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[Kohsaku Kawakami](https://orcid.org/0000-0002-3466-9365)

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[Roles of Supersaturation and Liquid–Liquid Phase Separation for Enhanced Oral Absorption of Poorly Soluble Drugs from Amorphous Solid Dispersions](https://mdr.nims.go.jp/datasets/ccb2cf24-3f9d-4adc-85bd-ddf5aa66a7b7)

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Roles of Supersaturation and Liquid–Liquid Phase Separation for Enhanced Oral Absorption of Poorly Soluble Drugs from Amorphous Solid DispersionsAcademic Editor: Guy Van denMooterReceived: 16 January 2025Revised: 9 February 2025Accepted: 14 February 2025Published: 16 February 2025Citation: Kawakami, K. Roles ofSupersaturation and Liquid–LiquidPhase Separation for Enhanced OralAbsorption of Poorly Soluble Drugsfrom Amorphous Solid Dispersions.Pharmaceutics 2025, 17, 262.https://doi.org/10.3390/pharmaceutics17020262Copyright: © 2025 by the author.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license(https://creativecommons.org/licenses/by/4.0/).ReviewRoles of Supersaturation and Liquid–Liquid Phase Separationfor Enhanced Oral Absorption of Poorly Soluble Drugs fromAmorphous Solid DispersionsKohsaku Kawakami 1,21 Research Center for Macromolecules and Biomaterials, National Institute for Materials Science, 1-1 Namiki,Tsukuba 305-0044, Ibaraki, Japan; kawakami.kohsaku@nims.go.jp2 Graduate School of Science and Technology, University of Tsukuba, 1-1-1 Tennodai,Tsukuba 305-8577, Ibaraki, JapanAbstract: Amorphous solid dispersion (ASD) is one of the most important enabling formu-lation technologies for the development of poorly soluble drugs. Because of its thermody-namically unstable nature in both solid and wet states, the evaluation and optimizationof the formulation performance involves some difficulties. The dissolution process is sen-sitively influenced by various factors, including the applied dose, medium composition,and pH. Supersaturated solutions can cause liquid–liquid phase separation (LLPS) and/orcrystallization, which complicates the comprehension of the dissolution process. However,LLPS should be evaluated carefully because it is closely related to oral absorption. As LLPSconcentration is analogous to amorphous solubility, it can be a key factor in predictingoral absorption from ASDs, if absorption is limited by solubility. Moreover, LLPS dropletsare expected to increase transmembrane flux by increasing the drug concentration nearthe epithelial cell membrane. In this review, recently updated knowledge on the dissolu-tion, membrane permeation, and oral absorption behaviors of ASDs is discussed with anemphasis on LLPS behavior.Keywords: amorphous solid dispersion; crystallization; supersaturation; liquid–liquidphase separation; membrane permeability; oral absorption1. IntroductionAmorphous solid dispersion (ASD) is widely recognized as a powerful formulationtechnology for poorly soluble drugs. Its usage is accelerating year by year. Between 2012and 2023, 48 drug products that contain ASD have been approved by the FDA [1]. Theamorphous state offers higher solubility than that of the crystalline state [2,3]; therefore,oral absorption of a drug may be enhanced if it is limited by solubility or dissolutionrate [4–8]. As solutions in the supersaturated state have solute concentrations that exceedequilibrium solubility, those solutions are not thermodynamically stable. Therefore, anappropriate formulation design is required for ASDs to effectively maintain supersaturation.For the crystalline solids, the dissolved drug concentration keeps increasing until theequilibrium state is achieved, whereas, for amorphous solids, the dissolution profile maydecrease after reaching maximum concentrations under non-sink conditions that have beendescribed as the “spring and parachute” [4]. The decrease in concentration is inducedeither by crystallization or liquid–liquid phase separation (LLPS) [9–11]. Understandingwhat happens during the dissolution process of ASDs is crucial for rational formulationdesign. The advantage of ASD is not expected if crystallization proceeds promptly duringPharmaceutics 2025, 17, 262 https://doi.org/10.3390/pharmaceutics17020262https://doi.org/10.3390/pharmaceutics17020262https://doi.org/10.3390/pharmaceutics17020262https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://www.mdpi.com/journal/pharmaceuticshttps://www.mdpi.comhttps://orcid.org/0000-0002-3466-9365https://doi.org/10.3390/pharmaceutics17020262https://www.mdpi.com/article/10.3390/pharmaceutics17020262?type=check_update&version=1Pharmaceutics 2025, 17, 262 2 of 17dissolution. If LLPS occurs, the activity of the solution is different from that of a “real”supersaturated solution, where all drug molecules are dissolved as solutes. A dissolutiontest is the first step in evaluating the potential of the formulations. However, both theselection of the experimental condition and the interpretation of the results are still quitechallenging for ASDs. In this review, the dissolution process of ASDs is discussed with anemphasis on the dissolved state of drug molecules and its impact on membrane permeationand oral absorption behavior.2. Supersaturation and LLPSSupersaturation and LLPS are important events for ASDs to improve oral absorptionof poorly soluble drugs, which must be appropriately evaluated for their development.Both events can be observed only under non-sink conditions in the dissolution test. Whenan amorphous drug or ASD is dissolved in a medium under the non-sink condition,three major scenarios are available in the dissolution process (Figure 1). (1) If the dissolutionis slow, the drug concentration may not reach amorphous solubility. In this case, furtherformulation efforts are required to accelerate the dissolution rate to achieve supersaturation.(2) If the dissolution is too rapid and/or the crystallization tendency of the drug is toohigh, and unless the stabilization effect of the polymer is sufficient, crystallization mayproceed after supersaturation is achieved. Such an ASD may work for drugs with highpermeability if absorption proceeds before crystallization occurs. However, efforts to retardcrystallization are generally preferred. (3) LLPS may occur after “real” supersaturation andcontinue for a long time under appropriate formulation design. This is the most favoredfor enhancing the oral absorption of poorly soluble drugs, as high drug concentration isexpected to be maintained in the gastrointestinal tract for a long duration. When crystalsappear, supersaturation/LLPS is destroyed. However, it may not happen immediatelyafter the appearance of crystals [12]. The dissolution behavior of ASDs depends on bothformulation properties and dissolution conditions. Thus, in addition to the appropriatedesign of ASDs, the appropriate design of test conditions, which requires an understandingof the environment of the gastrointestinal tract, is crucial for the successful evaluation anddevelopment of ASDs.Pharmaceutics 2025, 17, x FOR PEER REVIEW 2 of 18   design. The advantage of ASD is not expected if crystallization proceeds promptly during dissolution. If LLPS occurs, the activity of the solution is different from that of a “real” supersaturated solution, where all drug molecules are dissolved as solutes. A dissolution test is the first step in evaluating the potential of the formulations. However, both the selection of the experimental condition and the interpretation of the results are still quite challenging for ASDs. In this review, the dissolution process of ASDs is discussed with an emphasis on the dissolved state of drug molecules and its impact on membrane permea-tion and oral absorption behavior. 2. Supersaturation and LLPS Supersaturation and LLPS are important events for ASDs to improve oral absorption of poorly soluble drugs, which must be appropriately evaluated for their development. Both events can be observed only under non-sink conditions in the dissolution test. When an amorphous drug or ASD is dissolved in a medium under the non-sink condition, three major scenarios are available in the dissolution process (Figure 1). (1) If the dissolution is slow, the drug concentration may not reach amorphous solubility. In this case, further formulation efforts are required to accelerate the dissolution rate to achieve supersatura-tion. (2) If the dissolution is too rapid and/or the crystallization tendency of the drug is too high, and unless the stabilization effect of the polymer is sufficient, crystallization may proceed after supersaturation is achieved. Such an ASD may work for drugs with high permeability if absorption proceeds before crystallization occurs. However, efforts to re-tard crystallization are generally preferred. (3) LLPS may occur after “real” supersatura-tion and continue for a long time under appropriate formulation design. This is the most favored for enhancing the oral absorption of poorly soluble drugs, as high drug concen-tration is expected to be maintained in the gastrointestinal tract for a long duration. When crystals appear, supersaturation/LLPS is destroyed. However, it may not happen imme-diately after the appearance of crystals [12]. The dissolution behavior of ASDs depends on both formulation properties and dissolution conditions. Thus, in addition to the appro-priate design of ASDs, the appropriate design of test conditions, which requires an under-standing of the environment of the gastrointestinal tract, is crucial for the successful eval-uation and development of ASDs.  Figure 1. Typical patterns of ASD dissolution profiles. (a) Drug release is limited by dissolution. Small dots represent released drug molecules. Neither crystallization nor LLPS occurs in the solu-tion. Direct transformation from amorphous to crystal in the solid can proceed. (b) Supersaturation is immediately destroyed if crystals appear. The crystals that appeared from the supersaturated so-lution are indicated by the orange squares. Crystallization can also happen on solid surface. (c) If the dissolution is sufficiently rapid to induce LLPS and the state is stable, the LLPS concentration, which is analogous to amorphous solubility, is maintained. The green circles represent LLPS drop-lets. Figure 1. Typical patterns of ASD dissolution profiles. (a) Drug release is limited by dissolution.Small dots represent released drug molecules. Neither crystallization nor LLPS occurs in the solution.Direct transformation from amorphous to crystal in the solid can proceed. (b) Supersaturationis immediately destroyed if crystals appear. The crystals that appeared from the supersaturatedsolution are indicated by the orange squares. Crystallization can also happen on solid surface. (c) Ifthe dissolution is sufficiently rapid to induce LLPS and the state is stable, the LLPS concentration,which is analogous to amorphous solubility, is maintained. The green circles represent LLPS droplets.Pharmaceutics 2025, 17, 262 3 of 17Supersaturation achieved by dissolution of amorphous solids can be understood usingthe basic thermodynamic equation:∆G = RTlnx, (1)Here, ∆G and x are the Gibbs energy of dissolution and mole fraction, respectively. Rand T are the gas constant and temperature, respectively. As the amorphous state possessesa higher energy state relative to the crystals, a larger x is expected, which is analogous toamorphous solubility. However, the rapid dissolution of amorphous solids may offer ahigher concentration than the amorphous solubility, that is, “real” supersaturation, whichresults in crystallization or LLPS [9]. The crystallization rate depends on the degreeof supersaturation, the crystallization tendency of the drug, and the stabilization effectof the excipients. Figure 2a shows the precipitation/LLPS behavior of supersaturatedgriseofulvin (GF) solutions [13]. When the degree of supersaturation was marginal, theconcentration decreased slowly until reaching the LLPS concentration. As the degree ofsupersaturation increased, the concentration decreased more rapidly over time. When thedegree of supersaturation was sufficiently high, crystallization proceeded quickly to reachcrystalline solubility, which was much lower than the LLPS concentration. Consequently,the order of the dissolved concentration in the final state was completely opposite to thatof the initial concentration. In the presence of vinylpyrrolidone-vinyl acetate copolymer(PVPVA) (Figure 2b), supersaturation was effectively maintained relative to the solutionswithout the polymer. Nevertheless, the trend in reaching the LLPS concentration was thesame; that is, the decrease in concentration was faster for solutions with a higher degreeof supersaturation.Pharmaceutics 2025, 17, x FOR PEER REVIEW 3 of 18   Supersaturation achieved by dissolution of amorphous solids can be understood us-ing the basic thermodynamic equation: Δ𝐺 = 𝑅𝑇𝑙𝑛𝑥, (1)Here, ∆G and x are the Gibbs energy of dissolution and mole fraction, respectively. R and T are the gas constant and temperature, respectively. As the amorphous state pos-sesses a higher energy state relative to the crystals, a larger x is expected, which is analo-gous to amorphous solubility. However, the rapid dissolution of amorphous solids may offer a higher concentration than the amorphous solubility, that is, “real” supersaturation, which results in crystallization or LLPS [9]. The crystallization rate depends on the degree of supersaturation, the crystallization tendency of the drug, and the stabilization effect of the excipients. Figure 2a shows the precipitation/LLPS behavior of supersaturated griseo-fulvin (GF) solutions [13]. When the degree of supersaturation was marginal, the concen-tration decreased slowly until reaching the LLPS concentration. As the degree of super-saturation increased, the concentration decreased more rapidly over time. When the de-gree of supersaturation was sufficiently high, crystallization proceeded quickly to reach crystalline solubility, which was much lower than the LLPS concentration. Consequently, the order of the dissolved concentration in the final state was completely opposite to that of the initial concentration. In the presence of vinylpyrrolidone-vinyl acetate copolymer (PVPVA) (Figure 2b), supersaturation was effectively maintained relative to the solutions without the polymer. Nevertheless, the trend in reaching the LLPS concentration was the same; that is, the decrease in concentration was faster for solutions with a higher degree of supersaturation. Figure 2. Concentration–time curves of supersaturated GF solutions (phosphate buffer, pH7.0) in the absence (a) and presence of PVPVA (b) [13]. The supersaturation was created by adding con-centrated acetone solution of GF to the buffer. The concentrations presented in the figure represent initial GF concentration. The red and black dotted lines are apparent LLPS concentration and crys-talline solubility of GF, respectively. Figures are adopted from ref. [13] after modifications with per-mission from Elsevier. When LLPS occurs, assuming that the supersaturated solution is separated into con-centrated and diluted phases with concentrations of xH and xL, the Gibbs energy of mixing, ∆Gmix, can be calculated as follows: Δ𝐺 = 𝑓 𝑅𝑇𝑙𝑛𝑥 𝑓 𝑅𝑇𝑙𝑛𝑥 , (2)Figure 2. Concentration–time curves of supersaturated GF solutions (phosphate buffer, pH7.0) in theabsence (a) and presence of PVPVA (b) [13]. The supersaturation was created by adding concentratedacetone solution of GF to the buffer. The concentrations presented in the figure represent initialGF concentration. The red and black dotted lines are apparent LLPS concentration and crystallinesolubility of GF, respectively. Figures are adopted from ref. [13] after modifications with permissionfrom Elsevier.When LLPS occurs, assuming that the supersaturated solution is separated into con-centrated and diluted phases with concentrations of xH and xL, the Gibbs energy of mixing,∆Gmix, can be calculated as follows:∆Gmix = fH RTlnxH + fLRTlnxL, (2)Pharmaceutics 2025, 17, 262 4 of 17where f H and f L are the fractions of concentrated and diluted phases, respectively. Thisprocess is illustrated in Figure 3. When phase separation occurs, the sum of the Gibbsenergies of each phase falls on the gray tangent line in the figure and is, therefore, lowerthan the Gibbs energy without phase separation. When the drug concentration is betweentwo spinodal lines (i.e., inflection points), phase separation occurs via spinodal decomposi-tion. The concentration between the binodal and spinodal lines results in phase separationbased on the nucleation/growth of the concentrated phase (this does not mean the crys-tallization of drug molecules). The final composition after the phase separation does notdepend on the phase separation mechanism.Pharmaceutics 2025, 17, x FOR PEER REVIEW 4 of 18   where fH and fL are the fractions of concentrated and diluted phases, respectively. This process is illustrated in Figure 3. When phase separation occurs, the sum of the Gibbs energies of each phase falls on the gray tangent line in the figure and is, therefore, lower than the Gibbs energy without phase separation. When the drug concentration is between two spinodal lines (i.e., inflection points), phase separation occurs via spinodal decompo-sition. The concentration between the binodal and spinodal lines results in phase separa-tion based on the nucleation/growth of the concentrated phase (this does not mean the crystallization of drug molecules). The final composition after the phase separation does not depend on the phase separation mechanism.  Figure 3. Schematic representation of LLPS. When LLPS occurs after the dissolution of ASDs, a colloidal structure is obtained that cannot be explained solely by the thermodynamics mentioned above. The formation of dispersed LLPS droplets increases the Gibbs energy relative to the separation into two distinct phases because of the increase in interfacial energy. Small LLPS droplets with sizes in the order of hundreds of nanometers tend to be formed in the presence of charged polymers, such as Eudragit and hydroxypropylmethylcellulose acetate succinate (HPM-CAS) [14–17]. The decrease in the interfacial tension due to polymer adsorption and elec-tric/steric repulsion between the droplets are responsible for the stabilization of the small droplets. Thus, in the presence of neutral polymers or in the absence of polymers, the size of LLPS droplets is typically larger than 1 µm [13,15]. The LLPS concentration (xL) can be an important factor in determining transmem-brane flux and oral absorption, as described later. Table 1 summarizes examples of the LLPS concentrations for various drugs. In most cases, the LLPS concentration is higher than the crystalline solubility by more than one order of magnitude. Table 1. Equilibrium crystalline solubility and LLPS concentrations of poorly soluble drugs. Compound Temperature pH Crystalline Solubili-tyyanyi(µg/mL) LLPS Conc.yanyi(µg/mL) LLPS/Crystal yanyiSolubility Ratio Reference Albendazole 25 7.0 < 0.1 1.4 >14 [17] Clotrimazole 37 10.0 0.4 5.2 13 [18] Clozapine 37 10.0 8.8 136 15 [18] Danazol 25 6.8 0.9 13 14 [19] Diclofenac sodium 37 1.2 3.4 92 27 [20] Efavirenz 37 6.8 8.2 18.4 2.2 [18] Felodipine 37 6.8 0.94 9.8 10 [18] Fenofibrate 25 7.0 0.1 1.0 10 [15] Binodal LineSpinodal LinexL xHΔGmixDrug conc.xLxHFigure 3. Schematic representation of LLPS.When LLPS occurs after the dissolution of ASDs, a colloidal structure is obtainedthat cannot be explained solely by the thermodynamics mentioned above. The formationof dispersed LLPS droplets increases the Gibbs energy relative to the separation intotwo distinct phases because of the increase in interfacial energy. Small LLPS dropletswith sizes in the order of hundreds of nanometers tend to be formed in the presence ofcharged polymers, such as Eudragit and hydroxypropylmethylcellulose acetate succinate(HPMCAS) [14–17]. The decrease in the interfacial tension due to polymer adsorption andelectric/steric repulsion between the droplets are responsible for the stabilization of thesmall droplets. Thus, in the presence of neutral polymers or in the absence of polymers, thesize of LLPS droplets is typically larger than 1 µm [13,15].The LLPS concentration (xL) can be an important factor in determining transmembraneflux and oral absorption, as described later. Table 1 summarizes examples of the LLPSconcentrations for various drugs. In most cases, the LLPS concentration is higher than thecrystalline solubility by more than one order of magnitude.The presence of a small amount of polymer does not influence the LLPS concentrationin most cases. However, under competition of crystallization and supersaturation, theLLPS concentration can appear differently. HPMCAS is frequently reported to offer highapparent LLPS concentration [24] because of the strong ability of cellulose polymers toinhibit crystallization [19,25]. Although this concentration does not have thermodynamicsignificance, it is of practical importance for determining oral absorption. The polymersmay participate in the formation of nanodroplets. In particular, cellulose polymers are likelyto be preferentially distributed to the colloidal phase [16,19]. Moreover, polymers mayindependently solubilize the drug, as it is reported to occur with methacrylate polymers(Eudragit) [16].Pharmaceutics 2025, 17, 262 5 of 17Table 1. Equilibrium crystalline solubility and LLPS concentrations of poorly soluble drugs.Compound Temperature pH Crystalline Solubility(µg/mL)LLPS Conc.(µg/mL)LLPS/CrystalSolubility Ratio ReferenceAlbendazole 25 7.0 < 0.1 1.4 >14 [17]Clotrimazole 37 10.0 0.4 5.2 13 [18]Clozapine 37 10.0 8.8 136 15 [18]Danazol 25 6.8 0.9 13 14 [19]Diclofenacsodium 37 1.2 3.4 92 27 [20]Efavirenz 37 6.8 8.2 18.4 2.2 [18]Felodipine 37 6.8 0.94 9.8 10 [18]Fenofibrate 25 7.0 0.1 1.0 10 [15]Griseofulvin 37 7.0 12 38 3.2 [13]Indomethacin 37 2.0 3.0 30.4 10 [18]Ketoconazole 37 10.0 3.7 54.4 15 [18]Loratadine 37 6.8 1.6 7.6 4.8 [18]Nifedipine 37 6.8 1.4 45 32 [21]Naftopidil 37 6.8 10.0 58.3 5.8 [22]Posaconazole 37 6.5 1.7 12 7.1 [23]Ritonavir 37 6.8 1.3 18.8 14 [18]Surfactants can also be included both in formulations and dissolution medium. Ifsurfactant concentration is above critical micellar concentration, poorly soluble drugsare dissolved by micelles to increase the apparent LLPS concentration [15]. However, itseffect on the supersaturation/LLPS behavior is complicated, as the presence of surfactantsaccelerates both dissolution and crystallization rates.3. Dissolution of ASDsASDs typically contain hydrophilic polymers to improve the dissolution behaviorand storage stability of amorphous drugs. A small amount of surfactant may also beadded to improve stability and dissolution behaviors. Although a larger drug/polymerratio is favored from the viewpoint of pill burden, a sufficient amount of polymer isrequired to ensure congruent drug release and physical stability of the drug. Unless theamount of polymer is sufficient to achieve a congruent release, the polymer is preferentiallydissolved first, leaving the amorphous drugs concentrated in the formulation, which resultsin the slow dissolution of the drug (Figure 4a). Saboo et al. reported that the release ofnilvadipine [26] (Figure 4b) or indomethacin [27] from ASDs formulated with PVPVAproceeded with a congruent release mechanism only when the drug amount was lowerthan 10%. Ueda et al. investigated the release of nifedipine loaded at a concentration of25% with a mixture of HPMC and Eudragit S occurred via congruent release only whenthe Eudragit/HPMC ratio was higher than 50% [28]. Only the drug concentration hasbeen evaluated in most dissolution studies of ASDs reported. However, the measurementof the polymer concentration is also required to judge whether the dissolution is basedon congruent or incongruent release. From this aspect, the upper limit of drug loadingis called the limit of congruency (LoC) and is likely to be correlated with the strength ofthe interaction between the drug and the polymer [29]. However, the above discussionconcerns binary or trinary mixtures composed only of drugs and polymers. Dissolutionbehavior is influenced by the addition of disintegrants, including inorganic and effervescingsalts [30–32]. Also, the deposition of highly soluble components on the surface of ASD maywork to improve the dissolution behavior [33]. These efforts may improve the congruencyof the dissolution process of ASDs.Pharmaceutics 2025, 17, 262 6 of 17Pharmaceutics 2025, 17, x FOR PEER REVIEW 6 of 18    Figure 4. (a) Schematic representation of congruent and incongruent release from ASDs. (b) Disso-lution test of nilvadipine/PVPVA ASDs. The ratio of nilvadipine/PVPVA is 10/90 (blue) or 15/85 (red). Nilvadipine and PVPVA are shown as closed and open symbols, respectively. Data are taken from ref. [26] with permission from Elsevier. To reduce formulation size, great attention has recently been paid to the design of ASDs with high drug loading. One interesting approach is to include drug-rich nanopar-ticles in ASDs to facilitate the formation of LLPS droplets during the dissolution process [34,35]. In this attempt, the LLPS droplet size was determined beforehand and then nano-particles of identical sizes were dispersed in the ASD. This approach may be valid for avoiding the incongruent release problem mentioned above, although it should only be applicable to drugs with low crystallization tendency. As drug molecules are locally con-centrated in the formulation, unlike typical ASDs where drug and polymers are expected to be mixed at a molecular level, the physical stability of the ASD should be directly influ-enced by crystallization tendency of drug. The dissolution test is the simplest method for predicting oral absorption; however, the testing procedure and interpretation of the results for supersaturable formulations are not straightforward [36]. Moreover, the dissolution tests of supersaturable formulations frequently suffer from reproducibility issues [37], which should also be true for ASDs. Box 1 summarizes the points to consider when performing in vivo predictable dissolution tests for ASDs. It should be stressed that the discussion below is not for quality control pur-poses [3] but is aimed at predicting oral absorption. The use of a non-sink condition is inevitable, as supersaturation must be investigated for prediction. Conventional dissolu-tion tests under sink conditions only provide dissolution rates that are not necessarily correlated with in vivo absorption [38,39]. As the supersaturation behavior is sensitive to the degree of supersaturation, as shown in Figure 2, the selection of the applied dose is also important. “Sink index (SI)” is a convenient parameter for describing the degree of the sink conditions, which can be defined as 𝑆𝐼 = ⁄ , (3)where Cs, Dose, and V represent the crystalline solubility, applied dose, and medium vol-ume, respectively [40,41]. The appropriate SI range for the in vivo predictive dissolution test depends on the compound. For drugs with solubilities higher than 1 µg/mL, the SI of FDA-recommended or USP dissolution methods for selected ASD products ranges from 0.005 to 0.7, with some exceptions [41]. In our experience, SI values from 0.01 to 0.06 of-fered a good correlation with in vivo absorption [13,17,22], which corresponds to a slightly higher concentration relative to LLPS. Figure 4. (a) Schematic representation of congruent and incongruent release from ASDs. (b) Disso-lution test of nilvadipine/PVPVA ASDs. The ratio of nilvadipine/PVPVA is 10/90 (blue) or 15/85(red). Nilvadipine and PVPVA are shown as closed and open symbols, respectively. Data are takenfrom ref. [26] with permission from Elsevier.To reduce formulation size, great attention has recently been paid to the design of ASDswith high drug loading. One interesting approach is to include drug-rich nanoparticles inASDs to facilitate the formation of LLPS droplets during the dissolution process [34,35]. Inthis attempt, the LLPS droplet size was determined beforehand and then nanoparticles ofidentical sizes were dispersed in the ASD. This approach may be valid for avoiding theincongruent release problem mentioned above, although it should only be applicable todrugs with low crystallization tendency. As drug molecules are locally concentrated inthe formulation, unlike typical ASDs where drug and polymers are expected to be mixedat a molecular level, the physical stability of the ASD should be directly influenced bycrystallization tendency of drug.The dissolution test is the simplest method for predicting oral absorption; however,the testing procedure and interpretation of the results for supersaturable formulations arenot straightforward [36]. Moreover, the dissolution tests of supersaturable formulationsfrequently suffer from reproducibility issues [37], which should also be true for ASDs.Box 1 summarizes the points to consider when performing in vivo predictable dissolutiontests for ASDs. It should be stressed that the discussion below is not for quality controlpurposes [3] but is aimed at predicting oral absorption. The use of a non-sink condition isinevitable, as supersaturation must be investigated for prediction. Conventional dissolutiontests under sink conditions only provide dissolution rates that are not necessarily correlatedwith in vivo absorption [38,39]. As the supersaturation behavior is sensitive to the degreeof supersaturation, as shown in Figure 2, the selection of the applied dose is also important.“Sink index (SI)” is a convenient parameter for describing the degree of the sink conditions,which can be defined asSI =CsDose/V, (3)where Cs, Dose, and V represent the crystalline solubility, applied dose, and mediumvolume, respectively [40,41]. The appropriate SI range for the in vivo predictive dissolutiontest depends on the compound. For drugs with solubilities higher than 1 µg/mL, theSI of FDA-recommended or USP dissolution methods for selected ASD products rangesfrom 0.005 to 0.7, with some exceptions [41]. In our experience, SI values from 0.01 to0.06 offered a good correlation with in vivo absorption [13,17,22], which corresponds to aslightly higher concentration relative to LLPS.Pharmaceutics 2025, 17, 262 7 of 17Box 1. Points to be considered for in vivo predictable dissolution test for ASDs.✓ Non-sink conditions must be used for observing supersaturation. The degree of supersatura-tion is also an important factor.✓ Change in pH during transfer of ASD in the gastrointestinal tract must be considered, espe-cially when solubility of drug or polymer is pH-dependent. Attention to buffer capacity isalso required.✓ If LLPS occurs, attention to free concentration (activity) is required.✓ The dissolution test is a batch system. Crystallization/LLPS behavior is different if there is anabsorption sink, as it decreases degree of supersaturation.✓ In vivo components, especially bile acids, may influence the supersaturation behavior.Changes in pH during the transfer of ASD in the gastrointestinal tract must be consid-ered, especially when the solubility of a drug or polymer is pH-dependent. Experience inan acidic environment can significantly alter the dissolution of basic drugs and excipients.The dissolution behavior of acidic components can also be influenced by the experience ofan acidic condition. HPMCAS is not soluble in an acidic environment to form insoluble gelsthat inhibit the release of drugs; thus, the dissolution of the drug is suppressed comparedto the simple dissolution test at a neutral pH [15,42]. Attention to buffer species is alsorequired when ionizable components are included in ASDs. Phosphate buffers are typicallyused for dissolution studies; however, pH in the gastrointestinal tract is maintained bycarbonate buffer, which has a lower buffering capacity relative to the phosphate buffer.Thus, the dissolution rate of ionizable components may be overestimated by using phos-phate buffer [43]. In the case of HPMCAS, a very low concentration, 5–10 mM of phosphatebuffer (pH 6.5), was found to show a “biorelevant” dissolution rate [44].When LLPS occurs, the free concentration must be determined to estimate oral absorp-tion, as it is analogous to the activity of the drug. Many in vitro studies have revealed thatactivity is well-correlated with transmembrane absorption [45,46], although its correlationwith in vivo absorption requires consideration of other influential factors [47]. The removalof droplets/particles during the quantification process, which is usually performed byfiltration or ultracentrifugation, is sometimes difficult because the size of the LLPS particlesis too small to be separated [48,49]. Figure 5 shows the dissolution profiles of montelukast(MLK) ASDs, where two types of syringe filters with different pore sizes were used. WhenEudragit L100-55 was used as a polymeric excipient, the LLPS particles passed throughthe membrane with a 0.45 µm pore, resulting in apparent superior dissolution. However, adramatic decrease in concentration was found when a syringe filter with a 0.22 µm porewas applied. The apparent LLPS concentrations in the presence of PVPVA, Eudragit, and inthe absence of polymers were 2.0, 1.2, and 0.6 µg/mL, respectively. The final concentrationof MLK in the presence of PVPVA agreed with the LLPS concentration, whereas those in thepresence of Eudragit and in the absence of polymer shifted slightly to lower concentration,presumably because of the presence of excess solids to change equilibrium balance [50].The evaluation using a 0.22 µm syringe filter offered a good prediction of oral absorption,as shown later.Pharmaceutics 2025, 17, 262 8 of 17Pharmaceutics 2025, 17, x FOR PEER REVIEW 8 of 18    Figure 5. Non-sink dissolution study of MLK ASDs (25 µg/mL as equivalent of MLK). Eudragit L100-55 and PVPVA were used as excipients. All ASDs (MLK:polymer = 1:4) were prepared by freeze-drying. The test solution was filtrated using syringe filters with two different pore sizes (0.22 or 0.45 µm), as noted in the figure. PM represents the physical mixture of crystalline MLK and man-nitol. Unpublished results. The dissolution test is a batch system where the dissolved drug is not removed from the test medium throughout the study. As the LLPS/crystallization behavior is influenced by the degree of supersaturation, retention of the drug in the medium, which is different from conditions in the intestinal tract, may accelerate LLPS/crystallization to decrease the free drug concentration. Bevernage et al. revealed that the decrease in the concentration of supersaturated posaconazole solution was slower in the presence of an absorption sink, as the removal of the free drug through the membrane reduced the driving force of crys-tallization [51]. This result demonstrates the limitations of dissolution studies in predict-ing the oral absorption of supersaturable dosage forms. To overcome this problem, disso-lution tests that include absorption sinks have been proposed, as represented by a bipha-sic dissolution system [36,52,53]. In this system, the octanol phase is included in the pad-dle vessel to allow the transfer of the dissolved drug to the octanol phase. This system may work for predicting oral absorption from supersaturable dosage forms to some ex-tent; however, the limitations of this system include the absence of a transfer barrier to the octanol phase and difficulty in including the surface-active component in the medium. In vivo components may also influence the dissolution process of ASDs. The use of simulated gastric fluids for dissolution studies may work to some extent; however, the composition of the intestinal components is not limited to those in the simulated fluids, and in addition, the inter-individual variance of the intestinal components is substantial [54]. Intestinal components form various types of molecular assemblies, including mixed micelles, vesicles, and oil droplets, which can accommodate lipophilic drugs [54–56]. Moreover, surface-active agents can alter the dissolution and supersaturation behaviors of supersaturable formulations [13–15,23,45]. Nevertheless, LLPS behaviors in buffer so-lutions and human intestinal fluids were found to be similar [49]. If a crystalline drug is included in ASDs, even in trace amounts, it can destroy the supersaturation behavior. This is because the remaining crystals may act as templates for crystallization [57,58]. However, it was also observed that a trace amount of crystals did not impact the supersaturation behavior, as they may have dissolved immediately [59–61]. Thus, the impact of residual crystal seems to depend on many factors, including the Figure 5. Non-sink dissolution study of MLK ASDs (25 µg/mL as equivalent of MLK). EudragitL100-55 and PVPVA were used as excipients. All ASDs (MLK:polymer = 1:4) were prepared byfreeze-drying. The test solution was filtrated using syringe filters with two different pore sizes (0.22or 0.45 µm), as noted in the figure. PM represents the physical mixture of crystalline MLK andmannitol. Unpublished results.The dissolution test is a batch system where the dissolved drug is not removed fromthe test medium throughout the study. As the LLPS/crystallization behavior is influencedby the degree of supersaturation, retention of the drug in the medium, which is differentfrom conditions in the intestinal tract, may accelerate LLPS/crystallization to decrease thefree drug concentration. Bevernage et al. revealed that the decrease in the concentrationof supersaturated posaconazole solution was slower in the presence of an absorptionsink, as the removal of the free drug through the membrane reduced the driving forceof crystallization [51]. This result demonstrates the limitations of dissolution studies inpredicting the oral absorption of supersaturable dosage forms. To overcome this problem,dissolution tests that include absorption sinks have been proposed, as represented by abiphasic dissolution system [36,52,53]. In this system, the octanol phase is included inthe paddle vessel to allow the transfer of the dissolved drug to the octanol phase. Thissystem may work for predicting oral absorption from supersaturable dosage forms to someextent; however, the limitations of this system include the absence of a transfer barrier tothe octanol phase and difficulty in including the surface-active component in the medium.In vivo components may also influence the dissolution process of ASDs. The use ofsimulated gastric fluids for dissolution studies may work to some extent; however, thecomposition of the intestinal components is not limited to those in the simulated fluids, andin addition, the inter-individual variance of the intestinal components is substantial [54]. In-testinal components form various types of molecular assemblies, including mixed micelles,vesicles, and oil droplets, which can accommodate lipophilic drugs [54–56]. Moreover,surface-active agents can alter the dissolution and supersaturation behaviors of supersat-urable formulations [13–15,23,45]. Nevertheless, LLPS behaviors in buffer solutions andhuman intestinal fluids were found to be similar [49].If a crystalline drug is included in ASDs, even in trace amounts, it can destroy thesupersaturation behavior. This is because the remaining crystals may act as templates forcrystallization [57,58]. However, it was also observed that a trace amount of crystals didnot impact the supersaturation behavior, as they may have dissolved immediately [59–61].Thus, the impact of residual crystal seems to depend on many factors, including thecrystallization rate of the drug, the solubility of the drug, and the supersaturation stabilityPharmaceutics 2025, 17, 262 9 of 17of the formulation. Dissolution tests are sometimes more discriminative than other physicalcharacterization methods, including X-ray powder diffraction and differential scanningcalorimetry [61]. This means that the dissolution behavior of ASDs can be influenced evenwithout the obvious appearance of crystals in the formulation.4. Membrane Permeability of Supersaturated DrugVarious solubilization technologies are available for poorly soluble drugs. However,the use of solubilization agents may face a solubility–permeability interplay issue [47,62],as an increase in equilibrium solubility using solubilization agents does not increase theactivity of the drug. The interplay effect generally does not cause serious issues in vivo [47];however, it sometimes happens in the presence of strong interactions between the drugand its carrier [63,64]. A great advantage of ASD is that it is free from interplay issues, asit increases the activity of the drug, which can be assessed using membrane permeationstudies. The permeation rate is proportional to the difference in the activity of the drug inthe donor and acceptor phases, i.e.,dxadt= D(γdxd − γaxa), (4)where x and γ are the concentration and activity coefficient of the drug, respectively.Subscripts a and d represent the acceptor and the donor phases, respectively. t and D denotetime and diffusion coefficient, respectively. If the donor phase is saturated with excesssolids, xd may be replaced with the solubility under the assumption of a sufficiently fastdissolution rate. Thus, if the solution is supersaturated with amorphous solids, xd may bereplaced by the LLPS concentration, which generates a much stronger driving force forpermeation. Moreover, LLPS droplets may be regarded as drug reservoirs that immediatelyprovide lost drug molecules due to permeation.As discussed in the previous section, the ability of dissolution tests to predict thein vivo absorption is limited. Thus, the observation of membrane permeability provides agreater chance of prediction of oral absorption. When a simple polymeric membrane is usedfor the test, permeation is dominated by the activity of the drug in the donor phase [47]. Ifan artificial lipid membrane is used, the drug is distributed to the membrane, in additionto the donor and the acceptor phases. In this situation, the equations to describe the drugdistribution are as follows:dCmdt= kd(γmSmγdSd− γmCmγdCd)(5)dCadt= ka(γmCmγaCa− γmSmγaSa)(6)Equations (5) and (6) describe the drug distribution from the donor phase to themembrane and from the membrane to the acceptor phase, respectively. Here, γm, Sm,and Cm are the activity coefficient, solubility, and drug concentration in the membrane,respectively. kd and ka are the permeation coefficients into and out of the membrane,respectively. A sufficiently larger γmSm than γdSd is required for the effective distributionof the drug from the donor phase to the membrane, whereas an excessively large γmSmlimits the distribution of the drug from the membrane to the acceptor phase. Thus, thecomposition of the membrane significantly influences permeation results [65]. To facilitatedrug release from the membrane to the acceptor phase, solubilization agents must beincluded in the acceptor phase [66].A side-by-side cell equipped with an artificial lipid membrane may allow the predic-tion of oral absorption. Figure 6 shows an example where the oral absorption from GFPharmaceutics 2025, 17, 262 10 of 17ASDs was predicted. For the donor phase, GF dissolved in an organic solvent was addedto create a supersaturated solution, where various types of polymers were predissolved.The membrane permeability depended on the polymer type (Figure 6a). The results of theoral administration study of GF ASDs are presented in Figure 6b, which exhibited a goodcorrelation with the membrane permeation study.Pharmaceutics 2025, 17, x FOR PEER REVIEW 10 of 18   ASDs was predicted. For the donor phase, GF dissolved in an organic solvent was added to create a supersaturated solution, where various types of polymers were predissolved. The membrane permeability depended on the polymer type (Figure 6a). The results of the oral administration study of GF ASDs are presented in Figure 6b, which exhibited a good correlation with the membrane permeation study. Many membrane transport studies have suggested that transmembrane flux is dom-inated by the activity of the drug; however, a detailed investigation suggested that LLPS droplets may also contribute to enhanced drug permeation [66], which was explained by the particle drifting effect [67]. In this explanation, drug droplets/particles are assumed to approach the membrane effectively because they are carried as concentrated droplets/par-ticles. However, the presence of a mucus layer is not ignorable in living systems. Thus, another explanation for the contribution of LLPS droplets may be their effective penetra-tion through the mucus layer to approach the epithelial membrane. In fact, submicron particles are known to diffuse in the mucus layer more rapidly than general thought, de-pending on their surface properties [68,69].  Figure 6. (a) The side-by-side membrane permeation study of supersaturated GF solution. The GF concentration in the acceptor phase is presented. An artificial lipid membrane comprising lecithin and dodecane was used for the study. The initial concentrations of GF and polymer in the donor phase were 0.2 mg/mL and 5 mg/mL, respectively. The types of polymers used are indicated in the figure. Unpublished results. (b) Plasma GF concentration after administration of GF ASDs to fasted rats at a dose of 10 mg/kg. The polymers used for the ASDs are indicated in the figure. PM indicates the physical mixture of crystalline GF and mannitol. The figures were adopted from ref. [13] after modifications with permission from Elsevier. Surfactants are frequently included in both ASDs and dissolution medium. If the sur-factant concentration is above the critical micellar concentration, its negative influence on the membrane permeation of drugs is anticipated [47,62]. Biorelevant media contain large amounts of bile acids that dissolve poorly soluble drugs. Many in vitro membrane perme-ation studies have revealed that drug molecules dissolved in bile salt micelles cannot per-meate through the membrane [46]. However, bile salt micelles are unlikely to inhibit the permeation of the captured drug in vivo [47,70]. Presumably, the inhibitory effect of sur-factant micelles depends on the strength of the interaction between the drug and the mi-celles, which requires further verification. Figure 6. (a) The side-by-side membrane permeation study of supersaturated GF solution. The GFconcentration in the acceptor phase is presented. An artificial lipid membrane comprising lecithinand dodecane was used for the study. The initial concentrations of GF and polymer in the donorphase were 0.2 mg/mL and 5 mg/mL, respectively. The types of polymers used are indicated in thefigure. Unpublished results. (b) Plasma GF concentration after administration of GF ASDs to fastedrats at a dose of 10 mg/kg. The polymers used for the ASDs are indicated in the figure. PM indicatesthe physical mixture of crystalline GF and mannitol. The figures were adopted from ref. [13] aftermodifications with permission from Elsevier.Many membrane transport studies have suggested that transmembrane flux is domi-nated by the activity of the drug; however, a detailed investigation suggested that LLPSdroplets may also contribute to enhanced drug permeation [66], which was explainedby the particle drifting effect [67]. In this explanation, drug droplets/particles are as-sumed to approach the membrane effectively because they are carried as concentrateddroplets/particles. However, the presence of a mucus layer is not ignorable in livingsystems. Thus, another explanation for the contribution of LLPS droplets may be theireffective penetration through the mucus layer to approach the epithelial membrane. In fact,submicron particles are known to diffuse in the mucus layer more rapidly than generalthought, depending on their surface properties [68,69].Surfactants are frequently included in both ASDs and dissolution medium. If thesurfactant concentration is above the critical micellar concentration, its negative influenceon the membrane permeation of drugs is anticipated [47,62]. Biorelevant media containlarge amounts of bile acids that dissolve poorly soluble drugs. Many in vitro membranepermeation studies have revealed that drug molecules dissolved in bile salt micelles cannotpermeate through the membrane [46]. However, bile salt micelles are unlikely to inhibitthe permeation of the captured drug in vivo [47,70]. Presumably, the inhibitory effect ofsurfactant micelles depends on the strength of the interaction between the drug and themicelles, which requires further verification.Pharmaceutics 2025, 17, 262 11 of 175. Impact of LLPS on Oral AbsorptionASD technology is usually applied to BCS class II compounds, where the solubilityand/or dissolution rate can be limiting factors for oral absorption. As LLPS is analogous toamorphous solubility, LLPS concentration-limited absorption can occur. Figure 7a showsa non-sink dissolution study of fenofibrate (FEN) ASDs [15]. The solubility of FEN doesnot depend on pH; however, the solubility of Eudragit L100 and HPMCAS does. Thus,a pH shift was required to find a correlation between the dissolution test and the oraladministration study. The ASD with Eudragit exhibited the best dissolution, followed bythe PVPVA ASD. The HPMCAS ASD failed to improve the dissolution behavior comparedwith crystalline FEN, as HPMCAS formed a gel during the low pH period, which preventedthe release of FEN, even after the addition of Tween. Figure 7b shows the oral absorptionof FEN from ASDs in rats [15]. The ASD prepared using Eudragit exhibited the bestabsorption, followed by the PVPVA ASD. The ASD with HPMCAS did not improve theoral absorption relative to crystalline FEN. This order was in good agreement with thatof the dissolution study. Figure 7c shows the relationship between the apparent LLPSconcentration of FEN in the presence of each polymer and the area under concentration(AUC) of the plasma fenofibric acid concentration in the rat study [15]. A good correlationwas found except for HPMCAS ASD, which indicated the absorption from Eudragit andPVPVA ASDs were solubility (LLPS concentration)-limited. Absorption from the HPMCASASD appeared to be limited by the dissolution rate, as this ASD caused gelation duringthe dissolution process, retarding drug release. As the presence of HPMCAS offered thehighest LLPS concentration, if the HPMCAS ASD could be formulated appropriately, e.g.,by adding disintegrants, it should provide the best absorption.Pharmaceutics 2025, 17, x FOR PEER REVIEW 11 of 18   5. Impact of LLPS on Oral Absorption ASD technology is usually applied to BCS class II compounds, where the solubility and/or dissolution rate can be limiting factors for oral absorption. As LLPS is analogous to amorphous solubility, LLPS concentration-limited absorption can occur. Figure 7a shows a non-sink dissolution study of fenofibrate (FEN) ASDs [15]. The solubility of FEN does not depend on pH; however, the solubility of Eudragit L100 and HPMCAS does. Thus, a pH shift was required to find a correlation between the dissolution test and the oral administration study. The ASD with Eudragit exhibited the best dissolution, followed by the PVPVA ASD. The HPMCAS ASD failed to improve the dissolution behavior com-pared with crystalline FEN, as HPMCAS formed a gel during the low pH period, which prevented the release of FEN, even after the addition of Tween. Figure 7b shows the oral absorption of FEN from ASDs in rats [15]. The ASD prepared using Eudragit exhibited the best absorption, followed by the PVPVA ASD. The ASD with HPMCAS did not im-prove the oral absorption relative to crystalline FEN. This order was in good agreement with that of the dissolution study. Figure 7c shows the relationship between the apparent LLPS concentration of FEN in the presence of each polymer and the area under concen-tration (AUC) of the plasma fenofibric acid concentration in the rat study [15]. A good correlation was found except for HPMCAS ASD, which indicated the absorption from Eudragit and PVPVA ASDs were solubility (LLPS concentration)-limited. Absorption from the HPMCAS ASD appeared to be limited by the dissolution rate, as this ASD caused gelation during the dissolution process, retarding drug release. As the presence of HPM-CAS offered the highest LLPS concentration, if the HPMCAS ASD could be formulated appropriately, e.g., by adding disintegrants, it should provide the best absorption.  Figure 7. (a) pH-shift non-sink dissolution study of FEN ASDs. The test was initiated at pH 1.2, followed by an increase to pH 7.0 at 30 min. Tween 80 was also added at 30 min to reach a concen-tration of 0.1%. (b) Oral administration study of FEN ASDs in fasted rats at a dose of 7.5 mg/kg. Plasma fenofibric acid concentration, which is a major metabolite of FEN, is presented. (c) Correla-tion between apparent LLPS concentration in the presence of polymers and AUC of the oral admin-istration study. Polymer type: Eudragit L100 (purple), PVPVA (red), and HPMCAS (green). The blue symbol represents crystalline FEN. Figures are adopted from ref. [15] after modifications with permission from Elsevier. Figure 8 shows other examples where oral absorption could be explained by appar-ent LLPS concentration [17]. When albendazole (ALZ), a poorly soluble basic drug, was administered in the form of ASDs (Figure 8a), the order of the polymers that offered high exposure was PVPVA, HPMCAS, and Eudragit L100. The apparent LLPS concentrations of ALZ in the presence of these polymers were 7.2, 7.0, and 3.8 µg/mL, respectively. For Figure 7. (a) pH-shift non-sink dissolution study of FEN ASDs. The test was initiated at pH1.2, followed by an increase to pH 7.0 at 30 min. Tween 80 was also added at 30 min to reacha concentration of 0.1%. (b) Oral administration study of FEN ASDs in fasted rats at a dose of7.5 mg/kg. Plasma fenofibric acid concentration, which is a major metabolite of FEN, is presented.(c) Correlation between apparent LLPS concentration in the presence of polymers and AUC of theoral administration study. Polymer type: Eudragit L100 (purple), PVPVA (red), and HPMCAS (green).The blue symbol represents crystalline FEN. Figures are adopted from ref. [15] after modificationswith permission from Elsevier.Figure 8 shows other examples where oral absorption could be explained by apparentLLPS concentration [17]. When albendazole (ALZ), a poorly soluble basic drug, wasadministered in the form of ASDs (Figure 8a), the order of the polymers that offered highexposure was PVPVA, HPMCAS, and Eudragit L100. The apparent LLPS concentrations ofALZ in the presence of these polymers were 7.2, 7.0, and 3.8 µg/mL, respectively. For ALZin the absence of polymers, the concentration was 1.4 µg/mL. Thus, the order of absorptionand apparent LLPS concentrations agreed well. A similar observation was made for MLKPharmaceutics 2025, 17, 262 12 of 17ASDs (Figure 8b). MLK is a poorly soluble acidic drug. A significant improvement inoral absorption was observed for the PVPVA ASD, whereas the Eudragit ASD providedonly a marginal improvement. This absorption order also agreed well with the apparentLLPS concentrations. The dissolution test (Figure 4) could also predict oral absorption.These observations revealed that the oral absorption from ASDs can be predicted from theapparent LLPS concentration if the absorption is limited by solubility.Pharmaceutics 2025, 17, x FOR PEER REVIEW 12 of 18   ALZ in the absence of polymers, the concentration was 1.4 µg/mL. Thus, the order of ab-sorption and apparent LLPS concentrations agreed well. A similar observation was made for MLK ASDs (Figure 8b). MLK is a poorly soluble acidic drug. A significant improve-ment in oral absorption was observed for the PVPVA ASD, whereas the Eudragit ASD provided only a marginal improvement. This absorption order also agreed well with the apparent LLPS concentrations. The dissolution test (Figure 4) could also predict oral ab-sorption. These observations revealed that the oral absorption from ASDs can be predicted from the apparent LLPS concentration if the absorption is limited by solubility.  Figure 8. (a) Oral administration study of ALZ ASDs and physical mixtures of crystalline ALZ with mannitol in fasted rats at a dose of 10 mg/kg. The polymers used for the ASDs were PVPVA, HPM-CAS, and Eudragit L100. The plasma concentration of the major metabolite, ALZ sulfoxide, is pre-sented. The figure was reproduced from ref. [17] with modification under the Creative Commons license. (b) Oral administration study of MLK ASDs in fasted rats at a dose of 10 mg/kg. The poly-mers used were PVPVA and Eudragit L100-55. The ASDs (MLK:polymer = 1:4) were prepared by freeze-drying. The absorption from a physical mixture of crystalline MLK and mannitol is also pre-sented. Unpublished results. The prediction of plasma concentration profiles after oral administration of ASDs re-mains challenging. However, an attempt to include the particle drifting effect in conven-tional prediction protocols has been reported [71]. Figure 9a shows the predicted plasma concentration profile of itraconazole after the oral administration of SporanoxTM. By con-sidering the particle drifting effect, the predicted plasma concentrations increased to reach the experimental concentrations. A similar observation was made for the enzalutam-ide/PVPVA ASD (Figure 9b). Figure 8. (a) Oral administration study of ALZ ASDs and physical mixtures of crystalline ALZwith mannitol in fasted rats at a dose of 10 mg/kg. The polymers used for the ASDs were PVPVA,HPMCAS, and Eudragit L100. The plasma concentration of the major metabolite, ALZ sulfoxide, ispresented. The figure was reproduced from ref. [17] with modification under the Creative Commonslicense. (b) Oral administration study of MLK ASDs in fasted rats at a dose of 10 mg/kg. Thepolymers used were PVPVA and Eudragit L100-55. The ASDs (MLK:polymer = 1:4) were preparedby freeze-drying. The absorption from a physical mixture of crystalline MLK and mannitol is alsopresented. Unpublished results.The prediction of plasma concentration profiles after oral administration of ASDsremains challenging. However, an attempt to include the particle drifting effect in con-ventional prediction protocols has been reported [71]. Figure 9a shows the predictedplasma concentration profile of itraconazole after the oral administration of SporanoxTM.By considering the particle drifting effect, the predicted plasma concentrations increasedto reach the experimental concentrations. A similar observation was made for the enzalu-tamide/PVPVA ASD (Figure 9b).Numerous studies that claimed the enhancement of oral absorption using ASD tech-nology are available in the literature. However, the use of the amorphous state may nothave been a key factor in some cases. Basic drugs can be supersaturated if they are trans-ferred from an acidic to a neutral pH. Therefore, amorphization may not be required todesign supersaturable dosage forms. Figure 10a shows the oral absorption of ALZ fromASDs and physical mixtures (PMs) with an identical composition. Absorption from thePMs significantly increased with the addition of Eudragit L100-55 as an excipient. Theabsorption of ALZ from the 1:3 PM was slightly lower but almost identical to that from the1:3 ASD, suggesting that the supersaturation–maintenance effect of the polymer was likelyto be more important than the dissolution enhancement by amorphization. The absorptionenhancement was found to depend on the amount of the polymer added. This observationindicated that the oral absorption of basic drugs can be improved without amorphization ifa polymer that can maintain the supersaturation is used.Pharmaceutics 2025, 17, 262 13 of 17Pharmaceutics 2025, 17, x FOR PEER REVIEW 13 of 18    Figure 9. Comparison of model fitting to oral absorption data in rats using GastroPlusTM following conventional procedure (solid lines) and a model which considers particle drifting effect into un-stirred water layer (break lines) [71]. (a) The fitting to oral absorption of itraconazole from ASD (Sporanox). The absorption data was taken from ref. [72]. (b) The fitting to oral absorption of en-zalutamide from PVPVA ASD. The absorption data was taken from ref. [73]. Figures are adopted with permission from American Chemical Society. https://pubs.acs.org/doi/10.1021/acs.molpharma-ceut.9b00889, accessed on 16 January 2025. Numerous studies that claimed the enhancement of oral absorption using ASD tech-nology are available in the literature. However, the use of the amorphous state may not have been a key factor in some cases. Basic drugs can be supersaturated if they are trans-ferred from an acidic to a neutral pH. Therefore, amorphization may not be required to design supersaturable dosage forms. Figure 10a shows the oral absorption of ALZ from ASDs and physical mixtures (PMs) with an identical composition. Absorption from the PMs significantly increased with the addition of Eudragit L100-55 as an excipient. The absorption of ALZ from the 1:3 PM was slightly lower but almost identical to that from the 1:3 ASD, suggesting that the supersaturation–maintenance effect of the polymer was likely to be more important than the dissolution enhancement by amorphization. The ab-sorption enhancement was found to depend on the amount of the polymer added. This observation indicated that the oral absorption of basic drugs can be improved without amorphization if a polymer that can maintain the supersaturation is used. Another example presented below is a case where the evaluation of the reference crystal was inappropriate (Figure 10b). In the literature, the oral absorption of ibuprofen has been reported to be improved by using ASD technology, where poloxamer 188 was used as an excipient [74]. Absorption from the crystalline PM was extremely low in this study. Our similar attempt using ASDs yielded similar plasma concentration profiles as shown in the figure. However, as the absorption from crystalline PM was similar to that from ASDs, we could not find any improvement in absorption using ASDs. The difference between our study and the literature is the difference in the absorption from crystalline PM. The most likely reason for this is the difference in the particle sizes of the IBP crystals, as absorption of poorly soluble drugs can be limited by the dissolution rate if the particle size is too large. IBP does not require amorphization to improve its oral absorption. Figure 9. Comparison of model fitting to oral absorption data in rats using GastroPlusTM followingconventional procedure (solid lines) and a model which considers particle drifting effect into unstirredwater layer (break lines) [71]. (a) The fitting to oral absorption of itraconazole from ASD (Sporanox).The absorption data was taken from ref. [72]. (b) The fitting to oral absorption of enzalutamide fromPVPVA ASD. The absorption data was taken from ref. [73]. Figures are adopted with permissionfrom American Chemical Society. https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.9b00889,accessed on 16 January 2025.Pharmaceutics 2025, 17, x FOR PEER REVIEW 14 of 18    Figure 10. (a) Oral administration study of ALZ ASDs and physical mixtures (PM) of crystalline ALZ with Eudragit L100-55 or mannitol (shown as Crystal PM) in fasted rats at a dose of 10 mg/kg. Eudragit L100-55 was used for the ASDs and PMs at the mixing ratio (ALZ:Eudragit) shown in the figure. Unpublished results (except the crystal PM data). (b) Oral administration study of IBP ASDs in fasted rats at a dose of 25 mg/kg. The data presented by black symbols are taken from literature with permission from Elsevier, where poloxamer 188 was used as an excipient [74]. The IBP:polox-amer ratios were 2:1 (triangle) or 1:1 (square). The circles represent PM at a ratio of 1:10. Our data is presented by colored symbols. Eudragit L100 (purple squares) or PVP k30 (red triangles) was used as a polymer for ASDs at a mixing ratio of 1:4 (IBP:polymer). The ASDs were prepared by freeze-drying. Blue circles represent crystalline IBP, which had a particle size of a few µm. The error bars were omitted for clarity, but the deviations were significantly large for the literature data on ASDs. Our data are unpublished results. 6. Conclusions Recently updated knowledge on the dissolution, membrane permeation, and oral ab-sorption behaviors of ASDs is presented with a focus on LLPS behavior. As the dissolution process is sensitive to various factors, including the applied dose, medium composition, and pH, these factors must be considered when designing a dissolution test for predicting in vivo absorption. Supersaturated solutions can cause LLPS and/or crystallization. The LLPS concentration, which is analogous to the amorphous solubility, is typically higher than the crystalline solubility by more than an order of magnitude. Transmembrane flux is governed not by apparent solubility but by activity. Therefore, ASD has an advantage over other solubilization techniques that increase the equilibrium solubility to increase the flux. Moreover, LLPS droplets are expected to increase the transmembrane flux by increas-ing the drug concentration near the epithelial cell membrane, possibly through the particle drifting effect and/or effective permeation through the mucus layer. The LLPS concentra-tion can be a dominant factor in predicting oral absorption from ASDs if absorption is limited by solubility. A model prediction that considers the particle drifting effect in the unstirred water layer is also available. An appropriate formulation design is necessary for finding these advantages of ASDs. Funding: This research received no external funding. Institutional Review Board Statement: All animal experiments were approved by the Ethical Re-view Committee of Daiichi Sankyo RD Novare (Exp. No. 2017-024, 2017-027). Almost the same ex-perimental protocol was applied for all experiments, which is described in detail in ref. [13]. Informed Consent Statement: Not applicable. Figure 10. (a) Oral administration study of ALZ ASDs and physical mixtures (PM) of crystallineALZ with Eudragit L100-55 or mannitol (shown as Crystal PM) in fasted rats at a dose of 10 mg/kg.Eudragit L100-55 was used for the ASDs and PMs at the mixing ratio (ALZ:Eudragit) shown inthe figure. Unpublished results (except the crystal PM data). (b) Oral administration study of IBPASDs in fasted rats at a dose of 25 mg/kg. The data presented by black symbols are taken fromliterature with permission from Elsevier, where poloxamer 188 was used as an excipient [74]. TheIBP:poloxamer ratios were 2:1 (triangle) or 1:1 (square). The circles represent PM at a ratio of 1:10.Our data is presented by colored symbols. Eudragit L100 (purple squares) or PVP k30 (red triangles)was used as a polymer for ASDs at a mixing ratio of 1:4 (IBP:polymer). The ASDs were preparedby freeze-drying. Blue circles represent crystalline IBP, which had a particle size of a few µm. Theerror bars were omitted for clarity, but the deviations were significantly large for the literature dataon ASDs. Our data are unpublished results.Another example presented below is a case where the evaluation of the referencecrystal was inappropriate (Figure 10b). In the literature, the oral absorption of ibuprofenhas been reported to be improved by using ASD technology, where poloxamer 188 wasused as an excipient [74]. Absorption from the crystalline PM was extremely low in thishttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.9b00889Pharmaceutics 2025, 17, 262 14 of 17study. Our similar attempt using ASDs yielded similar plasma concentration profiles asshown in the figure. However, as the absorption from crystalline PM was similar to thatfrom ASDs, we could not find any improvement in absorption using ASDs. The differencebetween our study and the literature is the difference in the absorption from crystalline PM.The most likely reason for this is the difference in the particle sizes of the IBP crystals, asabsorption of poorly soluble drugs can be limited by the dissolution rate if the particle sizeis too large. IBP does not require amorphization to improve its oral absorption.6. ConclusionsRecently updated knowledge on the dissolution, membrane permeation, and oral ab-sorption behaviors of ASDs is presented with a focus on LLPS behavior. As the dissolutionprocess is sensitive to various factors, including the applied dose, medium composition,and pH, these factors must be considered when designing a dissolution test for predictingin vivo absorption. Supersaturated solutions can cause LLPS and/or crystallization. TheLLPS concentration, which is analogous to the amorphous solubility, is typically higherthan the crystalline solubility by more than an order of magnitude. Transmembrane flux isgoverned not by apparent solubility but by activity. Therefore, ASD has an advantage overother solubilization techniques that increase the equilibrium solubility to increase the flux.Moreover, LLPS droplets are expected to increase the transmembrane flux by increasing thedrug concentration near the epithelial cell membrane, possibly through the particle driftingeffect and/or effective permeation through the mucus layer. The LLPS concentration canbe a dominant factor in predicting oral absorption from ASDs if absorption is limited bysolubility. A model prediction that considers the particle drifting effect in the unstirredwater layer is also available. An appropriate formulation design is necessary for findingthese advantages of ASDs.Funding: This research received no external funding.Institutional Review Board Statement: All animal experiments were approved by the EthicalReview Committee of Daiichi Sankyo RD Novare (Exp. No. 2017-024, 2017-027). Almost the sameexperimental protocol was applied for all experiments, which is described in detail in ref. [13].Informed Consent Statement: Not applicable.Data Availability Statement: The raw data supporting the conclusions of this article will be madeavailable by the authors upon request.Acknowledgments: The dissolution study data of MLK ASDs (Figure 5), the oral absorption dataof MLK ASDs (Figure 8b), and the oral absorption data from ALZ PMs and IBP ASDs (Figure 10)were obtained as part of the activities under the Consortium of Biopharmaceutical Tools (CoBiTo) atRitsumeikan University. 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