AAPS PharmSciTech (2024) 25:192

https://doi.org/10.1208/s12249-024-02912-9

RESEARCH ARTICLE

Thermally‑Induced Supersaturation Approach for Optimizing Drug

Loading and Biopharmaceutical Properties of Supersaturated
Lipid‑Based Formulations: Case Studies with Ibrutinib
and Enzalutamide
Arvind Sirvi1 · Akash Janjal1 · Kajal Guleria1 · Mahesh Chand1 · Abhay T. Sangamwar1

Received: 7 June 2024 / Accepted: 31 July 2024

© The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists 2024

Abstract
Lipid-based formulations (LbFs) have demonstrated success in pharmaceutical applications; however, challenges persist
in dissolving entire doses of the drug into defined liquid volumes. In this study, the temperature-induced supersaturation
method was employed in LbF to address drug loading and pill burden issues. Supersaturated LbFs (super-LbF) were prepared
using the temperature-induced supersaturation method, where the drug load is above its equilibrium solubility. Further, the
influence of the drug’s physicochemical and thermal characteristics on drug loading and their relevance with an apparent
degree of supersaturation (aDS) was studied using two model drugs, ibrutinib and enzalutamide. All the prepared LbFs were
evaluated in terms of physical stability, dispersion, and solubilization capacity, as well as pharmacokinetic assessments.
Drug re-crystallization was observed in the lipid solution on long-term storage at higher aDS values of 2–2.5. Furthermore,
high-throughput lipolysis studies demonstrated a significant decrease in drug concentration across all LbFs (regardless of
drug loading) due to a decline in the formulation solvation capacity and subsequent generation of in-situ supersaturation.
Further, the in vivo results demonstrated comparable pharmacokinetic parameters between conventional LbF and super-LbF.
The short duration of the thermodynamic metastable state limits the potential absorption benefits. However, super-LbFs
of Ibr and Enz showed superior profiles, with 1.7-fold and 5.2-fold increased drug exposure compared to their respective
crystalline suspensions. In summary, this study emphasizes the potential of temperature-induced supersaturation in LbF for
enhancing drug loading and highlights the intricate interplay between drug properties, formulation characteristics, and in
vivo performance.

Keywords aqueous solubilization · lipid-based formulation · oral absorption · physical stability · temperature-induced
supersaturation

Introduction enhance the absorption profile of these drugs. Particu-

larly, lipid-based formulation (LbF) as a supersaturating
In recent years, advancements in drug discovery technolo- formulation has gained significant attention for the oral
gies and methodologies, such as high-throughput screen- delivery of drug molecules with limited solubility and
ing and computational chemistry, have led to the identifi- dissolution-limited characteristics [3, 4]. This approach
cation of a growing number of lipophilic drug candidates facilitates the direct delivery of drugs in a solution form
with poor absorption profiles [1, 2]. Therefore, there is to the gastric region, thereby bypassing the rate-limiting
a need to develop an efficient formulation approach to dissolution step [5]. In the literature, it has been demon-
strated that LbF can enhance absorption while reducing
the positive food effect and inter-subject variability [3].
* Abhay T. Sangamwar
Despite the aforementioned advantages, conventional LbFs
[email protected]; [email protected]
are unable to accommodate higher drug loading due to the
1
Department of Pharmaceutics, National Institute low oil solubility of drugs. This is particularly challenging
of Pharmaceutical Education and Research, Sector‑67, in preclinical dose escalation studies, where higher oral
S.A.S Nagar, Punjab 160062, India

Vol.:(0123456789)
192 Page 2 of 17 AAPS PharmSciTech (2024) 25:192

doses of drugs are required [6]. Additional factors, such propensity to supersaturate within a lipid vehicle affect
as drug precipitation during colloidal dispersions, physi- their physical stability [8, 19]. Therefore, investigating
cal instability, and inadequate in vitro-in vivo correlation, the influence of drug properties is essential for designing
have been recognized as obstacles to fully realizing the super-LbF.
therapeutic potential and commercial success of conven- Biopharmaceutical assessment of super-LbF serves as
tional LbFs [7]. a pivotal determinant that reflects their true potential for
Thus, significant advancements are ongoing in the field clinical success. To date, super-LbF are well explored for
of LbFs, including the exploration of supersaturated LbF the selection of excipients or different types of LbF using in
(super-LbF), intending to boost drug loading capacities vitro dispersion and digestion settings. In the literature, it has
and simultaneously reduce pill burden [8–12]. Super-LbF been observed that super-LbF generates in-situ supersatura-
elevates the drug concentration above its equilibrium solu- tion in the non-sink digestive condition due to a reduction
bility in lipid solution by employing temperature-based or in solubilization capacity upon dispersion [4]. However, the
ionic liquid-based supersaturation [7, 8, 13, 14]. Among rate and extent of supersaturation depend on several factors,
these methods, temperature-mediated supersaturation including lipid excipients, drug payload, the nature of the
is a highly considered and feasible approach to elevate drug, and interactions of the drug with solubilizing species
the drug solubility in the lipid solution. In this approach, in digestive conditions [5]. The imbalance of supersaturation
supersaturation is induced directly in LbF thermally, by in the intraluminal passage could affect the in vivo absorp-
heating the lipid solution to, for example, 60–65°C, satu- tion of drugs, leading to variable outcomes, as evidenced
rating the system with the drug, and subsequently cooling in the literature [7]. Ilie et al. observed a noteworthy 2.3 to
it down to ambient temperature. The apparent degree of 2.5-fold increase in celecoxib exposure following the oral
supersaturation (aDS) is often used to express supersatu- administration of type I based super-LbF compared to non-
ration terminology as well as define drug payload within supersaturated LbF formulations, attributed to the enhanced
LbF, represented in Eq. 1. Where C is the solubilized con- supersaturated state observed in digestion studies [9]. Simi-
centration in super-LbF and C* is its saturation solubility larly, Koehl et al. observed a significant increase in the oral
in particular lipid solution at ambient temperature [15]. bioavailability of venetoclax using type-I based super LbF
compared to lipid suspension in pigs [8]. In contrast, Sique-
C
aDS = (1) ira et al. observed that type III super-LbF (50 mg/g formu-
C*
lation) exhibited comparatively lower cinnarizine exposure
The aDS in LbF significantly influences critical quality when compared to conventional LbF (20 mg/g formulation).
attributes such as physical stability, dispersion capacity, These observations could be attributed to drug-specific fac-
aqueous solubilization, and subsequently in vivo absorp- tors, particularly the high propensity of cinnarizine to pre-
tion. To date, few studies have reported on the influence of cipitate after the dispersion and digestion of super-LbF [20].
aDS achieved through temperature-mediated supersatura- Recently, Paulus and colleagues investigated the influence of
tion. Schultz et al. observed a linear correlation between supersaturation on the absorption of cinnarizine from type
processing temperature and increased solubility in lipidic I and type II LbFs. They observed a complex relationship
vehicles [16]. Consistent with previous findings, Almasri between supersaturation and drug exposure, with supersatu-
et al. also reported a 4–sevenfold increase in fenofibrate ration improving drug exposure more distinctly for type I
solubility in medium-chain triglyceride-based oils when LbFs compared to type II [21, 22]. Thus, there is a need to
the processing temperature was set at 60°C compared to explore the correlation between in vitro findings and their
25°C [17]. Due to the high degree of supersaturation, the influence on pharmacokinetic parameters, particularly for
physical stability of liquid LbF is of significant interest. type III-based super-LbF formulations.
Bannow et al. demonstrated that the increased thermo- The current study aims to develop a super-LbF using
dynamic driving force is responsible for drug re-crystal- a temperature-based supersaturation method across vari-
lization within lipid solution due to the higher degree of ous drug loading levels for two model drugs, Ibrutinib
supersaturation (aDS value of 2.5) and the onset of pre- (Ibr) and Enzalutamide (Enz). These drugs were chosen
cipitation time was within 24 h [18]. Similarly, Ilie et al. for their distinct physicochemical (log P and ionization
also observed that high aDS values (> 1.35) trigger drug behavior) and thermal characteristics (melting tempera-
crystallization within the vehicle during 28 days of storage tures and T ­ m/Tg ratios) (Table I) [23–26]. The physical
period. These observations were found to be more depend- stability of super-LbF was comprehensively evaluated
ent on the type of drug rather than the type of lipid vehi- over an extended duration, using type III-A LbF as the
cle [19]. The physicochemical properties of drugs, such prototype formulation. The prototype type III-A LbF was
as lipophilicity, melting point, glass-forming ability, and selected based on the higher drug solubility. Subsequently,
the in vitro studies were conducted to assess the influence
AAPS PharmSciTech (2024) 25:192 Page 3 of 17 192

Table I  Physicochemical and Parameters Ibrutinib Enzalutamide

Thermal Properties of Ibrutinib
and Enzalutamide [25, 26] Molecular weight 440.5 g/mol 464.44 g/mol
Dissociation constant (pKa) 3.8 (weak basic) Neutral
Log P 4.0 2.98
Polar surface area 99.2 Å2 109 Å2
Aqueous solubility in phosphate buffer (pH 6.5) 3 μg/mL 2.9 μg/mL
Melting peak Temperature (­ Tm) 158.11°C 200.83°C
Enthalpy of fusion (δhfus) 80.29 j/g 77.75 j/g
Crystallization peak temperature during cooling N/A N/A
Midpoint glass transition temperature (Tg, midpoint) 82.48°C 93.65°C
Crystallization peaks upon re-heating (Tcryst, heat) N/A N/A
Tm/Tg ratio 1.92 2.14
Approved maximum daily dose in human 560 mg (4 × 140 mg 160 mg
capsules) (4 × 40 mg
capsules)

of supersaturation on emulsification, and drug distribu- HPLC Quantification Method

tion in the aqueous phase. The results of these in vitro
studies were then correlated to the absorption parameters Chromatographic separation for Ibr and Enz was carried out
obtained from pre-clinical pharmacokinetic studies in using the Waters HPLC separation module and a GL Sci-
rats. This will help to understand the interplay between ences reversed-phase C18 column (5 μm; 4.6 × 250 mm) at
the physical attributes of the formulation and their result- a temperature of 25 ℃. The chromatographic conditions for
ant in vivo efficacy. drug quantification of Ibr and Enz are provided in supporting
information. Empower Pro software (Waters Corporation,
California, USA) was used to acquire chromatographic data,
which was extracted and analyzed at 260 nm and 237 nm
Materials & Methods wavelengths for Ibr and Enz, respectively. The analytical
methods demonstrated a unidirectional, proportional rela-
Materials tionship between concentration and the area under the curve
for both drugs. The HPLC analytical method validation
Ibrutinib (Ibr) as a gift sample was obtained from MSN parameters are summarized in supporting information.
Laboratories Pvt. Ltd. (Hyderabad, India). Enzaluta-
mide (Enz) was obtained as a request sample from Zydus Solid‑State Characterization of Model Drugs
Lifesciences Ltd. (Ahmedabad, India). Abitec Corpo-
ration (Columbus, Ohio) provided ­C apmul ® MCM EP Solid-state properties of Ibr and Enz were evaluated
(Glycerol Monocaprylocaprate) as a gift sample. Gat- using polarized light microscopy, powder X-ray diffrac-
tefossé (Co., France) provided ­M aisine ® CC, Labrafac tion (PXRD), and differential scanning calorimetry (DSC).
­Lipophile® WL1349, L ­ auroglycol®, ­Peceol®, ­Labrasol® Polarized light microscopy was employed to identify the
and ­Transcutol® HP. PEG 200 (Polyethylene glycol) was microscopic features of the powder drug samples. A Leica
given by Thermo Fisher Scientific India Pvt. Ltd. (Mum- DMLP polarized microscope (Leica Microsystems Wetzlar
bai, India). TRIS maleate salt, sodium hydrogen phos- GmbH, Germany) was used for this purpose, and the sam-
phate, sodium taurocholate hydrate, calcium chloride, and ple micrographs were captured using a Leica JVC digital
lecithin were provided by HiMedia Laboratories Pvt. Ltd. camera and analyzed using Leica IM 50 software. The
(Mumbai, India). Sigma-Aldrich (St. Louis, MO, USA) PXRD analysis was performed using an X-ray powder
provided 4-bromophenylboronic acid, K ­ olliphor® RH40. diffractometer (Rigaku, Japan). The applied voltage and
Avra Synthesis Pvt. Ltd (Hyderabad, India) delivered current were 40 kV and 35 mA, respectively. The samples
Sodium hydroxide for the study. All experiments were were mounted on stainless steel sample holders and were
conducted using purified water obtained from a Milli-Q scanned in continuous mode between 3 − 40° (2θ), with
purification system (Millipore, Bedford, MA). Other addi- a step size of 0.02°, and a scanning speed of 5 degrees/
tional solvents and chemicals used in this study were of min. The same instrument conditions were used for pre-
analytical grade. cipitate analysis. Additionally, DSC (TA Q2000 DSC with
192 Page 4 of 17 AAPS PharmSciTech (2024) 25:192

a refrigerated cooling accessory, TA Instruments, New Table II  A detailed Description of Formulation Components of Con-
Castle, DE) was employed to investigate the solid-state ventional and Super-LbF
properties and T
­ m/Tg ratio for model drugs. The samples Ibrutinib formulations
were prepared in hermetically sealed pans and were heated Formulation code Conventional LbF Super-LbF A Super-LbF B
at 20°C/min to 10°C above the melting temperature, held Capmul® MCM EP 500 mg 500 mg 500 mg
isothermally for 3 min, cooled at a rate of 20°C/min to Labrasol® 300 mg 300 mg 300 mg
-20°C, and reheated at 20°C/min to just above the melt- PEG 200 200 mg 200 mg 200 mg
ing temperature in the heat-cool-heat mode. The sample Drug load (mg/g) 60 mg 120 mg 150 mg
weights for each repeat sample were within 2–3 mg. aDS value 1 2 2.5
Enzalutamide formulations
Formulation code Conventional LbF Super-LbF A Super-LbF B
Solubility Studies in Excipients and Lipid Solutions Capmul® MCM EP 500 mg 500 mg 500 mg
Labrasol® 300 mg 300 mg 300 mg
In this study, solubility investigations were conducted at Transcutol® 200 mg 200 mg 200 mg
37 ± 2°C using a variety of lipids, surfactants, and co-sol- Drug load (mg/g) 32 mg 64 mg 80 mg
vents, employing a previously reported method [27]. Differ- aDS value 1 2 2.5
ent lipids and surfactants based on their chain length were
used to prepare the different prototype formulations (com-
position details are represented in Table S1). Type II LbF is Conventional LbF
composed of 60% w/v lipids and 40% w/v surfactants, while
type III formulation is prepared with 50% w/v lipids, 30% Conventional LbF was formulated using 50% w/v of oil,
w/v surfactants, and 20% w/v co-solvent. The formulation is 30% w/v of surfactant, and 20% w/v of co-solvent. The drug
prepared by combining excipients in the aforementioned pro- payload of each model drug was maintained at 100% of its
portions, which are mixed and vortexed to form an isotropic equilibrium solubility (measured at 25 ± 2°C) in the lipid
clear solution [28]. In addition, a water shaker bath (Daihan solution. Following drug loading, the formulations were
Scientific, Korea) was used to determine drug solubility in vortexed to achieve a clear solution.
various excipients and prototype lipid solutions, operated
at 37 ± 2°C and 100 rpm for 48 h. The crystalline drug was Super‑LbF
added in excess to individual vials containing a measured
amount of each excipient or lipid solution (1 g). After an Super-LbF was formulated with high drug loading wherein
equilibrium period, an adequate amount of samples was drug concentration exceeded than its equilibrium solubil-
obtained, following the collection of supernatant after their ity in the lipid solution. Higher drug loading was attained
centrifugation for 10 min at 10,000 rpm using iFuge UCo2R, through a temperature-based supersaturation method. In this
(Neuation Technologies, Gujarat, India) [14]. Later, the col- process, the lipid solution was heated to 60 ± 2°C, facili-
lected supernatant was diluted with a 70:30% v/v mixture of tating drug supersaturation, followed by cooling to room
ethyl acetate and methanol solution. Further, the resultant temperature [10]. The procedure was repeated to obtain a
supernatant was diluted with methanol before being ana- clear solution. In this study, two different super-LbF were
lyzed for drug content using HPLC conditions as described prepared with varying drug loadings based on their equilib-
in the section ‘HPLC quantification method’. The prototype rium solubility in the lipid solution (Table II).
lipid solution was chosen based on the maximum equilib-
rium solubility of drugs [29]. Furthermore, an evaluation of Physical Stability Evaluation
the impact of ambient and high temperature (25 ± 2°C and
60 ± 2°C) on drug solubility in the chosen prototype lipid The physical stability of prepared formulations was moni-
formulations was performed [9, 30]. tored for 60 days of storage at 25 ± 2°C and 65 ± 5%RH.
The samples were stored in closed glass vials throughout
the stability analysis. Visual assessments and polarized
Preparation of Ibrutinib and Enzalutamide Lipid light microscopy were performed at pre-defined time points.
Formulations Samples were withdrawn from different locations within the
glass vial, placed on a curved glass slide, and observed under
The influence of supersaturation on liquid lipid formulation the polarized light microscope (Leica, Germany).
was evaluated by preparing LbF with three different drug Drug content was determined at each time interval to
loading. The detailed formulation compositions of lipid for- assess the chemical stability. Every collected sample was
mulations for Ibr and Enz are provided in Table II. centrifuged for 10 min at 10,000 rpm, to allow solid particles
AAPS PharmSciTech (2024) 25:192 Page 5 of 17 192

to settle down. Subsequently, 50 mg of the supernatant was monitored for their solid-state characteristics using powder
diluted with a 70:30% v/v mixture of ethyl acetate and meth- X-ray diffraction [18]. The precipitate characterization was
anol solution. Further dilution of resultant supernatant with performed for super-LbF B (as a representative super-LbF)
methanol before being analyzed for content of drug using to assess the physical form of the precipitate compared with
HPLC conditions as described in the section ‘HPLC quan- the unformulated crystalline drug. The instrumental condi-
tification method’. tions for PXRD analysis are provided in the section ‘Solid-
state characterization of model drugs.
In Vitro Dispersion Studies
In Vivo Pharmacokinetic Study
The prepared LbF were studied for droplet size, polydis-
persity index (PDI), and zeta potential using the Zetasizer Ethics Approval
Nano ZS instrument (Malvern Instruments, Worcestershire,
UK). For this analysis, 100 mg of each liquid formulation The animal study protocol received approval from the insti-
was diluted with milli-Q water up to 20 mL in a glass vial. tutional Animal Ethics Committee of the National Institute
An emulsion was created by gently reversing the vial mul- of Pharmaceutical Education and Research (NIPER), S.A.S.
tiple times. Subsequently, a 1 mL aliquot was extracted and Nagar, and is duly registered with the Committee for the Pur-
analyzed in triplicate using a disposable ZEN0040 cuvette pose of Control and Supervision of Experiments on Animals
for droplet size and a DTS1070 cuvette for zeta potential. (CPCSEA) under registration number 108/1999/CPCSEA.
The approval numbers for the study protocols related to Ibr
In Vitro Digestion Study and Enz are IAEC/22/47 and IAEC/23/72, respectively.
For pharmacokinetic studies, the animals involved in this
The high throughput lipolysis method reported by Kilic and study were accommodated in the Central Animal Facility
Dressman was used to assess the impact of aDS on the aque- at NIPER, S.A.S. Nagar. They were housed in plastic cages,
ous drug solubilization in digestive conditions [31]. Briefly, adhering to standard laboratory conditions, which include
each formulation was dispersed in a digestion buffer (36 mL) maintaining a natural 12-h light–dark cycle. The animals
to simulate human fasted-state intestinal conditions. The had ad libitum access to a conventional rodent dry diet and
composition details of the media are provided in Table S2. water. Each study group consists five number of animals.
The initial experimental period (0–10 min) focused on the
dispersion behavior of the formulation, which was obtained Ibrutinib
by stirring it in the digestion medium. Aliquots (~ 0.5 mL)
were removed from the media at predefined time points of In the pharmacokinetic evaluation of Ibr formulations,
2.5, 5, and 10 min relative to the start of dispersion. The female Sprague Dawley rats (weighing 180–220 g) received
samples were centrifuged at 37°C and 15,000 rpm for 10 oral administrations of crystalline drug suspension, conven-
min and analyzed for aqueous phase and pellet phase drug tional LbF, and super-LbF. super-LbF with the highest Ibr
concentration. After completion of the dispersion phase, the loading was selected for further pharmacokinetic evalua-
digestion process was initiated by adding 4 mL of pancre- tion to assess the impact of supersaturation and formulation
atic extract to the solution. The addition of 0.6 M NaOH quantity. Sodium carboxymethyl cellulose (0.2% w/v) was
solution to the media maintained a 6.5 pH throughout the used as a suspending agent to disperse the crystalline drug in
experiment. Aliquots (~ 0.5 mL) from the digestive media purified water. Each group of five animals received a 20 mg/
were collected at predefined time points of 5, 15, 30, 45, and kg dose of Ibr in a 10 mL/kg volume [32]. Following oral
60 min relative to the initiation of digestion. Further diges- administration, blood samples of approximately 0.2 mL were
tion was prevented by the prior addition of lipase inhibitor obtained from the tail vein at predetermined intervals (0.5,
(4-bromophenylboronic acid, 5 μL/mL of 1 M in metha- 1, 1.5, 2, 4, and 8 h). Subsequently, the collected samples
nol) to every micro-centrifuge sample tube. The samples underwent centrifugation at 5000 rpm for 5 min to separate
were then instantly centrifuged for 10 min at 15,000 rpm the plasma.
and 37°C using a laboratory centrifuge to collect an aque-
ous solubilized phase. The aqueous phase was analyzed for Enzalutamide
its drug concentration using the protocol mentioned in the
section ‘HPLC quantification method’. After phase separa- The pharmacokinetic assessment of Enz formulations was
tion by centrifuging the digestion media, the resultant pel- conducted in male Sprague Dawley rats weighing between
let was carefully extracted from polypropylene tubes. These 200–220 g. The study involved the oral administration of
pellets were then transferred onto a glass plate and air- crystalline drug suspension, conventional LbF, and super-
dried at ambient temperature. Then the dried samples were LbF. Super-LbF with the highest Enz loading was used for
192 Page 6 of 17 AAPS PharmSciTech (2024) 25:192

further pharmacokinetic evaluation to assess the impact structure observed in the polarized mode microscope (Fig-
of supersaturation. Each group of five animals received a ure S1B). The PXRD spectrum of Ibr and Enz (Figure S2)
30 mg/kg dose of Enz in a 10 mL/kg volume [24]. After exhibited several intense and sharp peaks, confirming its
administration, blood samples (approximately 0.2 mL) were crystalline nature. In this study, we also conducted thermal
collected at specified intervals (0.5, 1, 2, 4, 8, 12, 24, 48, and characterization of model drugs through DSC heating–cool-
72 h), followed by centrifugation at 5000 rpm for 5 min to ing-heating experiments. Figure S3 depicts the resulting
separate the plasma. DSC thermograms, and the characteristic temperatures,
enthalpies from observed thermal events, and the T­ m/Tg ratio
Plasma Sample Analysis for each compound are summarized in Table I. The DSC
heating curves (first heating cycle) revealed distinct endo-
For quantitative analysis, plasma samples underwent pro- thermic peaks for Ibr and Enz, occurring at 156–158°C and
cessing using the liquid–liquid extraction method. Spe- 198–201°C respectively [23, 26]. During the cooling and re-
cifically, 10 µL of the internal standard and 200 µL of heating cycles, no endothermic event was observed for either
acetonitrile: methanol (in a 50:50 ratio, used as a precipitat- of the drugs. Further, Enz exhibited a higher ­Tm/Tg ratio of
ing agent) were added to 100 µL of the collected plasma 2.14 compared to Ibr, which had a ratio of 1.92. Fridgeirs-
samples. Sorafenib and diclofenac were used as internal dottir et al. suggest that a higher ­Tm/Tg ratio is linked to
standards for the quantitative determination of Ibr and Enz, an elevated propensity for drug crystallization [33]. This
respectively. The mixture was vortexed for a few minutes finding suggests that Enz may have a greater tendency to
and then centrifuged for 10 min at 10,000 rpm using iFuge crystallize than Ibr, which could have implications for drug
UCo2R, (Neuation Technologies, Gujarat, India). The result- loading and the physical stability of the formulation.
ing supernatant was collected into a microcentrifuge tube,
followed by evaporation of the supernatant using a vacuum Solubility Study in Excipients and Lipid Solutions
concentrator. The final dried residue was re-dispersed with
160 µL of acetonitrile: water (9:1) and subjected to drug Solubility investigations were carried out at 37 ± 2°C using
quantification using HPLC. The detailed bioanalytical a range of lipids, surfactants, and co-solvents utilizing a pre-
method specifications and validation results are given in viously reported method [27]. A variety of lipid excipients
supporting information. were used, based on chain length long chain length (LC),
and medium chain (MC). Amongst the MC-based lipids,
Statistical Analysis investigated, ­Capmul® MCM EP demonstrated the high-
est solubility value. Specifically, it exhibited a significant
All the experiments were performed in triplicate, and the solubility of 44.07 ± 4.7 mg/g for Ibr and 12.91 ± 2.0 mg/g
results are presented as mean ± standard deviation. The stu- for Enz (Fig. 1a and c). In contrast, lipid vehicles based on
dent’s t-test and ANOVA were employed to identify statisti- long-chain triglycerides (LC) exhibited the lowest solubil-
cally significant differences between two groups and more ity for both drugs. The tendency of higher drug solubility
than two groups, respectively, using GraphPad Prism® (ver- in MC-based lipids than LC-based lipids can be ascribed to
sion 8.4.2), with a significance level set at p-value < 0.05. drug-specific interactions with the length of the lipid chains
The pharmacokinetic parameters based on the non-compart- and the nature of the lipid vehicle [29]. Evaluation of sur-
mental analysis were calculated by using the PK-Solver, a factants revealed promising solubility outcomes for both
freely available menu-driven add-in program for Microsoft drugs. Ibr exhibited solubility values of 35.72 ± 1.68 mg/g
Excel. in ­Labrasol® and 31.06 ± 3.84 mg/g in ­Kolliphor® RH 40,
while Enz demonstrated maximum solubility in L ­ abrasol®
®
(57.7 ± 5.6 mg/g) and T ­ ween 20 (24.4 ± 3.5 mg/g) (Fig. 1b
Result and Discussion and e). Furthermore, PEG 200 and T ­ ranscutol® were identi-
fied as optimum solubilizers based on their maximum solu-
Solid State Characterization of Model Drugs bilization capacity for Ibr and Enz, respectively, as depicted
in Fig. 1c and f. The results indicate that the compound's
The identification of the crystalline form of Ibr was carried thermal behavior significantly influences its solubility
out using different analytical tools, and irregularly shaped value. Using the selected excipients, prototype lipid formu-
crystals were observed in the optical mode of the micro- lations were prepared through the admixture method and
scope. In the polarized mode, birefringence was observed the detailed compositions of these prototype formulations
due to the anisotropic crystalline nature of the Ibr (Fig- are presented in Table I. The prototype formulations were
ure S1A). The Enz was found to be a white, non-hygroscopic selected based on criteria such as miscibility and solubility
crystalline powder with an irregular shape of crystalline for both drugs (Table S1, supporting information).
AAPS PharmSciTech (2024) 25:192 Page 7 of 17 192

Oil Surfactant 60 Co-solvent

60 40

Solubility value in mg/g

30

Solubility value in mg/g

Solubility value in mg/g

40
40
Ibrut inib

20

20 20

10

0 0 0
Maisine Peceol Phosal Capmul Labrafac Lauroglycol Kolliphor Kolliphor Tween 20 Tween 80 Span 80 Labrasol Ethanol Propylene Glycol Transcutol PEG 200
50 MCM 90 EL RH40

a b c
Oil Surfactant 100 Co-solvent
20 80

80

Solubility value in mg/g

Enzalutamide

15 60
Solubility value in mg/g
Solubility value in mg/g

60

10 40

40

5 20
20

0 0 0
Maisine Peceol Phosal Capmul Labrafac Lauroglycol Kolliphor Kolliphor Tween 20 Tween 80 Span 80 Labrasol Ethanol Propylene Glycol Transcutol PEG 200
50 MCM 90 EL RH40

d e f

Fig. 1  Equilibrium solubility of ibrutinib a, b & c and enzalutamide d, e & f in oil components, surfactants, and co-solvents at 37 ± 2°C

Table III  Temperature-Dependent Solubility and aDS Values for Ibru- mg/g at 25 ± 2°C. The observed trend highlights the impor-
tinib and Enzalutamide at 25 ± 2°C, 37 ± 2°C, and 60 ± 2°C tance of temperature-induced drug supersaturation within
Parameters Ibrutinib Enzalutamide lipid vehicles by overcoming crystal lattice energy. Specifi-
cally, lower crystallization lattice energies (in the case of
Solubility at 25 ± 2°C (mg/g) 60.15 ± 6.74 31.09 ± 0.55 Ibr) were found to promote greater drug dissolution in lipid
Solubility at 37 ± 2°C (mg/g) 73.68 ± 1.81 44.62 ± 6.14 vehicles due to weaker intermolecular forces within the crys-
Solubility at 60 ± 2°C (mg/g) 161.03 ± 9.19 75.15 ± 4.59
tal lattice [35].
aDS at 37 ± 2°C 1.22 ± 0.12 1.43 ± 0.18
Furthermore, determining the degree of supersaturation
aDS at 60 ± 2°C 2.68 ± 0.14 2.41 ± 0.15
in a lipid solution is crucial for establishing the drug load-
ing. aDS values were determined and the results are shown
in Table III. The chosen formulation demonstrated minimal
Influence of Temperature‑Induced Supersaturation enhancement in the aDS at 37°C for both drugs. In con-
on the Drug Loading in LbFs trast, higher aDS values of 2.68 ± 0.14 and 2.41 ± 0.15 were
achieved for Ibr and Enz, respectively, when the formulation
The study assessed the impact of temperature on the drug was prepared at a temperature of 60 ± 2°C. A similar trend
solubility in the chosen prototype formulation, which is was noted with celecoxib and cinnarizine, where aDS values
a type IIIA formulation (compositions are represented in ranging from 1.5 to 2.5 were achieved using the thermal-
Table II) [34]. The study results (Table III), indicate that induced supersaturation method [19]. Additionally, Bennett-
drug solubility increased significantly when examined at ele- Lenane et al. conducted a retrospective analysis of data from
vated temperatures compared to room temperature. Particu- 21 distinct super-LbFs. Their findings concluded that both
larly noteworthy was the 2.5 to 2.7-fold increase in Ibr con- MC-based and LC-based LbFs exhibited a similar degree of
centration observed when heated at 60 ± 2°C (161.03 ± 9.19 supersaturation (1–3.5) when heated at 60 ± 2°C [15]. Based
mg/g), in comparison to 25°C (60.15 ± 6.74 mg/g). A similar on the assessed aDS value, three distinct drug loading levels
trend was observed for Enz, where solubility increased to were identified for further examination. The detailed formu-
75.15 ± 4.59 mg/g at 60 ± 2°C compared to 31.09 ± 0.55 lation compositions are given in Table II. The drug loading
192 Page 8 of 17 AAPS PharmSciTech (2024) 25:192

for conventional LbF was equal to equilibrium solubility revealed a reduction in the original content after the 60-day
in the lipid solution while super-LbF A and super-LbF B time interval (p-value > 0.05), as depicted in Fig. 3a.
featured drug loads surpassing the equilibrium solubility of In the case of Enz formulations, conventional LbF
drugs. remained stable throughout the study period whereas both
super-LbFs exhibited drug crystallization after 60 days of
Influence of Degree of Supersaturation storage. The microscopic evaluation revealed the genera-
on the Physical Stability of LbF tion of non-uniformly shaped crystals for the Enz contain-
ing super-LbFs, as illustrated in Fig. 2. These observations
In this study, the conventional and super-LbFs were kept at suggest that drug crystallization within the liquid precon-
room temperature stability conditions (25°C/65% RH) for centrate is dependent upon the inherent characteristics of
60 days. The microscopic evolution was carried out under a the drug and the achieved aDS in the lipid solution [15,
light microscope to check the generated drug particle within 19]. The tendency of Enz to undergo re-crystallization in
the formulation and results are represented in Fig. 2. The the liquid LbF has been previously documented [36]. The
conventional formulation and super-LbF A of Ibr exhibited drastic reduction in drug content over the storage period is
no discernible signs of drug crystallization throughout the also evident in Fig. 3b (p-value < 0.05). Similarly, Bannow
60-day observation period. However, contrasting results et al. demonstrated that the increased thermodynamic driv-
were observed in the microscopic images of super-LbF B, ing force is responsible for drug re-crystallization within
revealing the presence of drug crystals within the formu- lipid solution due to the higher degree of supersaturation
lations at a 60-day time point. These findings align with (aDS value of 2.5) and the onset of precipitation time was
previously reported outcomes by Ilie et al. in 2020, where within 24 h [18]. Furthermore, the differences in the phys-
lipid formulations with a lower degree of supersaturation icochemical properties of Enz and Ibr, such as lipophilic-
within the range of 1–2 aDS values showed a low risk of ity and melting point, also affect their physical stability.
precipitation on short-term storage, and crystallization ten- Overall, these findings suggest that an enhanced degree
dency increased for higher degrees of supersaturation (> 2 of supersaturation within the formulation may pose the
aDS values) [19]. Additionally, the drug content analysis risk of drug precipitation over a longer period of storage.

Fig. 2  Evaluation of the physical stability of conventional LbF and super-LbF through microscopic evaluation. The microscopic images are cap-
tured at 50 × magnification power for 60-day samples
AAPS PharmSciTech (2024) 25:192 Page 9 of 17 192

150 Ibrutinib Formulations 150 Enzalutamide Formulations

Initial 60 Day Initial 60 Day

ns ns ns ns

100 100
% Drug Content

% Drug Content
50 50

0 0
Conventional Super LbF A Super LbF B Conventional Super LbF A Super LbF B
LbF LbF
a b

Fig. 3  Drug content determination for ibrutinib a and enzaluta- showed that ‘*’ represents a statistically significant difference
mide b formulations after 60 days of storage. A paired student t-test (p-value < 0.05), and "ns" denotes a non-significant difference with
analysis was conducted to assess statistical significance. The results initial values

Influence of Degree of Supersaturation and after a 2-h period of aqueous dispersion. In the case
on Dispersion Behavior of LbF of Ibr, conventional LbF exhibited a mean droplet size
of 245 ± 20 nm with 0.350 ± 0.02 as a PDI value at the
To examine the effect of the degree of supersaturation initial time of dispersion (Fig. 4a and b). No substan-
on dispersion behavior, the average droplet size, PDI tial differences in these values were detected after 2-h
value, and zeta potential were evaluated for both initial of dispersion, indicating the stability of the emulsion.

1500 1.5 -20

Initial 2h Initial 2h ns Initial 2h
(Intensity averaged distribution)

-15
ns
Polydispersity Index

Zeta Potential value

Droplet size in nm

1000 1.0
Ibrut inib

-10

ns
500 0.5 ns
ns
-5

0 0.0 0
Conventional Super LbF A Super LbF B Conventional Super LbF A Super LbF B Conventional Super LbF A Super LbF B
LbF LbF LbF
a b c
1500 1.0 -40
Initial 2h Initial 2h
Initial 2h
ns ns

1000
(Intensity averaged distribution)

0.8 ns
-30
Polydispersity Index

Zeta Potential value

Enzalutamide

Droplet size in nm

500
0.6 ns
ns
100 -20
ns ns
0.4
ns ns

50 -10
0.2

0 0.0 0
Conventional Super LbF A Super LbF B Conventional Super LbF A Super LbF B Conventional Super LbF A Super LbF B
LbF LbF LbF

d e f

Fig. 4  Dispersion behavior i.e. average droplet size a & d, polydis- period of aqueous dispersion. The results showed that ‘*’ represents a
persity index value b & e, and zeta potential c & f of conventional statistically significant difference (p-value < 0.05), and "ns" denotes a
LbF and super-LbFs for ibrutinib and enzalutamide. The sample non-significant difference with initial values
measurements (n = 3) were performed both initially and after a 2-h
 192 Page 10 of 17 AAPS PharmSciTech (2024) 25:192

The zeta potential values for the conventional lipid- Influence of Degree of Supersaturation on Aqueous
based formulation at the initial time of dispersion and Phase Drug Distribution of LbF
after 2-h were -11.17 ± 0.40 and -14.53 ± 2.48, respec-
tively (Fig. 4c). However, the increase in the aDS within The aqueous solubilization of drugs is a crucial factor that
the LbFs resulted in significant changes in dispersion influences the in vivo oral bioavailability of LbFs. Moreover,
parameters after a 2-h dispersion period (p value < 0.05) the characteristics of solubilizing species and drug precipi-
which might be due to dilution-mediated drug precipita- tates formed during lipolysis serve as pivotal determinants
tion [37]. As illustrated in Fig. 4, a drastic increase in governing in vivo performance. In this study, we assessed
droplet size and PDI values was observed for super-LbF the influence of drug loading in LbF on drug solubilization
after 2-h of dispersion. through an in vitro high throughput lipolysis experiment, as
For Enz, both conventional and super-LbF A main- reported by Kilic and Dressman [31].
tained consistently low droplet sizes and PDI values,
even after a 2-h aqueous dispersion. However, in con- Ibrutinib Solubilization in Digestive Conditions
trast, super-LbF B (with an aDS value of 2.5) exhibited
an increase in the droplet sizes and PDI values after 2-h of Figure 5a depicts a decreasing pattern in the solubilization
dispersion, as evidenced by Fig. 4d and e. The zeta poten- profiles of both conventional and super-LbFs under digestive
tial shifted from -19.10 ± 1.04 initially to -12.90 ± 2.54 environments. In the dispersion phase, all Ibr formulations
after 2-h (Fig. 4f). This shift is likely due to changes in exhibited a subtle decline in drug concentration, whereas a
particle interactions or aggregation within the dispersion rapid decrease was observed after the initiation of the diges-
[38]. In comparing both drug formulations, it appears tion process, regardless of the drug loading levels. The rapid
that the amount of drug load significantly influences the decline in drug concentration could be attributed to the dimin-
overall droplet size and dispersion parameters. This phe- ished solubilization capacity on digestion, particularly notable
nomenon may be due to a substantial quantity of drug for the type III formulation [39]. By completion of the study,
dissolved in liquid preconcentrate and a decrease in solu- the drug concentration moves toward its equilibrium solubility
bilization capacity upon dilution. The difference in zeta in the digestion media, reaching approximately 26 µg/mL. It
potential observations of super-LbF also signifies the was also noted that a low amount of lipid solution (for super-
physical instability of emulsion [38]. LbFs) did not show a significant impact on the aqueous drug
solubilization in the digestive condition (p-value > 0.05).

Ibrutinib Formulations

500 20 Conventional LbF

Conventional LbF
Ibrutinib aqueous phase concentration ( g/mL)

Super-LbF A Super-LbF A

400 Super-LbF B Super-LbF B

15
Supersaturation Ratio

300

10

200

100 5

0
-10 0 10 20 30 40 50 60 0
Time in min 0 min 30 min 60 min 0 min 30 min 60 min 0 min 30 min 60 min

a b

Fig. 5  a Aqueous drug phase distribution of conventional LbF and indicates the initiation of the digestion phase. b The supersaturation
super-LbFs in the digestive condition for ibrutinib. The blue region ratio (SR) value for ibrutinib was obtained at 0 min, 30 min, and 60
indicates the dispersion phase and the remaining profile represents min of the lipolysis process
the digestion phase. The vertical dotted line on the x-axis at 0 min
AAPS PharmSciTech (2024) 25:192 Page 11 of 17 192

The apparent equilibrium solubility of Ibr in post-diges- compared to their non-ionized counterparts, thereby reduc-
tive media was found to be 17.5 ± 2.6 µg/mL. The supersatu- ing their availability to solubilize drug molecules within
ration ratio was determined by calculating the amount of micelles [41, 42]. Second, the presence of surfactants and
solubilized drug concentration to the apparent equilibrium co-solvents in a liquid preconcentrate can reduce the con-
solubility of Ibr in post-digestive media at different time centration of the solubilized drug. This reduction occurs
points. During the dispersion stage, a higher supersatura- because the hydrophilic components are miscible with the
tion ratio (SR value ~ 11–15) was achieved for all the for- aqueous medium, leading to a loss in apparent solubility.
mulations. Devraj et al. previously reported similar obser- Consequently, this results in transient supersaturation within
vations, demonstrating that MC-based formulations induce the colloidal phase [4, 29, 43, 44].
the formation of a supersaturated concentration of the drug
in the dispersion phase [40]. However, during the digestion Enzalutamide Solubilization in Digestive Conditions
process, the solubilization capacity in the colloidal phase
decreased, resulting in lower SR values observed at 30 min The concentration profile of Enz in the aqueous phase under
as well as 60 min post-lipolysis, as represented in Fig. 5b. digestive conditions, as influenced by different levels of drug
There are two plausible mechanisms for the reduction in payload, is summarized in Fig. 6a. During the dispersion
the solubilization capacity of conventional LbF and super- phase, the concentration of Enz remained solubilized (at
LbF. First, during digestion, the hydrolysis of triglycerides 83.23 ± 14.38 µg/mL) for the conventional LbF formula-
releases free fatty acids into the gastrointestinal milieu. tion. Conversely, in the case of super-LbFs, the solubilized
These free fatty acids have a propensity to associate with Enz level experienced a rapid decline during the dispersion
drug molecules, enhancing their solubilization due to their phase. The calculated SR value for each formulation at dif-
amphiphilic nature and ability to form micelles. However, ferent intervals of dispersion and digestion phase is sum-
despite the initial enhancement in solubilization, the endoge- marized in Fig. 6b. Under the conditions of digestion, the
nous absorption of free fatty acids can subsequently lead to a conventional LbF formulation displayed a gradual decrease,
reduction in micellar solubilization capacity. This occurs as reaching a concentration of 18.52 ± 3.32 µg/mL at the end
a result of the increased presence of ionized free fatty acids of the experiment. Both super-LbF formulations exhibited
in the intestinal environment. The microclimate pH gradient comparable profiles despite differences in drug payload
between the unstirred water layer and the intestinal lumen and lipid amounts, with the concentration levels approach-
promotes the protonation of free fatty acids, converting ing near-equilibrium solubility concentration. The meas-
them into their ionized forms. The ionized form of free fatty ured equilibrium solubility of Enz was determined to be
acids exhibits greater solubility in aqueous environments 4.45 ± 0.98 µg/mL in the post-digestive medium.

Enzalutamide Formulations

150 25 Conventional LbF

Enzalutamide aqueous phase concentration ( g/mL)

Conventional LbF

Super-LbF A Super-LbF A

Super-LbF B 20 Super-LbF B

100
Supersaturation Ratio

15

10
50

0
-10 0 10 20 30 40 50 60 0
Time in min 0 min 30 min 60 min 0 min 30 min 60 min 0 min 30 min 60 min

a b

Fig. 6  a Aqueous drug phase distribution of conventional LbF and min indicates the initiation of the digestion phase. b The supersatura-
super-LbFs in the digestive condition for enzalutamide. The blue tion ratio (SR) value for enzalutamide was obtained at 0 min, 30 min,
region indicates the dispersion phase and the remaining profile rep- and 60 min of the lipolysis process
resents the digestion phase. The vertical dotted line on the x-axis at 0
192 Page 12 of 17 AAPS PharmSciTech (2024) 25:192

In this study, it was observed that the lipophilicity of the experiment and the PXRD overlay is presented in Fig. 7b.
drug, and the quantity of formulation substantially influ- The PXRD overlay confirms the crystalline nature of the
ence the distribution of the drug in the aqueous phase. The precipitated drug, as evidenced by the presence of charac-
amount of formulation used during the digestion study also teristic peaks resembling those of crystalline Enz. In this
governs solubilization by altering the colloidal structures. study, the effect of the physicochemical properties (ioniza-
In the case of the super-LbFs, the low quantity of lipids tion and melting point) of these drugs had a similar impact
may be accountable for the generation of a lower portion on the nature of the physical form of the precipitate. The
of the digestive product or colloidal species, resulting in crystalline nature of the precipitate could be detrimental to
lower solubility in the digestive phase. Conversely, a high drug absorption as it may reintroduce the dissolution step.
portion of lipid mass results in the generation of a lipid-rich However, these observations lack confirmatory validity, as
phase, which improves the association with lipophilic drugs the physical form of the precipitate may undergo alterations
and enhances the apparent solubilization [45]. Overall, the with increased sample processing time, indicating a limita-
amount and type of formulation, drug payload, and inherent tion of the current study. In the future, advanced real-time
characteristics (lipophilicity) of the drug are accountable analytical approaches can be used to identify the true nature
for the reduction in solubilization capacity, thereby leading of the precipitate without interfering with the sample [5, 46].
to the generation of transient supersaturation for both drugs
[43]. The transient supersaturated state is highly thermody- Pharmacokinetic Study
namically unstable, leading to the precipitation of the drug.
Hence, solid-state analysis was performed on the precipitate To validate the in vitro findings, an in vivo pharmacoki-
obtained after digestion to identify its nature. netic study was performed using Sprague Dawley rats after
every oral administration of the prepared formulation. In
Precipitate Characterization this study, conventional LbF and super-LbF (highest drug
payload) were employed to investigate the influence of drug
The physical characteristics of drug precipitates play a vital loading levels and enhanced drug solubilization under in
role in influencing drug absorption in the intestinal region vivo conditions. Crystalline aqueous suspensions were used
[44]. The diffraction patterns of the blank digest, crystalline as a control group for each drug.
Ibr, and obtained precipitate are shown in Fig. 7a. The crys-
talline Ibr showed characteristic diffraction peaks at 2θ val- In Vivo Fate of Ibrutinib After Oral Administration
ues of 5.60, 10.57, 12.24, 13.51, 16.01, 16.55, 18.21, 19.72,
21.16, 21.56, 23.39, 26.40 and 28.77 [32]. The obtained pre- Figure 8a illustrates the plasma concentration–time profiles
cipitate after the lipolysis study of Super-LbF (high drug after the oral administration of crystalline Ibr suspension,
loading level) also showed characteristic peaks of crystal- conventional LbF, and Super-LbF B. A Summary of calcu-
line Ibr with slight deviations. Similarly, the assessment lated pharmacokinetic parameters is represented in Table IV.
was conducted for Enz super-LbF following the digestion Crystalline Ibr demonstrated constrained pharmacokinetic

Super LbF precipitate

Super LbF precipitate
Intensity

Intensity

Blank digest precipitate

Blank digest precipitate

Crystalline ibrutinib
Crystalline enzalutamide

5 10 15 20 25 30 35 40 5 10 15 20 25 30 35 40
2θ 2θ

a b

Fig. 7  PXRD overlays of the precipitate obtained after the lipolysis the obtained precipitate after lipolysis of the super-LbF (highest drug
of super-LbF (highest drug payload) of ibrutinib a and enzalutamide payload) were compared
b. The diffraction patterns of the blank digest, crystalline drug, and
AAPS PharmSciTech (2024) 25:192 Page 13 of 17 192

Ibrutinib Formulations Enzalutamide Formulations

600 15000
Crystalline Ibrutinib Crystalline Enzalutamide
Plasma Concentration (in ng/mL)

Plasma Concentration (in ng/mL)

Convential LbF Convential LbF
Super-LbF Super-LbF

400 10000

Enzalutamide
Ibrutinib

200 5000

0 0
0 1 2 3 4 5 6 7 8 0 20 40 60 80
Time (in h) Time (in h)

a b

Fig. 8  a Plasma concentration–time profiles after the oral administra- rats. b Plasma concentration–time profiles after the oral administra-
tion of crystalline ibrutinib suspension, conventional LbF, and super- tion (30 mg/kg equivalent dose) of crystalline enzalutamide suspen-
LbF (highest drug load) at a dose of 20 mg/kg in Sprague Dawley sion, conventional LbF, and super-LbF (highest drug load)

Table IV  Pharmacokinetic Model drugs (administered dose) Orally administered Pharmacokinetic Parameters
Parameters After Oral formulations
Administration of Ibrutinib and Cmax (ng/mL) AUC (ng*h/mL)
Enzalutamide Formulations
Ibrutinib (20 mg/kg) Crystalline Ibr 147.49 ± 73.53 461.33 ± 168.83
Conventional LbF 398.27 ± 180.25* 666.39 ± 289.58#
Super-LbF B 458.42 ± 80.48* 797.28 ± 137.70*
Enzalutamide (30 mg/kg) Crystalline Enz 1372.79 ± 784.02 55,217.22 ± 33,917.81
Conventional LbF 9858.09 ± 2001.96* 300,176.52 ± 42,196.45*
Super-LbF B 7424.31 ± 897.28* 289,262.92 ± 65,008.87*

All the data are represented as mean ± standard deviation (no. of animal used for each group = 5)
“*” represents a significant difference when compared to respective crystalline drug suspension
“#” represents no significant difference was observed compared respective crystalline drug suspension

parameters, with a C ­ max of 147.49 ± 73.53 ng/mL and an parameters was identified between conventional and Super-
AUC value of 461.33 ± 168.83 ng*h/mL. Both lipid formula- LbFs in in vivo settings (p-value > 0.05). The upsurge in
tions demonstrated higher drug exposure, as evidenced by pharmacokinetic parameters within 2-h for both formula-
elevated ­Cmax and AUC values when compared to the crys- tions may be attributed to the initial phase of drug super-
talline suspension. The plasma concentration profile sug- saturation in the digestive conditions. Similar observations
gests that the initial phase of the pharmacokinetic profile, regarding intestinal absorption were also noted in the case
especially within the first 2-h time, is sensitive to formu- of fenofibrate-based type IIIA MC-based LbF, wherein the
lation changes, which impacts absorption. This sensitivity formulations demonstrated the capability to induce transient
includes factors such as drug supersaturation and the main- supersaturation for a shorter duration (up to 1-h). However,
tenance of a metastable state, impacting early drug absorp- in the later absorption phase, a marked difference was
tion and disposition. Among the lipid formulations, both the observed due to drug precipitation [41]. In contrast, Siqueira
conventional LbF and Super-LbF B demonstrated compa- et al. observed that type III Super-LbF (50 mg/g formula-
rable maximum concentration ­(Cmax) values, with specific tion) exhibited comparatively lower cinnarizine exposure
values of 398.27 ± 180.25 ng/mL and 458.42 ± 80.48 ng/ when compared to conventional LbF (20 mg/g formulation).
mL, respectively (p value > 0.05). AUC values were found These observations could be attributed to the high propen-
to be 666.39 ± 289.58 ng*h/mL and 797.28 ± 137.7 ng*h/ sity of cinnarizine to precipitate after the dispersion and
mL for conventional LbF and Super-LbF B, respectively digestion of Super-LbF. The reason for the aforementioned
(p value > 0.05). No major variation in pharmacokinetic finding could be drug-specific absorption and the imbalance
192 Page 14 of 17 AAPS PharmSciTech (2024) 25:192

of the supersaturated state in the intestinal region [20]. Over- and Siqueira et al. [48, 49]. The obtained pharmacokinetic
all, the short time duration of the thermodynamic metastable results of this study will further guide the improvement of
state during the digestive condition may impede the poten- formulation development to enhance the quality attributes
tial benefits of supersaturating formulations. The quantity of of Super-LbF.
lipid formulation used for the pharmacokinetic study did not Despite the advantages of superimposable pharmacoki-
significantly influence the drug exposure, as the low quantity netic profiles, maintaining the physical stability of super-
used in the Super-LbF B formulation resulted in similar drug LbF presents a significant challenge. This is primarily due
exposure levels to those observed with the conventional LbF, to the inherent high chemical potential associated with
despite the larger amounts administered in the conventional the supersaturated state, which drives the system towards
LbF [47]. achieving a more favorable energy state through spontane-
ous re-crystallization [21]. To address this issue, precipi-
In Vivo Fate of Enzalutamide After Oral Administration tation inhibitors can be used in future studies to improve
the stability of super-LbF by inhibiting drug nucleation and
Similarly, pharmacokinetic studies in Sprague–Dawley rats crystal growth in the liquid super-LbF [27]. These inhibi-
were conducted for the Enz formulations, and the plasma tors may act through different mechanisms, including vis-
concentration–time profile is depicted in Fig. 8B. Statistical cosity enhancement, increased drug solubility within the
analysis by one-way ANOVA suggests significantly higher liquid LbF matrix, and facilitating favorable intermolecular
exposure after administration of both the conventional LbF interactions [7]. These precipitation inhibitors also can help
and super-LbF of Enz compared to the free Enz suspension. to stabilize the in-situ supersaturation which is generated
(p-value < 0.05), as detailed pharmacokinetic parameters after dispersing the super-LbF in the aqueous media [18,
are provided in Table IV. However, the limited absorption 50, 51]. Formulation strategies such as the incorporation
observed with crystalline Enz is constrained by dissolu- of solidifying lipids, or adsorption on solid carriers, could
tion limitations and these results align well with previous serve as potential approaches to sustain drug supersaturation
reports [218]. Among the lipid formulations, Conventional for extended periods [16, 29]. In our study, we observed the
LbF exhibited a comparable rate and extent of absorption need for advanced in vitro tools, such as a combined lipoly-
(in terms of Cmax and AUC values) to Super-LbF with a sis-permeation setup, to improve the correlation between in
non-significant difference (p-value > 0.05). The conven- vitro and in vivo outcomes. This setup could be employed
tional LbF has a C ­ max and AUC value of 9.8 ± 2.0 µg/mL to achieve better predictive accuracy for super-LbF. Our
and 300.17 ± 42.19 µg*h/mL, respectively. While the ­Cmax research group has been working in the same direction, to
and AUC values for the Super-LbF B were 7.4 ± 0.8 µg/ establish super-LbF as a viable formulation technique for
mL and 289.26 ± 65.00 µg*h/mL respectively. However, use in preclinical, clinical, and manufacturing stages. From a
the observed variations in Enz solubilization behavior pre-clinical perspective, super-LbF provided flexibility in the
between conventional LbF and Super-LbF are not signifi- dose escalation studies, as well as simplicity of preparation
cantly reflected in the pharmacokinetic outcomes associated and dosage loading. This makes them useful for simplified
with these formulations. The observed differences could be formulations from bench to clinic. Additionally, they save
attributed to the variations between the in vitro setup and the effort required to bridge preclinical and clinical formu-
the in vivo gastrointestinal conditions in rats, including fac- lation types and provide for more flexibility in delivering
tors such as gastric emptying and luminal content, which dosage ranges during clinical studies. From a commercial
were not considered in this study. Additionally, the non-sink manufacturing perspective, the production process of super-
conditions employed in the in vitro setup may not accurately LbF is expected to be achievable through established simple
predict the in vivo luminal behavior [9, 48]. industrial methods (solution mixing and heating).
Overall, the pharmacokinetic results of Super-LbF
showed comparable results with a low volume of formula-
tion (due to high drug load) in comparison to conventional Conclusion
LbF (which required a higher formulation quantity due to
low drug load). These observations indicate that Super-LbF In summary, the limited drug loading capacity of LbF pre-
can deliver the desired dose with a low volume of formula- sents a significant challenge in achieving the desired dose
tion excipient, potentially reducing formulation costs. Super- for pre-clinical or clinical studies. In the present study, the
LbF has successfully addressed the challenge associated influence of the temperature-induced supersaturation method
with limited drug loading and higher pill burden providing was investigated to enhance the solubility of Ibr and Enz
comparable benefits with conventional LbF in preclinical within lipid solutions. The results indicate a significant
studies. Moreover, the observed pharmacokinetic results improvement in drug loading capacity compared to conven-
for both drugs are well corroborated with Thomas et al. tional LbF, with a 2.5-fold increase for both Ibr and Enz.
AAPS PharmSciTech (2024) 25:192 Page 15 of 17 192

However, the enhanced drug loading capacity comes with The protocol necessary for conducting a pharmacokinetic study on
the trade-off of decreased physical stability over an extended laboratory animals received approval from the Institutional Animal
Ethics Committee, NIPER S.A.S. Nagar, India.
period, leading to a risk of drug re-crystallization. The phys-
ical instability is influenced by the physicochemical proper-
Conflict of Interest The authors declare that they have no conflict of
ties of the drug and achieved aDS value. Also, super-LbF interest.
demonstrates significant changes in dispersion parameters,
including droplet size and zeta potential. Additionally, we
conducted a biopharmaceutical assessment to investigate the
interplay between drug payload and in-situ supersaturation, References
followed by pharmacokinetic evaluations. During lipolysis
1. Nadin A, Hattotuwagama C, Churcher I. Lead-oriented synthesis:
studies, both conventional and super-LbF showed the gen-
a new opportunity for synthetic chemistry. Angew Chem Int Ed.
eration of in-situ supersaturation for a shorter time period, 2012;51:1114–22. https://​doi.​org/​10.​1002/​anie.​20110​5840.
subsequently posing a risk of drug precipitation. Further- 2. Stegemann S, Moreton C, Svanbäck S, Box K, Motte G, Paudel A.
more, in vivo studies suggest that both conventional and Trends in oral small-molecule drug discovery and product devel-
opment based on product launches before and after the Rule of
super-LbF formulations exhibit comparable dose-normalized
Five. Drug Discovery Today. 2023;28: 103344. https://d​ oi.o​ rg/1​ 0.​
pharmacokinetic profiles. However, the in vivo performance 1016/j.​drudis.​2022.​103344.
of super-LbF suggests that maintaining a balance of super- 3. Feeney OM, Crum MF, McEvoy CL, Trevaskis NL, Williams HD,
saturation in the intestinal region is a pivotal factor. This can Pouton CW, Charman WN, Bergström CAS, Porter CJH. 50 years
of oral lipid-based formulations: Provenance, progress and future
be improved by formulation interventions such as the use
perspectives. Adv Drug Deliv Rev. 2016;101:167–94. https://​doi.​
of precipitation inhibitors (PIs), controlling lipid digestion, org/​10.​1016/j.​addr.​2016.​04.​007.
and solidifying super-LbF. Overall, these findings may guide 4. Williams HD, Trevaskis NL, Yeap YY, Anby MU, Pouton CW,
the improvement of formulation development to enhance the Porter CJH. Lipid-Based Formulations and Drug Supersaturation:
Harnessing the Unique Benefits of the Lipid Digestion/Absorption
quality attributes of super-LbF.
Pathway. Pharm Res. 2013;30:2976–92. https://​doi.​org/​10.​1007/​
s11095-​013-​1126-0.
Abbreviations aDS: Apparent degree of supersaturation; AUC : Area
5. Sirvi A, Debaje S, Guleria K, Sangamwar AT. Critical aspects
under the curve; 4-BPB: 4-Bromophenylboronic acid; Enz: Enzaluta-
involved in lipid dispersion and digestion: Emphasis on in vitro
mide; Seq: Equilibrium solubility; GIT: Gastrointestinal tract; Ibr: Ibru-
models and factors influencing lipolysis of oral lipid based for-
tinib; LbF: Lipid-based formulation; LC: Long-chain; MC: Medium-
mulations. Adv Coll Interface Sci. 2023;321: 103028. https://​doi.​
chain; PDI: Polydispersity index; PWSD: Poorly water-soluble drug;
org/​10.​1016/j.​cis.​2023.​103028.
PXRD: Powder X-ray diffraction:; SEDDS: Self-emulsifying drug
6. Alvebratt C, Dening TJ, Åhlén M, Cheung O, Strømme M, Gogoll
delivery systems; Super-LbF: Supersaturated LbF; super-SNEDDS
A, Prestidge CA, Bergström CAS. In Vitro Performance and
: Supersaturated self nano-emulsifying drug delivery systems;
Chemical Stability of Lipid-Based Formulations Encapsulated
SR: Supersaturation ratio
in a Mesoporous Magnesium Carbonate Carrier. Pharmaceutics.
2020;12:426. https://​doi.​org/​10.​3390/​pharm​aceut​ics12​050426.
Supplementary Information The online version contains supplemen-
7. Holm R, Kuentz M, Ilie-Spiridon A-R, Griffin BT. Lipid based
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 208/s​ 12249-0​ 24-0​ 2912-9.
formulations as supersaturating oral delivery systems: From cur-
rent to future industrial applications. Eur J Pharm Sci. 2023;189:
Acknowledgements The authors wish to acknowledge the Director of
106556. https://​doi.​org/​10.​1016/j.​ejps.​2023.​106556.
NIPER S.A.S. Nagar for providing the essential facilities and infra-
8. Koehl NJ, Henze LJ, Kuentz M, Holm R, Griffin BT. Supersatu-
structure for conducting the experimental work. The authors are also
rated Lipid-Based Formulations to Enhance the Oral Bioavail-
thankful to Mr. Ajay Lale for his assistance in manuscript proofreading.
ability of Venetoclax. Pharmaceutics. 2020;12:564. https://​doi.​
org/​10.​3390/​pharm​aceut​ics12​060564.
Author Contributions Arvind Sirvi: Conceptualization, methodology,
9. Ilie A-R, Griffin BT, Brandl M, Bauer-Brandl A, Jacobsen A-C,
investigation, data curation, validation, manuscript writing and review-
Vertzoni M, Kuentz M, Kolakovic R, Holm R. Exploring impact
ing. Akash Janjal: Methodology, formal data analysis, manuscript
of supersaturated lipid-based drug delivery systems of celecoxib
reviewing and editing. Kajal Guleria: Formal data analysis, manuscript
on in vitro permeation across PermeapadⓇ membrane and in vivo
reviewing and editing. Mahesh Chand:Methodology (Pharmacokinetic
absorption. Eur J Pharm Sci. 2020;152: 105452. https://​doi.​org/​
study). Abhay T. Sangamwar: Conceptualization, data review, supervi-
10.​1016/j.​ejps.​2020.​105452.
sion, validation, project administration, writing-review and editing.
10. Thomas N, Holm R, Müllertz A, Rades T. In vitro and in vivo
performance of novel supersaturated self-nanoemulsifying
Funding Not applicable.
drug delivery systems (super-SNEDDS). J Control Release.
2012;160:25–32. https://​doi.​org/​10.​1016/j.​jconr​el.​2012.​02.​027.
Data Availability Data will be made available on request.
11. Nora G-I, Venkatasubramanian R, Strindberg S, Siqueira-Jør-
gensen SD, Pagano L, Romanski FS, Swarnakar NK, Rades T,
Declarations Müllertz A. Combining lipid based drug delivery and amorphous
solid dispersions for improved oral drug absorption of a poorly
Ethics Approval All aspects of animal handling, care, and experi- water-soluble drug. J Control Release. 2022;349:206–12. https://​
mentation adhere to the ARRIVE guidelines and comply with the doi.​org/​10.​1016/j.​jconr​el.​2022.​06.​057.
UK Animals (Scientific Procedures) Act, 1986, along with associated 12. Panbachi S, Beranek J, Kuentz M. Hydrophobic deep eutectic
guidelines and the EU Directive 2010/63/EU for animal experiments. solvent (HDES) as oil phase in lipid-based drug formulations. Int J
192 Page 16 of 17 AAPS PharmSciTech (2024) 25:192

Pharm. 2024;661: 124418. https://d​ oi.o​ rg/1​ 0.1​ 016/j.i​ jphar​ m.2​ 024.​ poorly soluble drugs. Mol Pharmaceutics. 2020;17:180–9. https://​
124418. doi.​org/​10.​1021/​acs.​molph​armac​eut.​9b008​89.
13. Jadhav K, Sirvi A, Janjal A, Kashyap MC, Sangamwar AT. Uti- 26. Matsumura N, Ono A, Akiyama Y, Fujita T, Sugano K. Bottom-
lization of Lipophilic Salt and Phospholipid Complex in Lipid- up physiologically based oral absorption modeling of free weak
Based Formulations to Modulate Drug Loading and Oral Bio- base drugs. Pharmaceutics. 2020;12:844. https://​doi.​org/​10.​3390/​
availability of Pazopanib. AAPS PharmSciTech. 2024;25:59. pharm​aceut​ics12​090844.
https://​doi.​org/​10.​1208/​s12249-​024-​02780-3. 27. Sirvi A, Kuche K, Chaudhari D, Ghadi R, Date T, Katiyar SS, Jain
14. Sirvi A, Jadhav K, Sangamwar AT. Enabling superior drug load- S. Supersaturable self-emulsifying drug delivery system: A strat-
ing in lipid-based formulations with lipophilic salts for a brick egy for improving the loading and oral bioavailability of querce-
dust molecule: Exploration of lipophilic counterions and in vitro- tin. Journal of Drug Delivery Science and Technology. 2022;71:
in vivo evaluation. Int J Pharm. 2024;656:124108. https://​doi.​org/​ 103289. https://​doi.​org/​10.​1016/j.​jddst.​2022.​103289.
10.​1016/j.​ijpha​rm.​2024.​124108. 28. Thomas N, Müllertz A, Graf A, Rades T. Influence of Lipid
15. Bennett-Lenane H, O’Shea JP, Murray JD, Ilie A-R, Holm R, Composition and Drug Load on the In Vitro Performance of
Kuentz M, Griffin BT. Artificial Neural Networks to Predict the Self-Nanoemulsifying Drug Delivery Systems. J Pharm Sci.
Apparent Degree of Supersaturation in Supersaturated Lipid- 2012;101:1721–31. https://​doi.​org/​10.​1002/​jps.​23054.
Based Formulations: A Pilot Study. Pharmaceutics. 2021;13:1398. 29. Mondal S, Sirvi A, Jadhav K, Sangamwar AT. Supersaturating
https://​doi.​org/​10.​3390/​pharm​aceut​ics13​091398. lipid-based solid dispersion of atazanavir provides enhanced solu-
16. Schultz HB, Thomas N, Rao S, Prestidge CA. Supersaturated bilization and supersaturation in the digestive aqueous phase. Int J
silica-lipid hybrids (super-SLH): An improved solid-state lipid- Pharm. 2023;638: 122919. https://d​ oi.o​ rg/1​ 0.1​ 016/j.i​ jphar​ m.2​ 023.​
based oral drug delivery system with enhanced drug loading. Eur J 122919.
Pharm Biopharm. 2018;125:13–20. https://d​ oi.o​ rg/1​ 0.1​ 016/j.e​ jpb.​ 30. Michaelsen MH, Siqueira Jørgensen SD, Abdi IM, Wasan KM,
2017.​12.​012. Rades T, Müllertz A. Fenofibrate oral absorption from SNEDDS
17. Almasri R, Joyce P, Schultz HB, Thomas N, Bremmell KE, Pres- and super-SNEDDS is not significantly affected by lipase inhibi-
tidge CA. Prestidge, Porous Nanostructure, Lipid Composition, tion in rats. Eur J Pharm Biopharm. 2019;142(2019):258–264.
and Degree of Drug Supersaturation Modulate In Vitro Fenofi- https://​doi.​org/​10.​1016/j.​ejpb.​2019.​07.​002.
brate Solubilization in Silica-Lipid Hybrids. Pharmaceutics. 31. Kilic M, Dressman J. A simplified method to screen for in-vivo
2020;12:687. https://​doi.​org/​10.​3390/​pharm​aceut​ics12​070687. performance of oral lipid formulations. J Pharm Pharmacol.
18. Bannow J, Yorulmaz Y, Löbmann K, Müllertz A, Rades T. 2014;66:615–23. https://​doi.​org/​10.​1111/​jphp.​12182.
Improving the drug load and in vitro performance of super- 32. Qiu Q, Lu M, Li C, Luo X, Liu X, Hu L, Liu M, Zheng H,
saturated self-nanoemulsifying drug delivery systems (super- Zhang H, Liu M, Lai C, Song Y, Deng Y. Novel Self-Assembled
SNEDDS) using polymeric precipitation inhibitors. Int J Pharm. Ibrutinib-Phospholipid Complex for Potently Peroral Delivery
2020;575: 118960. https:// ​ d oi. ​ o rg/ ​ 1 0. ​ 1 016/j. ​ ijpha ​ r m. ​ 2 019.​ of Poorly Soluble Drugs with pH-Dependent Solubility. AAPS
118960. PharmSciTech. 2018;19:3571–83. https:// ​ d oi. ​ o rg/ ​ 1 0. ​ 1 208/​
19. Ilie A-R, Griffin BT, Kolakovic R, Vertzoni M, Kuentz M, Holm s12249-​018-​1147-4.
R. Supersaturated lipid-based drug delivery systems – explor- 33. Fridgeirsdottir GA, Harris R, Fischer PM, Roberts CJ. Support
ing impact of lipid composition type and drug properties on Tools in Formulation Development for Poorly Soluble Drugs.
supersaturability and physical stability. Drug Dev Ind Pharm. J Pharm Sci. 2016;105:2260–9. https://​doi.​org/​10.​1016/j.​xphs.​
2020;46:356–64. https://​doi.​org/​10.​1080/​03639​045.​2020.​17215​ 2016.​05.​024.
26. 34. Pouton CW. Formulation of poorly water-soluble drugs for oral
20. Siqueira SDVS, Müllertz A, Gräeser K, Kasten G, Mu H, Rades administration: Physicochemical and physiological issues and
T. Influence of drug load and physical form of cinnarizine in new the lipid formulation classification system. Eur J Pharm Sci.
SNEDDS dosing regimens: in vivo and in vitro evaluations. AAPS 2006;29:278–87. https://​doi.​org/​10.​1016/j.​ejps.​2006.​04.​016.
J. 2017;19:587–94. https://​doi.​org/​10.​1208/​s12248-​016-​0038-4. 35. Brinkmann J, Huxoll F, Luebbert C, Sadowski G. Solubility of
21. Paulus F, Bauer-Brandl A, Stappaerts J, Holm R. Digestion is pharmaceutical ingredients in triglycerides. Eur J Pharm Biop-
a critical element for absorption of cinnarizine from supersatu- harm. 2019;145:113–20. https://​doi.​org/​10.​1016/j.​ejpb.​2019.​10.​
rated lipid-based type I formulations. Eur J Pharm Sci. 2024;192: 012.
106634. https://​doi.​org/​10.​1016/j.​ejps.​2023.​106634. 36. Lee S-M, Lee J-G, Yun T-H, Cho J-H, Kim K-S. Enhanced
22. Paulus F, Holm R, Stappaerts J, Bauer-Brandl A. Absorption of Stability and Improved Oral Absorption of Enzalutamide with
cinnarizine from type II lipid-based formulations: Impact of lipid Self-Nanoemulsifying Drug Delivery System. Int J Mol Sci.
chain length, supersaturation, digestion, and precipitation inhi- 2024;25:1197. https://​doi.​org/​10.​3390/​ijms2​50211​97.
bition. Eur J Pharm Sci. 2024;197: 106765. https://​doi.​org/​10.​ 37. Suys EJA, Chalmers DK, Pouton CW, Porter CJH. Polymeric
1016/j.​ejps.​2024.​106765. Precipitation Inhibitors Promote Fenofibrate Supersaturation and
23. Wilson V, Lou X, Osterling DJ, Stolarik DF, Jenkins G, Gao W, Enhance Drug Absorption from a Type IV Lipid-Based Formu-
Zhang GGZ, Taylor LS. Relationship between amorphous solid lation. Mol Pharmaceutics. 2018;15:2355–71. https://​doi.​org/​10.​
dispersion in vivo absorption and in vitro dissolution: phase 1021/​acs.​molph​armac​eut.​8b002​06.
behavior during dissolution, speciation, and membrane mass 38. Francke NM, Bunjes H. Influence of drug loading on the physical
transport. J Control Release. 2018;292:172–82. https://​doi.​org/​ stability of phospholipid-stabilised colloidal lipid emulsions. Int
10.​1016/j.​jconr​el.​2018.​11.​003. J Pharm X. 2020;2: 100060. https://​doi.​org/​10.​1016/j.​ijpx.​2020.​
24. Ohtsu Y, Gibbons JA, Suzuki K, Fitzsimmons ME, Nozawa K, 100060.
Arai H. Absorption, Distribution, Metabolism, and Excretion of 39. Anby MU, Williams HD, McIntosh M, Benameur H, Edwards GA,
the Androgen Receptor Inhibitor Enzalutamide in Rats and Dogs. Pouton CW, Porter CJH. Lipid Digestion as a Trigger for Super-
Eur J Drug Metab Pharmacokinet. 2017;42:611–26. https://​doi.​ saturation: Evaluation of the Impact of Supersaturation Stabiliza-
org/​10.​1007/​s13318-​016-​0374-x. tion on the in Vitro and in Vivo Performance of Self-Emulsifying
25. Stewart AM, Grass ME. Practical approach to modeling the Drug Delivery Systems. Mol Pharmaceutics. 2012;9:2063–79.
impact of amorphous drug nanoparticles on the oral absorption of https://​doi.​org/​10.​1021/​mp300​164u.
AAPS PharmSciTech (2024) 25:192 Page 17 of 17 192

40. Devraj R, Williams HD, Warren DB, Mohsin K, Porter CJH, Pou- 47. Tanaka Y, Doi H, Katano T, Kasaoka S. The impact of quantity of
ton CW. In vitro assessment of drug-free and fenofibrate-con- lipid based formulations with different compositions on the oral
taining lipid formulations using dispersion and digestion testing absorption of ritonavir: A trade-off between apparent solubility
gives detailed insights into the likely fate of formulations in the and permeability. Eur J Pharm Sci. 2022;168: 106079. https://d​ oi.​
intestine. Eur J Pharm Sci. 2013;49:748–60. https://​doi.​org/​10.​ org/​10.​1016/j.​ejps.​2021.​106079.
1016/j.​ejps.​2013.​04.​036. 48. Thomas N, Richter K, Pedersen TB, Holm R, Müllertz A, Rades
41. Crum MF, Trevaskis NL, Pouton CW, Porter CJH. Transient T. In vitro lipolysis data does not adequately predict the in vivo
Supersaturation Supports Drug Absorption from Lipid-Based For- performance of lipid-based drug delivery systems containing
mulations for Short Periods of Time, but Ongoing Solubilization fenofibrate. AAPS J. 2014;16:539–49. https://​doi.​org/​10.​1208/​
Is Required for Longer Absorption Periods. Mol Pharmaceutics. s12248-​014-​9589-4.
2017;14:394–405. https://​doi.​org/​10.​1021/​acs.​molph​armac​eut.​ 49. Siqueira Jørgensen SD, Al Sawaf M, Graeser K, Mu H, Müllertz
6b007​92. A, Rades T. The ability of two in vitro lipolysis models reflect-
42. Suys EJA, Brundel DHS, Chalmers DK, Pouton CW, Porter CJH. ing the human and rat gastro-intestinal conditions to predict the
Interaction with biliary and pancreatic fluids drives supersatura- in vivo performance of SNEDDS dosing regimens. Eur J Pharm
tion and drug absorption from lipid-based formulations of low Biopharm. 2018;2018(124):116–24. https://​doi.​org/​10.​1016/j.​
(saquinavir) and high (fenofibrate) permeability poorly soluble ejpb.​2017.​12.​014.
drugs. J Control Release. 2021;331:45–61. https://​doi.​org/​10.​ 50. Koehl NJ, Henze LJ, Bennett-Lenane H, Faisal W, Price DJ, Holm
1016/j.​jconr​el.​2021.​01.​007. R, Kuentz M, Griffin BT. In Silico, In Vitro, and In Vivo Evalua-
43. Alskär LC, Keemink J, Johannesson J, Porter CJH, Bergström tion of Precipitation Inhibitors in Supersaturated Lipid-Based For-
CAS. Impact of Drug Physicochemical Properties on Lipolysis- mulations of Venetoclax. Mol Pharmaceutics. 2021;18:2174–88.
Triggered Drug Supersaturation and Precipitation from Lipid- https://​doi.​org/​10.​1021/​acs.​molph​armac​eut.​0c006​45.
Based Formulations. Mol Pharmaceutics. 2018;15:4733–44. 51. Ilie A-R, Griffin BT, Vertzoni M, Kuentz M, Kolakovic R, Pru-
https://​doi.​org/​10.​1021/​acs.​molph​armac​eut.​8b006​99. dic-Paus A, Malash A, Bohets H, Herman J, Holm R. Exploring
44. Sassene PJ, Knopp MM, Hesselkilde JZ, Koradia V, Larsen A, precipitation inhibitors to improve in vivo absorption of cinnar-
Rades T, Müllertz A. Precipitation of a Poorly Soluble Model izine from supersaturated lipid-based drug delivery systems. Eur J
Drug during In Vitro Lipolysis: Characterization and Dissolution Pharm Sci. 2021;159:105691. https://d​ oi.o​ rg/1​ 0.1​ 016/j.e​ jps.2​ 020.​
of the Precipitate. J Pharm Sci. 2010;99:4982–91. https://​doi.​org/​ 105691.
10.​1002/​jps.​22226.
45. Lee KWY, Porter CJH, Boyd BJ. The Effect of Administered Dose Publisher's Note Springer Nature remains neutral with regard to
of Lipid-Based Formulations on the In Vitro and In Vivo Perfor- jurisdictional claims in published maps and institutional affiliations.
mance of Cinnarizine as a Model Poorly Water-Soluble Drug. J
Pharm Sci. 2013;102:565–78. https://​doi.​org/​10.​1002/​jps.​23384. Springer Nature or its licensor (e.g. a society or other partner) holds
46. Stillhart C, Kuentz M. Trends in the Assessment of Drug Super- exclusive rights to this article under a publishing agreement with the
saturation and Precipitation In Vitro Using Lipid-Based Deliv- author(s) or other rightsholder(s); author self-archiving of the accepted
ery Systems. J Pharm Sci. 2016;105:2468–76. https://​doi.​org/​10.​ manuscript version of this article is solely governed by the terms of
1016/j.​xphs.​2016.​01.​010. such publishing agreement and applicable law.