Biphasic analyses for deconvoluting crystallization and drug absorption

Abstract

A system for evaluation of a pharmaceutical formulation in a biphasic test environment is provided. The components of the system can include a test apparatus, a measurement device, and a processor. The test apparatus can be configured to maintain a dissolution phase and an absorption phase. The measurement device can be configured to generate concentration data for a pharmaceutical analyte in the absorption phase over time. The processor can be configured to generate inferred concentration information for the pharmaceutical analyte in the dissolution phase from the concentration data and to generate an output indicative of behavior of the pharmaceutical formulation. Also provided are an apparatus for evaluation of a pharmaceutical formulation in a biphasic test environment and a non-transitory computer-readable medium storing instructions that, when executed by one or more processors of a system for evaluation of a pharmaceutical formulation in a biphasic test environment.

Description

25-021 (098121-00441)BIPHASIC ANALYSES FOR DECONVOLUTING CRYSTALLIZATION AND DRUG ABSORPTIONRELATED APPLICATIONS

[0001] The instant application claims priority to U.S. Provisional Application No. 63 / 776,645, filed on March 24, 2025, the entire contents of which are expressly incorporated by reference herein in their entirety.GOVERNMENT SUPPORT

[0002] This invention was made with government support under R35GM155235 awarded by National Institutes of Health. The government has certain rights in the invention.FIELD OF THE DISCLOSURE

[0003] The invention disclosed herein relates to crystallization dynamic analysis of materials, and in particular to the behavior of materials in a biphasic process.BACKGROUND

[0004] Pharmacokinetics is the study of the absorption, distribution, metabolism, and excretion of drugs within biological systems. One critical aspect of pharmacokinetics is the modeling of dissolution processes, as only dissolved drug molecules can permeate through biological membranes and get absorbed by the body. The dissolution performance of a pharmaceutical formulation, including both the amount of drug dissolved and the rate of dissolution, determines the amount and duration of drug absorption in the body. This information is essential for drug development, dosage optimization, and prediction of therapeutic efficacy.

[0005] Dissolution tests for oral formulations are usually performed in an aqueous solution, in which the dissolved drug concentration is measured as a function of time. Dissolution tests measure how much, how fast, and how reliable a drug is released from the formulation and enters the body. Several variations can be incorporated in dissolution tests, including buffer conditions with different additives to make the media body-fluid like, stirring under controlled conditions, and in some cases, the inclusion of an absorptive sink.

[0006] Dissolution modeling in a biphasic system addresses the transport of molecules between two phases, including release of the drug molecule from the formulation and transportPage 1 of 44MEl\60404268.v225-021 (098121-00441)into the absorptive sink. The dissolution of the drug occurs in different oral formulations such as tablets, capsules, and powders. The absorptive sink, such as that provided by an immiscible organic solvent layer mimics the fast drug absorption process in the body. Accurate modeling of these diffusion processes is essential for predicting drug bioavailability, controlling release kinetics, and optimizing therapeutic outcomes.

[0007] Current pharmacokinetic models incorporate dissolution and absorption processes using mathematical frameworks such as Fick's laws of diffusion, compartmental models, and physiologically based pharmacokinetic (PBPK) models. These approaches attempt to capture the dynamic exchange of drug molecules, considering parameters such as partition coefficients, diffusion coefficients, and membrane permeability. Currently, PBPK models have well-defined physiological parameters, and therefore, the accuracy of the pharmacokinetic model is largely dependent on formulation-relevant parameters, mostly on dissolution kinetics. For simple formulations, PBPK models often achieve satisfactory predictions. However, for more sophisticated formulations, their performance remains challenging to predict. Therefore, it is important to develop dissolution tests that can more accurately reflect the drug release kinetics in the body.

[0008] Supersaturating formulations are widely used as solubility-enhancing formulation strategies to increase bioavailability. Examples of such formulations include amorphous solid dispersions (ASDs), weakly basic drugs, salts, and lipid-based formulations. In supersaturated solutions, the solution contains more dissolved drug than its equilibrium solubility, rendering the solution thermodynamically unstable. Therefore, in such systems, the drug will crystallize with time, crash out from the solution, and return to its equilibrium solubility, leading to unpredictable or complete loss of bioavailability.

[0009] Different experimental techniques, such as the use of biorelevant media with different bile salts and biological surfactants, hollow-fiber membrane-based dissolution apparatuses and biphasic dissolution tests to mimic drug absorption, dynamic dissolution systems to simulate changing pH over time such as media transfer methods and TIM systems, are used to capture physiologically relevant dissolution performance of pharmaceutical formulations. Computational methods include advanced methods such as computational fluid dynamic simulations (CFD) to simulate hydrodynamics in the body affecting dissolution and population, and stochastic models to account for variability, empirical models such as the Higuchi model and Weibull model, mechanistic models such as the Noyes- Whitney equation,Page 2 of 44MEl\60404268.v225-021 (098121-00441)diffusion-layer and film models. Recent developments also integrate machine learning to improve predictive accuracy in dissolution and pharmacokinetic modeling.

[0010] Despite advancements, several challenges persist in accurately measuring and modeling dissolution, in particular, for supersaturating formulations. For example, crystallization usually occurs much faster in dissolution tests compared to that in the body, due to the lack of fast solute removal from drug absorption. For highly supersaturated solutions, amorphous precipitation and the formation of a cloudy solution hinder direct measurements of dissolved drug concentration and crystallization kinetics. Biological systems exhibit complex microenvironments with varying viscosity, pH, ionic strength, endogenous species such as bile salts and mucus, which affect dissolution and crystallization kinetics markedly. Empirical data fitting is not adequate as, for example, experimental techniques provide valuable insights but often require assumptions, leading to uncertainties in parameter estimation.

[0011] Thus, to enhance the predictive power and applicability of dissolution tests and modeling, improvements are needed. Addressing the challenges with the biphasic dissolution test and modeling will enable more accurate measurements and modeling of drug dissolution from supersaturating formulations, leading to better drug formulation strategies, bioavailability predictions, improved therapeutic efficacy, and enhanced patient outcomes. The present disclosure provides systems and methods for improved biphasic analysis.SUMMARY

[0012] The following brief summary is not intended to include all features and aspects of the present disclosure, nor does it imply that any claim must include all features and aspects discussed in this summary.

[0013] Accordingly, in one aspect, a system for evaluation of a pharmaceutical formulation in a biphasic test environment is provided. The components of the system include a test apparatus, a measurement device, and a processor. The test apparatus can be configured to maintain a dissolution phase and absorption phase. The measurement device can be configured to generate concentration data for a pharmaceutical analyte in the absorption phase over time. The processor can be configured to generate inferred concentration information for the pharmaceutical analyte in the dissolution phase from the concentration data and to generate an output indicative of behavior of the pharmaceutical formulation.Page 3 of 44MEl\60404268.v225-021 (098121-00441)

[0014] In one embodiment, the dissolution phase contains at least one of a buffered medium configured to maintain a selected pH, pure water, one or more surfactants, one or more lipids, or one or more polymers.

[0015] In one embodiment, the dissolution phase includes a buffer configured to maintain a pH that is configured to influence dissolution behavior, supers aturation behavior, crystallization behavior, induction time, or relative performance of the pharmaceutical formulation.

[0016] In one embodiment, the dissolution phase is an aqueous phase. In one embodiment, the absorption phase is an organic phase.

[0017] In one embodiment, the absorption phase contains at least one of a water-immiscible solvent and a non-polar organic solvent.

[0018] In one embodiment, the organic phase contains at least one of decanol, n-hexane, heptane, cyclohexane, benzene, n-butanol, butyl acetate, carbon tetrachloride, methyl-t-butyl ether, 2-butanone, pentane, diisopropyl ether, ethyl acetate, diethyl ether, toluene, trichloroethylene, dichloromethane, 1,2-dichloroethane, chloroform, 1 -octanol, 1 -dodecanol, oleic acid, isopropyl myristate, vegetable oil, soybean oil, castor oil, and olive oil.

[0019] In one embodiment, the pharmaceutical analyte can be selected from the group consisting of organic molecules, inorganic molecules, pharmaceutical molecules, biological molecules, amorphous materials, and environmental contaminants.

[0020] In one embodiment, the measurement device can be a concentration measurement device.

[0021] In one embodiment, the processor can be configured with at least one algorithm.

[0022] In one embodiment, the algorithm includes a Wagner-Nelson deconvolution algorithm, numerical deconvolution, point-area deconvolution, regularized deconvolution, nonparametric / spline-based methods, Wiener deconvolution algorithm, Richardson-Lucy algorithm, a Van Cittert deconvolution algorithm, other deconvolution methods, or a subtraction algorithm.Page 4 of 44MEl\60404268.v225-021 (098121-00441)

[0023] In another aspect, an apparatus for evaluation of a pharmaceutical formulation in a biphasic test environment is provided. The apparatus contains a vessel assembly, a concentration measurement device, and a controller. The vessel assembly is configured to maintain a dissolution phase and an absorption phase, wherein the dissolution phase comprises a pharmaceutical analyte. The concentration measurement device can be arranged to generate time-based concentration data for the pharmaceutical analyte in the absorption phase. The controller can be configured to receive the time-based concentration data and generate output indicative of behavior of the pharmaceutical formulation.

[0024] In one embodiment, the behavior includes at least one of dissolution behavior, supersaturation behavior, crystallization behavior, induction time, or relative performance of the pharmaceutical formulation.

[0025] In one embodiment, the concentration measurement device includes an inline spectroscopic device selected from the group consisting of a UV spectrophotometer, a UV-Vis spectrophotometer, a near-infrared spectrometer, a mid-infrared spectrometer, a Raman spectrometer, and a fluorescence spectrometer.

[0026] In one embodiment, the concentration measurement device can be an offline analytical device selected from the group consisting of a high-performance liquid chromatography system, a gas chromatography system, a mass spectrometer, and a nuclear magnetic resonance instrument.

[0027] In one embodiment, the vessel assembly further includes a mixing element configured to agitate at least the dissolution phase.

[0028] In one embodiment, the vessel assembly further includes a temperature control element configured to maintain the biphasic test environment at a selected temperature.

[0029] In one embodiment, the concentration measurement device is positioned to monitor the absorption phase through an optical path.

[0030] In another aspect, the present invention provides a non-transitory computer-readable medium storing instructions. The non-transitory computer-readable medium can be executed by one or more processors of a system for evaluation of a pharmaceutical formulation in a biphasic test environment. When executed by the processors, the non-transitory computer-Page 5 of 44MEl\60404268.v225-021 (098121-00441)readable medium, can cause the one or more processors to receive time-based concentration data for a pharmaceutical analyte in an absorption phase of the biphasic test environment. The processors can further generate, from the time-based concentration data, a concentration profile for the pharmaceutical analyte in the absorption phase, process the concentration profile using a deconvolution or subtraction algorithm to generate inferred concentration information for the pharmaceutical analyte in a dissolution phase of the biphasic test environment, generate output indicative of behavior of the pharmaceutical formulation based on the inferred concentration information, and, store the output in a library configured for post-processing analysis.

[0031] In one embodiment, the output is indicative of relative performance of the pharmaceutical formulation relative to other pharmaceutical formulations and is usable to rank, select, or exclude one or more of the pharmaceutical formulations.

[0032] In one embodiment, the dissolution phase and the absorption phase are selected to model transfer of the pharmaceutical analyte between biologically relevant compartments, and wherein the output is indicative of in vivo absorption performance of the pharmaceutical formulation.

[0033] In one embodiment, the output is indicative of crystallization behavior, induction time, or supersaturation behavior of the pharmaceutical formulation and is usable to evaluate a precipitation inhibitor, a crystallization modifier, an excipient system, a formulation dosage form, or a process condition for the pharmaceutical formulation.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The features and advantages of the invention are apparent from the following description taken in conjunction with the accompanying drawings in which:

[0035] FIG. 1 is a depiction of an apparatus disclosed herein, according to certain embodiments;

[0036] FIG. 2 is an abstraction regarding the transfer of solutes between the two phases, according to certain embodiments;

[0037] FIGs. 3A-3B are graphs showing absorption profiles of ketoconazole without (FIG.3A) and with crystallization (FIG. 3B), according to certain embodiments;Page 6 of 44MEl\60404268.v225-021 (098121-00441)

[0038] FIGs.4A-4B are graphs showing deconvoluted concentrations of ketoconazole without (FIG. 4A) and with crystallization (FIG. 4B), according to certain embodiments;

[0039] FIGs.5A-5B are graphs showing reduced donor solution concentration of ketoconazole due to drug absorption in the absence of crystallization (FIG. 5A) and actual crystallization kinetics in the case of crystallization (FIG. 5B), according to certain embodiments;

[0040] FIG. 6 is a graph showing the nucleation induction time as a function of fenofibrate concentration, according to certain embodiments;

[0041] FIG. 7 is a graph showing fenofibrate crystallization kinetics analyzed by a Michaelis-Menten type heterogenous nucleation model, according to certain embodiments;

[0042] FIG. 8 is a graph showing the nucleation induction time as a function of ketoconazole concentration, according to certain embodiments;

[0043] FIG. 9 is a graph shows ketoconazole crystallization kinetics analyzed by a Michaelis-Menten type heterogenous nucleation model, according to certain embodiments;

[0044] FIGs. 10A-10B are graphs show the impact of an absorption phase acting as absorption sink on ketoconazole (FIG. 10A) and enzalutamide (FIG. 10B) crystallization, according to certain embodiments;

[0045] FIGs. 11A-11F are graphs showing the impact of supersaturation on ketoconazole crystallization, according to certain embodiments;

[0046] FIGs. 12A-12D are graphs showing the impact of a stabilizer on ketoconazole crystallization, according to certain embodiments;

[0047] FIGs. 13A-13D are graphs showing ketoconazole immediate crystallization at 800 pg / mL in the presence of HPMC compared to PVPVA, according to certain embodiments;

[0048] FIG. 14 is a graph demonstrating dissolution of enzalutamide beyond its solubility in biphasic dissolution tests, according to certain embodiments;

[0049] FIG. 15 are graphs showing the results of biphasic dissolution tests indicating correct enzalutamide ASD formulation rank order, according to certain embodiments;Page 7 of 44MEl\60404268.v225-021 (098121-00441)

[0050] FIG. 16 are graphs showing the result of biphasic dissolution tests demonstrating comparable performance of fenofibrate ASD formulations containing hydroxypropyl methylcellulose acetate succinate (HPMCAS) HF and hydroxypropyl methylcellulose (HPMC) in the initial stage and rapid crystallization of a HPMCAS LF formulation (condition: 40 rpm, 20 mM bile salt, pH 6.8), according to certain embodiments.DETAILED DESCRIPTION

[0051] The technology disclosed herein provides a system for evaluation of a pharmaceutical formulation in a biphasic test environment. The system generates and analyzes data that describes dissolution and transfer of a pharmaceutical analyte between a dissolution phase and an absorption phase under selected test conditions. The data may include concentration data, time-based data, and derived data that may be used to identify dissolution behavior, supersaturation behavior, crystallization behavior, induction time, or relative performance of the pharmaceutical formulation. The system may support formulation screening and in vitro assessment of behavior that correlates with in vivo absorption performance, particularly where direct measurement in the dissolution phase is impaired by precipitation or crystallization. One advantage of the technology is the resultant reduction of animal use in the pharmaceutical industry.

[0052] In some embodiments, the technology may be embodied as a system for evaluation of a pharmaceutical formulation in a biphasic test environment. The system may include a test apparatus that maintains a dissolution phase and an absorption phase, a measurement device that generates data for a pharmaceutical analyte in the absorption phase over time, and a processor that generates derived data from the measured data. The derived data may indicate dissolution behavior, supersaturation behavior, crystallization behavior, induction time, or relative performance of the pharmaceutical formulation.

[0053] In some embodiments, the technology may be embodied as an apparatus for generation of analytical data from a pharmaceutical formulation in a biphasic test environment. The apparatus may include one or more vessels, one or more phase boundaries between a dissolution phase and an absorption phase, one or more mixing elements, and one or more measurement elements arranged to monitor concentration of a pharmaceutical analyte in the absorption phase. The apparatus may be configured to support collection of time-based concentration data under selected test conditions for subsequent analysis.Page 8 of 44MEl\60404268.v225-021 (098121-00441)

[0054] In some embodiments, the technology may be embodied as software stored in a non-transitory computer-readable medium or executed by one or more processors of an analytical instrument. The software may receive measured concentration data, generate time-based concentration profiles, apply one or more deconvolution models and / or subtraction models, and generate inferred concentration information for a pharmaceutical analyte in the dissolution phase. The software may further generate outputs that classify, rank, exclude, or otherwise evaluate pharmaceutical formulations according to observed or inferred behavior in the biphasic test environment.

[0055] In some embodiments, the technology may be embodied as a screening platform for development of pharmaceutical formulations. The screening platform may be used to compare candidate formulations, compare excipients, compare stabilizers, compare processing conditions, or identify conditions associated with crystallization, sustained supersaturation, or improved correlation with in vivo absorption behavior. The screening platform may therefore assist selection of formulations for further development, refinement, or in vivo study.

[0056] Advantageously, the technology disclosed herein may provide substantial benefit in the pharmaceutical industry, particularly in preformulation studies, formulation screening, process development, biopharmaceutics modeling, and comparative evaluation of pharmaceutical products. In many pharmaceutical programs, dissolution behavior, supers aturation behavior, and crystallization behavior affect bioavailability, dose consistency, and commercial viability. The present technology may be used to generate analytical data that assists identification of formulations that maintain dissolved analyte for a longer period, delay crystallization, or provide transfer behavior that better correlates with in vivo absorption performance. The technology may therefore support earlier and more informed selection of candidate formulations for further development.

[0057] In some embodiments, the technology may be used in development of supersaturating pharmaceutical formulations, including amorphous solid dispersions, lipid-based formulations, salt forms, weakly basic pharmaceutical formulations, and formulations that include precipitation inhibitors or crystallization modifiers. The technology may be used to compare candidate excipients, polymers, surfactants, solvents, process conditions, or dosage-form architectures under controlled biphasic test conditions. Data generated by the technology may reveal differences in dissolution rate, persistence of supers aturation, induction time, crystallization tendency, or relative transfer of analyte between phases. A user may thereforePage 9 of 44MEl\60404268.v225-021 (098121-00441)use the technology to rank formulations, eliminate poorly performing formulations, or identify formulation variables associated with improved performance.

[0058] In some embodiments, the technology may be used by pharmaceutical manufacturers, contract development and manufacturing organizations, contract research organizations, academic laboratories, and regulatory support groups. A manufacturer may use the technology during formulation development, scale-up, reformulation, lifecycle management, or comparative assessment of related products. A research organization may use the technology to screen a set of candidate formulations before animal studies or other resource-intensive testing. An academic or translational laboratory may use the technology to study mechanisms of dissolution, supersaturation, crystallization, and transfer behavior in complex pharmaceutical systems. The technology may therefore reduce experimental burden, improve screening efficiency, and provide data that supports technical decision making across multiple stages of product development.

[0059] The technology may also provide technical effects that improve interpretation of biphasic pharmaceutical testing. In some embodiments, the technology may provide a cleaner analytical path by generation of concentration data from an absorption phase when direct measurement in a dissolution phase is impaired by precipitation, turbidity, or crystallization. In some embodiments, the technology may generate derived data that reveals behavior in the dissolution phase from measured behavior in the absorption phase. In some embodiments, the technology may improve identification of crystallization events, induction time, or formulation-dependent transfer behavior that may not be readily apparent from direct observation alone. The technology may therefore improve characterization of pharmaceutical formulations and improve the predictive value of in vitro testing.

[0060] In some embodiments, the technology may be used as part of a broader screening platform that guides selection of formulations for additional work. For example, a user may use the technology to select between candidate formulations, to identify promising excipient systems, to select process conditions associated with delayed crystallization, or to prioritize formulations for in vivo study. The technology may also support comparison of a reference formulation and a modified formulation, comparison of formulations prepared by different manufacturing methods, or comparison of formulations under different media, temperature, agitation, or phase- volume conditions. The technology may therefore be integrated into routine formulation screening, investigational studies, or commercial development workflows.Page 10 of 44MEl\60404268.v225-021 (098121-00441)

[0061] In order to provide some context for the teachings herein, a non-limiting introduction for some related terminology is now provided.

[0062] Generally, as used here, “biphasic test environment” refers to an analytical environment that includes a first liquid phase and a second liquid phase maintained in contact during a test and arranged to permit transfer of an analyte between the phases under controlled conditions. In one example, the biphasic test environment includes a dissolution phase and an absorption phase that is immiscible or physically separated with the dissolution phase, wherein a pharmaceutical analyte enters the dissolution phase from a pharmaceutical formulation and thereafter transfers from the dissolution phase into the absorption phase. Similar examples include a vessel having vertically separated dissolution and absorption phases, a vessel having laterally separated phases with an interface therebetween, and a test chamber in which the phases are separated by a membrane or other boundary that permits analyte transfer. Other similar examples include two compartments, one donor compartment and one acceptor compartment, physically separated dissolution and absorption phases, which are integrated with a hollow-fiber membrane module. These examples are illustrative only and do not require any particular vessel or compartment shape, orientation, interface geometry, or transfer mechanism unless expressly recited.

[0063] Generally, as used here, “pharmaceutical formulation” refers to a composition that includes a pharmaceutical analyte and one or more additional components arranged for administration, dissolution testing, release testing, screening, or other performance evaluation. In one example, the pharmaceutical formulation is an amorphous solid dispersion that includes a drug and a polymeric stabilizer. Similar examples include tablet formulations, capsule formulations, lipid-based formulations, salt formulations, weakly basic pharmaceutical formulations, suspensions, and other dosage-form or intermediate formulations prepared for evaluation of dissolution, supersaturation, crystallization, or transfer behavior. These examples are illustrative only and do not require any particular dosage form, route of administration, excipient set, or manufacturing history unless expressly recited.

[0064] Generally, the pharmaceutical formulation may be introduced to the dissolution phase in any suitable physical form that permits release of a pharmaceutical analyte during testing. In some embodiments, the pharmaceutical formulation may be provided as a solid dosage form, a particulate material, a powder, a granule, a pellet, a tablet, a capsule, a suspension, a dispersion, a gel, an emulsion, a lipid-based formulation, a solution, or another dosage-form orPage 11 of 44MEl\60404268.v225-021 (098121-00441)intermediate presentation. In some embodiments, the pharmaceutical formulation is added to the dissolution phase, and the pharmaceutical analyte is released into the dissolution phase and thereafter transfers from the dissolution phase into the absorption phase. These examples are illustrative only and are not to be construed as limiting.

[0065] Generally, as used here, “pharmaceutical analyte” refers to a chemically identifiable component of a pharmaceutical formulation that is monitored directly or indirectly during a test to evaluate formulation behavior. In one example, the pharmaceutical analyte is an active pharmaceutical ingredient released from an amorphous solid dispersion into a dissolution phase and thereafter transferred into an absorption phase. Similar examples include a neutral drug, an ionizable drug, a salt-form drug species, a prodrug, or another therapeutically relevant molecular species released from a formulation during a biphasic test. These examples are illustrative only and do not require that the pharmaceutical analyte be the sole active component, the only monitored species, or present in any particular concentration range unless expressly recited. The pharmaceutical analyte may be present in various physical states, including solvated molecules, ions, or amorphous and crystalline particles.

[0066] Generally, as used here, “dissolution phase” refers to a simulated systemic environment in which a dosage of pharmaceutical formulation undergoes physical breakdown, dilution, and / or solubilization of the active pharmaceutical ingredient to a dissolution medium or physiological fluid. Similar examples of dissolution medium or physiological fluid in dissolution phase include buffered solutions, acidic media, basic media, water, gastric juice, intestinal fluid, saliva, lung surfactant / lining fluid, lachrymal fluid, and interstitial fluid. In some embodiments, the dissolution phase could be an aqueous phase.

[0067] Generally, as used here, “absorption phase” refers to a simulated systemic environment that accepts transfers of a pharmaceutical analyte during a biphasic test, from a physically separated dissolution phase. In some embodiments, an absorption phase could be an organic phase.

[0068] Generally, as used here, “organic phase” refers to a liquid phase that is distinct from and at least partially immiscible with a dissolution phase and that accepts transfer of a pharmaceutical analyte during a biphasic test. In one example, the organic phase includes decanol and functions as an absorptive sink for the pharmaceutical analyte. Similar examples include octanol and other pharmaceutically acceptable or analytically suitable organic liquidsPage 12 of 44MEl\60404268.v225-021 (098121-00441)that receive analyte from the dissolution phase and permit concentration measurement over time. These examples are illustrative only and do not require any specific solvent identity, polarity, density, or partition coefficient unless expressly recited. The organic phase can be further defined by its relative density, which determines its position as either the upper or lower layer in the biphasic system.

[0069] Generally, as used here, “absorptive sink” refers to a phase, region, apparatus, or medium that receives a pharmaceutical analyte from another phase in an amount effective to reduce concentration of the pharmaceutical analyte in the originating phase or to alter concentration behavior of the pharmaceutical analyte over time. In one example, an absorption phase functions as an absorptive sink by accepting dissolved pharmaceutical analyte from a dissolution phase during a biphasic dissolution test. Similar examples include sink phases that promote continued dissolution, delay crystallization, alter supers aturation behavior, or provide a measurable concentration profile associated with analyte transfer. These examples are illustrative only and do not require any particular sink strength, partition rate, or mechanistic explanation unless expressly recited.

[0070] Generally, as used here, “concentration data” refers to data that represents, directly or indirectly, amount of a pharmaceutical analyte in a selected phase, region, or sample at one or more times during a test. In one example, the concentration data includes optical measurement data from the absorption phase that is converted into analyte concentration values over time. Similar examples include spectroscopic data, chromatographic data, detector output, sampled concentration values, calibrated signal values, and derived concentration values associated with a selected phase of the test environment. These examples are illustrative only and do not require any particular detector type, calibration format, unit system, or sampling interval unless expressly recited.

[0071] Generally, as used here, “time-based concentration data” refers to concentration data associated with two or more different times during a test and arranged to describe temporal change in analyte behavior. In one example, the time-based concentration data includes absorption-phase analyte concentration values measured at a plurality of time points during dissolution of a pharmaceutical formulation. Similar examples include periodic measurements, continuous measurements, sampled profiles, or reconstructed profiles that permit evaluation of dissolution, transfer, supersaturation, or crystallization behavior over time. These examples arePage 13 of 44MEl\60404268.v225-021 (098121-00441)illustrative only and do not require equal spacing of measurements, continuous acquisition, or any particular minimum number of measurements unless expressly recited.

[0072] Generally, as used here, “deconvolution model” or “deconvolution” refers to a computational framework that uses measured concentration behavior in one phase to generate inferred concentration information for an analyte in another phase or otherwise infer less directly observable behavior of the analyte during the test. In one example, the deconvolution model applies a Wagner-Nelson approach to concentration data measured in the absorption phase to generate inferred concentration information for the pharmaceutical analyte in the dissolution phase. Similar examples include first-order absorption-based models, subtraction methods, and other deconvolution approaches that correlate measured transfer behavior with inferred concentration behavior in another phase. These examples are illustrative only and do not require use of Wagner-Nelson or any single mathematical approach unless expressly recited.

[0073] Generally, as used here, “inferred concentration information” refers to data generated from measured data by analytical processing to represent estimated analyte concentration behavior in a phase, region, or condition that is not directly measured in the same manner. In one example, the inferred concentration information includes an estimated aqueous-phase concentration profile generated from measured absorption-phase concentration data. Similar examples include reconstructed concentration curves, estimated supersaturation levels, estimated concentration maxima, and other analytically derived representations of analyte behavior that are produced from measured data and a model. These examples are illustrative only and do not require perfect reconstruction, direct observability of the inferred phase, or any particular confidence metric unless expressly recited.

[0074] Generally, as used here, “dissolution behavior” refers to behavior associated with release of a pharmaceutical analyte from a pharmaceutical formulation into a liquid phase over time. In one example, the dissolution behavior includes an increase in dissolved analyte concentration in a dissolution phase during initial exposure of an amorphous solid dispersion to the dissolution phase. Similar examples include delayed release, rapid release, sustained release, concentration plateau behavior, rapid release followed by liquid-liquid phase separation, and release behavior associated with different formulations, excipients, or process conditions. These examples are illustrative only and do not require complete dissolution, monotonic increase, or any particular release mechanism unless expressly recited.Page 14 of 44MEl\60404268.v225-021 (098121-00441)

[0075] Generally, as used here, “supersaturation behavior” refers to behavior associated with a dissolved analyte concentration that exceeds an equilibrium solubility level for at least a portion of a test period or otherwise reflects departure from equilibrium dissolution conditions. In one example, the supersaturation behavior includes generation of a transient aqueous-phase analyte concentration above an equilibrium solubility level following dissolution of a pharmaceutical formulation. Similar examples include sustained supersaturation, rapid loss of supersaturation, delayed loss of supersaturation, and formulation-dependent differences in maintenance of dissolved analyte above equilibrium concentration. These examples are illustrative only and do not require a specific solubility model, a specific threshold duration, or any single mechanism of supersaturation unless expressly recited.

[0076] Generally, as used here, “crystallization behavior” refers to behavior associated with formation, onset, progression, inhibition, delay, or other occurrence of crystalline material from a pharmaceutical analyte during a test. In one example, the crystallization behavior includes onset of crystal formation from a supersaturated dissolution phase after a period of delayed nucleation. Similar examples include immediate crystallization, delayed crystallization, heterogeneous crystallization, homogeneous crystallization, reduced crystallization in the presence of a stabilizer, and crystallization behavior inferred from a change in transfer profile or inferred concentration profile. These examples are illustrative only and do not require direct visual observation of crystals, any specific crystal form, or any single detection method unless expressly recited.

[0077] Generally, as used here, “induction time” refers to a period between establishment of a condition associated with possible crystallization and a detectable indication of crystallization behavior. In one example, the induction time is the interval between formation of a supersaturated condition and a detectable change in inferred concentration behavior that indicates crystallization onset. Similar examples include time to first measurable deviation in transfer rate, time to first detectable precipitation-related signal, and time to first analytical indication of a crystallization event. These examples are illustrative only and do not require direct microscopic detection, a specific threshold criterion, or any single analytical signal unless expressly recited.

[0078] Generally, as used here, “relative performance” refers to comparative behavior of two or more pharmaceutical formulations under one or more common test conditions. In one example, the relative performance includes rank ordering of candidate formulations accordingPage 15 of 44MEl\60404268.v225-021 (098121-00441)to maintenance of supersaturation, delay of crystallization, or extent of analyte transfer into an absorption phase during a defined test interval. Similar examples include comparison of formulations prepared with different polymers, excipients, process conditions, salt forms, or other dosage-form architectures. These examples are illustrative only and do not require absolute equivalence of all test conditions beyond those expressly selected for comparison, or any particular ranking algorithm, unless expressly recited.

[0079] Generally, as used here, “formulation screening” refers to evaluation of two or more candidate pharmaceutical formulations or formulation variables to identify one or more candidates for further development, modification, or testing. In one example, the formulation screening includes comparison of candidate amorphous solid dispersions to identify a formulation associated with delayed crystallization and greater analyte transfer into an absorptive sink phase. Similar examples include screening of excipient systems, polymer types, surfactants, process conditions, dosage forms, and media conditions. These examples are illustrative only and do not require high-throughput testing, elimination of all non-selected candidates, or any particular screening scale unless expressly recited.

[0080] Turning to FIG. 1, an example of a system 100 for evaluation of a pharmaceutical formulation in a biphasic test environment is shown. The components of the system include a test apparatus 120, a measurement device 150, and a processor 160. The test apparatus 120 may be configured to maintain a dissolution phase 140 and an absorption phase 130. The measurement device 150 may be configured to generate concentration data for a pharmaceutical analyte in the absorption phase over time. The processor 160 may be configured to generate inferred concentration information for the pharmaceutical analyte in the dissolution phase from the concentration data and to generate an output indicative of behavior of the pharmaceutical formulation. FIG. 2 shows an abstraction regarding pharmaceutical molecule transitions between the dissolution phase 140 and absorption phase 130.

[0081] As shown, the apparatus may include an interface 170, which may be a computer or other user-operated interface for controlling and / or observing functions of the apparatus. The interface 170 may show, for example, elapsed time, concentration data, temperature, inferred concentration information, or any other aspects of the operation or testing results.

[0082] In an exemplary method, the test apparatus 120 is assembled with a selected dissolution phase 140 and absorption phase 130. A pharmaceutical formulation with a pharmaceuticalPage 16 of 44MEl\60404268.v225-021 (098121-00441)analyte to be evaluated is added to the dissolution phase 140 according to a desired protocol. Mixing may be performed, and temperature may be controlled. The measurement device 150 is used either continuously or at intervals to generate concentration data for the pharmaceutical analyte in the absorption phase 130. In some embodiments, the processor 160 generates inferred concentration information for the pharmaceutical analyte in the dissolution phase 140 from the concentration data and generates an output indicative of behavior of the pharmaceutical formulation.

[0083] The dissolution phase can be an aqueous phase. The dissolution phase 140 can include a liquid medium or solution in which water or a polar solve mixture is the primary solvent component. The dissolution phase 140 may include one or more pharmaceutical formulations that are dissolved or partially dissolved therein. In one embodiment, the pharmaceutical formulation includes one or more water-soluble or water-dispersible components, including but not limited to salts, buffers, surfactants, stabilizers, or active pharmaceutical ingredients, and maintains a distinct phase boundary from a co-existing absorption phase. In one embodiment, the dissolution phase 140 includes a pharmaceutical analyte. In some embodiments, the pharmaceutical analyte is selected from one or more of the following: organic molecules, inorganic molecules, pharmaceutical molecules (e.g. fenofibrate, ketoconazole, enzalutamide), biological molecules (e.g. amino acid, protein, nucleic acid, pathogen fragment), amorphous materials (e.g. amorphous nanoparticles, silica, amorphous solid dispersions), environmental contaminants (e.g. organic pollutants, pesticides, volatile organic compounds, airborne contaminants), hydrophobic substances, hydrophilic substances, volatiles, and surfactants. In various embodiments, the dissolution phase 140 is provided or contains a pharmaceutical analyte (e.g., pharmaceutical analyte to be evaluated) or formulation containing the analyte at a concentration above its amorphous solubility. The concentration of the pharmaceutical analyte can provide, using the disclosed systems and methods, information about crystallization kinetics in the presence of amorphous precipitates and absorptive sink. In various embodiments, the dissolution phase 140 is provided or contains a pharmaceutical analyte (e.g., pharmaceutical analyte to be evaluated) or formulation containing the analyte at a concentration above its crystalline solubility but below or equal to amorphous solubility. The concentration of the pharmaceutical analyte can provide, using the disclosed systems and methods, information about crystallization kinetics of the pharmaceutical analyte in the presence of absorptive sink.Page 17 of 44MEl\60404268.v225-021 (098121-00441)

[0084] In some embodiments, the dissolution phase 140 is provided or contains a pharmaceutical analyte (e.g., pharmaceutical analyte to be evaluated) or formulation containing the analyte at a concentration above its crystalline solubility. The concentration of the pharmaceutical analyte can provide, using the disclosed systems and methods, information about shifted solubility boundaries in the presence of an absorptive sink.

[0085] In some embodiments, the dissolution phase 140 also includes one or more crystallization inhibitors (e.g, hydroxypropyl methylcellulose (HPMC) or polyvinyl alcohol (PVA)).

[0086] In some embodiments, the dissolution phase 140 also includes one or more biorelevant components such as bile salts.

[0087] In some embodiments, the dissolution phase 140 contains a buffered medium configured to maintain a selected pH. The term “buffered medium” as used herein refers to an aqueous solution comprising a buffering agent in an amount sufficient to resist changes in pH upon the addition of acidic or basic species. The buffered medium typically comprises a mixture of a weak acid and its conjugate base, or a weak based in its conjugate acid, which maintains the solution within a predetermined pH range.

[0088] In one embodiment, the buffered medium is added to the dissolution phase to maintain a selected pH range, in order to include dissolution behavior, including but not limited to influence dissolution behavior, supersaturation behavior, crystallization behavior, induction time, or relative performance of the pharmaceutical formulation.

[0089] The buffered medium within the dissolution phase may function as a chemical regulator of the hydrogen ion activity, thereby providing a stabilized pH environment that directly modulates the ionization state and solubility profile of the pharmaceutical analyte. By maintaining the dissolution phase at a predetermined pH value, the buffered medium provides an environment that may be useful to systematically influence the dissolution behavior and supersaturation behavior of the analyte, particularly by preventing localized pH fluctuations that would other lead to non-uniform precipitation. Consequently, aspects of the buffered medium acts may be adjusted to tune the physical transformation pathways of the pharmaceutical analyte, ensuring reproducible experimental outcomes and a comprehensive characterization of the kinetic properties of the pharmaceutical formation.Page 18 of 44MEl\60404268.v225-021 (098121-00441)

[0090] In one embodiment, the dissolution phase is an aqueous phase, and the absorption phase is an organic phase. In one embodiment, the organic phase contains at least one of a water-immiscible solvent and a non-polar organic solvent.

[0091] In one embodiment, the organic phase contains at least one of decanol, n-hexane, heptane, cyclohexane, benzene, n-butanol, butyl acetate, carbon tetrachloride, methyl-t-butyl ether, 2-butanone, pentane, diisopropyl ether, ethyl acetate, diethyl ether, toluene, trichloroethylene, dichloromethane, 1,2-dichloroethane, chloroform, 1 -octanol, 1 -dodecanol, oleic acid, isopropyl myristate, vegetable oil, soybean oil, castor oil, and olive oil. The absorption phase 130 can serve a number of purposes, including acting as an absorptive sink that mimic in vivo pharmaceutical analyte absorption.

[0092] In one embodiment, the absorption phase and dissolution phase are separated in two chambers, with a donor chamber and a receptor chamber, separated by the direct interface of the absorption phase and dissolution phase using a membrane. In one embodiment, the absorption phase and dissolution phase are both contained in a single vessel, with the absorption phase floating directly on top of the aqueous layer.

[0093] In one embodiment, the pharmaceutical analyte can be selected from the group consisting of organic molecules, inorganic molecules, pharmaceutical molecules, biological molecules, amorphous materials, and environmental contaminants.

[0094] In one embodiment, the pharmaceutical analyte includes pharmaceutical molecules. Examples of pharmaceutical molecules include, but are not limited to fenofibrate, ketoconazole, or enzalutamide.

[0095] The term “organic molecules” as used herein refers to any carbon-based chemical compound comprising at least one carbon atom covalently bonded to other atoms, typically including, but not limited to hydrogen, oxygen, nitrogen, sulfur, or halogens. The term encompasses, without limitation, aliphatic, aromatic, and heterocyclic structures, as well as saturated and unsaturated hydrocarbons, polymers, and synthetic intermediates. Exemplary organic molecules include, but are not limited, alkanes, alcohols, carboxylic acids, and esters.

[0096] The term “inorganic molecule” as used herein refers to a chemical compound that generally lacks carbon-hydrogen bonds. Exemplary inorganic molecules include, without limitation, water, sodium chloride, sulfuric acid, and various metal-coordinated complexes.Page 19 of 44MEl\60404268.v225-021 (098121-00441)

[0097] The term “pharmaceutical molecule” as used herein refers to a chemically or biologically active substance, or a precursor thereof, intended for use in the medical diagnosis, cure, treatment, or preventions of disease in humans or animals. A pharmaceutical molecule includes active pharmaceutical ingredients (APIs), prodrugs, metabolites, and salts thereof, whether small molecules or complex macromolecules. Exemplary pharmaceutical molecules include without limitation, antibiotics and hormones.

[0098] The term “biological molecule” or “biomolecule” as used herein refers to any organic compound that is produced by, or is a constituent of, a living organism a cellular system. The class of biological molecule or biomolecule include both primary and secondary metabolites as well as large macromolecules essential for biological signaling, structure, or catalysis. Exemplary biological molecules include, without limitation, proteins, peptides, nucleic acids (DNA and RNA), lipids, and polysaccharides.

[0099] The term “amorphous materials” as used herein refers to a solid-state substance characterized by a lack of long-range, repeating crystalline order or a defined molecular lattice. In an amorphous state, the constituent atoms or molecules are arranged in a substantially disordered or random fashion, often resulting in unique solubility, dissolution, and thermodynamic properties compared to a crystalline counterparts. Exemplary amorphous materials include, without limitation, polymers, amorphous solid dispersions of pharmaceutical analyte, and amorphous nanoparticles.

[0100] The term “environmental contaminants” as used herein refers to any chemical or biological species that is extraneous to the intended composition or is potentially deleterious to an ecosystem. The class of environmental contaminants may be present in dissolution phase, soil, or air, and covers substances originating from industrial, agricultural, or anthropogenic sources. Exemplary environmental contaminants include, without limitation, pesticides, herbicides, heavy metal ions, and perfluoro alkyl substances (PFAS).

[0101] The term “measurement device” as used herein refers to any analytical instrument, apparatus, or sensor-based system configured to detect, quantify, or characterize a physical, chemical, or biological property of a substance or a medium. A measurement device of the disclosed system may include at least one transducer or detector capable of generating a signal, such as an electrical, optical, or thermal signal, corresponding to a parameter of interest, including but not limited to concentration, pH, temperature, pressure, or molecular mass.Page 20 of 44MEl\60404268.v225-021 (098121-00441)Generally, the term “measurement device” is intended to encompass both standalone laboratory instruments and integrated sensor components, including, without limitation, spectrophotometers, chromatography systems, mass spectrometers, pH meters, and electrochemical sensors. The measurement device may further include data processing circuitry for the conversion of raw signals into a human-readable or machine-storable format. Exemplary measurement devices include, without limitations, UV-Visible (UV-Vis) Spectrophotometers, Fluorescence Spectrometers, Infrared Spectrometers, Refractometers, High-Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Ion Chromatography (IC), Mass Spectrometers (MS), Nuclear Magnetic Resonance (NMR) Spectrometers, pH Meters, Ion-Selective Electrodes (ISE), Conductivity Meters, X-Ray Diffractometers, Differential Scanning Calorimeters and other such devices as deemed suitable.

[0102] In one embodiment, the measurement device can be a concentration measurement device.

[0103] Generally, the term “concentration measurement device” as used herein refers to an analytical instrument or apparatus configured to quantify the amount of an analyte, including dissolved particles, suspended particles, or amorphous materials, within a sample solution or medium. A concentration measurement device may comprise at least one sensor or detector configured to detect or measure a physical, chemical, or optical property of the sample that indicates the concentration of a pharmaceutical analyte. The term “concentration measurement device” encompasses, without limitation, devices utilizing principles of spectroscopy, refractometry, electrochemistry, or mass spectrometry to generate a signal representative of the concentration. Exemplary concentration measurement device includes, without limitations, UV-Vis spectrophotometer, refractometer, conductivity meter, and High-performance liquid chromatography (HPLC).

[0104] The term “processor” as used herein refers to one or more electronic circuits, devices, or systems configured execute logical, arithmetic, or input / output operations upon data. A processor may be a single-core or multi-core unit and may include, without limitation, a central processing unit (CPU), a graphics processing unit (GPU), a digital signal processor (DSP), a field-programmable gate array (FPGA), or an application- specific integrated circuit (ASIC). In the disclosed technology, the processor is configured to receive signals from at least one measurement device, perform data transformation according to a pre-defined set ofPage 21 of 44MEl\60404268.v225-021 (098121-00441)instructions, and generate an output, such as a calculated concentration, a pH value, or a control signal for a biphasic system. As shown in FIG. 1, the processor 160 may be included with the interface 170 in a unitary computing device. Examples of suitable computing devices include a personal computer (PC) or similar device (such as a tablet computer). The computing device may therefore be provided with components and functionality as described herein and deemed appropriate.

[0105] In one embodiment, the processor can be configured with at least one algorithm.

[0106] In one embodiment, the algorithm includes a Wagner-Nelson deconvolution algorithm, numerical deconvolution, point-area deconvolution, regularized deconvolution, nonparametric / spline-based methods, Wiener deconvolution algorithm, Richardson-Lucy algorithm, a Van Cittert deconvolution algorithm, other deconvolution methods, or a subtraction algorithm.

[0107] In one embodiment, the disclosed system operates through the coordinated functional integration of the test apparatus, the measurement device and the processor. The test apparatus defines a reaction or extraction volume containing the dissolution phase and the absorption phase, maintaining a stable liquid-liquid interface for the partitioning of the pharmaceutical analyte. During operation, the measurement device is configured for inline analysis of a physical or chemical property, such as the concentration, through collection of a raw signal (e.g., an optical absorbance or electrochemical potential) of the sample and pharmaceutical analyte. The measurement device is further operatively connected with the processor, and transmit the raw signal to the processor. The processor, executing a set of predefined algorithms, transforms this raw signal into a quantitative value, such as a molar concentration of the pharmaceutical analyte, and generate an output that indicates the behavior of the pharmaceutical analyte.

[0108] In an alternative embodiment, the measurement device is configured for offline measurement. Exemplary offline measurement tools may include for example a gas chromatography system, a mass spectrometer, and a nuclear magnetic resonance instrument. In offline measurement, the absorption phase may be sampled at various intervals, e.g., via a fluid path in connection with the absorption phase and controlled by the processor 140.

[0109] In another embodiment, an apparatus for evaluation of a pharmaceutical formulation in a biphasic test environment is provided. In this example, the apparatus contains Page 22 of 44MEl\60404268.v225-021 (098121-00441)a vessel assembly, a concentration measurement device, and a controller. The vessel assembly can be configured to maintain the dissolution phase and the absorption phase during a test. The dissolution phase can be configured to receive a pharmaceutical analyte from the pharmaceutical formulation and the absorption phase can be configured to receive the pharmaceutical analyte from the dissolution phase. The concentration measurement device can be arranged to generate time-based concentration data for the pharmaceutical analyte in the absorption phase. The controller can be configured to receive the time-based concentration data and generate output indicative of behavior of the pharmaceutical formulation.

[0110] The term “vessel assembly” as used herein refers to a structural containment system configured to hold, mix, or process one or more liquid or gas phases, including the dissolution phase and the absorption phase of the disclosed system. The vessel assembly may include at least one reaction chamber or fluid reservoir constructed from a chemically insert material, including but not limited to glass, stainless steel, and a fluorinated polymer. The assembly may further include one or more functional sub-components, including but not limited to inlet and outlet ports for the introduction or removal of fluids, stirrers or impellers for phase dispersion, thermal jackets for temperature regulation, optical windows for interface monitoring, and pH sensor probe for pH monitoring. The shape, structure, material composition, and physical properties of the vessel assembly may be varied or optimized according to specific experimental or industrial requirements without departing from the scope of the disclosed technology.

[0111] The term “controller” as used herein refers to a hardware-based or software-implemented functional unit configured to manage, direct, or regulate the operation of one or more components of the disclosed system. The controller is operably connected to the concentration measurement device and the vessel assembly, and is configured to translate computational instructions into physical actions, including but not limited to, send and receipt of the measurement or detection signal, generating output data based on the measurement or detection signal, modulation of stirring speeds, adjustment of thermal parameters via a heating or cooling element, and monitoring of the pH value of the dissolution phase and absorption phase through pH measurement.

[0112] In one embodiment, the behavior includes at least one of dissolution behavior, supersaturation behavior, crystallization behavior, induction time, or relative performance of the pharmaceutical formulation.Page 23 of 44MEl\60404268.v225-021 (098121-00441)

[0113] In one embodiment, the vessel assembly further includes a mixing element configured to agitate at least the dissolution phase during the test.

[0114] Generally, the term “mixing element” as used herein refers to a mechanical or fluidic component configured to impart kinetic energy to a liquid medium such as the dissolution phase or the absorption phase, to facilitate mass transfer, dissolution, or phase dispersion. A mixing element of the disclosed system may comprise a movable member, including but not limited to an impeller, stirrer, magnetic stir bar, or rotor, or a stationary structure, including but not limited to a static mixer, baffle, or nozzle, designed to induce turbulent or laminar flow. In the disclosed system, the mixing element is configured to facilitate dissolution of the pharmaceutical analyte in at least the dissolution phase. The operational parameters of the mixing, such as rotational speed (RPM) or flow rate, may be regulated by a controller.

[0115] In one embodiment, the vessel assembly further includes a temperature control element configured to maintain the biphasic test environment at a selected temperature.

[0116] Generally, the term “temperature control element” as used herein refers to one or more components, devices, or systems configured to monitor, maintain, increase, or decrease the thermal energy of a medium, including but not limited to the dissolution phase and absorption phase, within a predetermined range. A temperature control element of the disclosed system comprises at least one hear exchange interface, including but not limited to a thermal jacket, heating mantle, immersion heater, Peltier device, or cooling coil, operably connected to a power source or a refrigerant supply. The temperature control element may further include a temperature sensor (e.g. a thermocouple or thermistor) that provides real-time thermal data to a controller or processor to facilitate a closed-loop feedback mechanism. In the disclosed system, the temperature control element is configured to stabilize the solubility of a pharmaceutical analyte, prevent the unwanted crystallization of amorphous materials, or regulate the viscosity of the buffered medium to ensure consistent mass transfer across the liquid-liquid interface.

[0117] In one embodiment, the concentration measurement device is positioned to monitor the absorption phase through an optical path.

[0118] Generally, the term “optical path” as used herein refers to the specific trajectory and physical distance traversed by a beam of electromagnetic radiation, including but not Page 24 of 44MEl\60404268.v225-021 (098121-00441)limited to ultraviolet, visible, or infrared light, as it propagates from a radiation source, through a medium of interest ,and to a detector. In the disclosed system, the optical path extends through at least one of the dissolution phase, or absorption phase. The length of the optical path, often referred to as the path length, is a defined parameter used by the measurement device to quantify the concentration of a pharmaceutical analyte according to the Beer-Lambert Law or related spectroscopic principles. The optical path may be modified or directed by one or more optical elements, including but not limited to lenses, mirrors, prisms, filters, or fiber optic waveguides, and may pass through optical windows integrated into the vessel assembly to allow for non-invasive, real-time monitoring of the chemical environment.

[0119] In one embodiment, the disclosed apparatus operates through the coordinated functional integration of the vessel assembly, the concentration measurement device and the controller to monitor and evaluate a pharmaceutical formulation in a biphasic test environment. The vessel assembly provides a chemically inert environment for the containment and interaction of the dissolution phase and the absorption phase, maintaining a stable liquid-liquid interface necessary for partitioning studies. During operation, the concentration measurement device can be configured to be integrated into or fluidically coupled with the vessel assembly. The concentration measurement device can further interrogate at least one of dissolution phase, or absorption phase along with a defined optical path or electrochemical interface to generate a raw signal corresponding to the amount of solvated molecules or amorphous materials present therein. This signal can be transmitted to the controller, which processes the data and, via a feedback loop, dynamically regulates the operational parameters of the apparatus and concentration measurement device, such as mixing speed of a mixing element, the thermal output of a temperature control element, to maintain the buffered medium at a predetermined pH value and stabilize the dissolution behavior, supersaturation behavior, crystallization behavior, or relative performance of the pharmaceutical analyte. The controller can further control continuous monitoring and measurement of concentration of the pharmaceutical analyte.

[0120] In some embodiments, a non-transitory computer-readable medium storing instructions is provided. The non-transitory computer-readable medium can be executed by one or more processors of the system for evaluation of the pharmaceutical formulation in a biphasic test environment. When executed by the processor(s), the instructions may cause the one or more processors to: receive time-based concentration data for a pharmaceutical analytePage 25 of 44MEl\60404268.v225-021 (098121-00441)in an absorption phase of the biphasic test environment, generate, from the time-based concentration data, a concentration profile for the pharmaceutical analyte in the absorption phase, process the concentration profile using a deconvolution model to generate inferred concentration information for the pharmaceutical analyte in a dissolution phase of the biphasic test environment, generate output indicative of behavior of the pharmaceutical formulation based on the inferred concentration information, and, storing the output in a library configured for post-processing analysis. The library may be useful, for example, as a standardized data resource that enables comparison of properties of various pharmaceutical formulations and analytes, thereby enabling selection for further research, a diagnostic, therapeutic or other regime (or cessation thereof).

[0121] For example, in some embodiments, the system includes a data library that stores measured concentration data, inferred concentration information, metadata for test conditions, and outputs generated from prior evaluations. The metadata may include identity of a pharmaceutical analyte, identity of a pharmaceutical formulation, composition of a dissolution phase, composition of an absorption phase, pH, temperature, agitation condition, measurement interval, detector type, and other variables associated with a biphasic test. The data library may therefore accumulate a set of historical test records that correspond to known formulations, known test conditions, and known analytical outcomes.

[0122] In some embodiments, the system may apply a machine-learning model, an artificial-intelligence model, or another pattern-recognition model to data in the data library to identify relationships between measured concentration behavior, inferred concentration behavior, and formulation performance. For example, the model may be trained with historical biphasic test data to identify patterns associated with delayed crystallization, rapid loss of supersaturation, stable transfer into an absorption phase, or improved correlation with in vivo absorption behavior. The model may therefore assist interpretation of new test data by comparison of a current dataset to stored datasets that exhibit known behavior under similar conditions.

[0123] In some embodiments, the system may use artificial intelligence to refine analytical treatment of newly acquired data. For example, the system may identify a preferred deconvolution model, identify a preferred parameter set, detect an outlier dataset, detect signal noise, identify a likely artifact, or recommend exclusion of a dataset that does not satisfy a model assumption. The system may also suggest a revised sampling interval, a revised testPage 26 of 44MEl\60404268.v225-021 (098121-00441)duration, a revised phase-volume ratio, a revised pH condition, or another revised test parameter based on comparison of current data to historical data in the data library. The artificial-intelligence function may therefore improve consistency of interpretation and may reduce error in selection of an analytical approach for a given pharmaceutical formulation.

[0124] In some embodiments, the system may generate a confidence value, a classification output, a ranking output, or a recommendation output based on combined use of newly acquired data and historical data in the data library. For example, the system may classify a pharmaceutical formulation as likely to exhibit delayed crystallization, likely to exhibit rapid precipitation, or likely to maintain supersaturation under selected conditions. The system may also rank candidate pharmaceutical formulations according to similarity to historical formulations associated with favorable transfer behavior or favorable in vivo correlation. The output may assist a user in selection of a formulation for further development, selection of a stabilizer or excipient system, or selection of a revised biphasic test condition for additional evaluation.

[0125] In some embodiments, the data library may be updated after each biphasic test so that the system continues to learn from additional measured concentration data, inferred concentration information, and confirmed formulation outcomes. The library may include data generated within a single laboratory, data generated across multiple instruments, or data generated across multiple development programs. The machine-learning or artificialintelligence model may therefore be retrained or refined over time to improve interpretation of concentration profiles, improve detection of crystallization-related events, and improve prediction of formulation performance from biphasic test data.

[0126] Generally, the term “non-transitory computer-readable medium” as used herein refers to any tangible article of manufacture, memory, or storage device configured to store data and computer-executable instructions in a non-transitory manner. This term encompasses, without limitation, magnetic storage media (e.g., hard disk drives, magnetic tape), optical storage media (e.g., CD-ROMs, DVDs), and semiconductor memory devices, including volatile and non-volatile memory such as Random Access Memory (RAM), Read-Only Memory (ROM), Erasable Programmable Read-Only Memory (EPROM), and flash memory. The non-transitory computer-readable medium stores a set of instructions that, when executed by a processor or controller, cause the system to perform specific operations, such as receiving detected signals from a measurement device, calculating the concentration of a pharmaceuticalPage 27 of 44MEl\60404268.v225-021 (098121-00441)analyte, regulating a temperature control element, or stabilizing the pH value of a buffered medium within the vessel assembly.

[0127] In one embodiment, the output is indicative of relative performance of the pharmaceutical formulation relative to other pharmaceutical formulations and is usable to rank, select, or exclude one or more of the pharmaceutical formulations.

[0128] In one embodiment, the dissolution phase and the absorption phase are selected to model transfer of the pharmaceutical analyte between biologically relevant compartments, and wherein the output is indicative of in vivo absorption performance of the pharmaceutical formulation. In one embodiment, the output is indicative of crystallization behavior, induction time, or supersaturation behavior of the pharmaceutical formulation and is usable to evaluate a precipitation inhibitor, a crystallization modifier, an excipient system, a formulation dosage form, or a process condition for the pharmaceutical formulation.

[0129] It is to be understood that the aspects described herein are not limited to specific embodiments, or examples, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.

[0130] Disclosed herein are systems, methods, computer program products, and apparatuses for deconvolution of crystallization and pharmaceutical analyte absorption data from in situ concentration measurements using a biphasic setup.

[0131] The present disclosure is illustrated and further described in more detail with reference to the following non-limiting examples.EXAMPLES

[0132] Example 1: Experimental Setup

[0133] The experimental setup is shown in FIG. 1. As intimated by FIG. 2, pharmaceutical analyte absorption from the dissolution phase to the absorption phase can be modeled using first-order kinetics.

[0134] Example 2: Deconvoluting drug crystallization kineticsPage 28 of 44MEl\60404268.v225-021 (098121-00441)

[0135] In the disclosed systems, a supersaturated solution of ketoconazole is in equilibrium with amorphous precipitates (nanoparticles) in the dissolution phase. The presence of these particles hindered direct concentration measurements in the dissolution phase. Measuring the concentration in the absorption phase may result in different absorption profiles, as shown in FIGs. 3A-3B, where FIG. 3A is the absorption profile for a system without crystallization, and FIG. 3B is the absorption profile for a system where the drug crystallized.FIG. 3A follows first order appearance, whereas FIG. 3B follows two-stage kinetics. Combined with experimental characterization, it can be concluded that the two-stage appearance kinetics pattern in FIG. 3B corresponds to crystallization.

[0136] After deconvolution using first order absorption kinetics assuming maximum solution concentration with no crystallization using the Wagner-Nelson deconvolution method, the data of FIGs. 4A-4B was collected. FIG. 4A shows a constant aqueous solution concentration as a function of time, and FIG.4B shows the onset of crystallization event where absorption was overestimated by assuming no crystallization. The excess amount of drug is due to the portion of drug crystallized that was overestimated by assuming maximum absorption. The onset gives crystallization induction time.

[0137] The first order absorption profile can then be calculated, assuming no crystallization and calculate the difference due to crystallization. Subsequently, the crystallization kinetics can be resolved and actual solution drug concentration changes identified (FIGs. 5A-5B). The advantage of the biphasic assay was systematically evaluated with different model drugs, as shown in the examples below.

[0138] Example 3: Crystallization induction time as a function of drug supersaturation

[0139] In this example, two model drugs, fenofibrate and ketoconazole, were examined for crystallization induction time using a biphasic experimental setup, with decanol used as the absorption phase. The decanol here acts as an absorptive sink mimicking in vivo drug absorption. Also, it helps with clean UV data collection when the dissolution phase is highly turbid with a mixture of crystalline and amorphous particles.

[0140] Using the algorithm and biphasic method developed, induction experiments were performed using fenofibrate and ketoconazole at concentrations above their amorphous solubility, to study the crystallization kinetics in the presence of amorphous precipitates. The control group was the neat drug without any additive. To test their ability to inhibit nucleation Page 29 of 44MEl\60404268.v225-021 (098121-00441)rate, 100 pg / mL polymeric crystallization inhibitor, hydroxypropyl methylcellulose (HPMC) or polyvinyl alcohol (PVA) were added.

[0141] For fenofibrate, the induction time as a function of drug concentration is shown in FIG. 6. In this graph, it can be seen that HPMC demonstrated better ability to inhibit heterogeneous nucleation than PVA. This data was further analyzed using a Michaelis-Menten type heterogeneous nucleation model:p Jp,maxWhere Jpis the nucleation rate originated from the surface of foreign particles (amorphous precipitates), JPimaxis the maximum nucleation rate when KMapproaches 0 (all the free drug are adsorbed on particle surfaces), [FP] is the concentration of foreign particles, and KMdescribes the affinity between the free drug and the particle surface.

[0142] Using the double-reciprocal plot:1 KM 1 ! 1Jp Jp.max [FP] JP,maxA straight line with a slope of '<Mand an intercept of — - — is achieved through the plot of Jp,max Jp,max1 1— versus - — -. As shown in FIG.7, all data follows a reasonably linear relationship. Therefore, Jp [Fp\nucleation induction time can be predicted using this mathematical model at other drug concentrations. Similarly, data for ketoconazole are shown in FIGs. 8-9, with similar rank order (HPMOPVA) at low drug concentrations obtained. For HPMC at low particle concentrations, a similar linear relationship was observed. At high drug concentrations, both stabilizers appear to behave similarly. For the control and PVA system, as well as HPMC at high particle concentrations, nucleation rate appeared to be insensitive to particle concentrations.

[0143] Example 4: Impact of absorption sink on crystallization kinetics on homogenous crystallization

[0144] Using the algorithm combined with biphasic experimental setup, crystallization induction time experiments were performed using two model drugs: ketoconazole andPage 30 of 44MEl\60404268.v225-021 (098121-00441)enzalutamide, at concentrations equal to or slightly below their amorphous solubility. Therefore, the system is a homogeneous clear solution to start with.

[0145] In a single-phase dissolution setup with pH 6.5 buffer, both drugs crystallized quickly, showing nearly immediate crystallization and decline in solution concentration for ketoconazole, and an average induction time (time before crystallization where solution concentration decline occurs) of about 100 min for enzalutamide. However, with the introduction of the absorption phase in a biphasic dissolution setup, neither drug crystallized. Deconvolved drug concentrations remained constant with time (FIGs. 10A-10B).

[0146] Therefore, the use of a biphasic setup can significantly alter the solution crystallization kinetics of supersaturated formulations. The use of a biphasic setup, by introducing an absorptive sink, closely mimic fast drug absorption in vivo, and therefore would give results that more relevant to in vivo conditions.

[0147] Example 5: Impact of absorption sink on crystallization kinetics in heterogenous crystallization - impact of drug supersaturation

[0148] Using the algorithm coupled with the biphasic experimental setup, crystallization kinetics of a heterogeneous system was determined at drug concentrations above amorphous solubility. Ketoconazole was used as a model drug at different concentrations in the absence of stabilizers. Above amorphous solubility, liquid-liquid phase separation occurs, leading to spontaneous formation of amorphous drug nanoparticles. It is challenging to perform in-line measurements in a turbid suspension in the presence of nanoparticles. Therefore, drug concentrations were measured in the decanol phase in the biphasic setup and analyzed the data using the algorithm disclosed herein.

[0149] FIG. HA shows measured drug concentration as a function of time using the biphasic experimental setup. A higher initial drug concentration of 200 pg / mL led to lower amount of drug absorbed into the decanol phase compared to 160 pg / mL of drug. Further analysis of the data revealed two distinct absorption rate (two slopes) for the 200 pg / mL data as shown in FIG. 11B. The transition point correlates with the onset of crystallization events, with an induction time of amount 48 minutes, reflected in the deconvolved drug concentration shown in FIG. HD and calculated drug crystallization kinetics in FIG. HF. In contrast, no crystallization event was detected in the 160 pg / mL solution, as reflected by the constantPage 31 of 44MEl\60404268.v225-021 (098121-00441)absorption rate (slope) in FIG. 11B, constant deconvolved drug concentration shown in FIG.11C, and no drug crystallized shown in FIG. HE.

[0150] Example 6: Impact of absorption sink on crystallization kinetics in heterogenous crystallization - Impact of polymer stabilizer

[0151] Ketoconazole at 500 pg / mL was also evaluated in the presence of two different polymer stabilizers, HPMC and HPMCAS. The HPMCAS system did not crystallize, as shown in FIG. 12A, with a total concentration of 500 pg / mL in decanol. The HPMC system crystallized, with a plateau observed in the diffusion profile shown in FIG. 12A. The initial diffusion profiles of the two systems overlapped until crystallization occurred at around 100 min. Further data analysis confirmed crystallization event at around 105 min, as shown in the decline in absorption rate (slope) in FIG. 12B, deconvoluted drug concentration in FIG. 12C, and crystallization kinetics shown in FIG. 12D.

[0152] Example 7: Immediate crystallization without induction time

[0153] Using the disclosed method, ketoconazole at 250 pg / mL and 800 pg / mL was evaluated in the presence of PVA and HPMC. The PVA system remained stable without crystallization, whereas the HPMC system crystallized at 800 pg / mL. As shown in FIG. 13A, the diffusional profiles are similar at 250 pg / mL with only slight differences in total drug concentration (within experimental error). However, at 800 pg / mL, the HPMC system showed much lower plateau drug concentration, with differences in drug transport rate observed from the beginning. Further analysis revealed that the initial absorption rate (slope) was different between the HPMC and PVA system (FIG. 13C), suggesting immediate crystallization and hence low absorption rate. The change in slope for both systems at around 500 min are caused by complete drug transport. Immediate crystallization with no delay was also confirmed in FIG. 13D.

[0154] Example 8: Predicting formulation performance in vivo - change of solubility phase boundary

[0155] In this example, the absorption profile of enzalutamide crystalline solids was evaluated. The crystalline solubility measured by the shake-flask method in pH 6.5 buffer was extremely low (1.16 pg / mL). However, with the presence of an absorptive sink in the biphasic setup, substantial increase was observed in the amount of drug dissolved in solution, with anPage 32 of 44MEl\60404268.v225-021 (098121-00441)average of ~45 |ig / mL, much higher than the solubility of the drug (FIG. 14). This is caused by rapid solute removal from the aqueous solution into the decanol phase, thus facilitating further drug dissolution in the aqueous solution. Therefore, the in vivo performance of a formulation would be much higher due to rapid absorption, and is not limited by the solubility limit of the drug.

[0156] Example 9: Rank order amorphous solid dispersion (ASD) formulations -competition between drug release and crystallization

[0157] The performance of 4 enzalutamide ASD formulations was evaluated using the disclosed biphasic setup. Due to the presence of the absorptive sink, this method was able to capture delayed (or absence) of crystallization and continued drug absorption beyond the drug’s solubility limit. Whereas in typical single-phase dissolution experiments, rapid crystallization and low drug release was observed, often leading to underestimation of formulation performance and incorrect rank order in in vitro tests. The same formulations were previously evaluated, but disconnect was observed between dissolution results and rat bioavailability data. Using biphasic dissolution experiments, crystallization was not observed in the PVPVA formulations, and the slower release kinetics of HPMCAS formulations (compared to PVPVA) was correctly captured (FIG. 15).

[0158] Previous studies have evaluated fenofibrate ASDs formulated with different polymers and has shown that in single phase dissolution, rapid crystallization was observed in all three formulations, with HPMC showing the fastest crystallization, followed by HPMCAS LF, and HPMCAS HF. However, in vivo rat data showed that the HPMC formulation exhibited the best performance and lack of crystallization, followed by HPMCAS HF (lack of crystallization, its slow release contributed to the lower performance), and rapid crystallization of the HPMCAS LF formulation. However, using biphasic dissolution experiments, delayed or absence of crystallization was seen in the HPMC and HPMCAS HF based formulations (FIG.16). HPMCAS LF formulation still showed rapid crystallization, consistent with the rat data. The HPMC formulation eventually crystallized in the disclosed test, suggesting potential risk of this formulation at higher degree of supersaturations (e.g. higher doses or in other animal species or humans with lower intestinal bile salt concentrations than rats). The suboptimal performance of HPMCAS HF formulation in rats was likely caused by its slow release, given that HPMCAS HF can only fully dissolve at pHs higher than the average pH of rat GI; whereasPage 33 of 44MEl\60404268.v225-021 (098121-00441)in current biphasic dissolution tests, a pH 6.8 buffer was used to ensure rapid release of this formulation.

[0159] In addition to the above examples, the disclosed system can also be used in studying liquid-liquid phase separation and particle drifting effect.

[0160] When introducing elements of the present invention or the embodiment(s) thereof, the articles “a,” “an,” and “the” are intended to mean that there are one or more of the elements. Similarly, the adjective “another,” when used to introduce an element, is intended to mean one or more elements. The terms “including” and “having” are intended to be inclusive such that there may be additional elements other than the listed elements. The term “exemplary” is not intended to be construed as a superlative example but merely one of many possible examples.

[0161] The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. By way of example, “an element” means one element or more than one element.

[0162] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0163] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise. Furthermore, the terms first, second, etc., as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers.

[0164] The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted.

[0165] The terms “about” or “approximately,” as used herein, is inclusive of the stated value and means within an acceptable range of deviation for the particular value as determined by one of ordinary skill in the art, considering the measurement in question and the error associated with measurement of the particular quantity (i.e., the limitations of the measurement system). For example, “about” can mean within one or more standard deviations, or within ±Page 34 of 44MEl\60404268.v225-021 (098121-00441)10% or 5% of the stated value. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1, 0.2, 0.3, 0.4 . . . 2.0. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.

[0166] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0167] As used herein, “continuous” or “continuously” will be understood to mean without interruption or at periodic intervals. For example, the devices described herein may continuously monitor analyte concentrations by measuring analytes without stopping or by measuring an analyte concentration periodically, e.g., every few seconds, every few minutes, or at intervals that are sufficient for the particular clinical indication.

[0168] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additionalPage 35 of 44MEl\60404268.v225-021 (098121-00441)unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0169] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0170] The phrase “one or more,” as used herein, means at least one, and thus includes individual components as well as mixtures / combinations of the listed components in any combination.

[0171] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients and / or reaction conditions are to be understood as being modified in all instances by the term “about,” meaning within 10% of the indicated number (e.g., “about 10%” means 9%- 11% and “about 2%” means 1.8%-2.2%).

[0172] All percentages and ratios are calculated by weight unless otherwise indicated. All percentages are calculated based on the total composition unless otherwise indicated. Generally, unless otherwise expressly stated herein, “weight” or “amount” as used herein with respect to the percent amount of an ingredient refers to the amount of the raw material comprising thePage 36 of 44MEl\60404268.v225-021 (098121-00441)ingredient, wherein the raw material may be described herein to comprise less than and up to 100% activity of the ingredient. Therefore, weight percent of an active in a composition is represented as the amount of raw material containing the active that is used and may or may not reflect the final percentage of the active, wherein the final percentage of the active is dependent on the weight percent of active in the raw material.

[0173] All ranges and amounts given herein are intended to include subranges and amounts using any disclosed point as an end point. Thus, a range of “1% to 10%, such as 2% to 8%, such as 3% to 5%,” is intended to encompass ranges of “1% to 8%,” “1% to 5%,” “2% to 10%, ” and so on. All numbers, amounts, ranges, etc., are intended to be modified by the term “about,” whether or not so expressly stated. Similarly, a range given of “about 1% to 10%” is intended to have the term “about” modifying both the 1% and the 10% endpoints. Further, it is understood that when an amount of a component is given, it is intended to signify the amount of the active material unless otherwise specifically stated.

[0174] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0175] All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein. Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art of this disclosure.

[0176] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group.Page 37 of 44MEl\60404268.v225-021 (098121-00441)

[0177] A significant change is any detectable change that is statistically significant in a standard parametric test of statistical significance such as Student’s t-test, where p < 0.05.

[0178] While the invention has been described with reference to an exemplary embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0179] All statements herein reciting principles, aspects, and embodiments of the disclosure, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents as well as equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure.

[0180] Various other components may be included and called upon for providing for aspects of the teachings herein. For example, additional materials, combinations of materials and / or omission of materials may be used to provide for added embodiments that are within the scope of the teachings herein. Adequacy of any particular element for practice of the teachings herein is to be judged from the perspective of a designer, manufacturer, seller, user, system operator or other similarly interested party, and such limitations are to be perceived according to the standards of the interested party.

[0181] In the disclosure hereof any element expressed as a means for performing a specified function is intended to encompass any way of performing that function including, for example, a) a combination of circuit elements and associated hardware which perform that function or b) software in any form, including, therefore, firmware, microcode or the like as set forth herein, combined with appropriate circuitry for executing that software to perform the function. Applicants thus regard any means which can provide those functionalities as equivalent to those shown herein. No functional language used in claims appended herein is toPage 38 of 44MEl\60404268.v225-021 (098121-00441)be construed as invoking 35 U.S.C. § 112( / ) interpretations as "means-plus-function" language unless specifically expressed as such by use of the words "means for" or "steps for" within the respective claim.Page 39 of 44MEl\60404268.v2

Claims

25-021 (098121-00441)CLAIMSWhat is claimed is:

1. A system for evaluation of a pharmaceutical formulation in a biphasic test environment, comprising:a test apparatus configured to maintain a dissolution phase and an absorption phase; a measurement device configured to generate concentration data for a pharmaceutical analyte in the absorption phase over time; anda processor configured to generate inferred concentration information for the pharmaceutical analyte in the dissolution phase from the concentration data and to generate an output indicative of behavior of the pharmaceutical formulation.

2. The system of claim 1, wherein the dissolution phase comprises at least one of a buffered medium configured to maintain a selected pH, pure water, one or more surfactants, one or more lipids, or one or more polymers.

3. The system of claim 1, wherein the dissolution phase comprises a buffer configured to maintain a pH that is configured to influence dissolution behavior, supersaturation behavior, crystallization behavior, induction time, or relative performance of the pharmaceutical formulation.

4. The system of claim 1,wherein the dissolution phase is an aqueous phase; andwherein the absorption phase is an organic phase.

5. The system of claim 4,wherein the organic phase comprises at least one of a water-immiscible solvent and a non-polar organic solvent; andwherein the organic phase comprises at least one of decanol, n-hexane, heptane, cyclohexane, benzene, n-butanol, butyl acetate, carbon tetrachloride, methyl-t-butyl ether, 2-butanone, pentane, diisopropyl ether, ethyl acetate, diethyl ether, toluene, trichloroethylene, dichloromethane, 1,2-dichloroethane, chloroform, 1 -octanol, 1 -dodecanol, oleic acid, isopropyl myristate, vegetable oil, soybean oil, castor oil, and olive oil.Page 40 of 44MEl\60404268.v225-021 (098121-00441)6. The system of claim 1, wherein the pharmaceutical analyte is selected from the group consisting of organic molecules, inorganic molecules, pharmaceutical molecules, biological molecules, amorphous materials, and environmental contaminants.

7. The system of claim 1, wherein the measurement device is a concentration measurement device.

8. The system of claim 1, wherein the processor is configured with at least one algorithm.

9. The system of claim 8, wherein the algorithm comprises a Wagner-Nelson deconvolution algorithm, numerical deconvolution, point-area deconvolution, regularized deconvolution, nonparametric / spline-based methods, Wiener deconvolution algorithm, Richardson-Lucy algorithm, a Van Cittert deconvolution algorithm, other deconvolution methods, or a subtraction algorithm.

10. An apparatus for evaluation of a pharmaceutical formulation in a biphasic test environment, the apparatus comprising:a vessel assembly configured to maintain a dissolution phase and an absorption phase, wherein the dissolution phase comprises a pharmaceutical analyte;a concentration measurement device arranged to generate time-based concentration data for the pharmaceutical analyte in the absorption phase; anda controller configured to receive the time-based concentration data and generate output indicative of behavior of the pharmaceutical formulation.

11. The apparatus of claim 10, wherein the behavior comprises at least one of dissolution behavior, supersaturation behavior, crystallization behavior, induction time, or relative performance of the pharmaceutical formulation.

12. The apparatus of claim 10, wherein the concentration measurement device comprises an inline spectroscopic device selected from the group consisting of a UV spectrophotometer, a UV-Vis spectrophotometer, a near-infrared spectrometer, a mid-infrared spectrometer, a Raman spectrometer, and a fluorescence spectrometer.

13. The apparatus of claim 10, wherein the concentration measurement device comprises an offline analytical device selected from the group consisting of a high-performance liquidPage 41 of 44MEl\60404268.v225-021 (098121-00441)chromatography system, a gas chromatography system, a mass spectrometer, and a nuclear magnetic resonance instrument.

14. The apparatus of claim 10, wherein the vessel assembly further comprises a mixing element configured to agitate at least the dissolution phase.

15. The apparatus of claim 10, wherein the vessel assembly further comprises a temperature control element configured to maintain the biphasic test environment at a selected temperature.

16. The apparatus of claim 10, wherein the concentration measurement device is positioned to monitor the absorption phase through an optical path.

17. A non-transitory computer-readable medium storing instructions that, when executed by one or more processors of a system for evaluation of a pharmaceutical formulation in a biphasic test environment, cause the one or more processors to:receive time-based concentration data for a pharmaceutical analyte in an absorption phase of the biphasic test environment;generate, from the time-based concentration data, a concentration profile for the pharmaceutical analyte in the absorption phase;process the concentration profile using an algorithm to generate inferred concentration information for the pharmaceutical analyte in a dissolution phase of the biphasic test environment;generate output indicative of behavior of the pharmaceutical formulation based on the inferred concentration information; and,storing the output in a library configured for post-processing analysis.

18. The non-transitory computer-readable medium of claim 17, wherein the output is indicative of relative performance of the pharmaceutical formulation relative to other pharmaceutical formulations and is usable to rank, select, or exclude one or more of the pharmaceutical formulations.

19. The non-transitory computer-readable medium of claim 17, wherein the dissolution phase and the absorption phase are selected to model transfer of the pharmaceutical analyte between biologically relevant compartments, and wherein the output is indicative of in vivo absorption performance of the pharmaceutical formulation.Page 42 of 44MEl\60404268.v225-021 (098121-00441)20. The non-transitory computer-readable medium of claim 17, wherein the output is indicative of crystallization behavior, induction time, or supersaturation behavior of the pharmaceutical formulation and is usable to evaluate a precipitation inhibitor, a crystallization modifier, an excipient system, a formulation dosage form, or a process condition for the pharmaceutical formulation.Page 43 of 44MEl\60404268.v2

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