A method for in vitro expansion and induced activation of iNKT cells

Abstract

The application provides a method for in-vitro expansion and induced activation of iNKT cells, and belongs to the technical field of immune cell preparations. The in-vitro expansion method comprises the following steps: S1, separating peripheral blood PBMCs cells; S2, adding a part of the PBMCs cells into culture medium I for stimulation culture, irradiation, and separation to obtain the irradiated PBMCs cells; and S3, resuspending another part of the PBMCs cells in culture medium II, adding alpha-GalCer and IL-2 for stimulation culture, then adding the irradiated PBMCs cells to expand the iNKT cells. The induced activation method comprises the following steps: after the iNKT cells are expanded, adding culture medium III and a dasatinib derivative to induce maturation of the iNKT cells. The method can quickly promote expansion and induced activation of the iNKT cells, has high expansion efficiency, and the obtained iNKT cells have high cytotoxicity; in addition, the method is safe and does not cause a graft-versus-host reaction.

Description

Technical Field

[0001] This invention relates to the field of immune cell preparation technology, specifically to a method for in vitro expansion and induction activation of iNKT cells. Background Technology

[0002] Natural killer T cells (NKT) are a new and special subset of T lymphocytes that branch out early in the development of T lymphocytes. Unlike traditional T cells, NKT cells are special T cells with innate immune response functions. They have both NK cell functions and T cell characteristics, and can express both NK cell surface markers and T cell receptors (TCRs). Based on the type of TCRs expressed and whether antigen recognition depends on CD1d molecules, NKT cells are generally classified into three types: Type I NKT: CD1d restricted, expressing one constant TCR-α chain (human Vα24) and one restricted TCR-β chain (human Vβ11), hence the name invariant NKT (iNKT), also known as classical NKT; Type II NKT: CD1d restricted, but expressing non-constant TCR-αβ chains, also known as diverse NKT (dNKT), or non-classical NKT, expressing multiple different TCR-αβ chains. In anti-tumor immunity, their effects antagonize those of Type I NKT, acting as a suppressor of immune surveillance cells, and can exert an inhibitory effect on tumor immunity by secreting IL-13; Type III NKT: non-CD1d restricted NKT, also known as NKT-like cells, expressing different types of TCRs and NK markers, the origin and function of which are still unclear.

[0003] Invariant natural killer T (iNKT) cells are a subset of T lymphocytes with a restricted T cell antigen receptor (TCR) spectrum and recognize glycolipid antigens presented by CD1d molecules. Upon antigen stimulation, iNKT cells respond rapidly and secrete a large number of inflammatory cytokines, including interferon-γ (IFN-γ) and interleukin-4 (IL-4). This early response influences the outcome of downstream immune responses, thus iNKT cells possess regulatory properties. Due to the diversity of their effector functions, iNKT cells participate in many pathological processes and play an important role in host defense against infection, prevention of autoimmune diseases, and prevention of cancer.

[0004] Currently, iNKT cells amplified in vitro exhibit low bioactivity and are difficult to successfully achieve. Chinese invention patent document CN116716246A discloses a method and application for the in vitro amplification and induced activation of umbilical cord blood iNKT cells. The method includes the following steps: Amplification stage: Umbilical cord blood containing iNKT cells is obtained and the cell pellet is resuspended in a cell culture flask. 1640 basal medium, IL-2, IL-15, KDM2B, and 10 vol% of the umbilical cord blood are added to the resuspended cell pellet to amplify iNKT cells. Induction and activation stage: After amplification of iNKT cells, X-VIVO 10 medium, ConA, and α-GalCer are added to induce iNKT cell activation and maturation. This invention separately cultures, amplifies, and induces activation of iNKT cells, greatly improving the amplification efficiency and bioactivity of iNKT cells. However, the overall amplification fold of NK cells amplified in vitro using this combination of cytokines (IL-2, IL-15) is still not ideal.

[0005] Therefore, developing an efficient method for in vitro expansion and induced activation of iNKT cells has promising application prospects. Summary of the Invention

[0006] The purpose of this invention is to provide a method for in vitro expansion and activation of iNKT cells, which can rapidly promote the expansion and activation of iNKT cells, with high expansion efficiency, and the obtained iNKT cells have high cytotoxicity. The method is safe and does not cause graft-versus-host disease.

[0007] The technical solution of this invention is implemented as follows: This invention provides a method for in vitro expansion and induced activation of iNKT cells, comprising: In vitro amplification methods: S1. Isolate peripheral blood PBMCs; S2. A portion of PBMCs were added to medium I for stimulation culture, irradiated, and the irradiated PBMCs were isolated. S3. Resuspend another portion of PBMCs in culture medium II, add α-GalCer and IL-2 to stimulate culture, and then add irradiated PBMCs to expand iNKT cells.

[0008] This invention uses autologous irradiated PBMCs as feeder cells. These cells do not divide or proliferate, but still maintain metabolic activity. The proliferation of iNKT cells is promoted by expressing ligands or secreting cytokines on their surface. Simultaneously, the invention exhibits high safety and does not induce graft-versus-host disease. Stimulation with autologous irradiated PBMCs significantly increases the iNKT cell proliferation rate and demonstrates good cytotoxic activity. Furthermore, α-GalCer and IL-2 effectively promote iNKT cell proliferation.

[0009] Unlike existing technologies, this invention first induces PBMCs in SCGM culture medium containing vitamin C, lipopolysaccharide, and AB-type serum. This not only enhances cell resistance but also strongly activates monocytes / macrophages in PBMCs, inducing them to produce high yields of cytokines such as IL-12, IL-15, and IL-18. Subsequently, these cells are irradiated to prevent them from dividing and proliferating. This allows for the rapid production of large amounts of cytokines beneficial to the rapid proliferation of iNKT cells in the culture system, and it is safer and does not affect the activity of iNKT cells.

[0010] Induction and activation method: After expanding iNKT cells, add culture medium III and dasatinib derivative to induce iNKT cell maturation; The structural formula of the dasatinib derivative is shown in Formula I: Formula I.

[0011] As a further improvement of the present invention, the culture medium I is SCGM culture medium containing 10-20 mg / L vitamin C, 20-40 ng / mL lipopolysaccharide, and 2-10 wt% AB type serum; the culture medium II is SCGM culture medium containing 2-10 wt% AB type serum; and the culture medium III is X-VIVO 10 culture medium.

[0012] As a further improvement of the present invention, the separation method described in step S1 is the Ficoll density gradient centrifugation method.

[0013] As a further improvement of the present invention, the seeding amount of PBMCs cells in culture medium I in step S2 is 1-3 × 10⁻⁶. 6 The irradiation dose is 30-70 Gy, the dose rate is 2.5 Gy / min, and the stimulation culture conditions are 36-38℃, CO2 concentration is 5%, and the time is 3-5 days.

[0014] As a further improvement of the present invention, the inoculum size of PBMCs in culture medium II in step S3 is 1-3 × 10⁻⁶. 6The concentrations of PBMCs after adding α-GalCer and IL-2 were 50-100 ng / mL and 100-200 ng / mL, respectively, and the seeding density of irradiated PBMCs was 5-8 × 10⁻⁸ cells / mL. 5 The stimulation culture conditions are 36-38℃, CO2 concentration of 5%, and time of 1-2 days. The amplification conditions are 36-38℃, CO2 concentration of 5%, and time of 10-12 days.

[0015] As a further improvement of the present invention, in the induced activation method, the concentration of the dasatinib derivative in the system is 10-20 nmol / L, and the activation maturation time is 48-72 h.

[0016] As a further improvement of the present invention, the preparation method of the dasatinib derivative is as follows: T1. A monosubstituted diethylene glycol is prepared by reacting diethylene glycol with p-toluenesulfonyl chloride. T2. Dasatinib was reacted with a monosubstituted diethylene glycol to prepare an intermediate having the structure shown in Formula II: Formula II; T3. React the intermediate with citric acid to obtain the product.

[0017] Dasatinib rapidly mediates NK cell mobilization and significantly increases the number of large granular lymphocytes, primarily NK cells. Therefore, it has a good effect on expanding iNKT cells and enhancing cytotoxicity. However, dasatinib has poor water solubility and some cytotoxicity. Therefore, it is modified by introducing an ethylene glycol structure to increase water solubility, reduce toxicity, decrease enzymatic degradation, and improve bioavailability. Simultaneously, by reacting with citric acid, the content of single-molecule dasatinib can be increased, thereby better exerting its efficacy, promoting further expansion and activation of iNKT cells, and activating them into more cytotoxic iNKT cells, thus exerting a more effective anti-tumor effect. This immunotherapy not only improves the anti-tumor effect but also has less impact on the patient, fewer side effects, and is safer.

[0018] As a further improvement of the present invention, the molar ratio of diethylene glycol and p-toluenesulfonyl chloride in step T1 is 1-1.2:1; the molar ratio of dasatinib and monosubstituted diethylene glycol in step T2 is 1:1-1.5; and the molar ratio of intermediate and citric acid in step T3 is 3-3.2:1.

[0019] The present invention further protects an expanded and induced activated iNKT cell obtained by the above method.

[0020] This invention further protects the use of the above-mentioned expanded and induced activated iNKT cells in the preparation of antitumor drug formulations.

[0021] The present invention has the following beneficial effects: 1. This invention modifies dasatinib to obtain a dasatinib derivative with improved water solubility and a high content of single-molecule dasatinib, which enhances the amplification and induction of iNKT cells and significantly improves the cytotoxicity of the obtained iNKT cells, thus exerting a more effective anti-tumor effect.

[0022] 2. This invention uses vitamin C and lipopolysaccharide to induce PBMCs, which improves the cells' resistance and strongly activates monocytes / macrophages in PBMCs, inducing them to produce high-yield cytokines such as IL-12, IL-15, and IL-18. Feeder cells obtained after irradiation can rapidly produce a large number of cytokines that are beneficial to the rapid proliferation of iNKT cells, rapidly and efficiently promoting the expansion of iNKT cells, and has high safety, without causing graft-versus-host disease. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The mass spectrum of the dasatinib derivative in the preparation example; Figure 2 This is a comparison diagram of the cytotoxic activities of each group in test example 2. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Preparation of dasatinib derivatives The synthesis route is as follows: The method is as follows: T1. Add 0.1 mol p-toluenesulfonyl chloride and 200 mL pyridine to a three-necked flask, stir well, and add 100 mL of pyridine solution containing 0.11 mol diethylene glycol dropwise in an ice-water bath. Heat to room temperature and stir for 2 hours. Add 200 mL of cold water and 50 mL of concentrated hydrochloric acid to the reaction mixture, and stir slowly for half an hour. Transfer the reaction mixture to a separatory funnel and extract twice with 200 mL of ethyl acetate. Combine the organic layers, wash with water until neutral, and dry with anhydrous sodium sulfate. Filter and concentrate to remove the solvent to obtain monosubstituted diethylene glycol, which can be used directly in the next reaction. T2. Add 0.1 mol dasatinib, 0.12 mol potassium carbonate, and 200 mL acetonitrile to a three-necked flask, then add 100 mL of acetonitrile solution containing 0.12 mol monosubstituted diethylene glycol. Reflux overnight, filter, remove solvent under reduced pressure, and separate by silica gel column chromatography (eluting with dichloromethane:methanol = 1:1) to obtain the intermediate; ESI-MS calculated value: C 26 H 35 ClN7O4S(M+H) + 577.11, measured value: 577.1, yield: 74%. NMR results: 1 H NMR (300MHz, CDCl3) δ 8.21 (s, 1H), 7.12 (d, J =7.2Hz, 1H), 6.95 (d, J =7.0Hz, 1H), 6.78 (m, 1H), 5.27 (s, 1H), 3.72 (t, 2H), 3.49-3.55 (m, 8H), 3.17 (m, 4H), 2.53-2.59 (m, 6H), 2.33-2.37 (m, 6H).

[0027] T3. Add 0.31 mol of the intermediate and 0.1 mol of citric acid to 200 mL of N,N-dimethylformamide, then add 0.05 mol of p-toluenesulfonic acid. Under nitrogen protection, heat to 120 °C and stir for 7 h. Wash the product with dichloromethane and methanol, centrifuge, and obtain the precipitate, which is the product. ESI-MS calculated value: C 84 H 105 Cl3N 21 O 16 S3(M+H) + 1867.41, measured value: 1867.4, yield: 52%. NMR results: 1H NMR(300MHz, CDCl3)δ 8.17 (s, 3H), 6.92-7.04 (m, 6H), 6.74 (m, 3H), 5.22 (s, 3H), 4.27 (t, 6H), 3.62 (t, 6H), 3 .44-3.50 (m, 18H), 3.12 (m, 12H), 2.71 (s, 4H), 2.51-2.54 (m, 18H), 2.33-2.36 (m, 18H).

[0028] Example 1 This embodiment provides a method for in vitro expansion and induced activation of iNKT cells, including: In vitro amplification methods: S1. Ficoll density gradient centrifugation was used to isolate peripheral blood PBMCs. S2. A portion of PBMCs were added to SCGM medium containing 10 mg / L vitamin C, 20 ng / mL lipopolysaccharide, and 10 wt% AB serum. The seeding density of PBMCs was 1 × 10⁻⁶ cells / mL. 6 Cells were cultured at 36°C and 5% CO2 for 5 days, then irradiated at a dose of 30 Gy and a dose rate of 2.5 Gy / min to obtain irradiated PBMCs. S3. Resuspend another portion of PBMCs in SCGM medium containing 10 wt% AB serum. The seeding density of PBMCs was 2 × 10⁶ cells / year. 6 Cells / mL were added to the system, and α-GalCer and IL-2 were added to achieve concentrations of 50 ng / mL and 100 ng / mL, respectively. The system was incubated at 36°C with a CO2 concentration of 5% for 2 days. Then, irradiated PBMCs were added, with a seeding density of 5 × 10⁶ cells / mL. 5 amplification was performed at 36℃ with a CO2 concentration of 5% for 12 days. Induction and activation method: After the above amplification system, discard all the supernatant of the cell culture flask, add X-VIVO 10 medium, add the dasatinib derivative prepared in Example 1 to the system to a concentration of 10 nmol / L, and induce iNKT cells to mature for 56 h under the original conditions.

[0029] Example 2 This embodiment provides a method for in vitro expansion and induced activation of iNKT cells, including: In vitro amplification methods: S1. Ficoll density gradient centrifugation was used to isolate peripheral blood PBMCs. S2. A portion of PBMCs were added to SCGM medium containing 20 mg / L vitamin C, 40 ng / mL lipopolysaccharide, and 2 wt% AB serum. The seeding density of PBMCs was 3 × 10⁻⁶ cells / mL. 6 Cells were cultured at 38°C and CO2 concentration of 5% for 3 days, then irradiated at a dose of 70 Gy and a dose rate of 2.5 Gy / min to obtain irradiated PBMCs. S3. Resuspend another portion of PBMCs in SCGM medium containing 2 wt% AB serum. The seeding density of PBMCs was 2 × 10⁶ cells / year. 6 Cells / mL were added to the system, and α-GalCer and IL-2 were added to achieve concentrations of 100 ng / mL and 200 ng / mL, respectively. The system was incubated at 38°C with a CO2 concentration of 5% for 2 days. Then, irradiated PBMCs were added, with a seeding density of 8 × 10⁶ cells / mL. 5 amplification was performed at 38℃ with a CO2 concentration of 5% for 12 days. Induction and activation method: After the above amplification system, discard all the supernatant of the cell culture flask, add X-VIVO 10 medium, add the dasatinib derivative prepared in Example 1 to the system to a concentration of 20 nmol / L, and induce iNKT cells to mature for 56 h under the original conditions.

[0030] Example 3 This embodiment provides a method for in vitro expansion and induced activation of iNKT cells, including: In vitro amplification methods: S1. Ficoll density gradient centrifugation was used to isolate peripheral blood PBMCs. S2. A portion of PBMCs were added to SCGM medium containing 15 mg / L vitamin C, 30 ng / mL lipopolysaccharide, and 5 wt% AB serum. The seeding density of PBMCs was 2 × 10⁻⁶ cells / mL. 6 Cells were cultured at 37°C and 5% CO2 for 4 days, then irradiated at a dose of 50 Gy and a dose rate of 2.5 Gy / min. The irradiated PBMCs were then isolated. S3. Resuspend another portion of PBMCs in SCGM medium containing 5 wt% AB serum. The seeding density of PBMCs was 2 × 10⁶ cells / year. 6 Cells / mL were added to the system, and α-GalCer and IL-2 were added to achieve concentrations of 70 ng / mL and 150 ng / mL, respectively. The system was cultured at 37°C with a CO2 concentration of 5% for 2 days. Then, irradiated PBMCs were added, with a seeding density of 6 × 10⁶ cells / mL.5 amplification was performed at 37℃ with a CO2 concentration of 5% for 12 days. Induction and activation method: After the above amplification system, discard all the supernatant of the cell culture flask, add X-VIVO 10 medium, add the dasatinib derivative prepared in Example 1 to the system to a concentration of 15 nmol / L, and induce iNKT cells to mature for 56 h under the original conditions.

[0031] Comparative Example 1 The difference from Example 3 is that the dasatinib derivative was replaced with an equal concentration of dasatinib.

[0032] Comparative Example 2 The difference from Example 3 is that no stimulation culture was performed in step S2; instead, direct irradiation was performed.

[0033] Specifically as follows: A portion of PBMCs were added to SCGM medium containing 5 wt% AB serum, with a PBMCs seeding density of 2 × 10⁶ cells / mL. 6 PBMCs were cultured at 37°C and 5% CO2 for 4 days, then irradiated at a dose of 50 Gy and a dose rate of 2.5 Gy / min.

[0034] Comparative Example 3 The difference from Example 3 is that the dasatinib derivatives are replaced by ConA and a-GalCer.

[0035] Specifically as follows: Induction and activation method: After the above amplification system, discard all the supernatant of the cell culture flask, add X-VIVO 10 medium, add ConA and α-GalCer, where ConA is added to a concentration of 10 ng / mL and α-GalCer is added to a concentration of 70 ng / mL, and induce iNKT cells to mature for 56 h under the original conditions.

[0036] Test Example 1 The cell suspensions obtained on day 0 of the methods in Examples 1-3 and Comparative Examples 1-3, after the amplification in step S3 (day 14), were used in the experiments.

[0037] After blocking 100 μL of cell suspension with anti-CD16 / 32 antibody at 4°C for 15 min, add AlexFlour488-labeled anti-CD3e antibody, APC-labeled anti-CD1d antibody and PE-labeled anti-CD69 antibody. After staining at 4°C in the dark for 40 min, wash away unbound antibodies with flow cytometry staining buffer and resuspend the cells.

[0038] In addition, take 20 μL of cell suspension from each cell, mix it with 20 μL of trypan blue solution at a 1:1 ratio, and add it to a cell counting plate.

[0039] Cells were collected and analyzed using flow cytometry. First, lymphocytes were selected using FSC and SSC. Then, CD3e cells were analyzed. + T lymphocyte populations were gated, and finally CD1d-PBS-57 Tetramer was used. + and CD69 + IFN-γ + IL-4 + IL-17 + IL-10 + IL-2 + Gating and analysis were performed, with 50,000 cells collected for each detection indicator.

[0040] Calculation of the absolute number of iNKT cells: The absolute number of iNKT cells = the concentration of live cells in the culture system (cells / mL) × total volume × percentage of iNKT cells.

[0041] The expansion fold was calculated based on the absolute number of iNKT cells, and the results are shown in Table 1.

[0042] Table 1

[0043] As can be seen from the table above, the methods in Examples 1-3 of this invention can rapidly promote the proliferation of iNKT cells.

[0044] Test Example 2 K562 and HO8910 cells in logarithmic growth phase were collected as target cells, and the cell number was adjusted to 1×10⁻⁶. 5 Cells cultured on days 0 and 21 were used as effector cells. Effector cells were mixed with target cells (induced mature iNKT cells prepared in Examples 1-3 and Comparative Examples 1-3, or commercially available iNKT cells, purchased from Beijing GeneQiMing Technology Co., Ltd.) at an effector / target ratio of 10:1. Corresponding target cell wells, effector cell wells, and culture medium blank control wells were prepared, with three replicates per group. The cells were incubated at 37°C in a 5% CO2 incubator. After 12 hours, 20 μL of MTT (5 mg / mL) was added to each well, and incubation continued for 4 hours. After centrifugation at 2000 rpm for 15 minutes, the supernatant was washed away, and 150 μL of dimethyl sulfoxide was added to each well. The mixture was shaken for 10 minutes, and the OD value at 570 nm was measured using a microplate reader. The average value of the three replicates was taken. The results are shown in Table 2 and [Table data would be inserted here]. Figure 2 .

[0045] Methods for calculating cytotoxic activity: Kill rate (%) = [1 - (OD value of test wells - OD value of effector cell wells) / OD value of target cell wells] × 100% Table 2

[0046] Note: * indicates P<0.05; ** indicates P<0.01 compared with the commercially available iNKT cell group.

[0047] As shown in the table above, the induced mature iNKT cells obtained in Examples 1-3 of this invention have high cell-killing activity.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for in vitro expansion and induced activation of iNKT cells, characterized in that, include: In vitro amplification methods: S1. Isolate peripheral blood PBMCs; S2. A portion of PBMCs were added to medium I for stimulation culture, irradiated, and the irradiated PBMCs were isolated. The culture medium I is SCGM medium containing 10-20 mg / L vitamin C, 20-40 ng / mL lipopolysaccharide, and 2-10 wt% AB type serum; the inoculum size of PBMCs in culture medium I is 1-3 × 10⁻⁶ cells / mL. 6 The irradiation dose is 30-70 Gy, the dose rate is 2.5 Gy / min, and the stimulation culture conditions are 36-38℃, CO2 concentration is 5%, and the time is 3-5 days. S3. Resuspend another portion of PBMCs in culture medium II, add α-GalCer and IL-2 to stimulate culture, and then add irradiated PBMCs to expand iNKT cells; Induction and activation method: After expanding iNKT cells, add culture medium III, add dasatinib derivative, and induce iNKT cell maturation; in the induction and activation method, the concentration of dasatinib derivative in the system is 10-20 nmol / L, and the maturation time is 48-72 h; The structural formula of the dasatinib derivative is shown in Formula I: Formula I; The preparation method of the dasatinib derivative is as follows: T1. A monosubstituted diethylene glycol is prepared by reacting diethylene glycol with p-toluenesulfonyl chloride. T2. Dasatinib was reacted with a monosubstituted diethylene glycol to prepare an intermediate having the structure shown in Formula II: Formula II; T3. The intermediate is reacted with citric acid to obtain the product; Culture medium II is SCGM medium containing 2-10 wt% AB type serum; culture medium III is X-VIVO 10 medium.

2. The method for in vitro expansion and induced activation of iNKT cells according to claim 1, characterized in that, The separation method described in step S1 is Ficoll density gradient centrifugation.

3. The method for in vitro expansion and induced activation of iNKT cells according to claim 1, characterized in that, The seeding density of PBMCs in culture medium II in step S3 is 1-3 × 10⁻⁶. 6 The concentrations of PBMCs after adding α-GalCer and IL-2 were 50-100 ng / mL and 100-200 ng / mL, respectively, and the seeding density of irradiated PBMCs was 5-8 × 10⁻⁸ cells / mL. 5 The stimulation culture conditions are 36-38℃, CO2 concentration of 5%, and time of 1-2 days. The amplification conditions are 36-38℃, CO2 concentration of 5%, and time of 10-12 days.

4. The method for in vitro expansion and induced activation of iNKT cells according to claim 1, characterized in that, The molar ratio of diethylene glycol to p-toluenesulfonyl chloride in step T1 is 1-1.2:1; the molar ratio of dasatinib to monosubstituted diethylene glycol in step T2 is 1:1-1.5; and the molar ratio of the intermediate to citric acid in step T3 is 3-3.2:

1.

5. An expanded and induced activated iNKT cell prepared by the method of any one of claims 1-4.

6. The use of the expanded and induced activated iNKT cells as described in claim 5 in the preparation of antitumor drug formulations.

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