Container for in vitro drug testing

Abstract

The present disclosure relates to a container for in vitro drug testing. The container includes a reservoir having a wall made of a gel composition. The reservoir has an opening and is configured to receive a pharmaceutical formulation through the opening. The gel composition is selected to mimic one or more properties of tissue.

Description

Technical Field

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

[0002] Extravascular (including, for example, muscle or subcutaneous tissue) administration is a rapid and patient-friendly method for delivering pharmaceutical substances to the human body that requires minimal supervision by healthcare professionals. For many formulations administered by these routes of administration, their bioavailability depends to a large extent on various factors such as their diffusion behavior. Therefore, during development, it is crucial to test them to determine their performance. While animal testing remains the ultimate tool for comparing formulations, in vitro testing provides a faster and more cost-effective alternative.

[0003] In vitro performance determination is an evaluation technique that is used to obtain important information about the bioavailability and other preclinical relevant properties of pharmaceutical substances and dosage forms by utilizing a controlled in vitro environment rather than a living organism. This method has the potential to reduce the number of expensive and time-consuming animal and human tests. However, due to the lack of established standards, in vitro performance determination designed for extravascular dosage forms (including those for subcutaneous or intramuscular administration) faces numerous challenges. In addition, there are significant deficiencies in emphasizing feasibility and quality control testing, which hinder the development of reliable methods for evaluating the bioavailability of pharmaceutical substances.

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

[0005] Another in vitro performance assay involves using an instrument called Subcutaneous Injection Site Simulator N3 (SCISSOR N3). The SCISSOR N3 instrument uses a syringe filled with hyaluronic acid (HA) that acts as a donor medium. The syringe is separated from the surrounding receptor medium by a membrane. This setup attempts to simulate the microenvironment of the extracellular matrix, which provides diffusion resistance to the viscous HA. However, this system does not meet the international standards that are crucial for drug regulation. Such technical standards are provided by the national pharmacopoeia, which provides a unified structure for drug testing. In addition, for such highly specialized equipment, the instrument is quite costly and does not allow for flexible alteration of the in vitro environment, such as by changing the flow rate or stirring rate.

[0006] Other in vitro performance assays also utilize laboratory instruments or tools, such as shake flasks and continuous flow-through cells, or use materials such as hydrogels to simulate conditions within tissues. However, despite the subcutaneous formulation offering exciting alternatives and opportunities for existing and future drug substances, there is still a lack of suitable in vitro models that can reliably and rapidly predict the bioavailability of these substances in the human body and follow a clearly structured technical framework.

[0007] Accordingly, there is a need to provide a device that aims to address the above problems or provide a useful alternative. Summary of the Invention

[0008] According to a first aspect of the present invention, there is provided a container for in vitro drug testing, the container comprising:

[0009] A receptacle having a wall, wherein the receptacle has an opening and is configured to receive a drug formulation through the opening, and wherein the wall is made of a gel composition.

[0010] The concentration of the gel composition can be selected to simulate one or more properties of a tissue.

[0011] The gel composition may include one or more polysaccharides selected from the group consisting of agarose and hyaluronic acid.

[0012] The gel composition may include 0 - 5% by weight of agarose gel.

[0013] The gel composition may include 0 - 3% by weight of hyaluronic acid.

[0014] The gel composition may include one or more polypeptides selected from the group consisting of collagen, gelatin, and peptone.

[0015] The gel composition may include 0 - 1% by weight of collagen.

[0016] The gel composition may comprise 0-5% by weight of gelatin.

[0017] The gel composition may comprise one or more lipids selected from the group consisting of triglycerides and phospholipids.

[0018] The gel composition may comprise ions.

[0019] The gel composition may comprise vesicles.

[0020] The gel composition may comprise serum proteins.

[0021] The container may further comprise a stopper for sealing the opening of the reservoir.

[0022] The container may further comprise a ring surrounding the periphery of the opening of the reservoir for injecting a pharmaceutical preparation into the reservoir.

[0023] The ring may be made of a chemically inert material.

[0024] The chemically inert material may comprise a polymer or a metal.

[0025] The ring may comprise a collar that surrounds the inner wall thereof near the base of the ring, thereby forming a recess for receiving the periphery of the opening of the reservoir.

[0026] The ring may comprise a textured area that surrounds the inner wall thereof near the top of the ring.

[0027] According to a second aspect of the present invention, there is provided a method for preparing a container for in vitro drug testing, the method comprising the steps of:

[0028] forming a reservoir having a wall, wherein the wall is made of a gel composition; and

[0029] depositing a pharmaceutical preparation into the reservoir.

[0030] Forming a reservoir having a wall may comprise the steps of:

[0031] attaching the ring to a mold;

[0032] pouring the gel composition into the mold; and

[0033] inserting an inner punch into the gel composition to form the reservoir such that the ring surrounds the periphery of the opening of the reservoir.

[0034] According to a third aspect of the present invention, there is provided a testing device comprising a container as defined in the first aspect.

[0035] According to a fourth aspect of the present invention, there is provided a testing method comprising:

[0036] Deposit the pharmaceutical preparation into a container as defined in the first aspect;

[0037] Immerse the container in a medium within a United States Pharmacopeia (USP) Type IV flow-through cell; and

[0038] Monitor the diffusion of the pharmaceutical preparation between the container and the medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Embodiments of the present invention are provided by way of example only, and those skilled in the art will better understand and appreciate these embodiments from the following written description and drawings, in which:

[0040] Figure 1A FIG. shows a schematic diagram of a container for in vitro drug testing according to an exemplary embodiment.

[0041] Figure 1B FIG. shows Figure 1A a photograph of the container shown.

[0042] Figure 1C FIG. shows Figure 1A and 1B top views (left) and bottom views (right) of the ring shown.

[0043] Figure 2 FIG. shows a schematic diagram of a modified flow-through cell including a container according to another exemplary embodiment.

[0044] Figure 3 FIG. shows two flowcharts, each depicting the breakdown of a diffusion experiment conducted on a separate version of a hydrogel-based diffusion container.

[0045] Figure 4A FIG. shows a hydrogel container (left) and a modified flow-through cell (right) used as Figure 3 a proof-of-concept model for the diffusion experiment shown in the first flowchart of.

[0046] Figure 4B FIG. shows the assay setup for a proof-of-concept model for in vitro drug testing of Figure 4A .

[0047] Figure 5 FIG. shows the assay setup of a hydrogel container in a USP Apparatus IV for the diffusion experiment shown in the second flowchart of Figure 3 .

[0048] Figure 6A FIG. shows a depiction of and at reference Figure 4A and 4BLine graph of the diffusion curve in the explained experiment.

[0049] Figure 6B Illustrates a plot depicting the reference Figure 4A and 4B Line graph of the results of the explained reference experiment.

[0050] Figure 7A Illustrates a plot depicting the reference Figure 5 Line graph of the diffusion curve of the caffeine stock solution in the explained experiment.

[0051] Figure 7B Illustrates a plot depicting the reference Figure 5 in the explained experiment and Line graph of the diffusion curve in PBS solution.

[0052] Figure 7C Illustrates a plot depicting the reference Figure 5 in the explained experiment and Line graph of the diffusion curve in SIB solution.

[0053] Figure 7D Illustrates a plot depicting the reference Figure 5 Line graph of the results of the explained reference experiment.

[0054] Figure 7E Illustrates a plot depicting the reference Figure 5 in the explained experiment and heparin - treated Line graph of the diffusion curve in PBS solution. Detailed Description

[0055] The present invention relates to a container that acts as a release and diffusion adapter for in vitro testing of pharmaceutical formulations intended for subcutaneous administration. The container includes a reservoir having an opening and a stopper for sealing the opening of the reservoir. The container may further include a ring surrounding the perimeter of the opening of the reservoir to act as an injection port for delivering the pharmaceutical formulation into the reservoir. In use, the container is integrated into the flow - through cell of the United States Pharmacopeia (USP) Apparatus IV.

[0056] Figure 1A Illustrates a schematic view of a container 100 for in vitro drug testing according to an exemplary embodiment. Figure 1B Illustrates Figure 1AA photograph of the container 100 as shown. The container 100 is approximately 40 mm in height and 19 mm in width. The container 100 includes a reservoir 102 having an elongated cylindrical body with a uniform radius, which forms an interior pocket 104 for holding the pharmaceutical formulation 106. The reservoir 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 the upper end of the opening 112. The thickness of these walls 108 and 110 is approximately 2.5 mm and serves as a barrier between the pharmaceutical formulation 106 and the medium in the flow cell.

[0057] The bottom wall 108 and the side wall 110 of the reservoir 102 are made of a gel composition, and the concentration of the gel composition 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 and serves as a diffusion resistance and a membrane. In an embodiment, the gel composition includes one or more polysaccharides, such as agarose, hyaluronic acid, or other polysaccharides. For example, the gel composition may include agarose gel at a concentration of 0-5% by weight and / or hyaluronic acid at a concentration of 0-3% by weight. For use in the USP Apparatus IV, the agarose concentration can be slightly increased by about 3% to increase the firmness of the gel so that it can withstand the elevated shear forces within the flow cell. The gel composition may also include ions, vesicles, and / or serum proteins.

[0058] In another embodiment, the gel composition includes one or more polypeptides, such as collagen, gelatin, and peptone. For example, the gel composition may include collagen at a concentration of 0-1% by weight and / or gelatin at 0-5% by weight. In yet another embodiment, the gel composition includes one or more lipids selected from triglycerides and phospholipids.

[0059] The container 100 further includes a ring 114 that surrounds the perimeter of the opening 112 of the reservoir 102. The top of the ring 114 includes an opening to allow the pharmaceutical formulation 106 to be injected into the interior pocket 104 of the reservoir 102. Additionally, the container 100 includes a plug 116 that is designed to seal the opening of the ring 114 after the pharmaceutical formulation 106 has been injected into the interior pocket 104 of the reservoir 102, thereby effectively sealing the opening 112 of the reservoir 102. In an embodiment, the plug 116 is made of a rubber material to ensure a tight seal.

[0060] In Figure 1A and 1BIn the exemplary embodiments described, container 100 is used for in vitro testing of small molecule drugs for subcutaneous administration. In alternative embodiments, container 100 can also be used to test biomolecules such as proteins, peptides, or nucleic acids.

[0061] In Figure 1A and 1B the exemplary embodiments described, reservoir 102 has an elongated cylindrical body and a circular base. Those skilled in the art will appreciate that reservoir 102 can have different shapes such as a cube, sphere, oval, etc.

[0062] In Figure 1A and 1B the exemplary embodiments described, the bottom wall 108 and side wall 110 of reservoir 102 are entirely composed of a gel composition. In alternative embodiments, only a portion of reservoir 102 is made of the gel composition to allow diffusion of the drug formulation, and the remaining portion can be made of other materials. For example, a portion of the base of reservoir 102 is made of the gel composition and the remaining portion is made of an impermeable material. In another example, side wall 110 is made of the gel composition while bottom wall 108 is made of an impermeable material.

[0063] In Figure 1A and 1B the exemplary embodiments described, container 100 includes a ring 114 surrounding the perimeter of opening 112 of reservoir 102. In alternative embodiments, container 100 can be configured without a ring. In such a case, plug 116 is directly attached to opening 112 of reservoir 102 to seal it.

[0064] In Figure 1A and 1B the exemplary embodiments described, container 100 is created for integration into USP device IV. In alternative embodiments, container 100 can also be created for use with other devices such as USP device II, dispersion release device, extraction cell, etc.

[0065] Figure 1C Illustrated Figure 1A and 1B are a top view (left) and a bottom view (right) of ring 114 as shown. 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 during the preparation of container 100 to form reservoir 102. This opening 118 of ring 114 also serves as an injection port allowing drug formulation 106 to be injected into the inner pocket 104 of reservoir 102.

[0066] The ring 114 has a plurality of ridges 120 near its top that surround the inner wall of the ring 114. The ridges 120 enable a firm grip on the plug 116, preventing the plug 116 from shifting. As shown in the bottom view, the ring 114 has a collar 122 near its base that surrounds the inner wall of the ring 114. The collar 122 forms a recess that allows the liquid gel composition to fit therein. In other words, once the gel composition hardens, the recess can provide additional space to accommodate the perimeter of the opening 112 of the reservoir 102, thereby securing the ring 114 to the reservoir 102. In an embodiment, the ring 114 is made of a chemically inert material such as a polymer or a metal.

[0067] During the preparation of the container 100, the ring 114 is mounted onto a mold. The liquid gel composition is poured into the mold through the opening 118 at the top of the ring 114. An inner punch is inserted through the opening 118 of the ring 114 into the gel composition to form the inner pocket 104, thereby forming the reservoir 102, where the ring 114 surrounds the perimeter of the opening 112 of the reservoir 102. When preparing the container 100 for in vitro drug testing, the drug formulation 106 is injected into the reservoir 102 through the opening 118 of the ring 114, and then the opening 118 of the ring 114 is sealed with the plug 116, thereby sealing the opening 112 of the reservoir 102.

[0068] In Figure 1C the exemplary embodiment described, the ring 114 has a plurality of ridges 120 that surround the inner wall of the ring 114 near the top of the ring. In an alternative embodiment, the ring 114 can have other textural features to provide a good grip, such as a threaded pattern, embossing, a knurled surface, etc.

[0069] The present disclosure presents a novel in vitro testing method that combines the well-established USP Apparatus IV with a tissue-like matrix as a diffusion barrier to mimic specific characteristics of the subcutaneous microenvironment. This innovative method provides a validated, reproducible, discriminatory, biopredictive, and standardized setup for evaluating current and future subcutaneous formulations. Importantly, the method incorporates the use of the hydrogel container 100, which aids in accurately assessing the diffusion behavior of the drug formulation. This correlation enables predicting its in vivo performance.

[0070] In use, the container 100 serves as a physicochemical barrier to the diffusion of the pharmaceutical formulation. Due to the specificity of this setup with a continuous flow of a medium that perfuses the gel matrix, the diffusion of the pharmaceutical formulation can be used to distinguish different pharmaceutical formulations. Additionally, this setup can distinguish pharmaceutical formulations based on their diffusion and binding affinity to the matrix, rather than just based on their dissolution behavior, which greatly affects the absorption of the pharmaceutical formulation from the subcutaneous tissue. Compared to an assay that directly injects the formulation into a bulk media, this setup also significantly reduces the consumption of the available media in in vitro drug testing. This mimics the reduced direct availability of the fluid within the subcutaneous tissue.

[0071] The container 100 is sized to be consistent with the dimensions of the United States Pharmacopeia Apparatus IV. This advantageously enables performance testing in a well-defined and consistent environment, leading to a wider application to drugs and biomolecules. The gel composition used to make the reservoir 102 is customizable and can be prepared in a simple molding process. Thus, the biorelevance of the container 100 can be adjusted to ensure the feasibility and biopredictability of the assay. It also allows the pharmaceutical formulation to be fully encapsulated inside the container 100, avoiding drug leakage and direct media contact. Additionally, the ring 114 is a simple and reusable retainer that allows for the convenient injection of the pharmaceutical formulation using the original injection system provided by the manufacturer.

[0072] Figure 2 A schematic diagram is illustrated showing a modified flow-through cell 200 including a container 202 according to another exemplary embodiment. The container 202 includes a reservoir 204 made of a gel composition and a ring 206 surrounding the perimeter of an opening 208 of the reservoir 204. Different from the containers described above with reference to Figure 1A and 1B the container 202 in this embodiment does not have a plug. The ring 206 is configured to be attached to a lid 210 of the modified flow-through cell 200. Thus, the opening of the ring 208 is located inside the modified flow-through cell 200 and does not face the surrounding medium. This can effectively eliminate the use of a plug while allowing the injection of the pharmaceutical formulation 212 into or withdrawal from the reservoir 204 through the modified flow-through cell 200 throughout the experiment.

[0073] Figure 3 Two flowcharts 300A and 300B are illustrated, each flowchart depicting a breakdown of a diffusion experiment conducted on a separate version of a hydrogel-based diffusion container.

[0074] For the diffusion experiment, various insulin formulations were selected As model drugs, their diffusion behaviors were tested and compared in vitro. For this purpose, regular insulin, rapid-acting insulin, and long-acting insulin were considered. m-Cresol (a common preservative in insulin formulations), as a small molecule reference molecule, was monitored and measured together with the insulin formulations throughout the experiment. Table 1 below summarizes the composition and characteristics of the drug products studied.

[0075]

[0076] Table 1

[0077] Throughout the experiment, the diffusion of insulin and m-cresol through an agarose hydrogel in phosphate-buffered saline (PBS) was monitored. It was expected that m-cresol would diffuse faster than the peptide. It reached a stable level after several hours and could be used to monitor intercell-variability and the integrity of the hydrogel. Caffeine was selected as the second diffusion marker, which functioned similarly to m-cresol. These two molecular entities, which showed differences in their protein interactions, could be used as markers for albumin to penetrate from the release medium into the hydrogel.

[0078] 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 and in PBS to evaluate the diffusion behaviors of human soluble insulin, insulin lispro, and m-cresol present in the insulin formulation. In step 304A, a reference experiment without using the hydrogel container was conducted to evaluate the interaction of the insulin formulation with the pump device and identify its potential losses inside the device. These steps will be described in more detail below with reference to Figure 4A and 4B

[0079] The second diffusion experiment involved testing the second hydrogel container (version 2.0), which was an improved version of version 1.0. This second hydrogel container was as shown in Figure 1A and 1B and was designed and manufactured specifically for integration into the perfusion system of the USP apparatus IV.

[0080] In step 302B, the container was leak-tested with caffeine in PBS to evaluate the functionality of the cell inside the USP apparatus IV and the integrity of the hydrogel. In step 304B, with and ​The containers were tested to evaluate the diffusion behavior of human soluble insulin, insulin lispro, and m-cresol present in the insulin formulations. In step 306B, a reference experiment was conducted in the flow cell of the USP apparatus IV in the presence of the hydrogel container to evaluate the interaction of the insulin formulation with the pump device and to identify its potential loss within the device. In step 308B, the containers were tested using and to evaluate the diffusion behavior of human soluble insulin, insulin lispro, and m-cresol present in the insulin formulations. In step 310B, the containers were tested using in PBS to evaluate the diffusion behavior of the insulin formulation. Subsequently, heparin was added to to trigger insulin release. These steps will be described in more detail with reference to Figure 5 .

[0081] Chemical

[0082] Vials containing 10 mL of 100 IU / mL (equivalent to 3.5 mg regular human insulin / mL) and 10 mL of 100 IU / mL (equivalent to 3.5 mg isophane (NPH) insulin / mL) (from Novo Nordisk A / S, Bagsvaerd, Denmark) were purchased from the National University Hospital of Singapore, as well as 3 mL of 100 IU / mL (equivalent to 3.5 mg insulin glulisine / mL) prefilled pens ( Pens, from Sanofi (Paris, France). m-Cresol (99%) and caffeine reference standards were purchased from Sigma-Aldrich (Missouri, USA). Agarose (molecular biology grade) was purchased from Vivantis Technologies Sdn Bhd (Shah Alam, Malaysia). Buffer salts Na2HPO4·7H2O, KH2PO4, and KCl were purchased from Avantor (Pennsylvania, USA). NaCl was purchased from VWR International (Pennsylvania, USA), and Tris base was purchased from Vivantis Technologies Sdn Bhd (Shah Alam, Malaysia). Tris HCl, CaCl2, MgSO4·7H2O, CH3COONa, and NaHCO3 were purchased from Sigma-Aldrich (Missouri, USA). Hydrochloric acid was purchased from VWR International (Pennsylvania, USA). For HPLC quantification, methanol was purchased from Fisher Scientific (New Hampshire, USA), and acetonitrile was purchased from Avantor (Pennsylvania, USA). Regarding other additives: Polysorbate 80 (Tween 80) and porcine intestinal mucosa heparin sodium salt (≥150 IU / mg) were purchased from Sigma-Aldrich (Missouri, USA). All experiments were performed using purified water from a Milli-Q deionized water purification unit.

[0083] Preparation of Phosphate Buffered Saline (PBS)

[0084] PBS was prepared using NaCl, KCl, Na2HPO4·7H2O, and KH2PO4, with ion concentrations of 157 mM Na + 、4.5 mM K + 、140 mM Cl - and 10 mM HPO4 2- (see Table 2 below). After adjusting the buffer pH to 7.4, the buffer was vacuum filtered through a 0.45 μm membrane. Subsequently, it was degassed according to a modified degassing method recommended by the United States Pharmacopeia (USP), where the buffer was heated to approximately 40°C and vigorously stirred for 5 minutes. For some setups, 0.01% (w / v) Tween 80 was subsequently added to reduce surface adsorption.

[0085] Preparation of Subcutaneous Interstitial Buffer (SIB)

[0086] To prepare a more biorelevant medium SIB, Tris base and Tris HCl are first dissolved. Then NaCl, Na2HPO4·7H2O, KCl, KH2PO4, CH3COONa, MgSO4·7H2O, and CaCl2 are added, and finally NaHCO3 is added to avoid precipitation of insoluble carbonates. The ionic concentrations are 136 mM Na + , 3.9 mM K + , 1.3 mM Ca 2+ , 0.5 mM Mg 2+ , 114.9 mM Cl - , 20.6 mM HCO3 - , 1 mM HPO4 2- , 0.5 mM SO4 2- (see Table 2 below). After adjusting the buffer to pH 7.4 at 34 °C, the buffer is vacuum filtered through a 0.45 μm membrane. This is an important step because the pH of Tris is temperature-dependent. Next, a modified USP degassing method is employed, where the buffer is heated to approximately 40 °C and vigorously stirred for 5 minutes. The buffer is freshly prepared for each run to avoid precipitation and pH changes over time.

[0087]

[0088] FBS = fetal bovine serum; HBSS = Hanks balanced salt solution; PBS = phosphate buffered saline; SBF = simulated body fluid; SIB = subcutaneous interstitial buffer; SSIF = simulated subcutaneous interstitial fluid

[0089] Table 2

[0090] Quantification of Insulin and m-Cresol

[0091] The Chromaster high performance liquid chromatography (HPLC) system (VWR Hitachi, Tokyo, Japan) was used. The conventional settings included an HPLC pump (No. 5160), a column oven (No. 5310), an autosampler (No. 5260), and an ultraviolet-visible detector (No. 5420). A Hypersil BDS C18 column with dimensions of 100 x 4.6 mm (ThermoScientific, New Hampshire, USA) was used. The mobile phase consisted of 30% acetonitrile and 70% water, both acidified with 0.1% TFA. Over 9 minutes, the composition was gradually changed to 40% acetonitrile and 60% water and then gradually returned to its initial value at 10 minutes. The mobile phase was pumped at a flow rate of 1 mL / min, and the needle was washed with 30% acetonitrile before each injection. All samples were diluted with the mobile phase before measurement. The insulin concentration in each sample was measured by detecting the absorbance of monochromatic light at a wavelength of 214 nm (injection volume of 20 μL). A total running time of approximately 10 minutes was required, with the m-cresol peak appearing at 4 minutes, while and the insulin peak of

[0092] Quantification of Caffeine

[0093] appeared approximately 5 minutes later. Caffeine was quantified at a wavelength of 290 nm using a U-5100 ultraviolet / visible spectrophotometer from Hitachi (Tokyo, Japan). A 50 μl quartz cuvette was selected, and the samples were measured in triplicate without dilution.

[0094] Proof-of-Concept Model

[0095] Figure 4A Figure shows the hydrogel container 402 (left) and the modified flow cell 404 (right), which were used as Figure 3 a proof-of-concept model for the diffusion experiment shown in the first flow chart 300A of (regular human insulin, hexamer) and (insulin glulisine) were selected as the model peptide formulations for the initial setup. m-Cresol, which was present in both formulations, was also quantified as an internal standard.

[0096] With the help of a custom-made plastic reservoir, a cylindrical gel that forms both a physicochemical barrier and a pouch is prepared. The gel consists of 2% (w / w) agarose dissolved in PBS. After heating the gel to boiling point, a homogeneous mixture is obtained under constant stirring. The evaporated water is then replenished. The hot mixture is poured into a cylindrical plastic reservoir and an internal cylindrical plastic punch is inserted to form a pouch from which the drug formulation will be dispensed. After cooling at room temperature, the gel is formed and ready for use. A hydrogel container 402 with a gel thickness of approximately 2.5 mm was prepared.

[0097] The modified flow cell 404 was prepared from a 50 mL centrifuge tube (115 mm x 30 mm x 30 mm), roughly inspired by the flow cell of USP apparatus IV. The inlet port 406 and the outlet port 408 were formed using a Luer lock syringe needle after drilling the corresponding holes in the tube. The Luer lock syringe needle for the upper outlet port 408 was capped with a wire cutter, while the needle tip for the lower inlet port 406 was bent to direct the inflowing liquid to the bottom of the Falcon tube, from where the medium can flow to the outlet port 406 in an upward flow. The inserted needle was further secured with silicone sealant, threadseal tape, and sealing film to prevent leakage. In order to ensure that the drug formulation in the prepared gel pouch does not come into direct contact with the medium, a stainless steel wire cage 410 was made. The cage 410 was fixed to the inserted gel so that its opening was higher than the height of the upper outlet port 408. Because the level of the medium within the modified flow cell 404 does not rise above the upper outlet port 408, the drug formulation is separated from the medium and must choose a path through the hydrogel container 402. The modified flow cell 404 also includes a cap 412 to prevent accidental spillage from the cell 404.

[0098] Figure 4B Diagram of the in vitro drug testing Figure 4A Assay setup for a proof-of-concept model. Continuous media flow was achieved by connecting the modified flow cell 404 to a CP7-35 piston pump 414 (Sotax AG, Basel, Switzerland). A Luer lock adapter allows standard 1 / 4”-28UNF threads to be connected to the custom-made flow cell 404. At the 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 cell 404 in a closed-loop setup. The flow cell 404 was placed in a water bath 418 and the media temperature was raised to 37° ± 0.5C. To clear air bubbles from the tubing and to increase the temperature, the media was pumped through the flow cell 404 for at least 30 minutes. Once the desired temperature was reached, 1.5 mL of or (Equivalent to 150 IU or 5.25 mg of insulin) was injected into the coagulation capsule bag and then placed in cage 410. Samples of the medium with a volume of 0.2 mL were manually collected every hour during the first 7 hours, and then fresh medium was replenished. Then, insulin and m-cresol in the medium were quantified by HPLC. Subsequently, the cumulative diffusion of the model drug was calculated by comparing the amount of diffusion with the initially added drug concentration.

[0099] Subsequently, a reference experiment was conducted by adding insulin to the medium. This was to identify potential degradation or adsorption of insulin in the perfusion system. All experiments were repeated three times.

[0100] Figure 5 The figure shows the determination setup of the hydrogel container 100 in the USP apparatus IV 502 for the diffusion experiment shown in the second flowchart 300B for Figure 3 . To further improve and develop a diffusion determination that meets internationally recognized standards, the hydrogel container 100 was created for integration into the USP apparatus IV 502. The pharmacopoeial flow cell 504 of the USP apparatus IV 502 is a closed system with a heating jacket that allows for controlled flow of the medium from the bottom to the top of the flow cell 504.

[0101] Before finalizing the design of the current hydrogel container 100, multiple versions were created. Fusion 360 TM modeling software (Autodesk, California, USA) was used for the initial sketch of the 3D printed ring 114 that serves as the gel holder.

[0102] For fabricating the ring 114 of the container 100, a stereolithography (SLA) printer Form 2 TM (Formlabs, Massachusetts, USA) and HighTemp V2 TM resin (Formlabs, Massachusetts, USA) were used. This specific resin was chosen because it has high heat deflection and good compatibility with aqueous solvents. A layer thickness of 0.050 mm ensured high resolution, and with the addition of conventional printing support structures, the printing time was approximately 3 hours. After printing the ring 114, it was washed with isopropyl alcohol for 15 minutes and processed under ultraviolet light in an Asiga Flash TM curing station (Asiga, Alexandria, Australia) for several hours. The printed ring 114 was proven to be very durable and chemically resistant.

[0103] The ring 114 was installed onto a cylindrical mold, and the gel composition was filled into the mold. A punch was used to form an internal pocket of a similar size. Then, the drug formulation 106 was injected into the internal pocket of the reservoir 102, and the ring 114 was sealed with a rubber stopper 116.

[0104] The USP Apparatus IV 502 and various flow cells 504 have been described in various pharmacopoeias and related literature. For this diffusion experiment, the SOTAX CP7-35 piston pump 506, fraction collector 508, and SOTAX CE7 dissolution system were used. The medium 510 was injected into the reservoir 512, and the magnetic stirrer was operated at 150 rpm. The dissolution apparatus was operated in a closed-loop configuration, and the constant flow rate for all experiments was 8 ± 0.4 mL / min. Before inserting the flow cell 504 with an inner diameter of 22.6 mm, the system was set to bypass mode to purge the air in the capillary.

[0105] A single 5-mm ruby glass sphere and approximately 2.4 g of 1-mm-diameter glass beads 514 were filled into the flow cell 504 to ensure laminar flow. The heating jacket 516 maintained the temperature close to that found in subcutaneous tissue, and the temperature for all settings was 34 °C ± 0.5 °C. The corresponding drug formulation 106 was injected into the newly prepared container 100, sealed with a rubber stopper 116, and carefully placed on top of the glass beads 514. Subsequently, the flow cell 504 was assembled and placed in the main system.

[0106] The diffusion experiment was first conducted in container 100 with a caffeine stock solution (4 mg / mL) to evaluate the functionality of the new setup and the integrity of the hydrogel. 100 mL of PBS 510 was selected as the medium volume. The initial injection volume of the test drug was 1 mL. The experiment lasted for 8 hours, and samples with a volume of 0.5 mL were collected 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 a UV-Vis spectrometer. Subsequently, the cumulative diffusion of caffeine was calculated by comparing the amount of diffusion with the initially added drug concentration. All experiments were repeated three times.

[0107] In the next step, and were used to evaluate the new container 100. This was to assess the ability of the assay to distinguish compounds with different molecular sizes. PBS or SIB 510 with a volume of 60 mL was used respectively. 1 mL of each formulation 106 was injected into the reservoir 102 again. Samples with a volume of 0.5 mL were collected at 2, 4, 6, 8, 12, 16, 20, and 24 hours. The medium 510 was not replenished. HPLC was used to quantify insulin and m-cresol. Subsequently, the cumulative diffusion of insulin and m-cresol was calculated by comparing the amount of diffusion with the initially added drug amount.

[0108] In subsequent reference experiments, the two types of insulin were added to the receptor medium 510 in the presence of the container 100 to study the adsorption and degradation (recovery) of insulin over time. All experiments were repeated three times.

[0109] is a sustained-release formulation (depot formulation) of insulin. The suspension includes microcrystalline insulin in the presence of zinc and protamine. The performance test was conducted under the conditions described previously (using PBS as the release medium 510). In subsequent studies, an excess of sodium heparin (1 mg / mL) was added to the formulation 106. Heparin acts as a complexing agent, which triggers the release of insulin by forming a heparin-protamine complex.

[0110] Figure 6A illustrates a depiction and in the above reference Figure 4A and 4B a line graph of the diffusion curves in the experiments explained. Figure 6B illustrates a line graph depicting the results of the reference experiments in the above reference Figure 4A and 4B explained. The horizontal axis represents time (in hours), and the vertical axis represents the concentration percentage (%) of the formulation components. The first line 602 represents the data of regular human insulin from , the second line 604 represents the data of m-cresol from , the third line 606 represents the data of glulisine insulin from , and the fourth line 608 represents the data of m-cresol from .

[0111] Using this proof-of-concept model, the first experiment studied the and diffusion in PBS supplemented with 0.01% (w / v) Tween 80. HPLC analysis can quantify insulin (regular human insulin and glulisine insulin) as well as m-cresol. The cumulative diffusion is calculated as a percentage of the injected dose ( Figure 6A)。To quantify the significance of the differences shown in the diffusion behavior, the difference factor (f1) and the similarity factor (f2) were calculated. The f1 and f2 factors were developed by Moore and Flanner and later adopted by the FDA's immediate-release solid oral dosage form guidelines. The closer the f1 factor is to zero and the higher the calculated f2 factor, the more similar the two release curves are. To avoid any ambiguity during scale-up production and post-approval changes, the FDA has proposed limits of f1 ≤ 15 and f2 ≥ 50 to conclude that the releases of two selected formulations are similar. The FDA has specified several criteria that must be met before calculation, some of which are not applicable to the current setup, such as testing at least 12 individual dosage forms of both products.

[0112] and The diffusion curves of show that the cumulative diffusion of insulin and m-cresol increases over time, and both insulins were first detected after 3 hours. Regular human insulin from reached a peak of 9.4 ± 0.2% at 7 hours, while insulin lispro from

[0113] had a higher diffusion rate and reached 20 ± 1.0% at 7 hours (f1 = 119, f2 = 56). The f1 factor indicates a significant difference, while considering the FDA-specified threshold, the f2 factor would consider the two curves similar. In this case, if only one factor meets the given limit, the two formulations will be considered dissimilar, thanks to the ability of this assay to distinguish between the two insulin formulations. Meanwhile, their m-cresol counterparts diffused at a much faster rate and both reached a plateau at approximately 7 hours. The m-cresol in

[0114] appeared to diffuse to a smaller plateau at 84 ± 1.0%, while the m-cresol in Figure 6B reached a maximum at 88 ± 0.2%, but the difference was not significant (f1 = 5, f2 = 71). In addition, a reference experiment was conducted in which the pure formulations were injected into the medium ( ). The experiment showed that the insulin concentration decreased slightly over time, indicating that the adsorption or degradation process caused the recovery of regular human insulin in

[0115] Figure 7A to decrease from 95 ± 0.7% after 1 hour to 91 ± 4.7% after 7 hours, and the recovery of insulin lispro in

[0115] Figure 7A to decrease from 90 ± 1.5% after 1 hour to 84 ± 3.3% after 7 hours. Meanwhile, both m-cresol curves fluctuated around 100 ± 1.8%. The recovery curves of insulin and m-cresol were very similar (insulin: f1 = 5, f2 = 64; m-cresol: f1 = 1, f2 = 88).

[0115] Figure 7A illustrates the depiction of the referenceFigure 5 Line graph of the diffusion curve of the caffeine stock solution in the experiment explained above. Initially, caffeine was used as a small molecule and a stable model compound. The experiment was conducted in PBS for 8 hours, and it was found that the drug concentration in the receptor chamber increased steadily, where caffeine slowly diffused through the gel of container 100 and accumulated to approximately 101 ± 3% at 8.25 hours.

[0116] Figure 7B The illustration depicts the reference Figure 5 in the experiment explained above and line graph of the diffusion curve in PBS solution 510. Figure 7C The illustration depicts the reference Figure 5 in the experiment explained above and line graph of the diffusion curve in SIB solution 510. The horizontal axis represents time (in hours), and the vertical axis represents the concentration percentage (%) of the formulation components. The first line 702 represents the data of regular human insulin from and the second line 704 represents the data of m-cresol from the third line 706 represents the data of glulisine insulin from and the fourth line 708 represents the data of m-cresol from

[0117] Under the specified conditions, and were tested in PBS and the more biorelevant SIB. Similar to the test results found in the proof-of-concept study, hexameric human insulin had a slower diffusion rate than monomeric glulisine insulin during the first few hours. In PBS and SIB, the diffusion rate of insulin was the same and was first detected after 4 hours. The experiment was conducted for 24 hours, and no visible change occurred in the integrity of the hydrogel. In PBS, glulisine insulin reached 67 ± 0.2%, and its diffusion rate was faster than that of regular human insulin, whose peak was at 50 ± 4% (f1 = 44, f2 = 45).

[0118] Using the hydrogel container 100, two f - factors were calculated and found to exceed the given threshold, thus incontrovertibly indicating a significant difference between the two curves. Their internal standards reached a plateau at a similar rate, indicating that there was no significant loss of the 510 medium due to leakage in USPIV 502 throughout the run. In contrast, the tests in SIB 510 led to almost identical results (regular insulin: f1 = 2, f2 = 97; glulisine insulin: f1 = 3, f2 = 89), and both f1 and f2 tests were consistent with their significance. In SIB, glulisine insulin reached its peak after 24 hours at 67 ± 1.4%, while regular human insulin had a slower peak rate at 50 ± 3.2% (f1 = 49, f2 = 43). m - Cresol also reached a stable plateau here.

[0119] Figure 7D The figure shows a line graph depicting the reference Figure 5 results of the reference experiment explained above. The first line 710 represents data from regular human insulin, and the second line 712 represents data from glulisine insulin.

[0120] The reference experiment was conducted in and the case of directly injecting the receptor medium 510 into the flow - through cell 504. The hydrogel container 100 was also placed in this setup to determine that the interaction was covered by the recovery study in the presence of the agarose hydrogel. Figure 7D The recovery of the two insulins in PBS 510 is shown. A small but appreciable insulin loss was detected in both cases, with 78 ± 12% of regular insulin and 72 ± 24% of glulisine insulin recovered after 24 hours. The standard deviation was quite large compared to the usual diffusion runs. It can be assumed that insulin diffused into the gel present in this reference experiment, so 100% recovery was not achieved.

[0121] Figure 7E The figure shows a line graph depicting the reference Figure 5 in the experiment explained above and the diffusion curves of heparin - treated in PBS solution 510. The first line 714 represents data from untreated insulin, the second line 716 represents data from untreated m - cresol, the third line 718 represents data from treated insulin, and the fourth line 720 represents data from treated m - cresol.

[0122] It is an isotropic (NPH) human insulin suspension. Without further treatment, only 5.5 ± 3.8% of insulin was detected after 12 hours, and there was no appreciable increase thereafter. To release insulin from the suspension, an excess of heparin was added in subsequent experiments. The diffusion curve was more similar to that of hexameric human insulin, as a peak at 32 ± 49% was reached after 24 hours, although the similarity was not considered significant given the standard deviation. The m-cresol standard reached its stable plateau smoothly.

[0123] The preliminary proof-of-concept study (version 1.0) reflected the expected order of ranking, where the diffusion rate of hexameric insulin was slower than that of glulisine insulin. Contains zinc, a cation that can stabilize the hexameric state, while glulisine insulin has been engineered to reduce oligomer formation. Through two amino acid substitutions, the assembly of hexamers was reduced. The sequence of human insulin was altered at positions B3 (asparagine replaced by lysine) and B29 (lysine replaced by glutamate). In addition, is zinc-free. Due to the molecular weight difference, the molecular mobility and absorption rate of monomers are expected to be faster than those of dimers and hexamers. Apparently, glulisine insulin is marketed as a rapid-acting insulin, showing faster absorption in vivo.

[0124] At the same time, m-cresol was quantified to detect possible errors caused by gel preparation. Since m-cresol is a stable and small molecule, it was observed that they diffused faster than the two larger insulin molecules, which was consistent with expectations. In addition, the high recovery indicated no leakage in the perfusion cycle. However, non-specific interactions between m-cresol and proteins and existing excipients are known. This could explain and the reason why the m-cresol release curves reached a steady state at different levels, as their compositions were slightly different. Changes in the barrier properties caused by the interaction of hexameric insulin with the hydrogel may also affect the diffusion of m-cresol. Therefore, future evaluation of its diffusion with another small molecule compound is considered a beneficial addition to the characterization. Similar diffusion behavior can rule out the possibility of changes in the hydrogel structure.

[0125] Reference experiments suggest that insulin is lost or degraded over time. Due to the presence of a large number of hydrophobic surfaces, insulin (especially its monomeric form) adsorbs onto these surfaces, leading to aggregation and ultimately denaturation. Since insulin is directly injected into the medium in one go, a large amount of insulin comes into contact with these surfaces at a much faster rate than during normal operation. Adsorption onto these surfaces can explain why neither insulin was ever fully recovered, even at the earlier sampling points. Additionally, lysine insulin, which is mainly monomeric, also seems to be more prone to loss, which again demonstrates the adsorption of monomeric insulin. Further experiments may see the addition of higher concentrations of other surfactants, such as Tween 20.

[0126] As previously mentioned, the integration of the hydrogel container 100 into the USP apparatus IV 502 has been achieved, and the fraction collector enables the automation of release experiments, thus eliminating any human sampling errors that were more evident in experiments involving proof-of-concept models. The initial caffeine experiments confirmed its highly consistent and reproducible diffusion behavior with a low standard deviation. Caffeine had previously been used as a marker molecule to characterize agarose gels and exhibited extremely low plasma protein binding.

[0127] Using the United States Pharmacopeia apparatus IV 502 and the hydrogel container 100, the results of the initial design were successfully reproduced. In fact, the hydrogel container 100 showed more significant discriminative power. The differences in the rate and extent of the measured diffusion behavior were more pronounced.

[0128] A comparison between the release tests conducted in SIB and PBS indicates that the medium has no significant effect on the diffusion behavior. The ionic composition does not affect the permeation of insulin. Since these tests were only carried out using pure agarose gels, this may vary depending on the gel composition. The last experiment aimed to evaluate more complex drug formulations in the hydrogel container 100. is a microcrystalline suspension of insulin in the presence of protamine and zinc. In a physiological environment, protamine is broken down by enzymes in the subcutaneous tissue, releasing hexameric insulin from its complex. Alternatively, release can also be triggered in vitro. The use of an excess of heparin to form a stable complex with protamine results in the release of hexameric insulin. Future experiments may consider adding enzymes such as trypsin to achieve a higher level of biological relevance.

[0129] Overall, the hydrogel container 100 provides a reliable setup with optimal properties for further testing compounds in the USP apparatus IV. High sensitivity, reproducibility, and the ability to distinguish between different formulations have been achieved.

[0130] Based on the reported observations, the biopredictivity of the assay can be systematically evaluated and optimized. To understand the in vitro conditions of the donor and receptor chambers and to address the technical challenges posed by drug degradation through protein shear and adsorption, computational fluid dynamics (CFD) or other suitable software can be used to simulate the diffusion process in USP Apparatus IV.

[0131] Biopredictivity is achieved by systematically evaluating the changes in the observed in vitro diffusion rates in response to changes in the gel and media components, along with repeated benchmarking against the observed absorption rates in vivo. To this end, an in vitro-in vivo correlation (IVIVC) can be established to relate the data collected in vitro to the observed in vivo responses. Initially, the reported subcutaneous formulation pharmacokinetic profiles will be analyzed using MonolixSuite TM 2021 (Lixoft, Zug, Switzerland). To design a suitable computer model that allows simulation, Stella Architect TM (isee systems, Inc., New Hampshire, USA) can be used. To increase the level of biorelevance, the gel can be modified, for example, by using extracellular matrix components such as collagen, peptone, and hyaluronic acid. Serum proteins known to have stabilizing effects on other proteins will also be added to the media.

[0132] Those skilled in the art will appreciate that various changes and / or modifications can be made to the present invention as shown in the specific embodiments without departing from the broad scope of the present invention. Accordingly, the present embodiments are to be considered illustrative rather than restrictive in all respects.

Claims

1. A container for in vitro drug testing, the container comprising: A reservoir having a wall, wherein the reservoir has an opening and is configured to receive a pharmaceutical formulation through the opening, and wherein the wall is made of a gel composition.

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

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

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

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

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

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

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

9. The container according to any one of the preceding claims, wherein, The gel composition comprises one or more lipids selected from the group consisting of triglycerides and phospholipids.

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

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

12. The container according to any one of the preceding claims, wherein, The gel composition comprises serum proteins.

13. The container according to any one of the preceding claims, wherein, The container further comprises a stopper for sealing the opening of the reservoir.

14. The container according to any one of the preceding claims, further comprising a ring surrounding the perimeter of the opening of the reservoir for injecting the pharmaceutical preparation into the reservoir.

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

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

17. The container according to any one of claims 14 to 16, wherein, The ring comprises a collar that surrounds its inner wall near the base of the ring, thereby forming a recess to accommodate the perimeter of the opening of the reservoir.

18. The container according to any one of claims 14 to 17, wherein, The ring comprises a textured area that surrounds its inner wall near the top of the ring.

19. A method for preparing a container for in vitro drug testing, the method comprising the following steps: Forming a reservoir having a wall, wherein the wall is made of a gel composition; and Depositing a pharmaceutical formulation into the reservoir.

20. The method according to claim 19, wherein, Forming a reservoir having a wall comprises the steps of: Attaching a ring to a mold; Pouring the gel composition into the mold; and Inserting an inner punch into the gel composition to form the reservoir such that the ring surrounds the perimeter of the opening of the reservoir.

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

22. A test method, comprising: Depositing a pharmaceutical formulation into the container according to any one of claims 1 to 18; Immersing the container in a medium within a United States Pharmacopeia (USP) Type IV flow-through cell; And Monitoring the diffusion of the pharmaceutical formulation between the container and the medium.

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