In vitro drug testing container

Abstract

The present disclosure relates to a container for in vitro drug testing. The container includes a receptacle having a wall made from a gel composition. The receptacle has an opening and is configured to receive a drug formulation therethrough.

Description

[Technical Field]

[0001] The present disclosure relates broadly, but not exclusively, to containers for in vitro drug testing, methods for preparing the containers, testing devices including the containers, and testing methods. [Background technology]

[0002] Extravascular drug administration (e.g., intramuscular or subcutaneous) is a rapid and patient-friendly method for delivering pharmaceutical substances to the human body, requiring minimal oversight from medical professionals. For many formulations administered using these routes, bioavailability is highly dependent on several factors, such as their diffusion behavior. Therefore, during the development process, it is important to test them to determine their performance. While animal testing remains the ultimate means for comparing formulations, in vitro testing offers a more rapid and cost-effective alternative.

[0003] In vitro performance assays are an evaluation technique for obtaining important information about the bioavailability and other preclinically relevant properties of pharmaceutical substances and dosage forms by using a controlled in vitro environment rather than a living organism. This approach has the potential to reduce the number of costly and time-consuming animal and human clinical trials. However, in vitro performance assays designed for extravascular dosage forms (including those for subcutaneous or intramuscular administration) face several challenges due to a lack of established standards. Furthermore, significant deficiencies in emphasizing feasibility and quality control testing have hindered the development of reliable methods for assessing the bioavailability of pharmaceutical substances.

[0004] One such in vitro performance assay involves using a dispersion releaser (DR) that utilizes a biorelevant medium to mimic the interstitial fluid in subcutaneous tissue and a dialysis-based setup to analyze drug release. However, this method cannot mimic certain aspects of the physiological environment that can affect the performance of pharmaceutical substances and dosage forms. These factors may include biorelevant diffusion, tissue retention, and fluid dynamics. This can affect the reliability and predictive power of the test results.

[0005] Another in vitro performance assay involves using an instrument called the Subcutaneous Injection Site Simulator N3 (SCISSOR N3). The SCISSOR N3 instrument utilizes an injection cartridge filled with hyaluronic acid (HA) to act as the donor medium. This cartridge is separated from the surrounding acceptor medium by a membrane. This setup attempts to mimic the microenvironment of the extracellular matrix, which provides viscous HA for diffusion resistance. However, this system does not comply with international standards important for pharmaceutical regulation. Such technical standards are provided, for example, by national pharmacopoeias, which provide a harmonized structure for pharmaceutical testing. Furthermore, this instrument is rather expensive for such highly specialized equipment and does not allow the in vitro environment to be flexibly modified (e.g., by changing the flow rate or agitation speed).

[0006] Other in vitro performance assays have also utilized laboratory devices or tools, such as shake flasks and continuous flow-through cells, or materials, such as hydrogels, to mimic conditions within tissues. However, despite the exciting alternatives and opportunities that subcutaneous formulations offer for existing and future pharmaceutical substances, suitable in vitro models that can reliably and rapidly predict the bioavailability of these substances in the human body within a well-structured technical framework are lacking. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, there is a need to provide a device that attempts to address the above problems or provide a useful alternative. [Means for solving the problem]

[0008] According to a first aspect of the present invention, there is provided a container for in vitro drug testing, comprising: A receptacle having a wall, the receptacle having an opening and configured to receive a drug formulation therethrough, the wall being made from a gel composition. A container is provided comprising:

[0009] The concentration of the gel composition can be selected to simulate one or more properties of tissue.

[0010] The gel composition may include one or more polysaccharides selected from the group consisting of agarose and hyaluronic acid.

[0011] The gel composition may contain 0 to 5% by weight of agarose gel.

[0012] The gel composition may contain 0-3% by weight of hyaluronic acid.

[0013] The gel composition may comprise one or more polypeptides selected from the group consisting of collagen, gelatin and peptone.

[0014] The gel composition may contain 0 to 1% collagen by weight.

[0015] The gel composition may contain 0 to 5% by weight of gelatin.

[0016] The gel composition may include one or more lipids selected from triglycerides and phospholipids.

[0017] The gel composition may include ions.

[0018] The gel composition may include vesicles.

[0019] The gel composition may include serum proteins.

[0020] The container may further include a plug for sealing the opening of the receptacle.

[0021] The container may further include a ring surrounding the periphery of the opening of the receptacle for injection of the drug formulation into the receptacle.

[0022] The ring may be made of a chemically inert material.

[0023] The chemically inert material may include a polymer or a metal.

[0024] The ring may include a collar surrounding its inner wall adjacent the base of the ring to create a recess for receiving around the opening of the receptacle.

[0025] The ring may include a textured region surrounding its inner wall adjacent the top of the ring.

[0026] According to a second aspect of the present invention, there is provided a method for preparing a container for in vitro drug testing, comprising the steps of: forming a container having a wall, the wall being made from a gel composition; and placing the drug formulation in the receptacle.

[0027] Forming a container having a wall includes: attaching the ring to a mold; pouring the gel composition into a mold; inserting an inner punch into the gel composition to form a receptacle such that the ring surrounds the periphery of the opening of the receptacle; may include:

[0028] According to a third aspect of the present invention there is provided a testing device comprising a container as defined in the first aspect.

[0029] According to a fourth aspect of the present invention, there is provided a testing method comprising the steps of: placing a drug formulation in a container as defined in the first aspect; immersing the container in a medium within a United States Pharmacopeia (USP) IV flow-through cell; monitoring the diffusion of the drug formulation between the container and the medium; A test method is provided, comprising: [Brief explanation of the drawings]

[0030] Embodiments of the present invention are provided by way of example only and will be better understood and readily apparent to those skilled in the art from the following written description and drawings.

[0031] [Figure 1A] FIG. 1A is a schematic diagram illustrating a container for in vitro drug testing, according to an exemplary embodiment. [Figure 1B] FIG. 1B illustrates a photograph of the container shown in FIG. 1A. [Figure 1C] FIG. 1C illustrates a top view (left) and a bottom view (right) of the ring shown in FIGS. 1A and 1B. [Figure 2] FIG. 2 illustrates a schematic diagram showing a modified flow-through cell including a vessel according to another exemplary embodiment. [Figure 3] Figure 3 shows two flow charts, each illustrating the breakdown of the diffusion experiments performed on individual versions of the hydrogel-based diffusion container. [Figure 4A] FIG. 4A illustrates the hydrogel container (left) and modified flow-through cell (right) used as a proof-of-concept model for the diffusion experiments shown in the first flowchart of FIG. [Figure 4B] FIG. 4B illustrates the assay setup of the proof-of-concept model of FIG. 4A for in vitro drug testing. [Figure 5]FIG. 5 illustrates the assay setup of the hydrogel container in the USP Apparatus IV for the diffusion experiment shown in the second flow chart of FIG. [Figure 6A] FIG. 6A illustrates a line graph showing the diffusion profiles of Actrapid® and Apidra® in the experiment described with reference to FIGS. 4A and 4B. [Figure 6B] FIG. 6B illustrates a line graph showing the results of the reference experiment described with reference to FIGS. 4A and 4B. [Figure 7A] FIG. 7A illustrates a line graph showing the diffusion profile of the caffeine stock solution in the experiment described with reference to FIG. [Figure 7B] FIG. 7B illustrates a line graph showing the diffusion profiles of Actrapid® and Apidra® in PBS solution in the experiment described with reference to FIG. [Figure 7C] FIG. 7C illustrates a line graph showing the diffusion profiles of Actrapid® and Apidra® in SIB solution in the experiment described with reference to FIG. [Figure 7D] FIG. 7D illustrates a line graph showing the results of the reference experiment described with reference to FIG. [Figure 7E] FIG. 7E illustrates a line graph showing the diffusion profiles of Insulatard® and heparin-treated Insulatard® in PBS solution in the experiment described with reference to FIG. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention relates to a container that functions as a release and diffusion adapter for in vitro testing of drug formulations intended for subcutaneous administration. The container includes a receptacle with an opening and a plug for sealing the receptacle opening. The container may also include a ring surrounding the receptacle opening to function as an injection port for administering the drug formulation to the receptacle. During use, the container is incorporated into the flow-through cell of a United States Pharmacopeia (USP) apparatus IV.

[0033] FIG. 1A illustrates a schematic diagram showing a container 100 for in vitro drug testing according to an exemplary embodiment. FIG. 1B illustrates a photograph of the container 100 shown in FIG. 1A. The dimensions of the container 100 are approximately 40 mm in height and 19 mm in width. The container 100 includes a receptacle 102 having an elongated cylindrical body with a consistent radius that forms an interior pocket 104 for holding a drug formulation 106. The receptacle 102 has a bottom wall 108 as its base and a side wall 110 that surrounds the bottom wall 108 and extends from the bottom wall 108 to an opening 112 at the top of the side wall 110. These walls 108, 110 are approximately 2.5 mm thick and function as a barrier between the drug formulation 106 and the medium within the flow-through cell.

[0034] The bottom wall 108 and side wall 110 of the receptacle 102 are made of a gel composition whose concentration is selected to simulate one or more properties of tissue. The three-dimensional porous structure of the walls 108, 110 simulates the properties of subcutaneous tissue, acting as a diffusion resistance and membrane. In one embodiment, the gel composition includes one or more polysaccharides, such as agarose and hyaluronic acid. For example, the gel composition may contain an agarose gel concentration of 0-5% by weight and / or a hyaluronic acid concentration of 0-3% by weight. For use with the USP Apparatus IV, a slightly increased agarose concentration of approximately 3% is selected to increase the gel's firmness, enabling it to withstand high shear forces within the flow-through cell. The gel composition may further include ions, vesicles, and / or serum proteins.

[0035] In another embodiment, the gel composition comprises one or more polypeptides, such as collagen, gelatin, and peptone. For example, the gel composition may comprise a concentration of 0-1% by weight collagen and / or 0-5% by weight gelatin. In yet another embodiment, the gel composition comprises one or more lipids selected from triglycerides and phospholipids.

[0036] Container 100 further includes a ring 114 that surrounds the periphery of opening 112 of receptacle 102. The top of ring 114 includes an opening that allows drug formulation 106 to be injected into interior pocket 104 of receptacle 102. Container 100 further includes a plug 116 designed to seal the opening of ring 114 after drug formulation 106 has been injected into interior pocket 104 of receptacle 102, effectively sealing opening 112 of receptacle 102. In one embodiment, plug 116 is made of a rubber material for a secure seal.

[0037] 1A and 1B, the container 100 is used for in vitro testing of subcutaneously administered small molecule drugs. In an alternative embodiment, the container 100 can also be used for testing biomolecules such as proteins, peptides, or nucleic acids.

[0038] 1A and 1B, the receptacle 102 has an elongated cylindrical body and a rounded base. It will be understood by those skilled in the art that the receptacle 102 may have different shapes, such as a cube, a sphere, an oval, etc.

[0039] 1A and 1B, the bottom wall 108 and side wall 110 of the receptacle 102 are composed entirely of the gel composition. In alternative embodiments, only a portion of the receptacle 102 may be made of the gel composition that allows diffusion of the drug formulation, with the remaining portion being made of another material. For example, a portion of the base of the receptacle 102 may be made of the gel composition, and the remaining portion may be made of an impermeable material. In another example, the side wall 110 may be made of the gel composition, and the bottom wall 108 may be made of an impermeable material.

[0040] 1A and 1B, the container 100 includes a ring 114 that surrounds the periphery of the opening 112 of the receptacle 102. In an alternative embodiment, the container 100 may be constructed without a ring. In that case, a plug 116 is attached directly to the opening 112 of the receptacle 102 to seal it.

[0041] 1A and 1B, the container 100 is constructed for integration into a USP Apparatus IV. In alternative embodiments, the container 100 may be constructed for use with other apparatus, such as a USP Apparatus II, a dispersed release apparatus, an extraction cell, etc.

[0042] 1A and 1B. As shown in the top view, the top of ring 114 has an opening 118 through which a liquid gel composition can be filled into a mold to form receptacle 102 during the process of preparing container 100. This opening 118 in ring 114 also functions as an injection port, allowing drug formulation 106 to be injected into interior pocket 104 of receptacle 102.

[0043] The ring 114 has several ridges 120 surrounding the inner wall of the ring 114 adjacent its top. The ridges 120 provide a secure grip on the plug 116 to prevent it from becoming dislodged. As shown in the bottom view, the ring 114 has a collar 122 surrounding the inner wall of the ring 114 adjacent its base. The collar 122 creates a recess for the liquid gel composition to fit into. In other words, the recess provides additional space to accommodate the periphery of the opening 112 of the receptacle 102 once the gel composition hardens, thereby securing the ring 114 to the receptacle 102. In one embodiment, the ring 114 is made of a chemically inert material, such as a polymer or metal.

[0044] During the preparation process of container 100, ring 114 is mounted on a mold. A liquid gel composition is poured into the mold through opening 118 at the top of ring 114. An inner punch is inserted into the gel composition through opening 118 in ring 114 to form inner pocket 104, which, together with ring 114, forms receptacle 102 surrounding opening 112 of receptacle 102. During preparation of container 100 for in vitro drug testing, drug formulation 106 is injected into receptacle 102 through opening 118 in ring 114, followed by sealing opening 118 in ring 114 with plug 116, thereby sealing opening 112 of receptacle 102.

[0045] 1C, the ring 114 has several ridges 120 surrounding the inner wall of the ring 114 adjacent its top. In alternative embodiments, the ring 114 may have other textured features to provide a good grip, such as a threaded pattern, embossing, knurled surface, etc.

[0046] This disclosure presents a novel in vitro testing method that uses a tissue-like matrix as a diffusion barrier in conjunction with the well-established USP Apparatus IV to emulate the specific characteristics of the subcutaneous microenvironment. This innovative approach provides a validated, reproducible, discriminatory, biopredictive, and standardized setup for evaluating current and future subcutaneous formulations. Importantly, the methodology incorporates the use of a hydrogel container 100, which facilitates accurate assessment of the diffusion behavior of drug formulations. This correlation allows for prediction of their in vivo performance.

[0047] During use, the container 100 acts as a physicochemical barrier to the diffusion of drug formulations due to the specifics of the setup, which involves a continuous flow of medium perfusing the gel matrix, and can be used to differentiate drug formulations. Additionally, this setup can be used to differentiate drug formulations based on their diffusion and binding affinity to this matrix, rather than solely on their solubility behavior, which can significantly affect their absorption from the subcutaneous tissue. This setup also significantly reduces the consumption of accessible medium during in vitro drug testing compared to assays in which the formulation is directly injected into bulk medium. This mimics the reduced direct availability of fluid within the subcutaneous tissue.

[0048] The dimensions of the container 100 are consistent with the size of the USP IV apparatus. This advantageously enables performance testing in a well-defined and harmonized environment for a broader range of drug and biomolecule applications. The gel composition used to create the receptacle 102 is customizable and can be prepared through a simple molding process. Therefore, the biological relevance of the container 100 can be tailored to ensure assay viability and biopredictability. It also allows for complete containment of the drug formulation within the container 100, avoiding drug leakage and direct media contact. Furthermore, the ring 114 is a simple, reusable holder that allows for convenient injection of the drug formulation using the original injection system provided by the manufacturer.

[0049] FIG. 2 is a schematic diagram illustrating a modified flow-through cell 200 including a container 202 according to another exemplary embodiment. The container 202 includes a receptacle 204 made of a gel composition and a ring 206 surrounding an opening 208 in the receptacle 204. In contrast to the containers described above with reference to FIGS. 1A and 1B, the container 202 in this embodiment does not have a plug. The ring 206 is configured to attach to a cap 210 of the modified flow-through cell 200. Thus, the opening of the ring 208 is inside the modified flow-through cell 200 and does not face the surrounding medium. This may effectively eliminate the use of a plug and also allow a drug formulation 212 to be injected into or withdrawn from the receptacle 204 via the modified flow-through cell 200 throughout an experiment.

[0050] FIG. 3 shows two flow charts 300A, 300B, each showing a breakdown of diffusion experiments performed on a particular version of the hydrogel-based diffusion container.

[0051] For the diffusion experiments, various insulin formulations were selected as model drugs, and their diffusion behavior (Actrapid®, Apidra®, Insulatard®) was tested and compared in vitro. For this, regular, rapid-acting, and long-acting insulins were considered. m-Cresol, a common preservative present in insulin formulations, was monitored as a small molecule reference molecule and measured throughout the experiments along with the insulin formulations. Table 1 below summarizes the composition and properties of the investigated pharmaceuticals.

[0052] [Table 1]

[0053] Throughout the experiment, we monitored the diffusion of insulin and m-cresol through agarose hydrogels in phosphate-buffered saline (PBS). m-cresol was expected to diffuse more rapidly than peptides. It reached a plateau after several hours and can be used to monitor cell-to-cell variability and hydrogel integrity. Caffeine was chosen as a second diffusion marker to serve a similar purpose to m-cresol. These two molecular entities, which exhibit differences in protein interactions, serve as markers of albumin permeation from the release medium into the hydrogel.

[0054] The first diffusion experiment involved testing the first hydrogel container (version 1.0) in an isolated environment as a proof-of-concept model. In step 302A, the container was tested with Actrapid® and Apidra® in PBS to evaluate the diffusion behavior of human soluble insulin, insulin glulisine, and m-cresol present in the insulin formulation. In step 304A, a reference experiment was performed without the hydrogel container to evaluate the interaction of the insulin formulation with the pump device and identify their potential losses within the device. These steps are described in further detail below with reference to Figures 4A and 4B.

[0055] The second diffusion experiment involved testing a second hydrogel container (version 2.0), an improved version of version 1.0, illustrated in Figures 1A and 1B, specifically designed and fabricated for integration into the perfusion system of the USP Apparatus IV.

[0056] In step 302B, the container was tested for leakage using caffeine in PBS to evaluate the functionality of the cell and the integrity of the hydrogel inside the USP Apparatus IV. In step 304B, the container was tested with Actrapid® and Apidra® in PBS to evaluate the diffusion behavior of the human soluble insulin, insulin glulisine, and m-cresol present in the insulin formulation. In step 306B, a reference experiment was performed in the presence of the hydrogel container in the flow-through cell of the USP Apparatus IV to evaluate the interaction of the insulin formulation with the pump device and identify their potential losses within the device. In step 308B, the container was tested with Actrapid® and Apidra® in subcutaneous interstitial buffer (SIB) to evaluate the diffusion behavior of the human soluble insulin, insulin glulisine, and m-cresol present in the insulin formulation. In step 310B, the container was tested with Insulatard® in PBS to evaluate the diffusion behavior of the insulin formulation. This is followed by the addition of heparin to the Insulatard® to trigger the release of insulin. These steps are described in more detail below with reference to Figure 5.

[0057] chemicals Vials containing 10 mL of Actrapid® 100 IU / mL (equivalent to 3.5 mg regular human insulin / mL) and 10 mL of Insulatard® 100 IU / mL (equivalent to 3.5 mg isophane (NPH) insulin / mL) from NovoNordisk A / S (Bagsvaerd, Denmark), and 3 mL of Apidra® SoloSTAR® Pen 100 IU / mL (equivalent to 3.5 mg insulin glulisine / mL) from Sanofi (Paris, France) were purchased from the National University Hospital of Singapore. m-Cresol (99%) and caffeine reference standards were obtained from Sigma-Aldrich (Missouri, USA). Agarose (molecular biology grade) was obtained from Vivantis Technologies Sdn Bhd (Shah Alam, Malaysia). The buffer salts NaHPO·7H2O, KHPO, and KCl were obtained from Avantor (Pennsylvania, USA). NaCl was obtained from VWR International (Pennsylvania, USA), and Tris base was obtained from Vivantis Technologies Private Limited (Shah Alam, Malaysia). TrisHCl, CaCl, MgSO·7H2O, CHCOONa, and NaHCO3 were obtained from Sigma-Aldrich (Missouri, USA). Hydrochloric acid was obtained from VWR International (Pennsylvania, USA). For HPLC quantification, methanol was obtained from Fisher Scientific (New Hampshire, USA), and acetonitrile was obtained from Avantor (Pennsylvania, USA). Regarding further additives, polysorbate 80 (Tween 80) and heparin sodium salt from porcine intestinal mucosa (≥150 IU / mg) were obtained from Sigma-Aldrich (Missouri, USA). Purified water from a Milli-Q deionization unit was used for all experiments.

[0058] Preparation of phosphate-buffered saline (PBS) PBS, 157 mM Na + , 4.5 mM K + , 140 mM Cl - , 10 mM HPO4 2- The buffer was prepared using NaCl, KCl, NaHPO 7H O, and KHPO at an ionic concentration of 0.01% (see Table 2 below). After adjusting the pH to 7.4, the buffer was vacuum filtered through a 0.45 μm membrane. This was followed by a modified degassing method recommended by the USP, which involves heating the buffer to approximately 40°C and vigorously stirring for 5 minutes. In some setups, 0.01% (w / v) Tween 80 was added later to reduce surface adsorption.

[0059] Preparation of subcutaneous interstitial buffer (SIB) To prepare a more biorelevant medium, SIB, Tris base and TrisHCl were dissolved first. Then, NaCl, NaHPO 7H O, KCl, KH PO, CH COONa, MgSO 7H O, and CaCl were added. NaHCO was included last to avoid precipitation of poorly soluble carbonates. The ionic concentration was 136 mM Na. + , 3.9 mM K + , 1.3 mM Ca 2+ , 0.5mM Mg 2+ , 114.9 mM Cl - , 20.6 HCO3 - , 1 mM HPO4 2- , 0.5 mM SO4 2- The pH was adjusted to 7.4 at 34°C (see Table 2 below). After adjusting the pH to 7.4, the buffer was vacuum filtered through a 0.45 μm membrane. This is a critical step because the pH of Tris is temperature dependent. A modified USP degassing method was then used, in which the buffer was heated to approximately 40°C and vigorously stirred for 5 minutes. The buffer was prepared fresh for each run to avoid precipitation and pH changes over time.

[0060] [Table 2]

[0061] Quantification of insulin and m-cresol A Chromaster high-performance liquid chromatography (HPLC) system (VWR Hitachi, Tokyo, Japan) was used. The general setup included an HPLC pump (No. 5160), a column oven (No. 5310), an autosampler (No. 5260), and a UV-Vis detector (No. 5420). A Hypersil BDSC 18 column with dimensions of 100 × 4.6 mm was used (Thermo Scientific, New Hampshire, USA). The mobile phase consisted of 30% acetonitrile and 70% water, both acidified with 0.1% TFA. The composition was gradually changed to 40% acetonitrile and 60% water over 9 min, then gradually returned to its initial value over 10 min. The mobile phase was pumped at a flow rate of 1 mL / min, and the needle was rinsed with 30% acetonitrile before each injection. All samples were diluted with the mobile phase before measurement. The concentration of insulin in each sample was measured by detecting monochromatic absorbance at a wavelength of 214 nm in a 20 μL injection volume. A total run time of approximately 10 minutes was required, with the m-cresol peak appearing at 4 minutes and the insulin peaks for Actrapid®, Apidra®, and Insulatard® appearing after approximately 5 minutes.

[0062] Caffeine quantification Caffeine was quantified at a wavelength of 290 nm using a Hitachi U-5100 UV / visible spectrophotometer (Tokyo, Japan). A 50 μl quartz cuvette was selected, and samples were measured in triplicate without dilution.

[0063] Proof-of-Concept Model Figure 4A shows the hydrogel container 402 (left) and modified flow-through cell 404 (right) used as a proof-of-concept model for the diffusion experiment shown in the first flowchart 300A of Figure 3. The proof-of-concept model was designed to confirm the discriminatory power of agarose hydrogel under continuous medium flow. Because it serves as a physicochemical barrier between the drug and the medium, it is expected to generate data confirming differences in the diffusion behavior and gel interactions of closely related drug formulations. To this end, Actrapid® (regular human insulin, hexamer) and Apidra® (insulin glulisine) were selected as model peptide formulations for initial setup. m-Cresol, present in both formulations and serving as an internal standard, was also quantified.

[0064] A cylindrical gel, which forms both a physicochemical barrier and a pocket, was prepared using a custom-made plastic receptacle. The gel consisted of 2% (w / w) agarose dissolved in PBS. After heating the gel to the boiling point and under constant stirring, a homogeneous mixture was obtained. The evaporated water was then replenished. The hot mixture was poured into a cylindrical plastic receptacle, and an inner cylindrical plastic punch was inserted to form a pocket into which the drug formulation was dispensed. After cooling at room temperature, the gel was formed and ready for use. A hydrogel container 402 with a gel thickness of approximately 2.5 mm was fabricated.

[0065] Loosely inspired by the flow-through cell of the USP Apparatus IV, a modified flow-through cell 404 was prepared from a 50 mL centrifuge tube (115 mm x 30 mm x 30 mm). After respective holes were drilled in the tube, an inlet port 406 and an outlet port 408 were created from a Luer lock syringe needle. The Luer lock syringe needle for the upper outlet port 408 was capped with wire cutters, while the needle tip for the lower inlet port 406 was bent to direct the incoming fluid toward the bottom of the Falcon tube, allowing the media to flow upward toward the outlet port 406. The inserted needle was further secured with silicone sealant, thread seal tape, and parafilm to prevent leakage. A stainless steel wire cage 410 was created to ensure that the drug formulation inside the prepared gel pocket did not come into direct contact with the media. The cage 410 was secured at a height such that the inserted gel had its opening above the upper outlet port 408. Because the media level in the modified flow-through cell 404 does not rise above the top exit port 408, the drug formulation had to be separated from the media and routed through the hydrogel container 402. The modified flow-through cell 404 further includes a cap 412 to prevent accidental spillage from the cell 404.

[0066] Figure 4B shows the assay setup of the proof-of-concept model in Figure 4A for in vitro drug testing. Continuous media flow was achieved by connecting a modified flow-through cell 404 with a CP7-35 piston pump 414 (Sotax AG, Basel, Switzerland). A Luer lock adapter allowed a standard ¼-inch-28 UNF screw thread to be connected to the custom-made cell 404. At a selected flow rate (8 ± 0.4 mL / min in this setup), 50 mL of PBS media 416 containing 0.01% (w / v) Tween 80 was pumped through the flow-through cell 404 in a closed-loop setup. The cell 404 was placed in a water bath 418 to raise the media temperature to 37 °C ± 0.5 °C. Media was pumped through the cell 404 for at least 30 min to purge the tubing from air bubbles and allow the temperature to rise. Once the desired temperature was reached, 1.5 mL of either Actrapid® or Apidra® (equivalent to 150 IU or 5.25 mg of insulin) was injected into the gel pocket, which was then placed in the cage 410. Samples with a volume of 0.2 mL of medium were manually collected hourly for the first 7 hours before being replenished with fresh medium. Quantification of insulin and m-cresol in the medium was then performed by HPLC. The cumulative diffusion amount of the model drug was then calculated by comparing the diffusion amount with the initially added drug concentration.

[0067] Subsequently, a reference experiment was performed by adding insulin to the medium to identify potential degradation or adsorption of insulin in the perfusion system. All experiments were performed in triplicate.

[0068] Figure 5 shows the assay setup of the hydrogel container 100 in a USP Apparatus IV for the diffusion experiment shown in the second flowchart 300B of Figure 3. To further improve and develop the diffusion assay in line with internationally recognized standards, the hydrogel container 100 was prepared for integration into a USP Apparatus IV 502. The compendial flow-through cell 504 of the USP Apparatus IV 502 is an enclosed system with a heating mantle that allows for controlled media flow from the bottom to the top of the cell 504.

[0069] Multiple versions were created before the design of the current hydrogel container 100 was finalized. Fusion360™ modeling software (Autodesk, California, USA) was used for the initial sketch of the 3D printed ring 114 that would serve as the gel holder.

[0070] To manufacture the rings 114 of the container 100, a stereolithography (SLA) printer Form2™ (Formlabs, Massachusetts, USA) was used with HighTempV2™ resin (Formlabs, Massachusetts, USA). This particular resin was chosen for its high thermal displacement and good compatibility with aqueous solvents. A layer thickness of 0.050 mm ensured high resolution, and with the addition of typical printing support structures, a printing time of approximately 3 hours was required. After printing the rings 114, they were washed with isopropanol for 15 minutes and cured under UV light for several hours in an AsigaFlash™ curing station (Asiga, Alexandria, Australia). The printed rings 114 proved to be very durable and chemically resistant.

[0071] The ring 114 was placed in a cylindrical mold, and the gel composition was filled into the mold. A punch was used to create an internal pocket of similar size. The drug formulation 106 was then injected into the internal pocket of the receptacle 102, and the ring 114 was sealed with a rubber plug 116.

[0072] The USP Apparatus IV 502 and various flow-through cells 504 are described in various pharmacopoeias and related literature. For this diffusion experiment, a SOTAX CP7-35 Piston Pump 506 equipped with a fraction collector 508 and a SOTAX CE7 dissolution system was used alone. The medium 510 was loaded into the reservoir 512 with a magnetic stirrer operating at 150 rpm. The dissolution apparatus was operated in a closed-loop configuration at a constant flow rate of 8 ± 0.4 mL / min for all experiments. Before the flow-through cell 504 with an internal diameter of 22.6 mm was inserted, the system was set to bypass mode to purge air from the capillary.

[0073] One 5 mm ruby ​​glass sphere and approximately 2.4 g of 1 mm diameter glass beads 514 were loaded into the flow-through cell 504 to ensure laminar flow. A heating jacket 516 maintained a temperature approximating that found in subcutaneous tissue at 34°C ± 0.5°C for all settings. Freshly prepared containers 100 were injected with the respective drug formulations 106, sealed with rubber plugs 116, and carefully placed over the glass beads 514. The flow-through cell 504 was then assembled and placed into the main system.

[0074] Diffusion experiments were initiated with a caffeine stock solution (4 mg / mL) in container 100 to evaluate the functionality of the new setup and the integrity of the hydrogel. A medium volume of 100 mL of PBS 510 was selected. The initial injection volume of the test drug was 1 mL. The run was conducted over an 8-hour period, with samples collected in 0.5 mL volumes at 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.75, 3.25, 3.75, 4.25, 5.25, 6.25, and 8.25 hours. The medium 510 was not replenished. Caffeine was quantified using an ultraviolet-visible (UV / Vis) spectrometer. The cumulative amount of caffeine diffusion was then calculated by comparing it with the initially added drug concentration. All experiments were performed in triplicate.

[0075] In the next step, the new container 100 was evaluated with Actrapid® and Apidra® to assess the assay's ability to distinguish between compounds of different molecular sizes. 60 mL of PBS or SIB 510, respectively, was used. Again, 1 mL of each formulation 106 was injected into the receptacle 102. Samples with a volume of 0.5 mL were collected after 2, 4, 6, 8, 12, 16, 20, and 24 hours. The medium 510 was not replenished. Quantification of insulin and m-cresol was achieved by HPLC. The cumulative amount of insulin and m-cresol diffused was then calculated by comparing it with the amount of drug initially added.

[0076] In the next reference experiment, both insulins were added to the acceptor medium 510 in the presence of the container 100 to investigate the adsorption and degradation (recovery) of insulin over time. All experiments were performed in triplicate.

[0077] Insulatard® is a depot formulation of insulin. The suspension contains microcrystalline insulin in the presence of zinc and protamine. Performance testing was performed under the conditions described above (PBS was used as the release medium 510). In a follow-up study, excess heparin sodium salt (1 mg per mL) was added to the formulation 106. Heparin functions as a complexing agent, triggering the release of insulin by forming a heparin-protamine complex.

[0078] Figure 6A shows a line graph illustrating the diffusion profiles of Actrapid® and Apidra® in the experiment described with reference to Figures 4A and 4B. Figure 6B shows a line graph illustrating the results of the reference experiment described with reference to Figures 4A and 4B. The horizontal axis represents time in hours, and the vertical axis represents the concentration of the formulation components in percentage (%). A first line 602 represents data for regular human insulin from Actrapid®, a second line 604 represents data for m-cresol from Actrapid®, a third line 606 represents data for insulin glulisine from Apidra®, and a fourth line 608 represents data for m-cresol from Apidra®.

[0079] In this proof-of-concept model, initial experiments investigated the diffusion of Actrapid® and Apidra® in PBS supplemented with 0.01% (w / v) Tween 80. HPLC analysis allowed for the quantification of insulin (regular human insulin and insulin glulisine) and m-cresol. Cumulative diffusion was calculated as a percentage of the injected dose (Figure 6A). To quantify the significance of the displayed differences in diffusion behavior, the difference coefficient (f1) and similarity coefficient (f2) were calculated. The f1 and f2 factors were developed by Moore and Flanner before being adopted by the FDA guidelines for immediate-release solid oral dosage forms. The closer the f1 coefficient is to zero and the higher the calculated f2 coefficient, the more similar the two release profiles will be. To avoid any ambiguity during scale-up and post-approval changes, the FDA suggested limits of 15 or less for f1 and 50 or more for f2 to conclude that the release of the two selected formulations is similar. The FDA specified several criteria that must be met before calculation, and some criteria, such as having to test at least 12 individual dosage forms for both products, did not apply in the current setting.

[0080] The diffusion profiles of Actrapid® and Apidra® showed that the cumulative diffusion of both insulin and m-cresol increased over time, with both insulins first detected after 3 hours. Regular human insulin from Actrapid® peaked at 9.4 ± 0.2% at 7 hours, while insulin glulisine diffusion from Apidra® occurred at a higher rate, at 20 ± 1.0% at 7 hours (f1 = 119, f2 = 56). The f1 factor indicates a significant difference, while the f2 factor would indicate that both profiles were similar if the thresholds set by the FDA were considered. In this case, when only one factor reached the given limit, the two formulations would be considered different, giving credence to the assay's ability to distinguish between both insulin formulations.

[0081] Their m-cresol counterparts, on the other hand, diffused at a much faster rate, both reaching a plateau at approximately 7 hours. m-cresol in the Actrapid® formulation appeared to diffuse toward a smaller plateau at 84 ± 1.0%, while m-cresol in Apidra® reached a maximum at 88 ± 0.2%, although the difference was not significant (f1 = 5, f2 = 71).

[0082] Additionally, a reference experiment was performed in which the pure formulations were injected into the medium (Figure 6B). The experiment showed a slight decrease in insulin concentration over time: for regular human insulin in Actrapid®, from 95 ± 0.7% after 1 hour to 91 ± 4.7% after 7 hours, and for insulin glulisine in Apidra®, from 90 ± 1.5% after 1 hour to 84 ± 3.3% after 7 hours, indicating adsorption or degradation processes that reduced recovery. Meanwhile, both m-cresol profiles fluctuated around 100 ± 1.8%. The recovery profiles of both insulin and m-cresol were significantly similar (insulin: f1 = 5, f2 = 64; m-cresol: f1 = 1, f2 = 88).

[0083] Figure 7A illustrates a line graph showing the diffusion profile of the caffeine stock solution in the experiment described with reference to Figure 5. Initially, caffeine was used as a small, stable model compound. Experiments in PBS over 8 hours resulted in a steady increase in drug concentration in the acceptor compartment, with caffeine slowly diffusing through the gel of container 100, peaking at approximately 101 ± 3% at 8.25 hours.

[0084] Figure 7B shows a line graph illustrating the diffusion profiles of Actrapid® and Apidra® in PBS solution 510 in the experiment described above with reference to Figure 5. Figure 7C shows a line graph illustrating the diffusion profiles of Actrapid® and Apidra® in SIB solution 510 in the experiment described above with reference to Figure 5. The horizontal axis represents time in hours, and the vertical axis represents the concentration of the formulation components in percentage (%). A first line 702 represents data for regular human insulin from Actrapid®, a second line 704 represents data for m-cresol from Actrapid®, a third line 706 represents data for insulin glulisine from Apidra®, and a fourth line 708 represents data for m-cresol from Apidra®.

[0085] Actrapid® and Apidra® were tested in PBS and in more biologically relevant SIBs under the specified conditions. Similar to the results found in the proof-of-concept study, hexameric human insulin (Actrapid®) diffused at a slower rate compared to monomeric insulin glulisine (Apidra®) during the first few hours. In PBS and SIBs, insulin diffused at the same rate and was first detected after 4 hours. The experiment was carried out for 24 hours without any visible change in hydrogel integrity. In PBS, insulin glulisine diffused at a faster rate than regular human insulin, reaching 67 ± 0.2% (f1 = 44, f2 = 45), which peaked at 50 ± 4%.

[0086] In the hydrogel container 100, both f-factors were calculated and found to be outside the given thresholds, making it undeniable that both profiles are significantly different. The internal standards reached a plateau at similar rates, suggesting no significant loss of medium 510 due to leakage of USPIV 502 throughout the run. In comparison, testing in SIB 510 yielded nearly identical results (regular insulin: f1 = 2, f2 = 97; insulin glulisine: f1 = 3, f2 = 89), with both f1 and f2 tests consistent in significance. In SIB, insulin glulisine peaked at 67 ± 1.4% after 24 hours, while regular human insulin peaked at a slower rate of 50 ± 3.2% (f1 = 49, f2 = 43). m-Cresol again reached a stable plateau.

[0087] Figure 7D illustrates a line graph showing the results of the reference experiment described with reference to Figure 5. A first line 710 represents data for regular human insulin from Actrapid®, and a second line 712 represents data for insulin glulisine from Apidra®.

[0088] A reference experiment was performed in which Actrapid® and Apidra® were injected directly into the acceptor medium 510 in the flow-through cell 504. A hydrogel container 100 was placed in the setup to ensure that the interaction in the presence of agarose hydrogel was covered by the recovery study. The recovery of both insulins in PBS 510 is shown in Figure 7D. A small but significant loss of insulin was detected in both cases, with 78 ± 12% of normal insulin and 72 ± 24% of insulin glulisine recovered after 24 hours. The standard deviations are very large compared to normal diffusion runs. Since insulin diffuses into the gel present in this reference experiment, it can be assumed that 100% recovery will not be reached.

[0089] Figure 7E shows a line graph illustrating the diffusion profiles of Insulatard® and heparin-treated Insulatard® in PBS solution 510 in the experiment described with reference to Figure 5. A first line 714 represents the insulin data from untreated Insulatard®, a second line 716 represents the m-cresol data from untreated Insulatard®, a third line 718 represents the insulin data from treated Insulatard®, and a fourth line 720 represents the m-cresol data from treated Insulatard®.

[0090] Insulatard® is a suspension of human insulin in isophane (NPH). Without further processing, only 5.5 ± 3.8% of the insulin was detected after 12 hours, with no significant increase thereafter. In a follow-up experiment, excess heparin was added to release insulin from the suspension. The diffusion curve more closely resembles that of hexameric human insulin, peaking at 32 ± 49% after 24 hours, although the similarity is not considered significant enough at the given standard deviation. The m-cresol standard reached a stable plateau without issue.

[0091] The initial proof-of-concept study (version 1.0) reflected the expected ranking order, with hexameric insulin diffusing slower than insulin glulisine. Actrapid® contains zinc, a cation that stabilizes the hexameric state, while insulin glulisine (Apidra®) is engineered to reduce oligomer formation. Hexameric assembly is reduced by two amino acid substitutions. The human insulin sequence is altered at positions B3 (asparagine replaced with lysine) and B29 (lysine replaced with glutamic acid). Note that Apidra® does not contain zinc. Due to the difference in molecular weight, the molecular mobility and absorption rate of the monomer are expected to be faster compared to the dimer and hexamer. Notably, insulin glulisine has been touted as a rapid-acting insulin that exhibits faster absorption in vivo.

[0092] m-Cresol was also quantified to detect potential errors arising from gel preparation. Consistent with expectations, m-cresol was observed to diffuse more rapidly than the two larger insulin molecules, due to its stable and small size. Furthermore, the high recovery rate indicates no leakage from the perfusion cycle. Furthermore, m-cresol is known to interact nonspecifically with proteins and excipients present. This could explain the plateauing release profiles of Actrapid® and Apidra®, due to their slightly different compositions. Changes in barrier properties resulting from the interaction of hexameric insulin with the hydrogel could also affect m-cresol diffusion. Therefore, evaluating the diffusion of other small molecules in the future would be a useful addition to the characterization. Similar diffusion behavior would rule out the possibility of changes in the hydrogel structure.

[0093] The reference experiments suggested a loss or degradation of insulin over time. When many hydrophobic surfaces are present, insulin, especially in its monomeric form, adsorbs to these surfaces, leading to aggregation and eventual denaturation. Because insulin was injected directly into the medium all at once, much of the insulin came into contact with these surfaces much more quickly than in normal operation. Adsorption to these surfaces could explain why both insulins were not fully recovered, even at earlier sample points. Furthermore, primarily monomeric insulin glulisine was also more readily lost, which also appears to support adsorption of the monomeric form of insulin. Further experiments may involve the inclusion of higher concentrations of other surfactants, such as Tween 20.

[0094] As outlined in the previous section, integration of the hydrogel container 100 into the USP IV502 instrument was achieved, along with the fraction collector, allowing for automation of the release experiment and eliminating any human sampling error that was more pronounced in experiments involving proof-of-concept models. Initial experiments with caffeine confirmed highly consistent and reproducible diffusion behavior with low standard deviations. Caffeine has previously been used as a marker molecule for the characterization of agarose gels and exhibits very low plasma protein binding.

[0095] The results of the initial design were successfully replicated using the USP Apparatus IV502 and the hydrogel container 100. In fact, the hydrogel container 100 demonstrated even greater discrimination. The differences in the rate and extent of the measured diffusion behavior were more significant.

[0096] Comparison of release tests conducted in SIB and PBS showed that the medium did not significantly affect the diffusion behavior. The ionic composition did not affect insulin penetration. This may change with different gel compositions, as these tests were conducted only in pure agarose gels. The final experiments were dedicated to evaluating more complex drug formulations within the hydrogel container 100. Insulatard® is a microcrystalline suspension of insulin in the presence of protamine and zinc. In a physiological setting, protamine is degraded by enzymes within the subcutaneous tissue, resulting in the release of hexameric insulin from its complex. Alternatively, release can be induced in vitro. Excess heparin was used to form a stable complex with protamine, resulting in the release of hexameric insulin. Future experiments may consider the addition of enzymes such as trypsin to reach a higher level of biological relevance.

[0097] Overall, the hydrogel container 100 provides a reliable setup with optimal characteristics for further testing of compounds in a USP Apparatus IV. High sensitivity, reproducibility, and the ability to distinguish between different formulations are achieved.

[0098] Based on the reported observations, the biological predictability of the assay can be systematically evaluated and optimized. To understand the in vitro conditions in the donor and acceptor compartments and address technical challenges arising from drug degradation due to shear and protein adsorption, the diffusion process in the USP apparatus IV can be modeled using computational fluid dynamics (CFD) or other appropriate software.

[0099] Biopredictability is achieved through systematic evaluation of observed changes in in vitro diffusivity in response to changes in gel and vehicle composition, along with repeated benchmarking against absorption rates observed in vivo. To this end, an in vitro-in vivo correlation (IVIVC) can be established to correlate collected in vitro data with observed in vivo responses. First, the reported pharmacokinetic profile for the subcutaneous formulation is analyzed using MonolixSuite™ 2021 (Lixoft, Zug, Switzerland). Stella Architect™ can be used to design an appropriate in silico model that enables simulations (iseesystems, New Hampshire, USA). To improve the level of biological relevance, the gel can be modified using components of the extracellular matrix, such as collagen, peptone, and hyaluronic acid. The vehicle can be further supplemented with serum proteins known to have a stabilizing effect on other proteins.

[0100] It will be understood by those skilled in the art that numerous changes and / or modifications may be made to the invention as illustrated in the specific embodiments without departing from the scope of the invention as broadly described, and the present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

1. 1. A container for in vitro drug testing, comprising: A receptacle having a wall, the receptacle having an opening and configured to receive a drug formulation through the opening, the wall being made from a gel composition. The container.

2. The container of claim 1 , wherein the concentration of the gel composition is selected to simulate one or more properties of tissue.

3. 3. The container of claim 1 or 2, wherein the gel composition comprises one or more polysaccharides selected from the group consisting of agarose and hyaluronic acid.

4. 4. The container of claim 3, wherein the gel composition comprises 0 to 5% by weight of agarose gel.

5. 4. The container of claim 3, wherein the gel composition comprises 0 to 3% by weight of hyaluronic acid.

6. The container according to any one of claims 1 to 5, wherein the gel composition comprises one or more polypeptides selected from the group consisting of collagen, gelatin, and peptone.

7. 7. The container of claim 6, wherein the gel composition comprises 0 to 1% by weight of collagen.

8. 7. The container of claim 6, wherein the gel composition comprises 0 to 5% by weight of gelatin.

9. 9. The container of claim 1, wherein the gel composition comprises one or more lipids selected from triglycerides and phospholipids.

10. The container of any one of claims 1 to 9, wherein the gel composition comprises ions.

11. The container according to any one of claims 1 to 10, wherein the gel composition comprises vesicles.

12. The container of any one of claims 1 to 11, wherein the gel composition comprises a serum protein.

13. The container of any one of claims 1 to 12, further comprising a plug for sealing the opening of the receptacle.

14. The container according to any one of claims 1 to 13, further comprising a ring surrounding the periphery of the opening of the receptacle for injection of the drug formulation into the receptacle.

15. 15. The container of claim 14, wherein the ring is made of a chemically inert material.

16. 16. The container of claim 15, wherein the chemically inert material comprises a polymer or a metal.

17. 17. A container according to any one of claims 14 to 16, wherein the ring includes a collar surrounding its inner wall adjacent the base of the ring to create a recess for receiving the periphery of the opening of the receptacle.

18. A container according to any one of claims 14 to 17, wherein the ring comprises a textured area surrounding its inner wall adjacent the top of the ring.

19. 1. A method for preparing a container for in vitro drug testing, comprising: forming a receptacle having a wall, the wall being made from a gel composition; disposing of the drug formulation into said receptacle; The method comprising:

20. forming a receptacle having a wall; attaching the ring to a mold; pouring the gel composition into the mold; inserting an inner punch into the gel composition to form the receptacle so that the ring circumscribes the opening of the receptacle; 20. The method of claim 19, comprising:

21. A testing device comprising a container according to any one of claims 1 to 18.

22. 1. A test method comprising: placing a drug formulation in a container according to any one of claims 1 to 18; immersing the container in a medium within a United States Pharmacopeia (USP) IV flow-through cell; monitoring the diffusion of the drug formulation between the container and the medium; The test method includes:

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