal administration, drugs reach the brain via the bipolar olfactory cells of the nasal olfactory epithelium along the olfactory nerve, thus circumventing the blood-brain barrier against protein-based drugs. Therefore, intranasal administration is a highly effective method of drug delivery that acts on the central nervous system. [Overview of the project] [Problems that the invention aims to solve]

[0006] In response to the shortcomings of the prior art, the present invention provides the use of a combination of Achyranthes japonica polypeptide and human umbilical cord mesenchymal stem cell exosomes in the preparation of drugs for the prevention and / or treatment of Parkinson's disease. It has been found that the combination of Achyranthes japonica polypeptide and human umbilical cord mesenchymal stem cell exosomes can restore the number of dopaminergic neurons in the substantia nigra, improve the expression of tyrosine hydroxylase in the midbrain, alleviate motor and olfactory dysfunction in PD model mice, reduce the activation of microglia and astrocytes in the olfactory bulb and midbrain, and improve the activity of neurons in the olfactory bulb. [Means for solving the problem]

[0007] The technical solutions provided by this invention are as follows:

[0008] This invention provides the use of a combination of Achyranthes japonica polypeptide and human umbilical cord mesenchymal stem cell exosomes in the preparation of drugs for the prevention and / or treatment of Parkinson's disease.

[0009] Furthermore, the aforementioned drug is 1-2 × 10 8 It contains human umbilical cord mesenchymal stem cell exosomes at a concentration of 1 / kg and 50-100 μg / kg of Achyranthes japonica polypeptide.

[0010] Furthermore, the Hinatainokozuchi polypeptide is obtained by chopping dried Hinatainokozuchi roots, then mixing the chopped dried Hinatainokozuchi roots with water and steaming to obtain a mixture, adding ammonium sulfate to the mixture to precipitate it, centrifuging it, and freeze-drying it to obtain a crude extract of Hinatainokozuchi polypeptide. The preparation is carried out by dissolving a crude extract of Achyranthes japonica polypeptide in water to prepare a solution, and then purifying the solution by high-performance liquid chromatography to obtain the Achyranthes japonica polypeptide.

[0011] Furthermore, the steaming process is performed at a temperature of 80-850°C for 40-60 minutes.

[0012] Furthermore, the conditions for the high-performance liquid chromatography are as follows: C18 reversed-phase column, mobile phase: water / acetonitrile containing 0.1% trifluoroacetic acid (volume ratio of water to acetonitrile is 4:1), flow rate: 1.0 mL / min, and ultraviolet detector with a wavelength of 220 nm.

[0013] Furthermore, the human umbilical cord mesenchymal stem cell exosomes are Primary human umbilical cord mesenchymal stem cells are subcultured in serum-containing medium until the density of third-generation human umbilical cord mesenchymal stem cells reaches 80%. Then the medium is replaced with serum-free medium and cultured for 48 hours, after which the supernatant is collected. The supernatant is centrifuged at 400-500g to remove cells and cell debris, and then filtered through a 0.22μm filter. The umbilical cord mesenchymal stem cell exosomes are obtained by treating the filtered supernatant with an exosome isolation kit, separating and purifying it using a specific buffer and a centrifugation step.

[0014] Furthermore, the aforementioned use is, To improve motor function and olfactory perception, Increasing the number of dopaminergic neurons in the brain, To suppress the abnormal activation of microglia and astrocytes in the brain, This includes any of the following: improving the activity of olfactory bulb neurons.

[0015] Furthermore, the drug is administered intranasally.

[0016] Furthermore, the drug is a solution.

[0017] Furthermore, the drug further comprises pharmaceutically acceptable auxiliary materials. [Effects of the Invention]

[0018] In the present invention, mice are treated by combining hinata ino kochi polypeptide and human umbilical cord mesenchymal stem cell exosomes, during which a Parkinson's disease model (PD) is modeled for the mice using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). It has been found that this treatment can significantly alleviate PD-related symptoms, such as restoring the number of dopaminergic neurons in the substantia nigra, improving the expression of tyrosine hydroxylase in the midbrain, alleviating the motor and olfactory function impairments of PD model mice, reducing the activation of microglia and astrocytes in the olfactory bulb and midbrain, and improving the activity of neurons in the olfactory bulb.

[0019] In intranasal administration, since the present invention reaches the brain through bipolar olfactory cells in the nasal olfactory epithelium along the olfactory nerve, it can avoid the barrier effect of the blood-brain barrier on protein drugs.

Brief Description of the Drawings

[0020] [Figure 1] Preparation of hinata ino kochi polypeptide. A: Preparation process, B: High performance liquid chromatography purification, C: Lyophilized hinata ino kochi polypeptide. [Figure 2] Electron micrograph of human umbilical cord mesenchymal stem cell exosomes. The arrow indicates exosomes. Scale bar: 200 nm. [Figure 3] (A) Modeling and drug intervention process of PD model mice. i.p.: Intraperitoneal injection, i.n.: Intranasal administration, (B) Pole climbing test, (C) Olfactory test. ***p < 0.001, compared with the control group; #p < 0.05, ##p < 0.01, p < 0.001, compared with the MPTP group; &p < 0.05, compared with the MPTP + hinata ino kochi polypeptide group; $p < 0.05, compared with the MPTP + exosome group. [Figure 4](A) Immunofluorescence detection shows that treatment with a combination of Achyranthes japonica polypeptide and human umbilical cord mesenchymal stem cell exosomes enhances tyrosine hydroxylase (TH) expression in the substantia nigra of the midbrain in PD model mice. Scale bar: 50 mm. (B) Quantitative analysis of TH-positive cell count in the substantia nigra. ***p<0.001, compared to control group;##p<0.01, ###p<0.001, compared to MPTP group;&&p<0.01, compared to MPTP + Achyranthes japonica polypeptide group;$p<0.05, compared to MPTP + exosome group. [Figure 5] (A) Immunofluorescence detection shows that treatment with a combination of Achyranthes japonica polypeptide and human umbilical cord mesenchymal stem cell exosomes reduces Iba-1 expression in the substantia nigra of the midbrain in PD model mice. Scale bar: 50 mm. (B) Quantitative analysis of Iba-1 fluorescence intensity in the substantia nigra. **p<0.01, compared to control group; #p<0.05, compared to MPTP group; &p<0.05, compared to MPTP + Achyranthes japonica polypeptide group; $p<0.05, compared to MPTP + exosome group. [Figure 6] (A) Immunofluorescence detection shows that treatment with a combination of Achyranthes japonica polypeptide and human umbilical cord mesenchymal stem cell exosomes reduces GFAP expression in the substantia nigra of the midbrain in PD model mice. Scale bar: 50 mm. (B) Quantitative analysis of GFAP fluorescence intensity in the substantia nigra. ***p<0.001, compared to the control group; #p<0.05, ##p<0.01, compared to the MPTP group; &p<0.05, compared to the MPTP + Achyranthes japonica polypeptide group; $p<0.05, compared to the MPTP + exosome group. [Figure 7]Immunofluorescence detection has shown that treatment with the Hinata no Kozuchi polypeptide in combination with human umbilical cord mesenchymal stem cell exosomes improves the expression of Phospho-S6 Ribosomal protein in the lateral and central regions of the olfactory bulb of PD model mice. Red indicates Phospho-S6 Ribosomal protein, and DAPI (blue) indicates the cell nucleus. Scale bar: 25 mm. ***p < 0.001, compared with the control group; #p < 0.05, ##p < 0.01, p < 0.001, compared with the MPTP group; &p < 0.05, compared with the MPTP + Hinata no Kozuchi polypeptide group; $p < 0.05, compared with the MPTP + exosome group. [Figure 8] Immunofluorescence detection has shown that treatment with the Hinata no Kozuchi polypeptide and human umbilical cord mesenchymal stem cell exosomes reduces the expression of Iba-1 (red) in the lateral and central regions of the olfactory bulb of PD model mice. DAPI (blue) indicates the cell nucleus. Scale bar: 25 mm. ***p < 0.001, compared with the control group; #p < 0.05, ##p < 0.01, p < 0.001, compared with the MPTP group; &p < 0.05, compared with the MPTP + Hinata no Kozuchi polypeptide group; $p < 0.05, compared with the MPTP + exosome group. [Figure 9] Immunofluorescence detection has shown that treatment with the combination of the Hinata no Kozuchi polypeptide and human umbilical cord mesenchymal stem cell exosomes reduces the expression of GFAP (red) in the lateral region of the olfactory bulb of PD model mice. DAPI (blue) indicates the cell nucleus. Scale bar: 25 mm. **p < 0.01, compared with the control group; #p < 0.05, ##p < 0.01, compared with the MPTP group; &p < 0.05, compared with the MPTP + Hinata no Kozuchi polypeptide group; $p < 0.05, compared with the MPTP + exosome group.

Mode for Carrying Out the Invention

[0021] Hereinafter, the present invention will be described in more detail with reference to the drawings.

[0022] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods.

[0023] The reagents and materials used in this example are commercially available. All quantitative experiments in the examples were repeated at least three times, and the average result was taken.

[0024] The dried roots of Achyranthes japonica used are purchased from Eiseido Pharmacy.

[0025] The primary human umbilical cord mesenchymal stem cells used will be purchased from Nanjing Taisheng Biotechnology Co., Ltd.

[0026] The C57BL / 6J mice used will be purchased from the Experimental Animal Center of Nantong University.

[0027] The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) used is purchased from Sigma-Aldrich Company under product number M0896.

[0028] Example 1 1. Preparation of Achyranthes japonica polypeptide (Figure 1) Dried roots of Achyranthes japonica were chopped and decocted in a water bath (temperature 80-85°C, time 40-60 min). The aqueous extract was precipitated twice with ammonium sulfate and centrifuged at high speed. The precipitate was freeze-dried to obtain a crude extract of Achyranthes japonica polypeptide. The obtained crude extract of Achyranthes japonica polypeptide was dissolved in water and further purified by high-performance liquid chromatography (HPLC). Chromatography conditions: C18 reversed-phase column, mobile phase: water / acetonitrile containing 0.1% trifluoroacetic acid (4:1, volume ratio), flow rate: 1.0 mL / min, ultraviolet detector (220 nm).

[0029] 2. Preparation of human umbilical cord mesenchymal stem cell exosomes (Figure 2) Human umbilical cord mesenchymal stem cells were cultured in DMEM / F12 (Corning) containing 10% fetal bovine serum in a cell culture incubator at 37°C and 5% CO2 saturated humidity. When the cell density reached 80%, the cells were digested with 0.25% trypsin (Gibco) containing EDTA and subcultured. When the density of third-generation human umbilical cord mesenchymal stem cells reached 80%, the medium was replaced with serum-free medium and cultured for 48 hours. The supernatant was collected and ultracentrifuged at 400g for 10 minutes to remove dead cells and cell debris, and then filtered through a 0.22 μm filter (Millipore). Exosomes were isolated from 15 ml of cell culture supernatant using the exoEasy kit (QIAGEN). The filtered supernatant was transferred to a new test tube, and then an equal volume of buffer XBP was added to the supernatant. The total mixture of 30 ml was then added to an exoEasy spin column and centrifuged at 500g for 1 minute. After discarding the liquid, the spin column was returned to the same collection tube. The above steps were repeated until the supernatant was gone. Then, 10 ml of Buffer XWP was added to the spin column and centrifuged at 5000 g for 5 min to remove any remaining buffer. The spin column was transferred to a new collection tube. 400 μl of Buffer XE was added to the membrane and incubated for 1 min. Centrifuged at 500 g for 5 min and collected the eluate. The eluate was added to the spin column and incubated again for 1 min. Finally, the eluate was collected after centrifuging at 5000 g for 5 min and stored at -80°C until use.

[0030] Example 2 1. Creation and administration of an animal model of Parkinson's disease. PD model mice were induced in 8-week-old C57BL / 6J male mice by intraperitoneal administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP: N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). Prior to treatment, the mice underwent three days of behavioral training, including pole climbing. To avoid the influence of individual differences, mice with nearly equivalent levels of motor ability were selected and divided into a control group and a model group. Next, the model group mice were intraperitoneally injected with MPTP (25 mg / kg / day) for one week, after which their behavior was evaluated (Figure 3A). Mice that showed a significant decrease in behavior compared to the control group mice (injected with the same volume of saline) were considered to be successful PD model mice and were used in subsequent experiments.

[0031] Successful PD model mice were divided into four groups: MPTP group, MPTP + Achyranthes japonica polypeptide group, MPTP + exosome group, and MPTP + Achyranthes japonica polypeptide / exosome group. Mice were treated by intranasal administration. Achyranthes japonica polypeptide and exosomes were dissolved in PBS, and 2 × 10⁶ exosomes were administered. 8 The mice in the MPTP group were treated similarly and administered an equal volume of PBS solution.

[0032] 2. Pole Climbing Test A 1cm diameter, 50cm long pole was constructed with a 1.5cm diameter cork ball attached to its tip, and wrapped in gauze to increase friction. The pole was set up vertically, and mice were placed on the ball at the top of the homemade climbing pole. The time it took for the mouse to start moving, turn its head completely downwards, and return to the bottom of the pole was recorded. This was done a total of three times at 5-minute intervals, and the average value was calculated. Mice were subjected to pole climbing training at the same time every day starting three days before tail vein injection. To reduce experimental error, mice showing significant variability were excluded.

[0033] 3. Olfactory Test Mice were fasted for 24 hours, and small pieces of cheese were placed in the environment during the fasting period to allow the mice to become accustomed to the smell and taste so that they could find it during the experiment. A clean cage (42 cm long, 24 cm wide, 15 cm high) was prepared, and pieces of cheese were placed in five locations on the clean bedding: center, upper left, lower right, upper right, and lower left, buried 1 cm below the surface of the bedding. Mice were placed quietly in the center of the experimental cage, and the time it took each mouse to find the cheese was recorded. If a mouse could not find the cheese within 300 seconds, the time was recorded as 300 seconds. Statistical analysis was performed after discarding the minimum and maximum values.

[0034] 4. Immunofluorescence analysis of the substantia nigra and olfactory bulb The experimental steps were as follows: (1) Mouse brains fixed overnight with 4% paraformaldehyde were dehydrated at 4°C with 20% and then 30% sucrose. After the brains were completely dehydrated and sank to the bottom, they were removed. (2) After embedding the brain mass in OCT, 12 μm thick serial coronal sections of the substantia nigra of the mouse midbrain were prepared using a cryomicrotome. They were dried at 37°C and prepared for use. (3) The slides were placed in 0.01 M PBS and washed three times at room temperature for 10 minutes each. (4) A blocking solution containing 5% BSA and 0.5% Triton X700 was added and the mixture was blocked at room temperature for 1 hour. (5) The blocking solution was aspirated and discarded, and primary antibodies diluted according to the ratio were added dropwise and incubated overnight at 4°C. Commonly used primary antibody ratios: chicken anti-TH (1:500, ab76442, Abcam), rabbit anti-Iba-1 (1:500, ab7260, Abcam), mouse anti-GFAP (1:500, MAB360, Millipore), rabbit anti-pS6 ribosomal protein (1:50, 4858S, Cell Signaling Technology). (6) Washed three times for 10 minutes each with 0.01 M PBS at room temperature. (7) Secondary antibodies diluted with 0.01 M PBS according to the ratio were added dropwise and incubated at room temperature for 90 minutes in the dark. Commonly used secondary antibody ratios: goat anti-rabbit IgG H&L (Alexa Fluor@647) (1:500, A-31573, Invitrogen), goat anti-chicken IgY H&L (Alexa Fluor@555) (1:500, A-21437, Invitrogen), goat anti-mouse IgG H&L (Alexa Fluor@488) (1:500, A-11001, Invitrogen). (8) Washed three times with 0.01M PBS at room temperature, protected from light, for 10 minutes each. (9) Mounted by dropping antifluorescence quenching mounting solution containing DAPI. (10) Observed under a fluorescence microscope and photographed. (11) Fluorescence intensity of the images was measured using ImageJ software.

[0035] 5. Test Results The results showed that the combination of Achyranthes japonica polypeptide and human umbilical cord mesenchymal stem cell exosomes could significantly alleviate motor dysfunction in Parkinson's disease model mice. For example, in the pole climbing test, treated mice had a significantly shorter time to complete the climb compared to mice treated with MPTP (Figure 3B). This treatment also significantly improved olfactory function in Parkinson's disease mice, showing a significant reduction in the time it took the treated group to find hidden food (Figure 3C). The results of the above behavioral tests indicate that the combination therapy has a more pronounced effect compared to treatment with Achyranthes japonica polypeptide or exosomes alone. Histological analysis revealed a significant decrease in tyrosine hydroxylase (TH) expression in the substantia nigra of PD model mice. On the other hand, treatment with the combination of Achyranthes japonica polypeptide and human umbilical cord mesenchymal stem cell exosomes significantly improved TH expression and showed an increase in the number of dopaminergic neurons (Figure 4A, B). Furthermore, this treatment could also inhibit the activation of microglia and astrocytes in the midbrain, resulting in a decrease in the number of Iba-1 and GFAP-positive cells (Figure 5A, B; Figure 6A, B). Histological analysis of the olfactory bulb revealed that treatment with a combination of Achyranthes japonica polypeptide and human umbilical cord mesenchymal stem cell exosomes significantly improved the expression of phosphorylated S6 ribosomal protein (Phospho-S6 Ribosomal Protein), suggesting improved olfactory bulb neuronal activity (Figure 7). In addition, microglia activation in the lateral and central regions of the olfactory bulb was inhibited, resulting in a decrease in Iba-1-positive cells after treatment (Figure 8). Furthermore, astrocyte activation in the lateral region of the olfactory bulb was also inhibited, resulting in a decrease in GFAP-positive cells after treatment (Figure 9). The above morphological experimental results indicate that the combination therapy has a more significant effect than treatment with Achyranthes japonica polypeptide or exosomes alone.

[0036] The above embodiments have illustrated the present invention in detail, but the invention is not limited to the above embodiments. Those skilled in the art can make other modifications and improvements based on the teachings of the present invention, all of which fall within the scope of protection of the present invention.

Claims

1. Use of a combination of Achyranthes japonica polypeptide and human umbilical cord mesenchymal stem cell exosomes in the preparation of drugs for the prevention and / or treatment of Parkinson's disease.

2. The aforementioned drug is 1 to 2 x 10 ８ The use according to claim 1, characterized by comprising human umbilical cord mesenchymal stem cell exosomes at a concentration of 1 / kg and 50-100 μg / kg of Achyranthes japonica polypeptide.

3. The aforementioned Achyranthes japonica polypeptide is obtained by chopping dried Achyranthes japonica roots, then mixing the chopped dried roots with water and steaming to obtain a mixture, adding ammonium sulfate to the mixture to precipitate it, centrifuging it, and freeze-drying it to obtain a crude extract of Achyranthes japonica polypeptide. The use according to claim 1 or 2, characterized in that it is prepared by dissolving a crude extract of Achyranthes japonica polypeptide in water to prepare a solution, and then purifying the solution by high-performance liquid chromatography to obtain the Achyranthes japonica polypeptide.

4. The use according to claim 3, characterized in that the steaming is performed at a temperature of 80 to 850°C for a duration of 40 to 60 minutes.

5. The use according to claim 3, characterized in that the conditions for the high-performance liquid chromatography are a C18 reversed-phase column, mobile phase: water / acetonitrile containing 0.1% trifluoroacetic acid (volume ratio of water to acetonitrile is 4:1), flow rate: 1.0 mL / min, and ultraviolet detector with a wavelength of 220 nm.

6. The aforementioned human umbilical cord mesenchymal stem cell exosomes are Primary human umbilical cord mesenchymal stem cells are subcultured in serum-containing medium until the density of third-generation human umbilical cord mesenchymal stem cells reaches 80%. Then the medium is replaced with serum-free medium and cultured for 48 hours, after which the supernatant is collected. The supernatant is centrifuged at 400-500 g to remove cells and cell debris, and then filtered through a 0.22 μm filter. The use according to claim 1 or 2, characterized in that it is produced by a method of treating the filtered supernatant with an exosome isolation kit, separating and purifying it with a specific buffer and centrifugation step to obtain the umbilical cord mesenchymal stem cell exosomes.

7. The aforementioned use is, To improve motor function and olfactory perception, Increasing the number of dopaminergic neurons in the brain, To suppress the abnormal activation of microglia and astrocytes in the brain, The use according to claim 1, characterized by comprising any of the following: improving the activity of olfactory bulb neurons.

8. The use according to claim 1, characterized in that the method of administering the drug is intranasal administration.

9. The use according to claim 1, characterized in that the drug is a solution.

10. The use according to claim 1, characterized in that the drug further comprises pharmaceutically acceptable auxiliary materials.

Images