Polymeric Precipitation Inhibitors Promote Fenofibrate Supersaturation

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Polymeric Precipitation Inhibitors Promote Fenofibrate Supersaturation and Enhance Drug Absorption from a Type IV Lipid-Based Formulation Estelle J.A. Suys, David K Chalmers, Colin W. Pouton, and Christopher J.H. Porter Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00206 • Publication Date (Web): 16 Apr 2018 Downloaded from http://pubs.acs.org on April 16, 2018

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Molecular Pharmaceutics

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Polymeric Precipitation Inhibitors Promote Fenofibrate Supersaturation

2

and Enhance Drug Absorption from a Type IV Lipid-Based Formulation

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Estelle J.A. Suys1, 2, David K. Chalmers3, Colin W. Pouton1*, Christopher J.H. Porter1, 2*

4 5 6

1

Drug Delivery, Disposition and Dynamics, 2ARC Centre of Excellence in Convergent Bio-Nano Science and Technology and 3Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Pde Parkville, Victoria 3052, Australia.

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Abstract Graphic

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10 11 12

Abstract

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The ability of lipid based formulations (LBFs) to increase the solubilization, and prolong the

14

supersaturation, of poorly water-soluble drugs (PWSDs) in the gastro-intestinal (GI) fluids has

15

generated significant interest in the last decade. One mechanism to enhance the utility of

16

LBFs is to prolong supersaturation via the addition of polymers to the formulation that inhibit

17

drug precipitation (polymeric precipitation inhibitors or PPIs). In this work, we have evaluated

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the performance of a range of PPIs and have identified PPIs that are sufficiently soluble in LBF

19

to allow the construction of single phase formulations. An in vitro model was first employed

20

to assess drug (fenofibrate) solubilization and supersaturation on LBF dispersion and digestion.

21

An in vitro-in situ model was subsequently employed to simultaneously evaluate the impact of

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PPI enhanced drug supersaturation on drug absorption in rats. The stabilizing effect of the

23

polymers was polymer specific, and most pronounced at higher drug loads. Polymers that

24

were soluble in LBF allowed simple processing as single phase formulations, while

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formulations containing more hydrophilic polymers required polymer suspension in the

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formulation. The lipid soluble polymers Eudragit (EU) RL100 and poly-(propylene glycol) bis(2-

27

aminopropyl ether) (PPGAE) and the water soluble polymer hydroxypropylmethyl cellulose

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(HPMC) E4M were identified as the most effective PPIs in delaying fenofibrate precipitation in 1 Environment ACS Paragon Plus

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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vitro. An in vitro model of lipid digestion was subsequently coupled directly to an in situ single

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pass intestinal perfusion assay to evaluate the influence of PPIs on fenofibrate absorption

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from LBFs in vivo. This coupled model allowed for real-time evaluation of the impact of

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supersaturation stabilization on absorptive drug flux, and provided better discrimination

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between the different PPIs and formulations. In the presence of the in situ absorption sink,

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increased fenofibrate supersaturation resulted in increased drug exposure and a good

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correlation was found between the degree of in vitro supersaturation, and in vivo drug

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exposure. Improved in vitro-in vivo correlation was apparent when comparing the same

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formulation under different supersaturation conditions. These observations directly exemplify

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the potential utility of PPIs in promoting drug absorption from LBF, via stabilization of

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supersaturation, and further confirm that relatively brief periods of supersaturation may be

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sufficient to promote drug absorption, at least for highly permeable drugs such as fenofibrate.

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KEYWORDS: fenofibrate, lipid-based formulation, drug flux, in vivo absorption, in vitro

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digestion, in situ perfusion, polymer precipitation inhibitors, supersaturation

16 17

Introduction

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Favorable solubility-permeability behavior in the gastro-intestinal (GI) tract is a prerequisite

19

for oral drug absorption. Drug candidates with low water solubility and/or low permeability,

20

however, are increasingly frequent products of contemporary drug discovery programs and

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this has dictated the need to employ bioavailability-enabling formulations to mask

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problematic drug properties. For drugs with low water solubility, lipid-based formulations

23

(LBFs) circumvent the dissolution limitations of traditional solid formulations and, by piggy-

24

backing onto endogenous lipid digestion and absorption pathways, provide a means to

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enhance drug solubilization in the GI tract.1, 2 For LBFs, increased drug absorption results from

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an increase in apparent drug solubility in the GI tract due to drug solubilization in the colloids

27

formed by dispersion and digestion of the formulation. However, solubilization is also

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accompanied by a reduction in thermodynamic activity. Simplistically this may be viewed as a

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reduction in the free drug concentration in the GI fluids (since most of the drug is solubilized

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in colloidal particles). In spite of the drop in free concentration, however, drug absorption is

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typically maintained, albeit potentially sub-optimally, since the equilibrium controlling 2 Environment ACS Paragon Plus

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transfer from the solubilized reservoir to the free drug fraction is rapidly re-established on

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drug absorption.

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Formulation processing events that promote transient drug supersaturation in the colloids in

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the GI tract, result in transient increases in thermodynamic activity and an increase in free

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drug concentration. Formulation designs that promote drug supersaturation in the GI fluids

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therefore provide an opportunity to enhance drug absorption.3-8 One means by which drug

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supersaturation can be enhanced is to formulate using polymers that delay drug precipitation

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– so called polymeric precipitation inhibitors or PPIs. PPIs have historically been demonstrated

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to sustain periods of transient, metastable supersaturation that are long enough to improve

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the absorption of PWSDs. Most commonly, PPIs have been used in amorphous solid

11

dispersion formulations.9-14 More recently, the concept and understanding of PPIs has been

12

transferred to LBFs.15, 16 However, improvements in supersaturation with polymers observed

13

in vitro have not always translated to large increases in oral bioavailability.16-20 This suggests

14

that our understanding of the beneficial utility of PPIs is incomplete.

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Several polymers have been found to delay the onset of drug precipitation from

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supersaturated solutions9, 15, 21. Hydroxypropylmethyl cellulose (HPMC), appears to be one of

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the most effective PPIs with respect to kinetic stabilization of supersaturation both in solid

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dispersions22-25 and in LBFs16, 17, 19. However, HPMC is not soluble in LBFs17 and this raises the

19

question as to whether a PPI that can be dissolved in LBF can be effectively employed as PPI in

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LBF, or whether PPI work more effectively when more soluble in the aqueous phase of the GI

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content (and are relatively insoluble in LBF). This may be a key aspect of formulation design,

22

since ideally all components in the formulation should be soluble in the formulation to allow

23

for the generation of isotropic systems (which typically have improved physical stability when

24

compared to two phase formulations).

25

Evaluation of PPI performance in vitro has historically been performed using (relatively) high

26

throughput screening methods such as pH-shift or solvent-shift studies with drug precipitation

27

measured by visual or microscopic inspection,26,

28

nephelometric turbidity measurements,15, 31 X-ray scattering techniques,32 or in-line Raman

29

spectroscopy.33 . Interpretation of the results from these high throughput methods using

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simplified models is often difficult, however, especially when applied to LBFs, since the

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models lack bile components and do not allow for the simultaneous process of LBF digestion

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by lipase/colipase.

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UV-spectrophotometry,28 HPLC,29,

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Assessment of PPI performance in LBFs can be conducted under more biorelevant conditions

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using an in vitro digestion apparatus that better mimics conditions in the small intestine. This

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model accounts for the presence of the formulation and lipid digestion products, and allows

4

for an estimation of drug partitioning between the aqueous, oil and solid precipitate phases

5

that are formed during formulation digestion.34, 35

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Although the in vitro digestion model gives improved insight into LBF performance, efforts to

7

correlate the in vitro data from this model with in vivo exposure have proved elusive.1 This

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likely reflects the simplicity of the in vitro digestion experiment when compared to the

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intricacies of the in vivo environment.36, 37 One of the biggest shortcomings of the ‘closed’ in

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vitro model is the absence of an absorption sink that serves to remove drug and digestion

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byproducts from the GI fluids. Removal of these components changes the GI solubilization

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capacity and the drug concentration, simultaneously altering the degree of drug

13

supersaturation and the drivers for drug precipitation. The lack of an absorption sink in most

14

in vitro models typically increases supersaturation and, in doing so, provides an artificial

15

trigger for drug precipitation. As such, the in vitro digestion model likely underestimates drug

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solubilization and drug absorption. To overcome this limitation, a coupled in vitro digestion-in

17

vivo absorption model has recently been developed within our laboratory for the assessment

18

of PWSD absorption from LBFs.38 In this ‘open’ model the simulated intestinal fluids contained

19

within the vessel used to conduct the in vitro digestion test, are continuously pumped through

20

an externalized segment of rat jejunum. This coupled model then allows for blood collection

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from the mesenteric vein of the rat and direct measurement of drug flux through the

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enterocytes that line the small intestine. Previous studies using fenofibrate and simple LBFs38,

23

39

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solubility, supersaturation and permeability in drug absorption from LBFs.

25

The current study builds on our previous work to evaluate the impact of PPIs on drug

26

absorption using the coupled in vitro digestion-in vivo absorption model. We investigated a

27

range of polymers for their ability to promote and stabilize the supersaturation of fenofibrate

28

and probed the importance of polymer solubility in the LBF versus polymer solubility in

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simulated intestinal fluids. The better performing PPIs were subsequently tested in the

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coupled model to evaluate whether sustained supersaturation in vitro translates to more

31

effective drug transport through the absorptive membrane. The data suggest that, at least for

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fenofibrate, PPIs that are soluble in lipid media (and therefore more suitable as LBF

show that the coupled model can improve our understanding of the interplay between


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components) are capable of precipitation inhibition, and that prolonged in vitro

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supersaturation is correlated with increased absorptive drug flux.

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Materials and Methods Materials

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Fenofibrate

(2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoic

7

fenofibric acid and meclofenamic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA).

8

Captex® 300, a medium-chain triglyceride (composed mainly of caprylic and capric acid) and

9

Capmul® MCM, a blend of partially digested medium-chain glycerides, were donated by

10

Abitec Corporation (Columbus, OH). Kolliphor® EL (polyoxyl 35 hydrogenated castor oil) was

11

donated by BASF Corporation (Washington, NJ, USA). Transcutol® HP (diethylene glycol

12

monoethyl

13

taurodeoxycholate >95% (NaTDC), 4-bromophenylboronic acid (4- BPB) and porcine

14

pancreatin extract (P7545, 8 x USP specifications activity) were all obtained from Sigma-

15

Aldrich (St. Louis, MO, USA). Phosphatidylcholine (PC) (Lipoid E PC S, approximately 99.2%

16

pure, lecithin from egg yolk) was obtained from Lipoid (Lipoid GmbH, Ludwigshafen,

17

Germany). Sodium hydroxide (NaOH) was purchased from Merck (Darmstadt, Germany).

18

Water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA).

19

All other chemicals and solvents were of analytical purity or high performance liquid

20

chromatography (HPLC) grade. Hypergrade solvents were used for UPLC-MS/MS analysis.

ether)

was

supplied

by

Gattefossé

(St.

acid

Priest,

isopropyl

France).

ester),

Sodium

21 22

The following polymers were obtained from Sigma Aldrich Pty Ltd., Australia: hydroxylethyl

23

cellulose ethoxylate quaternized (HECEQ), poly(2-acrylamido-2-methyl-1-propanesulfonic acid)

24

(PAAMPS), polyanetholesulfonic acid (PAESA), poly(bis(2-chloroethyl) ether-alt-1,3-bis(3-

25

(dimethylamino)propyl)urea) (PCEDPU), poly(2-ethyl 2- oxazoline) (PEOX), poly-(propylene

26

glycol) bis(2-aminopropyl ether) (PPGAE), poly-(sodium 4-styrene sulfonate) (PSSS), poly(4-

27

styrenesulfonic acid-co-maleic acid) (PSAMA), polyvinylpyrrolidone (PVP-40), poly(1-

28

vinylpyrrolidone-co-2 dimethylaminoethyl methacrylate) (PVPDAM), Poly-(1→4)-β-N-acetyl-D-

29

glucosamine (Chitin, from shrimp shells), Buckminsterfullerene (Fullerene(C60)). Eudragit E 100,

30

E PO, L 100 and RL 100 were supplied by Evonik Degussa Pty Ltd., Australia. Hydroxyethyl

31

cellulose 30000S (HEC) was supplied by Shandong Head Co Ltd. Hydroxypropylmethyl



1

cellulose (HPMC) E4M and hydroxypropylmethyl cellulose acetate succinate (HPMCAS:HF)

2

were supplied by ShinEtsu Chemical Co.Ltd., c/o ANZChem Pty Ltd., Australia.

3 4

A complete listing of the abbreviations and structures used to identify the polymers can also

5

be found in the Supporting Information (Table S1).

6 7

Preparation of LBFs containing fenofibrate

8

LBFs consisted of the excipients and ratios presented in Figure 1 and were prepared as

9

previously described.38

10 11

Figure 1. Schematic representation of the composition of the three LBF types employed during the in vitro

12

digestion experiment (% content is % w/w). The Type IIIA and Type IIIB formulations contained a medium chain

13

lipid phase comprising a mixture of Captex® 300 and Capmul® MCM, based on the LFCS classification of LBFs

14

proposed by Pouton et al. 40

15 16

Fenofibrate was incorporated into the formulations at 40% w/w or 85% w/w of the saturated

17

solubility of the drug in a specific formulation (based on measured solubility values at 37 °C).

18

Drug solubility in each of the formulations was assessed using standard methods and all

19

experiments were performed in triplicate with samples taken at 2, 12, 24, 48 and 72 h.41

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Equilibrium solubility was defined as the value attained when at least three consecutive

21

solubility sample values varied by less than 5%. For fenofibrate, this value was typically

22

reached between 24 h and 48 h, in agreement with previously reported time frames.38

23

Polymer-containing formulations were made by adding the stated concentrations (% w/w) of

24

polymers to pre-assembled and equilibrated blank formulations. Formulations were vortex-

25

mixed and stored at 37 °C to equilibrate for at least 24 h prior to use.42

26 27

In vitro (polymer) screening


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Drug solubilization and supersaturation during formulation dispersion and

2

digestion

3

In vitro experiments were conducted using previously reported methods, conditions and

4

apparatus.43 Briefly, 1.1 g of drug-loaded LBF was dispersed using an overhead propeller

5

stirrer (length 96 mm) rotating at 450 min-1 (speed setting +3) in a jacketed and

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thermostatically controlled glass reaction vessel (Metrohm® AG, Hersiau, Switzerland)

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containing 40 mL of digestion buffer at a constant temperature (37°C). The buffer was made

8

in advance (equilibrated overnight) and pre-heated to 37°C prior to the dispersion-digestion

9

experiment and consisted of 2 mM Tris-maleate, 1.4 mM CaCl2.2H2O, 150 mM NaCl adjusted

10

to pH 6.5 to which 3 mM taurodeoxycholate (NaTDC) and 0.75 mM phosphocholine (PC) was

11

added. The pH was manually adjusted during the initial dispersion phase (15 min) using 0.1 M

12

of NaOH or HCl. Digestion was initiated after formulation dispersion by adding 4 mL of

13

pancreatin extract to the digestion medium. Pancreatin extract was prepared as described

14

previously38 and contained pancreatic lipase (and other pancreatic enzymes) and had a

15

pancreatic lipase activity of ~40,000 TBU (to provide approximately 1000 TBU per mL of

16

digest). Aliquots were removed from the media throughout the in vitro experiment at

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timepoints of -10, -5, 0, 5, 15, 30, 45, 60 min relative to the start of digestion. Lipase inhibitor

18

(4-BPB, 5 μL/mL of a 1.0 M in methanol) was added to the Eppendorf sample tubes prior to

19

the experiment to prevent further lipolysis after sampling. Hereafter samples were

20

immediately centrifuged at 37°C for 10 min using a bench-top centrifuge at 21,000 g (Heraeus

21

Fresco 21®, ThermoScientific, Langenselbold, Germany) in order to spin down any precipitated

22

material formed upon dispersion and/or digestion and to generate an aqueous solubilized

23

phase and a precipitated pellet phase. During the digestion phase (60 min) the pH was

24

continuously monitored and adjusted to a set point of pH 6.5 using a pH stat titrator via

25

automatic addition of 0.2 M or 0.6 M NaOH for Type IV LBF and medium-chain LBF (Type IIIA

26

and Type IIIB) respectively.

27 28

Fenofibrate solubility in aqueous pre- (dispersion) and post-digestion phase

29 30

The solubility of crystalline fenofibrate in the aqueous phase generated during dispersion and

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digestion of each of the emulsified blank (i.e. drug-free) LBF was assessed under the same in

32

vitro conditions as described above. To generate blank aqueous phase, aliquots were taken 7 Environment ACS Paragon Plus


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from the digestion vessel after 5 min and 10 min dispersion of blank formulations and after 5,

2

15, 30 and 60 min post digestion initiation. Samples were centrifuged (10 min, 21,000 g, 37°C,

3

Heraeus Fresco 21®), supernatants were pooled and 1 mL of this aqueous solution was added

4

to an excess of fenofibrate (10-15 mg) in three polypropylene tubes for each time point. These

5

were vortexed and placed in an orbital shaker incubator (Orbital Mixer Incubator, Ratek

6

Instruments, Melbourne, Australia) at 37°C. Samples were subsequently taken over 6 h at 1 h

7

intervals, vortex mixed and centrifuged for 10 min. The supernatant was sampled and diluted

8

with acetonitrile prior to HPLC analysis. The apparent equilibrium solubility of fenofibrate in

9

the aqueous colloidal phase obtained after dispersion (APDISP) and digestion (APDIGEST) was

10

defined as the mean solubility value over the last 2 time points, for which the change in

11

solubility was less than 5% (typically achieved after 2-3 h incubation). A measure of how

12

effectively supersaturation was generated and maintained over the duration of the

13

experiment was gained by comparing the AUC for the solubilized drug concentration (during

14

dispersion and digestion) as a function of time, divided by the AUC of the apparent

15

equilibrium solubility of fenofibrate in the AP during dispersion (APDISP) and digestion

16

(APDIGEST). These values were used to calculate the supersaturation ratio (S) as described by

17

Anby et al.17

18 19 20

HPLC-UV quantification of fenofibrate in formulations and in samples from in vitro experiments

21 22

Aqueous dispersion and digestion samples from the in vitro experiment were diluted 1:100

23

(v/v) with acetonitrile and mobile phase prior to HPLC-UV analysis. Drug quantification was

24

performed using a Waters Alliance 2695 Separation Module and a Waters 486 tunable

25

absorbance detector (Waters Alliance Instruments, Milford, MA, USA) with a reverse-phase

26

C18 column (150×3.9 mm, 5 μm, Waters Symmetry®) coupled to a C18 guard cartridge (4×2.0

27

mm, Phenomenex, Torrence, CA, USA). The column was maintained at ambient temperature.

28

The injection volume was 50 μL and UV absorbance was monitored at 286 nm. The mobile

29

phase consisted of acetonitrile and water in an 80:20 v/v ratio with 0.1% (v/v) formic acid and

30

was pumped through the column at a flow rate of 1 mL/min for 6 min. Fenofibrate eluted at 4

31

min.

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The assay was validated by multi-day matrix analysis of n = 6 (per concentration group) quality

33

control (QC) standards at low (1 μg/mL), medium (12.5 μg/mL) and high (50 μg/mL) 8 Environment ACS Paragon Plus

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concentrations on three different days. Intra-assay validation was accurate to 101.4, 94.9 and

2

96.1% and precise to 2.9, 1.9 and 2.9% RSD for 1.0, 12.5 and 50.0 μg/mL QC samples

3

respectively. Inter‐assay validation (over 3 days) was accurate to 101.9, 99.1 and 98.8% and

4

precise to ± 2.7, 2.7 and 3.5% RSD for 1.0, 12.5 and 50 μg/mL respectively. Linearity, R2 = 1;

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matrix recovery, assessed from blank digest samples (n = 6), was oil 91%, pellet 107%,

6

aqueous phase 96.5% (Type IIIA) and 96.9% (Type IV); specificity, no interfering peaks at RT of

7

fenofibrate; system precision, 1.9% RSD (n = 6).

8 9

Polarized light microscopy for solid-state analysis of precipitated fenofibrate

10 11

The pellet from dispersion (0 min) and digestion (30 min) samples was carefully removed from

12

polypropylene tubes after phase separation by centrifugation, transferred onto a microscope

13

slide and analyzed using a Zeiss Axiolab microscope (Carl Zeiss, Oberkochen, Germany)

14

equipped with crossed polarizing filters. Images were obtained using a Canon PowerShot A70

15

digital camera (Tokyo, Japan) and processed using ImageJ (Fiji).44 Pellets were analyzed within

16

1 h of sampling to minimize the risk of any changes in drug solid state properties due to

17

storage.

18 19

In vitro digestion-in vivo absorption model

20

Based on the results from the in vitro dispersion-digestion experiment, formulations

21

containing different polymers were chosen for in situ perfusion experiments to examine if the

22

addition of the PPIs led to increased drug flux across the intestinal membrane. All surgical

23

procedures and model parameters were as described by Crum et al.38, 39

24

Animals

25

All animal studies were performed in accordance with the Australian and New Zealand Council

26

for the Care of Animals on Research and Teaching guidelines and approved by the institutional

27

animal experimentation ethics committee (Monash Institute of Pharmaceutical Science –

28

Monash University). Male Sprague–Dawley rats (290–330 g) were allowed to acclimatize in

29

the institutional animal housing facility for at least three days with free access to standard

30

food and water ad libitum. Animals were fasted overnight (12–18 h) prior to surgery.

31

Anaesthesia was induced in rats by subcutaneous injection (1.0 mL/kg) of anaesthetic

32

“Cocktail I” (37.3 mg/mL ketamine, 9.8 mg/mL xylazine, 0.4 mg/mL acepromazine in saline) 9 Environment ACS Paragon Plus


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and maintained throughout the study with subcutaneous doses (0.44 mL/kg) of “Cocktail II”

2

(90.0 mg/mL ketamine, 0.9 mg/mL acepromazine in saline) when required.45 Rats were

3

maintained on a 37 °C heated pad throughout surgery and experiments conducted under

4

general anaesthesia. At the end of all experiments, rats were euthanized via an intravenous or

5

intracardiac injection of sodium pentobarbitone (100 mg) (Virbac Pty. Ltd., Milperra, New

6

South Wales, Australia).

7 8

Surgical Procedures

9

In situ rat jejunal perfusion experiments with simultaneous mesenteric blood collection were

10

conducted to assess drug flux across the corresponding isolated segment of a rat jejenum

11

without the confounding effects of hepatic first-pass metabolism. For animals undergoing in

12

situ intestinal perfusion with mesenteric cannulation, a vertical incision was made above the

13

right breastbone (above the right collarbone) to provide access to the jugular vein, which was

14

subsequently cannulated to enable infusion of donor blood via a peristaltic pump (as

15

described previously38, 46). To expose the small intestine, a mid-line incision was made in the

16

abdomen and a section of the jejunum was externalized and moistened with saline. Incisions

17

were made at the proximal and distal ends of the jejunum segment (~ 10 cm) by

18

electrocautery and the segment flushed using pre-warmed saline to remove intestinal

19

contents. This segment was cannulated with elbow propylene fittings and ligated using silk

20

sutures. The mesenteric vein draining the cannulated jejunum segment was prepared for

21

catheterization by blunt dissection of the surrounding connective tissue and flushed with

22

heparinized saline (90 IU/kg) through the jugular vein prior to mesenteric vein cannulation. A

23

fluorinated ethylene propylene catheter (BD Angiocath i.v. catheter, 24G) attached to silicone

24

tubing was employed to cannulate the mesenteric vein and collect venous blood draining

25

from the cannulated segment. This blood was continuously collected into heparinized

26

polypropylene tubes. The externalized jejunum section was kept moist (with warm saline) and

27

the rat was maintained at 37°C using a heated pad and heated light source. Fresh heparinized

28

donor blood was collected from donor rats immediately before surgery by cardiac puncture

29

and employed for infusion of donor blood throughout the experiment at 0.3 ml/min.

30 31

Assessment of fenofibric acid absorptive flux into the mesenteric vein

32


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After surgery, animals were equilibrated for 30 min, during which time heparinized (5

2

Units/mL) donor rat blood was infused via the jugular vein at a rate of 0.3 mL/min via an

3

infusion pump (Pump 11, Harvard Apparatus, Holliston, MA). Experiments were performed in

4

a heated cabinet and during re-equilibration, blood from the cannulated mesenteric vein was

5

collected for re-infusion through the jugular vein while saline (37°C) solution was pumped

6

through the jejunum segment to clear any residual content. In vitro dispersion and digestion

7

experiments were carried out as detailed above and the contents of the dispersion/digestion

8

vessel were pumped through the inlet tube of the jejenum using a peristaltic pump (Pump P-1,

9

Pharmacia Biotech, Amersham Biosciences, Piscataway, NJ) at a flow rate of 0.4 mL/min. The

10

infusion was started at the time of digestion initiation. Samples (0.5 mL) were taken from the

11

digestion vessel to quantify fenofibrate concentrations during dispersion and digestion.

12

Samples were also collected from the perfusate after passage through the jejenum to

13

measure the fenofibrate concentrations in the perfusate post absorption. These samples were

14

analyzed by HPLC using the conditions and methods detailed above. Blood from the

15

cannulated mesenteric vein was continuously collected into pre-weighed Eppendorf tubes

16

over 5 min intervals. These samples were immediately centrifuged (6708 g, Minispin™,

17

Thermo Fisher Scientific, Massachusetts, US) for 10 min to separate the plasma from whole

18

blood and stored at -80°C until sample analysis by UPLC-MS2 for fenofibric acid (FFA)

19

concentrations.

20 21

Validation of jejunum membrane integrity

22 23

The integrity of the jejunum membrane of the rat absorption model was validated using

24

radiolabeled passive permeability markers, for para- (mannitol) and trans-cellular (antipyrine)

25

routes. The permeability of these validation markers was assessed during perfusion of the

26

Type IV LBF loaded at 85% fenofibrate saturation in the presence of 5%w/w of HPMC E4M,

27

PPGAE and EU RL100, to investigate the potential disruptive effect of the formulation and the

28

polymers on the intestinal membrane. The validation study was conducted using 5 µCi [14C]-

29

antipyrine and 0.5 µCi [3H]-mannitol with 1 mM of unlabeled antipyrine and mannitol, in the

30

digestion media in addition to the LBF. The digestion experiment was performed as described

31

above. Samples were withdrawn from the digestion vessel at -10 and 0 min (prior to initiation

32

of digestion) and 10, 20, 30, 40, 50, 60 min (post-digestion). Blood from the mesenteric vein

33

was continuously collected over 5 min intervals as described above and plasma was separated

34

from whole blood as described above. For quantification of 14C and 3H radiolabel within the 11 Environment ACS Paragon Plus


Page 12 of 49

1

perfusate and plasma samples, 200 µL of the sample was placed in liquid scintillation vials to

2

which 2 mL of scintillation liquid (Ultima Gold™, PerkinElmer) was added and vortexed for 10 s.

3

Quantification of 14C and 3H radiolabel concentration in the perfusate and blood samples was

4

achieved using a Packard Tri-Carb 2000CA liquid scintillation analyzer (Packard, Meriden, CT).

5 6

The apparent permeability coefficients of the markers were calculated as described previously

7

for perfusion models,47, 48 using the appearance of the markers in the mesenteric vein blood,

8

as per Eq. 1 below.46 Appearance Papp =

9

dCr dt

V

× A·C

(1) d

10

where V (cm3) is the volume in the receptor chamber (in this case, the plasma volume in the

11

scintillation vials), A (cm2) is the surface area of the perfused jejunum segment, Cd is the initial

12

activity in the donor chamber (cpm) and dCr/dt is the rate of change of permeant

13

concentration in the receptor chamber (in this case, venous blood) determined from the

14

linear portion of a cumulative receptor chamber activity versus time plot. The surface area of

15

perfusion was standardized to 10 cm2. The apparent permeability coefficients of antipyrine

16

and mannitol from the appearance in venous blood are listed in Table 1 and were within the

17

range of previously reported values from the literature.38, 47, 49

18 19

Table 1. Apparent permeability coefficients of antipyrine and mannitol based on the appearance in the

20

mesenteric venous blood in the absence and presence of 5%w/w PPGAE, EU RL100 and HPMC E4M. Formulation

Mannitol Papp -6

Antipyrine Papp

(x 10 cm.sec )

(x 10-5 cm.sec-1)

3.43

1.62

+ PPGAE

3.74

1.35

+ EU RL100

3.01

1.62

+ HPMC E4M

3.39

1.07

Type IV, 85% fenofibrate

-1

21 22

Plasma sample preparation

23 24

Calibration standards for FFA were prepared by spiking 50 µL aliquots of blank plasma with 5

25

µL of an acetonitrile solution containing 0.47 - 30 µg/ml FFA. This provided spiked plasma

26

concentrations in the range of 15.6 – 1000 ng/mL FFA. Five microliters of an internal standard

27

(IS) solution of meclofenamic acid (MFA) (150 µg/mL) was also added to each 50 µL plasma 12 Environment ACS Paragon Plus

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1

sample (unknown) or standards, the tubes were vortexed, and 90 µL ACN was added (an

2

additional 5 µL ACN was added to unknown samples to match the spiking volume in the

3

plasma standards of 150 µL). The tubes were vortexed again and centrifuged for 10 min at

4

21,000 g (20°C, Fresco 21, Heraeus, Thermo Fisher Scientific, Massachusetts, US). The

5

supernatant (organic phase) was transferred into another Eppendorf, diluted 10-fold with

6

mobile phase, and centrifuged again, after which the supernatant was transferred into

7

autosampler vials and injected onto the LC-MS/MS.

8 9

Quantification of fenofibric acid in plasma by UPLC-MS2

10 11

Previous studies have shown that the hydrolysis of fenofibrate to fenofibric acid occurs rapidly

12

in the enterocyte with only fenofibric acid detectable in the portal blood and systemic blood

13

circulation.50,

14

concentrations.

51

Plasma samples were thus prepared to quantitate fenofibric acid

15 16

Plasma samples were analyzed on a LCMS-8050 triple quadrupole mass spectrometer

17

(Shimadzu, Kyoto, Japan), including a LC-30AD binary pump, a SiL30AC refrigerated

18

autosampler, a mobile phase vacuum degassing unit (DGU-20A5R) and a temperature-

19

controlled column compartment (CTO-20AC), coupled to a triple quadrupole mass

20

spectrometric (MS) detector equipped with an electrospray ionization source. The

21

autosampler temperature was 4 °C and column was heated to 40 °C. Chromatographic

22

separation was achieved using a Kinetex C18 column (50 × 2.1 mm, 2.6 μm) (Phenomenex,

23

Torrence, CA), coupled to a Kinetex C18 guard cartridge (4 × 2.0 mm). The injection volume

24

was 2 μL and the mobile phase consisted of 0.1% v/v formic acid in Milli Q water (A) and 0.1%

25

formic acid in acetonitrile (B). Under these conditions the retention times of FFA and the

26

internal standard, MFA, were 1.85 min and 2.96 min, respectively. Samples were eluted using

27

gradient elution at a constant flow rate of 0.25 mL/min and conducted as follows: The initial

28

composition was 50% solvent B and this was linearly increased to 95% B over 3.7 min and

29

then held at 95% B. After 4.9 min solvent B was decreased to 30% over 0.5 min and then

30

returned back to 50% by the end of the 6.4 min run time to equilibrate. The optimized MS/MS

31

conditions are displayed in Table 2, the nebulizing gas flow was set to 2.2 L/min, the

32

desolvation line temperature to 225°C, the interface temperature to 300 °C and the interface

33

voltage to 3.5 kV and heat block temperature to 400°C. Data acquisition and peak integration 13 Environment ACS Paragon Plus


Page 14 of 49

1

were performed using LabSolutions LCMS version 5.6 software package for LCMS 8050.

2

Unknown plasma concentrations were determined by interpolation from a weighted

3

(1/concentration) calibration curve of FFA:IS peak response plotted as a function of fenofibric

4

acid concentration.

5 6

Table 2. Experimental optimized MS/MS conditions on the LCMS 8050 for fenofibric acid (FFA) and

7

meclofenamic acid (MFA), with Q1 and Q3 being respectively the first and the third quadrupole mass filters

8

and CE being the collision energy. Scan Compound

structure

Exact mass

Parent

Product

Q1

Q3

m/z

m/z

(V)

(V)

317.00

231.00

16.0

16.0

21.0

258

9

19

14

214

15

7

18

mode

CE

(+/-)

FFA

318.065887

-

MFA (internal

295.016684

-

294.10

standard)

9 10

The plasma assay was validated using n=6 spiked plasma QC standards, prepared at low (15.6

11

ng/mL), medium (250ng/mL) and high (1000 ng/mL) concentrations. Intra-assay variability

12

was accurate to 104.1, 90.8 and 93.0% and precise to ± 3.0, 2.5 and 5.9% of the low, medium

13

and high QC samples respectively. Inter-assay variability was assessed over two separate days

14

and was accurate to 87.0, 98.7 and 98.5% and precise to 10.3, 4.4 and 4.1% at low, medium

15

and high QC respectively. For the assay specificity, no interfering peaks were observed in

16

blank plasma extracts at the retention time of FFA and MFA. To calculate the fenofibric acid

17

flux, i.e. the total transport into blood, the mean blood:plasma ratio was determined (0.78 ±

18

0.03) and was used to convert plasma concentrations into blood concentrations.

19 20

Statistical Analysis

21 22

All statistical analysis was performed using Graphpad Prism for windows (Version 7.01,

23

Graphpad Software Inc., CA, USA). Statistically significant differences were determined by


Page 15 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

one-way ANOVA followed by a Dunnett's multiple comparisons test at a significance level of α

2

= 0.05.

3 4 5

Results In vitro screen of PPIs

6 7

The equilibrium solubility of fenofibrate in the three formulations is shown in Table 3 and is

8

comparable to the results obtained previously for fenofibrate in similar LBFs.38 Solubility

9

values ranged from 128.2 mg/g in the Type IIIA formulation to 154.4 mg/g in the Type IV.

10



Page 16 of 49

1 2 3

Table 3. Equilibrium solubility of fenofibrate in the three examined LBFs, attained after 72 h at 37°C and the

4

equilibrium solubility of fenofibrate in blank dispersed and digested aqueous phase obtained during lipid

5

digestion [mean ± SD, n = 3]. equilibrium

apparent equilibrium solubility (mg/ml) in aqueous phase

solubility in

during lipid dispersion and digestion

Formulation types formulation (mg/g)

APDISP (-10 min)

APDIGEST (5min)

APDIGEST (60 min)

Type IIIA - MC

128.2 ± 4.3

1.63 ± 0.04

0.37 ± 0.03

0.26 ± 0.03

Type IIIB- MC

144.4 ± 3.7

1.05 ± 0.06

0.83 ± 0.03

0.62 ± 0.02

Type IV (no PPI)

154.4 ± 3.1

0.34 ± 0.02

0.39 ± 0.03

0.24 ± 0.05

+ EU RL100

0.29 ± 0.02

0.15 ± 0.07

0.14 ± 0.05

+ HPMC E4M

0.25 ± 0.01

0.31 ± 0.01

0.32 ± 0.05

+ PPGAE

0.30 ± 0.06

0.31 ± 0.08

0.23 ± 0.08

6 7

The increase in equilibrium solubility from the Type IIIA to the Type IV formulation suggests a

8

trend toward increasing drug solubility with increasing LBF polarity. It is perhaps

9

counterintuitive that a highly lipophilic drug, such as fenofibrate, is more soluble in

10

formulations containing a smaller amount of lipid (i.e. Type IV), however this observation has

11

been reported previously35, 43 and is consistent with the fact that fenofibrate is highly soluble

12

in cosolvents.52 The presence of surfactant (Kolliphor EL) also assists in drug solubility in the

13

formulation. Formulations were subsequently made up at drug loads of 40% and 85% of

14

saturated solubility in the formulation to investigate the effect of drug loading on PPI

15

performance. The effective drug load was confirmed by formulation assay, resulting in loading

16

at 95.2, 96.4 and 99.4% of target for the Type IIIA, Type IIIB and Type IV, respectively, and

17

98.1, 95.1 and 98.4% of target for the 40% and 85% drug loads, respectively. The apparent

18

equilibrium solubility values of fenofibrate in the aqueous dispersion and digestion phases

19

obtained from drug-free LBFs (Table 3) show that solubility is generally lower in the digested

20

colloids than in the colloids generated during dispersion. This can be attributed to the lower

21

solubilization capacity of mixed micelles containing lipid digestion products such as fatty acids

22

(FA) and monoglycerides (MG), compared to micro-emulsion droplets containing triglycerides

23

(TG) and diglycerides (DG). During dispersion of the Type IV formulation, water-miscible

24

components such as surfactant and cosolvent are diluted, which consequently leads to an

25

immediate drop in solubilization capacity of the colloids, resulting in a low apparent 16 Environment ACS Paragon Plus

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1

equilibrium solubility upon dispersion when compared to the Type III formulations that

2

contain larger quantities of non-water miscible lipids. Since much of the Type IV formulation is

3

non digestible only small changes in equilibrium solubility are evident after digestion of the

4

Type IV formulation.

5 6 7

Effect of drug saturation on the in vitro performance of LBFs containing PPIs

8 9

Three PPIs (Eudragit E100, HPMCAS:HF and HPMC E4M) were initially selected to provide

10

proof of concept that PPI were able to effectively prolong the duration of fenofibrate

11

supersaturation for each LBF type and drug load. By way of example, fenofibrate solubilization

12

profiles during dispersion and digestion of LBFs containing 1% w/w E100 are displayed in

13

Figure 2.

14 15

The solubility of fenofibrate in the colloids produced during formulation dispersion and

16

digestion is detailed in Table 2 and shown Figure 2 as the black dashed line. Figure 2 also

17

shows the drug concentrations measured during kinetic evaluation of drug solubilization

18

during lipid formulation digestion in the absence (grey) and presence (blue) of polymer (in this

19

example Eudragit E100). Concentrations above the black dashed line are therefore

20

‘supersaturated’. To give an indication of the thermodynamic instability generated by

21

formulation dispersion or digestion, the ratio of the APMAX (blue dashed line – representing

22

the maximum AP concentration that would be attained if no drug precipitated on formulation

23

dispersion) and the equilibrium solubility of the drug in the aqueous phase after dispersion

24

and digestion (APDISP or APDIGEST, black dotted line) was calculated as a measure of the maximal

25

degree of supersaturation during dispersion and digestion (i.e. SM). SM provides an indication

26

of the maximal driver for precipitation. The SM values are annotated in Figure 2 for both

27

dispersion (SMDISP) and digestion (SMDIGEST) phases. From previous reports (in the absence of

28

PPIs), it has been established that supersaturation is typically stable (and therefore

29

precipitation avoided) when the SM, is below ~3,17, 53 and that SM values during the digestion

30

phase (Figure 2) are generally higher than those calculated during the dispersion phase (since

31

the solubilization capacity of digested colloids is typically lower than that of dispersed colloids

32

containing undigested glycerides). Consistent with these previous results, loading

33

formulations at lower drug saturation (40% saturation, upper panels) resulted in the



1

generation of lower degrees of supersaturation (and lower SM) and therefore lower

2

precipitation. In contrast, at higher drug loads (85% saturation, lower panels), SM was higher

3

and precipitation more evident. Formulation dispersion also led to lower supersaturation than

4

that obtained after initiation of digestion (except for the Type IV formulation where digestion

5

was limited). Formulations containing drug at high (85 %) drug loading showed high initial

6

levels of supersaturation (significantly greater than the previously observed limit of 3) and as

7

such this was followed by rapid precipitation towards the apparent equilibrium solubility. In

8

general, however, even though drug precipitation was rapid from the formulations loaded at

9

85% saturation, the increased drug load did lead to the attainment of higher solubilized drug

10

concentrations when compared to the formulations loaded at 40% saturation – at least at

11

early time points. The solubilization capacity of the Type IIIA and IIIB formulations dropped

12

sharply after initiation of digestion, while the Type IV formulation rapidly lost solubilization

13

capacity upon dispersion. This is consistent with previously published solubilization profiles of

14

fenofibrate for Type IIIB and Type IV formulations at high drug load.38, 43

15

16 17

Figure 2. In vitro evaluation of drug solubilization profile of fenofibrate during dispersion and digestion of three LBFs,

18

[mean ± SD (n = 3)] in the presence and absence of Eudragit E100 (EU E100). Fenofibrate was incorporated at 40%

19

(upper row) and 85% (lower row) of the equilibrium solubility in the formulation and in the absence (grey circles) and

20

presence (blue circles) of 1% w/w EU E100. The horizontal (blue) dashed line represents the APMAX and the black

21

circles (dashed black line) represents the apparent equilibrium solubility of fenofibrate in the aqueous colloidal phase,

22

produced during drug-free LBF dispersion (APDISP) and digestion (APDIGEST). The blue shaded section represents the

23

experimental duration of the dispersion phase.


Page 18 of 49

Page 19 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

The Type IV formulation did not follow the same general trend where higher drug loads

3

generated higher solubilized drug concentrations. Instead, the Type IV formulation with a

4

drug load of 85% resulted in very rapid precipitation over the first 5 minutes of formulation

5

dispersion. When the drug load was lowered to 40%, the formulation was able to retain drug

6

in a supersaturated state for 30 min during digestion prior to subsequent precipitation over

7

the last 15 min of digestion. A similar observation for a Type IV formulation at various drug

8

loading was made by Williams et al.43 From the solubilization profiles in Figure 2 in the

9

absence of PPI (grey symbols) it is possible to establish a rank order of formulation

10

solubilization performance where in general Type IIIA> Type IIIB> Type IV.

11 Type IIIA

Type IIIB

Type IV AUC 40 % AUC 85 % S 40% S 85%

12 13 14

Figure 3 Fenofibrate solubilisation and supersaturation during in vitro dispersion and digestion of LBFs. The AUC of

15

solubilized fenofibrate during in vitro dispersion and digestion, calculated using the linear trapezoidal method is

16

shown in the bars, with data obtained using formulations loaded at 40% of saturation solubility in the open bars and

17

data at 85% saturation in the grey bars (left axis). The degree of fenofibrate supersaturation during dispersion and

18

digestion is shown as the blue squares (data at 40% saturation loading) or blue triangles (data at 85% saturation

19

loading) (right axis). Data expressed as the supersaturation ratio (S) and calculated with AUCDISPandDIGEST of the

20

solubilization profiles, [mean ± SD (n = 3)].

21 22

At high drug load, the addition of a PPI led to a delay in onset of drug precipitation for the

23

Type IIIB and Type IV formulations that contained lower lipid/surfactant+cosolvent ratios. The 19 Environment ACS Paragon Plus


1

trend towards greater proportional prolongation of supersaturation for Type IV LBFs was not

2

replicated for the formulations loaded at 40% saturation, where the addition of a PPI only

3

slightly increased periods of supersaturation and in some cases even led to increased drug

4

precipitation (E 100 and HPMC E4M). HPMCAS:HF and HPMC E4M followed a similar trend,

5

whereby the increase in fenofibrate solubilisation and supersaturation ratio was more

6

pronounced in the Type IV formulation at 85 % drug loading. The impact of the three PPIs on

7

fenofibrate solubilization during dispersion and digestion of the three LBFs is summarized in

8

Figure 3.

9 10

The high initial solvent capacity of the Type IV formulation for FFB, the loss of solvent capacity

11

on dispersion and digestion and the potential ability of the PPI to protect against that loss, at

12

least at high drug loading, made the Type IV formulation an attractive starting point to assess

13

the potential beneficial effect of the addition of a broader range of PPIs. Therefore, the Type

14

IV formulation at 85% saturation was chosen as a candidate to conduct a screening study to

15

explore the effect of polymer (and polymer solubility) on performance. A fixed drug saturation

16

ratio, rather than a fixed drug concentration, was employed in the screening experiments to

17

ensure consistent initial drug thermodynamic activity across the formulations.

18 19

Effect of polymer solubility on supersaturation prolongation

20 21

Whilst PPI are well described to limit drug precipitation from lipid formulations,19, 20 it is less

22

clear whether the ‘site of action’ of the polymer is in the aqueous phase, the lipid phase or at

23

the interface. To investigate whether PPIs perform more effectively when they are soluble in

24

the LBF (lipid) or in the micellar solution (aqueous), a range of PPIs was selected that had

25

previously been reported as being ‘superior’ precipitation inhibitors for non-electrolyte drugs,

26

such a fenofibrate.15, 31 Each polymer was assessed to determine whether it was soluble in the

27

Type IV LBF at a limit of 1% w/w, and whether the equivalent mass was soluble in the volume

28

of 40 mL of micellar solution employed in the digestion test. The solubility data for the PPIs

29

tested is summarized in Figure 4.


Page 20 of 49

Page 21 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure 4. Venn diagram showing the solubility of PPIs in the Type IV LBF (yellow) and micellar solution (blue)

3

respectively. Polymers displayed outside the diagram were not soluble in either of the media, whereas those in the

4

overlapping region were soluble in both media. Polymer solubility was tested at 1% w/w in the formulation and the

5

corresponding mass was used to assess solubility in 40 mL micellar solution.

6 7

The impact of polymer solubility on the ability to promote supersaturation is summarized in

8

Figure 5 for the Type IV loaded at 85% fenofibrate saturation solubility. The figure shows the

9

increase in drug solubilization afforded by the presence of PPI across a range of polymers and

10

these are color coded to display those polymers that are soluble in the formulation (and

11

therefore were dissolved in the formulation), those that were only soluble in micelles (and

12

therefore pre-dispersed in the micellar solution), those that were not soluble in the lipid

13

formulation and were therefore suspended in the LBF. An overview of the in vitro

14

solubilization profiles of the individual polymers is shown in the Supporting Information (Fig.

15

S1).

16 17

Suspensions of HPMCAS:HF resulted in a high-viscosity system containing polymeric

18

aggregates, making sampling difficult. Suspensions of HPMC E4M in LBFs were more stable

19

and were therefore employed in later experiments. Even though HPMC E4M was not soluble

20

in the Type IV LBF, it formed a stable suspension, where polymer particles did not sink to the

21

bottom of the vial, but rather remained suspended. This observation was not apparent for the

22

other lipid-insoluble polymers. The polymethacrylates, such E100, were not soluble in the

23

micellar solution, which was unexpected since previous studies suggest that they are soluble

24

in a simple aqueous buffer (phosphate buffered saline).15,

25

composition of the fasted intestinal fluid used in the current series of experiments, appears to

26

decrease polymer solubility. A formulation dependent solubility phenomenon was observed


31

Interestingly therefore, the


1

for HPMCAS:HF where increasing solubility was apparent with decreasing lipid content of the

2

formulations (Type IIIA < Type IIIB < Type IV).

3 4

Figure 5. In vitro evaluation of the impact of the presence of PPIs pre-dispersed in different media on fenofibrate

5

solubilization and supersaturation after dispersion (15 min) and digestion (60 min) in a Type IV formulation at 85%

6

saturated solubility [mean ± SD (n ≥ 3)]. Panel A: Shows the % drug solubilized over the period of formulation

7

dispersion and digestion, calculated by dividing the AUC of the concentration versus time profile obtained in the in

8

vitro test by the AUC of the APMAX over the same time period (representing 100% solubilized). The horizontal black

9

dotted line represents the extent of drug solubilization obtained from the formulation in the absence of polymer (also

10

shown as the black (control) bar). Data above the line represent formulations where polymer addition leads to lower

11

drug precipitation during formulation dispersion and digestion (and therefore greater drug solubilization). The green

12

panel highlights the area where the % solubilized fenofibrate exceeds that of the formulation without polymer

13

(control). Panel B: Mean supersaturation ratio (S) obtained over the period of dispersion and digestion of the

14

formulations, data grouped for polymers with different solubility profiles in the LBF.

15 16

Data above the dashed line in Figure 5A show polymers that generated increased drug

17

solubilization compared to the control formulation. The addition of PPIs, whether pre-

18

dispersed in the formulation or in the micellar solution, in all cases led to a reduction in drug

19

precipitation and maintained drug solubilization for an extended period of time. Interestingly

20

not all polymers from the same ‘class’ performed equally well. For example HEC and HECEQ,

21

both cellulose-based polymers, were not as effective as HPMC E4M or HPMCAS:HF. This was

22

also apparent for the polymethacrylates (Eudragit), where EU RL100 prolonged

23

supersaturation approximately twice as effectively as EU E100 or L100. Polymers containing a

24

sulfonate functional group, such as PAESA, PSSS, PAAMPS resulted in variable performance.

25

Thus, PSSS pre-dispersed in the formulation, was able to increase fenofibrate solubilization to

26

a greater extent than PAESA (which did not perform much better than the control formulation)


Page 22 of 49

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1

even though the two polymers are structurally similar. Likewise, PPGAE which contains two

2

amino groups, was one of the best performing polymers whereas other amino containing

3

polymers such as PVP-DAM or PVP-40 were much less effective. The utility of both PPGAE

4

(positively charged) and PSSS (negatively charged) suggests that charge had little specific

5

effect on the ability of fenofibrate to interact with the polymers and promote supersaturation,

6

consistent with the lack of ionization sites on fenofibrate.

7 8

To provide a broad indication of the impact of polymer solubility on PPI performance, the

9

ability of PPIs to maintain supersaturation throughout dispersion and digestion (as indicated

10

by the supersaturation ratio (S)) was averaged for polymers that were either soluble in

11

micellar solution, soluble in the LBF or suspended in the LBF. These data are summarized in

12

Figure 5B. Polymers that were soluble in the LBF exhibited the larger supersaturation ratio

13

(mean of ~3), however no statistically significant difference was obtained when compared to

14

the two other groups, i.e. PPIs pre-dissolved in micellar solution and PPIs suspended in LBF.

15

None of the PPIs was able to fully maintain supersaturation over the duration of the digestion

16

phase (60 min). A full list of the drug solubilization profiles obtained in the presence of the

17

individual polymers is provided in Figure S1.

18 19

From this in vitro screen, three polymers (EU RL100, HPMC E4M and PPGAE) were identified

20

as PPIs that increased fenofibrate solubilization by more than 35%, and were chosen to probe

21

the impact of polymer concentration on the duration of drug supersaturation. Across these

22

three polymers, only HPMC E4M, was used as a suspension in the LBF, while EU RL100 and

23

PPGAE were soluble and used as a solution. In light of the fact that pre-dispersion in the

24

formulation versus dissolution in the micellar solution had no significant impact on

25

performance, polymers were pre-dispersed in the LBF since this more closely reflects the

26

likely mode of use in a drug product. To evaluate the optimal polymer concentrations, a Type

27

IV formulation was loaded at two different polymer concentrations, i.e. 1% w/w and 5% w/w,

28

and the drug solubilization advantage obtained was calculated over the dispersion (15 min)

29

and digestion phase (60 min) (Figure 6). To investigate whether the increased drug

30

solubilization obtained by enhanced supersaturation in the presence the PPIs, reflected a

31

kinetic stabilization of the supersaturated state or changes to the inherent solubilization

32

capacity of the post-digestion AP, the apparent equilibrium solubility of fenofibrate in blank

33

(drug-free) AP was also measured in the presence of these polymers at the experimental 23 Environment ACS Paragon Plus


1

concentrations (Table 1). The addition of 5% w/w EU RL100 to the Type IV LBF resulted in a

2

highly viscous formulation. However no significant difference was evident in drug solubility in

3

the presence and absence of the three PPIs, loaded at 5% w/w. The impact of the polymer on

4

drug solubilization during digestion was therefore to stabilize supersaturation via a kinetic

5

mechanism rather than to enhance intrinsic solubility (i.e. to change system thermodynamics).

6 7

8 9

Figure 6. Impact of PPI concentration (1 and 5% w/w) on the extent of fenofibrate solubilization. Percentage (%) drug

10

solubilized over the period of formulation dispersion and digestion was calculated by dividing the AUC of the

11

concentration versus time profile obtained in the in vitro test by the AUC of the APMAX over the same time period

12

(representing 100% solubilized). PPIs were incorporated at 1 and 5% w/w, pre-dispersed in the LBF (Type IV) [mean ±

13

SD (n = 3)].

14 15

Figure 6 shows that a higher polymer concentration did not correlate with improved drug

16

solubilization for PPGAE and EU RL 100. In this case 1% w/w polymer loading resulted in

17

similar or better drug solubilisation than 5% w/w. Formulations loaded with 5% w/w HPMC

18

E4M resulted in a greater delay in precipitation compared to a 1% w/w polymer loading,

19

consistent with previous studies.17, 20 Based on these results, polymer concentrations of 1, 5

20

and 1% w/w were chosen for EU RL100, HPMC E4M and PPGAE respectively to pursue further

21

in vivo studies. At these loading levels, HPMC E4M was not soluble in the formulation (Type

22

IV). As such the formulations employed allowed further examination of the impact of polymer

23

solubility in the formulation on precipitation inhibition and in vivo ability to increase drug

24

absorption across the intestinal membrane.

25 26 27

In situ absorption evaluation

28 29

The in vitro solubilization profiles of fenofibrate during the conduct of in vitro digestion-in situ

30

absorption studies studies for the Type IV LBF containing the three selected polymers are

31

shown in Figure 7. 24 Environment ACS Paragon Plus

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1 2

**** ****

4 **** 3 2 1 0 control

EU RL100 HPMC E4M

PPGAE

3 4

Figure 7. In vitro impact of PPIs on drug solubulization and supersaturation. Panel A: Drug solubilization profiles of

5

fenofibrate in a Type IV formulations at 85% fenofibrate loading pre (orange open symbols) - and post-perfusion of

6

the jejunum during dispersion and digestion in the absence (grey circles) and presence (black circles) of three different

7

polymers pre-dispersed in the LBF [mean ± SD (n = 4-5)]. Polymer concentrations in the LBFs were 1, 5 and 1% w/w for

8

EU RL100, HPMC E4M and PPGAE respectively. Blue panels represent the dispersion stage. The black circles and

9

dashed line shows fenofibrate solubility in the aqueous colloidal phase produced by dispersion (APDISP) and digestion

10

(APDIGEST) of the formulation and the upper horizontal dotted black line represents the APMAX. Panel B: Fenofibrate

11

supersaturation in the absence and presence of polymers, expressed by the supersaturation ratio (S) calculated with

12

AUCDISPandDIGEST of the solubilization profiles in panel A, [mean ± SD]. **** Statistically significant difference (p
4-fold increase in absorption

13

(AUC, Figure 8B), with PPGAE being the most effective, consistent with the most effective

14

prolongation of supersaturation in vitro. This resulted in a significant increase in drug flux (p =

15

0.0282) with PPGAE, whereas variability in absorption precluded attainment of significance for

16

the other PPIs (p= 0.0631 and 0.1320 for HPMC E4M and EU RL100 respectively) compared to

17

the Type IV control (p-values for a one-way ANOVA with Dunnett's multiple comparisons test

18

with comparison to control). The addition of the PPIs to the formulation prolonged absorptive

19

flux for up to 60 min, although peak absorption rates reduced from 20 min onwards.

20

Fenofibric acid levels in the mesenteric blood were therefore increased for a longer period

21

than the period of prolongation of supersaturation in vitro (15 min). As described previously,

22

transient periods of supersaturation therefore appear to be able to promote ongoing

23

absorption for up to 1 hour.39

24

From Figure 8C it can be seen that there is a reasonable correlation between fenofibrate

25

absorption and the degree of supersaturation of fenofibrate during in vitro dispersion and

26

digestion (R2=0.74). This was particularly true when evaluating the effect of PPI on the same

27

formulation (eg the Type IV formulation), resulting in an R2 value of 0.87. Interestingly, the

28

additions of the PPIs to the Type IV formulation resulted in improved performance such that

29

the absorptive flux obtained was greater than that obtained from the Type IIIB LBF in the

30

absence of PPI. Attribution of the increases in flux to increases in supersaturation are

31

supported by the fact that the flux of the passive permeability markers antipyrine and

32

mannitol was unchanged in the presence of the formulation, consistent with limited effects

33

on intrinsic permeability.



1 2

To investigate whether changes in drug flux, obtained in the presence of PPIs in the LBFs,

3

reflected a propensity towards increased phase separation (precipitation) of drug in the

4

amorphous form in the presence of polymer, and therefore the potential for enhanced re-

5

dissolution of amorphous fenofibrate precipitate, centrifuged pellet phases collected during

6

dispersion and digestion were examined by crossed-polarized light microscopy. Figure 9

7

shows the physical form in which fenofibrate precipitated during in vitro dispersion and

8

digestion when formulated in a Type IV LBF in the presence of the polymers HPMC E4M, EU

9

RL100 and PPGAE. Characterization of the pellet phase confirms the presence of crystalline

10

fenofibrate. An increasing number of crystals can be observed from dispersion (0 min) to

11

digestion, consistent with the solubilization profiles. The crystals obtained during the

12

dispersion phase were smaller than those obtained in the digestion phase.

13

14 15

Figure 9. Crossed-polarized light micrographs of precipitated fenofibrate generated during dispersion and digestion

16

of a Type IV formulation loaded at 85% saturation, in the presence of PPIs. Images of the pellet phase were

17

obtained at 0 min (end of dispersion phase) and at 30 min after initiation of digestion.

18 19

Discussion


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1

Formulation technologies that increase the proportion of the drug dose that is delivered to the

2

systemic circulation after oral administration (oral bioavailability), and therefore reduce the

3

required dose, are actively sought. The low absorption of PWSDs, resulting from low aqueous

4

solubility and slow dissolution, dictates that PWSDs are common targets for such technologies. In

5

recent years, increasing attention has been directed towards formulation approaches that provide

6

a ‘spring and parachute’ approach to enhance drug solubility in the GI tract. In this method some

7

means of rapidly enhancing drug solubilisation, often above the solubility limit, (the spring) is

8

employed, followed by a mechanism to maintain drug solubilization and prevent precipitation (the

9

parachute).54, 55 Inclusion of PPIs in drug delivery systems as a means to delay the onset of

10

precipitation has received increasing attention over the past decade, although to this point the use

11

of PPIs in LBFs has been studied less thoroughly. The ability of a PPI to induce and kinetically

12

stabilize a supersaturated state, is typically screened in vitro, due to higher throughput and lower

13

cost. Whilst in vitro evaluation methods have identified polymers with appreciable precipitation

14

inhibition abilities,15,

15

behavior, and, more precisely, how effective they are in estimating the propensity for

16

precipitation or absorption in vivo. The transferability of in vitro estimates of drug solubilization

17

patterns to in vivo absorption is hampered by several factors1, with the lack of an absorption sink

18

being suggested as a key limitation to in vitro model utility. To address this, in the current study, a

19

new model, developed by Crum et al.38, has enabled evaluation of the impact of PPI on drug

20

solubilization in vitro and drug absorption in vivo after administration of a range of LBFs. This

21

coupled in vitro digestion-in vivo absorption model enables drug flux quantitation through an

22

intact, vascularized jejunum segment, and, unlike many in vitro cell based systems, is able to

23

tolerate relatively high concentrations of bile salts, surfactants and lipids. The mechanisms by

24

which supersaturation from LBFs is triggered in vivo include loss of formulation solubilization

25

capacity upon dispersion, initiation of lipid digestion and entry in the unstirred water layer.57, 58 A

26

biorelevant model is thus required to fully capture the intraluminal events that are expected to

27

contribute to the balance between precipitation and increased absorption after transient

28

supersaturation. However, since these models require relatively complex surgical procedures, they

29

are not well suited to high throughput screening of large numbers of formulations. Screening of

30

the ability of different PPI to prolong the duration of fenofibrate supersaturation was therefore

31

initially conducted using an in vitro digestion model.35

28, 56

it is unclear how predictive these assays are of intraluminal drug

32 33

Effect of drug saturation on LBF performance with PPI 29 Environment ACS Paragon Plus


1 2

HPMCAS:HF, HPMC E4M and Eudragit E 100, were initially chosen as representative PPIs to assess

3

the impact of drug load in the formulation on drug precipitation after formulation dispersion and

4

digestion, and the potential for PPIs to protect against drug precipitation during this process. The

5

choice of these polymers was based on the chemical diversity of E 100, HPMCAS:HF and HPMC

6

E4M and the fact that they have previously been shown to maintain drug supersaturation for

7

extended periods in vitro.15, 31, 59

8 9

A significant body of work has been published describing the properties of cellulose-based

10

polymers and these have been widely used for film coating and more recently as PPI, not only in

11

amorphous and solid dispersion formulations,60 but also in LBFs.16, 17, 19 Various characteristics of

12

these polymers have been reported to be responsible for precipitation inhibition, such as

13

hydrogen bonding capacity,61 van der Waals interactions and hydrophobic interactions.62 The

14

acetate succinate analogue of HPMC (HPMCAS), that was originally developed as an enteric

15

polymer for aqueous dispersion coating,63 has received increasing attention in drug delivery and

16

has been reported to be an effective PPI for many structurally different drugs covering a wide

17

variety of physical properties. To this point, however, evidence of utility has been provided

18

primarily with polymeric amorphous solid dispersion formulations.24, 64, 65 According to Friesen et

19

al., HPMCAS:HF has unique characteristics that underpin superior utility as a drug precipitation

20

inhibitor in solid dispersion formulations.66 Firstly, the high glass transition temperature of the

21

polymer results in low drug mobility and hence improved physical stability of amorphous drug

22

within the formulation. Secondly, above pH 5, HPMCAS:HF is at least partially ionized (pKa of

23

succinate groups ~ 5), and this reduces the formation of large aggregates and instead stabilizes the

24

polymer as nanosized amorphous drug-polymer aggregates. This inhibits conversion of drug to the

25

low-energy-low-solubility crystalline form. Finally, the amphiphilic nature of this PPI enables PWSD

26

to interact with the hydrophobic regions of the polymer, and these association structures remain

27

stable as colloidal species in aqueous solution. HPMCAS:HF therefore has many characteristics that

28

make it an ideal PPI. Its utility in LBFs, however, is restricted by very low solubility in LBFs, at least

29

in medium-chain lipid-rich formulations, such as the Type IIIA and Type IIIB formulations employed

30

here. This requires the generation of non-homogenous suspensions of polymer in LBFs. This

31

drawback complicates homogeneous sampling, a limitation alleviated when using HPMC, which

32

although poorly soluble in LBF, results in a stable suspension of the polymer in all LBFs used.

33

Polymethacrylates (eg Eudragits), that were also originally used for enteric coatings (moisture 30 Environment ACS Paragon Plus

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1

protection and odor/taste masking), have also recently been shown to possess precipitation

2

inhibition properties.15, 31 Polymethacrylates have the advantage of supply in highly reproducible

3

forms (when compared to some cellulosic derivatives which may have different physicochemical

4

properties depending on the source of raw material), are relatively easy to polymerize and have

5

significant synthetic flexibility such that the ratio and composition of the side chains can be varied.

6

Eudragit E100 was initially employed here since it is one of a limited number of polymers that have

7

some solubility in LBFs. Surprisingly, in our study, whilst E100 was soluble in the formulation, it

8

was not soluble at the levels examined in simulated fasted intestinal fluid (whereas previous

9

studies suggest solubility in aqueous buffers). Takemura et al. have previously reported an

10

interaction of E100 with taurocholate bile salt, and this may explain its observed precipitation in

11

the micellar solution employed here.67 Takemura et al. attribute this detrimental interaction to an

12

electrostatic and/or hydrophobic contact between the anionic amphiphile (bile salt) and the

13

hydrophobic backbone of the cationionic copolymer.

14 15

The polymers E 100, HPMCAS:HF and HPMC E4M were therefore employed in initial experiments

16

to determine whether drug loading significantly affected PPI performance. The in vitro

17

solubilization profiles of fenofibrate after dispersion and digestion of the three formulations (Type

18

IIIA-MC, IIIB-MC, and IV) at high drug load (85% saturation), resulted in more precipitation than

19

those obtained at low (40% saturation) drug load, in agreement with previous studies using

20

fenofibrate

21

reached at higher drug load, which increases the maximum supersaturation ratio (SM) and

22

therefore the pressure applied on the formulation to maintain supersaturation without drug

23

precipitation. The other determinant of supersaturation is the apparent equilibrium solubility of

24

the drug in the colloids formed by formulation dispersion and digestion. In this regard, Type IIIA

25

formulations typically provide intestinal colloidal species with higher intrinsic drug solubilities

26

when compared to Type IIIB or Type IV formulations. In addition, the lower ratio of surfactant and

27

cosolvent to lipid in Type IIIA formulations usually results in lower drug solubility in the

28

formulation and therefore a lower APMAX. Together, the reduction in APMAX and increase in

29

equilibrium solubility (APDIGEST and APDISP) often reduce SM, and decrease the potential for

30

precipitation during dispersion and digestion of Type IIIA LBF. In contrast, the presence of

31

surfactants and cosolvents in Type IIIB and Type IV formulations, whilst beneficial in increasing

32

drug load, also result in higher APMAX and reduced equilibrium solubility, thereby increasing SM

33

values and increasing the potential for precipitation. The lower equilibrium solubility of drugs in

38, 39, 43, 68

and other PWSDs17, 53, 69 in LBFs. This can be attributed to the higher APMAX



1

the colloids produced by dispersion and digestion of Type IV and Type IIIB formulations reflects

2

the water miscibility of many of the formulation components and the lower lipid content. As such,

3

on dilution in the GI fluids, solubilization capacity is typically lost. This is especially true of Type IV

4

formulation that typically have the highest capacity to load drug into the formulation, but lowest

5

ability to retain drug in a solubilized state on dispersion and digestion.

6 7

When comparing drug precipitation profiles in the presence and absence of polymer, the impact

8

of the PPIs was highly dependent on drug load and the formulation employed. Thus, the effect of

9

PPIs on formulation performance was limited for the Type IIIA formulation, which was the most

10

effective formulation in the absence of polymer. In the case of the Type IIB and Type IV

11

formulations, where drug precipitation was more evident, the PPIs had little effect at 40% drug

12

load (again where performance was reasonable in the absence of polymer). At high drug loads

13

(80%), however, where drug precipitation was most significant, the PPIs had the largest effect.

14

This is the opposite trend to that reported previously for the PWSD danazol, where beneficial PPI

15

performance in LBFs was more apparent at lower drug loads.59 The data suggest that PPIs need to

16

be treated as drug-specific tools and should be assessed on a case-by-case basis, rather than

17

regarded as general, non-specific tools.

18 19

In light of the data obtained, further studies focused on the ability of PPIs to overcome the

20

limitations to performance for the Type IV LBFs (poor ongoing solubilization) at high drug load.

21 22

Effect of polymer solubility on supersaturation prolongation

23 24

Having identified a test system to explore in more detail, subsequent studies focused on the

25

importance of drug solubility in either the formulation or the aqueous phase of the in vitro digest.

26

These studies were conducted to test the hypothesis that polymer solubility in the GI fluids may be

27

a critical determinant of utility and that those polymers with limited or no aqueous solubility may

28

be poor PPI. The data also sought to evaluate whether PPI that were soluble in the LBF (and

29

therefore more practical formulation excipients) may be less effective PPIs by virtue of segregation

30

from the aqueous phase. Finally, the studies evaluated whether water soluble polymers that were

31

suspended in a LBF were as effective as they were when pre-dissolved in the digestion media.

32

From Figure 5A it is apparent that all the polymers tested were able to inhibit fenofibrate 32 Environment ACS Paragon Plus

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1

precipitation from the Type IV formulation (85% saturation) to some extent and that the solubility

2

characteristics of the PPI in aqueous versus lipidic media did not significantly affect performance

3

(Figure 5B). The supersaturation ratio (S) was used to quantify supersaturation stabilization (Figure

4

5B) and similar mean values were obtained for polymers with differing solubility characteristics

5

(~2.5-3). The screening method employed here therefore suggests that the location of the

6

polymer was not a critical determinant of PPI utility and that even polymer suspended in LBFs

7

performed well.

8 9

The in vitro screen of PPI for fenofibrate provided a rank order classification for the PPI and

10

identified 3 superior polymers (Eudragit RL 100, HPMC E4M and PPGAE) that could be dissolved or

11

stably suspended in the LBFs for in vivo studies. Subsequent studies examined the likely optimal

12

concentration at which the PPI should be employed and revealed that higher levels of polymer

13

were not necessarily ideal, perhaps resulting in precipitation seeding. The polymer concentrations

14

employed in the in vivo experiments were therefore 1% w/w for EU RL100 and PPGAE, and 5%

15

w/w for HPMC E4M. In all cases, whilst the addition of PPI was able to significantly enhance the

16

degree of supersaturation, stabilization only persisted for relatively brief timescales (15-30 mins,

17

Figure 7A). Across these polymers, supersaturation values of up to 3.8 were evident over the

18

period of dispersion and digestion (Figure 7B). This approximates the value of 3 that has previously

19

been suggested to represent the limit beyond which significant precipitation is usually evident in

20

the absence of PPI.17, 53

21 22

The extent of precipitation inhibition observed here is substantially less than that observed

23

previously using solvent shift methods of supersaturation generation15,

24

studies, for example, the rates of precipitation of the non-electrolyte drugs danazol,

25

carbamazepine and ethinylestradiol were reduced by 100 fold compared to control (no PPI), in the

26

presence of low concentrations (0.001 and 0. 1% w/v) of E 100, HPMCAS:HF and PSSS. Other

27

studies, on the other hand, reported similar values to the current study for in vitro polymer-

28

mediated supersaturation performance for non-electrolyte drugs, such as fenofibrate,30benzamide

29

and phenacetin56. All these studies, however, utilized solvent-shift methods to promote

30

supersaturation generation (i.e. dilution of solutions of drug in cosolvents into aqueous buffer),

31

did not employ biorelevant media and did not include the dispersion and digestion of LBFs. These

32

discrepancies in in vitro polymer performance raise the question as to whether PPI-mediated


31

. In these previous


1

stabilization of solubilized drug concentrations is dependent on the technique employed to

2

generate the initial supersaturation, i.e. solvent-shift versus formulation digestion.

3 4 5

In situ absorption evaluation

6 7

The polymer loaded formulations were subsequently examined in the coupled in vitro digestion-in

8

situ perfusion model to gain a better mechanistic insight into the relationship between prolonged

9

supersaturation and enhancements in drug flux. In all cases, delaying precipitation in vitro resulted

10

in higher fenofibrate absorption during the infusion period of 60 min. The data suggest that whilst

11

the PPIs led to relatively moderate increases in supersaturation in vitro that this was able to

12

maintain supersaturation sufficiently to increase thermodynamic activity and therefore absorption

13

in vivo (Figure 8A). This is consistent with the findings of Crum et al. in which even transient

14

supersaturation (in the absence of PPI) was sufficient to maintain drug flux up to 1 h.39

15 16

The PPIs used in this study had no effect on membrane disruption, as demonstrated by the lack of

17

impact on the permeability of the para-and transcellular markers antipyrine and mannitol, and

18

also had no impact on the polymorphic form of precipitated drug. This supports the contention

19

that the PPIs enhance drug absorption by promoting supersaturation and that increases in

20

thermodynamic activity rather than changes to intestinal permeability, or crystal form are

21

responsible39,70,

22

supersaturation ratio (Figure 8) provides additional support for this contention. The improved

23

IVIVC obtained for the Type IV formulations alone (R2 0.87), rather than the combination of Type

24

IIIB and Type IV is highly consistent with recent data from McEvoy et al. in which the correlation

25

between absorption and supersaturation was better within a formulation type rather than across

26

different formulations.72 The current data also suggest that the addition of PPIs to Type IV LBF is

27

sufficient to ‘recover’ performance to levels similar to e.g. Type IIB formulations where drug

28

loading may be lower.

71

The (albeit limited) IVIVC between absorptive drug flux and in vitro

29 30

Conclusion

31

Previous studies have demonstrated the utility of polymers in delaying the onset of drug

32

precipitation (‘parachute’ behaviour) from formulations that promote supersaturation. The 34 Environment ACS Paragon Plus

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1

majority of these studies have been performed with solid dispersion formulations as the ‘spring’ to

2

promote supersaturation.12, 73, 74 A more limited number have started to explore the utility of PPIs

3

where supersaturation is generated via the use of LBFs.

4

extended to better understand the ability of PPI to promote drug absorption from LBFs using an in

5

vitro digestion-in situ perfusion model. The data suggest that PPI can support prolonged drug

6

supersaturation and that this results in improved absorptive drug flux in vivo. The data also

7

suggest that polymers that can be dissolved in LBFs can work equally well as PPI when compared

8

to those that are readily water soluble. This provides an additional benefit in formulation design

9

since the need to suspend polymers in LBFs leads to additional downstream stability concerns.

16, 18, 20, 59

Here, these studies have been

10 11

ASSOCIATED CONTENT

12

Supporting Information

13

The supporting information contains the structures of the individual polymers and the individual

14

solubilization profiles obtained during the in vitro digestion experiment with each polymer. This

15

material is available free of charge via the Internet at http://pubs.acs.org.

16 17

AUTHOR INFORMATION

18

Corresponding Author

19

*(C.J.H.P.) Address: Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal

20

Parade, Parkville, Victoria 3052, Australia. Phone: +61 3 9903 9649. Fax: +61 3 9903 9583. E-mail:

21

[email protected]

22

* (C.W.P) Address: Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal

23

Parade, Parkville, Victoria 3052, Australia. Phone: +61 3 9903 9562. Fax: +61 3 9903 9638. E-mail:

24

[email protected]

25

ORCID

26

Estelle J.A. Suys: 0000-0002-4285-0332

27

Christopher J. H. Porter: 0000-0003-3474-7551

28

Colin W. Pouton: 0000-0003-0224-3308

29

Notes



1

The authors declare no competing financial interest.

2

ACKNOWLEDGMENT

3

Funding support from Monash Graduate Research (MGR) for a scholarship to Ms Estelle J.A. Suys

4

and grant funding from the ARC Centre of Excellence in Convergent Bio Nano Science and

5

Technology (CE140100036) is gratefully acknowledged.

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

References 1. Feeney, O. M.; Crum, M. F.; McEvoy, C. L.; Trevaskis, N. L.; Williams, H. D.; Pouton, C. W.; Charman, W. N.; Bergstrom, C. A.; Porter, C. J. H. 50years of oral lipid-based formulations: Provenance, progress and future perspectives. Adv. Drug Deliv. Rev. 2016, 101, 167-94. 2. Suys, E. J. A.; Warren, D. B.; Porter, C. J. H.; Benameur, H.; Pouton, C. W.; Chalmers, D. K. Computational Models of the Intestinal Environment. 3. The Impact of Cholesterol Content and pH on Mixed Micelle Colloids. Mol Pharm 2017. 3. Brouwers, J.; Brewster, M. E.; Augustijns, P. Supersaturating drug delivery systems: the answer to solubility-limited oral bioavailability? J Pharm Sci 2009, 98, (8), 2549-72. 4. Thi, T. D.; Van Speybroeck, M.; Barillaro, V.; Martens, J.; Annaert, P.; Augustijns, P.; Van Humbeeck, J.; Vermant, J.; Van den Mooter, G. Formulate-ability of ten compounds with different physicochemical profiles in SMEDDS. Eur J Pharm Sci 2009, 38, (5), 479-88. 5. Gao, P.; Shi, Y. Characterization of Supersaturatable Formulations for Improved Absorption of Poorly Soluble Drugs. AAPS J. 2012, 14, (4), 703-713. 6. Mu, H. L.; Holm, R.; Mullertz, A. Lipid-based formulations for oral administration of poorly watersoluble drugs. International Journal of Pharmaceutics 2013, 453, (1), 215-224. 7. Li, P.; Hynes, S. R.; Haefele, T. F.; Pudipeddi, M.; Royce, A. E.; Serajuddin, A. T. M. Development of Clinical Dosage Forms for a Poorly Water-Soluble Drug II: Formulation and Characterization of a Novel Solid Microemulsion Preconcentrate System for Oral Delivery of a Poorly Water-Soluble Drug. J. Pharm. Sci. 2009, 98, (5), 1750-1764. 8. Serajuddin, A. T. M.; Li, P.; Haefele, T. F., In Development of lipid-based drug delivery systems for poorly water-soluble drugs as viable oral dosage forms - Present status and future prospects, 2008; Vol. 11. 9. DiNunzio, J. C.; Miller, D. A.; Yang, W.; McGinity, J. W.; Williams, R. O. Amorphous Compositions Using Concentration Enhancing Polymers for Improved Bioavailability of Itraconazole. Mol. Pharmaceutics 2008, 5, (6), 968-980. 10. Jackson, M. J.; Kestur, U. S.; Hussain, M. A.; Taylor, L. S. Dissolution of Danazol Amorphous Solid Dispersions: Supersaturation and Phase Behavior as a Function of Drug Loading and Polymer Type. Mol. Pharmaceutics 2016, 13, (1), 223-231. 11. Kothari, K.; Ragoonanan, V.; Suryanarayanan, R. The role of drug-polymer hydrogen bonding interactions on the molecular mobility and physical stability of nifedipine solid dispersions. Mol. Pharmaceutics 2015, 12, (1), 162-70. 12. Miller, J. M.; Beig, A.; Carr, R. A.; Spence, J. K.; Dahan, A. A Win-Win Solution in Oral Delivery of Lipophilic Drugs: Supersaturation via Amorphous Solid Dispersions Increases Apparent Solubility without Sacrifice of Intestinal Membrane Permeability. Mol. Pharmaceutics 2012, 9, (7), 2009-2016. 13. Van Ngo, H.; Nguyen, P. K.; Van Vo, T.; Duan, W.; Tran, V. T.; Tran, P. H.; Tran, T. T. Hydrophilichydrophobic polymer blend for modulation of crystalline changes and molecular interactions in solid dispersion. Int. J. Pharm. 2016, 513, (1-2), 148-152. 14. Franca, M. T.; Nicolay, R. P.; Kluppel Riekes, M.; Munari Oliveira Pinto, J.; Stulzer, H. K. Investigation of novel supersaturating drug delivery systems of chlorthalidone: The use of polymer-surfactant complex as an effective carrier in solid dispersions. Eur J Pharm Sci 2017, 111, 142-152. 36 Environment ACS Paragon Plus

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15. Warren, D. B.; Benameur, H.; Porter, C. J. H.; Pouton, C. W. Using polymeric precipitation inhibitors to improve the absorption of poorly water-soluble drugs: A mechanistic basis for utility. J. Drug Target. 2010, 18, (10), 704-731. 16. Gao, P.; Rush, B. D.; Pfund, W. P.; Huang, T. H.; Bauer, J. M.; Morozowich, W.; Kuo, M. S.; Hageman, M. J. Development of a supersaturable SEDDS (S-SEDDS) formulation of paclitaxel with improved oral bioavailability. J. Pharm. Sci. 2003, 92, (12), 2386-2398. 17. Anby, M. U.; Williams, H. D.; McIntosh, M.; Benameur, H.; Edwards, G. A.; Pouton, C. W.; Porter, C. J. H. Lipid Digestion as a Trigger for Supersaturation: Evaluation of the Impact of Supersaturation Stabilization on the in Vitro and in Vivo Performance of Self-Emulsifying Drug Delivery Systems. Mol. Pharmaceutics 2012, 9, (7), 2063-2079. 18. Gao, P.; Guyton, M. E.; Huang, T. H.; Bauer, J. M.; Stefanski, K. J.; Lu, Q. Enhanced oral bioavailability of a poorly water soluble drug PNU-91325 by supersaturatable formulations. Drug Dev. Ind. Pharm. 2004, 30, (2), 221-229. 19. Wei, Y. H.; Ye, X. L.; Shang, X. G.; Peng, X.; Bao, Q.; Liu, M. C.; Guo, M. M.; Li, F. Z. Enhanced oral bioavailability of silybin by a supersaturatable self-emulsifying drug delivery system (S-SEDDS). Colloids Surf., A 2012, 396, 22-28. 20. Gao, P.; Akrami, A.; Alvarez, F.; Hu, J.; Li, L.; Ma, C.; Surapaneni, S. Characterization and Optimization of AMG 517 Supersaturatable Self-Emulsifying Drug Delivery System (S-SEDDS) for Improved Oral Absorption. J. Pharm. Sci. 2009, 98, (2), 516-528. 21. Schram, C. J.; Taylor, L. S.; Beaudoin, S. P. Influence of Polymers on the Crystal Growth Rate of Felodipine: Correlating Adsorbed Polymer Surface Coverage to Solution Crystal Growth Inhibition. Langmuir 2015, 31, (41), 11279-11287. 22. Yamashita, K.; Nakate, T.; Okimoto, K.; Ohike, A.; Tokunaga, Y.; Ibuki, R.; Higaki, K.; Kimura, T. Establishment of new preparation method for solid dispersion formulation of tacrolimus. Int. J. Pharm. 2003, 267, (1-2), 79-91. 23. Usui, F.; Maeda, K.; Kusai, A.; Nishimura, K.; Yamamoto, K. Inhibitory effects of water-soluble polymers on precipitation of RS-8359. Int. J. Pharm. 1997, 154, (1), 59-66. 24. Yin, L. G.; Hillmyer, M. A. Preparation and Performance of Hydroxypropyl Methylcellulose Esters of Substituted Succinates for in Vitro Supersaturation of a Crystalline Hydrophobic Drug. Mol. Pharmaceutics 2014, 11, (1), 175-185. 25. Chen, Y. J.; Wang, S. J.; Wang, S.; Liu, C. Y.; Su, C.; Hageman, M.; Hussain, M.; Haskell, R.; Stefanski, K.; Qian, F. Sodium Lauryl Sulfate Competitively Interacts with HPMC-AS and Consequently Reduces Oral Bioavailability of Posaconazole/HPMC-AS Amorphous Solid Dispersion. Mol. Pharmaceutics 2016, 13, (8), 2787-2795. 26. Raghavan, S. L.; Trividic, A.; Davis, A. F.; Hadgraft, J. Crystallization of hydrocortisone acetate: influence of polymers. Int. J. Pharm. 2001, 212, (2), 213-221. 27. Christfort, J. F.; Plum, J.; Madsen, C. M.; Nielsen, L. H.; Sandau, M.; Andersen, K.; Mullertz, A.; Rades, T. Development of a video-microscopic tool to evaluate the precipitation kinetics of poorly-water soluble drugs: A case study with tadalafil and HPMC. Mol. Pharmaceutics 2017. 28. Vandecruys, R.; Peeters, J.; Verreck, G.; Brewster, M. E. Use of a screening method to determine excipients which optimize the extent and stability of supersaturated drug solutions and application of this system to solid formulation design. Int. J. Pharm. 2007, 342, (1–2), 168-175. 29. Bevernage, J.; Brouwers, J.; Clarysse, S.; Vertzoni, M.; Tack, J.; Annaert, P.; Augustijns, P. Drug Supersaturation in Simulated and Human Intestinal Fluids Representing Different Nutritional States. J. Pharm. Sci. 2010, 99, (11), 4525-4534. 30. Bevernage, J.; Forier, T.; Brouwers, J.; Tack, J.; Annaert, P.; Augustijns, P. Excipient-Mediated Supersaturation Stabilization in Human Intestinal Fluids. Mol. Pharmaceutics 2011, 8, (2), 564-570. 31. Warren, D. B.; Bergstrom, C. A. S.; Benameur, H.; Porter, C. J. H.; Pouton, C. W. Evaluation of the Structural Determinants of Polymeric Precipitation Inhibitors Using Solvent Shift Methods and Principle Component Analysis. Mol. Pharmaceutics 2013, 10, (8), 2823-2848. 32. Khan, J.; Hawley, A.; Rades, T.; Boyd, B. J. In Situ Lipolysis and Synchrotron Small-Angle X-ray Scattering for the Direct Determination of the Precipitation and Solid-State Form of a Poorly Water-Soluble Drug During Digestion of a Lipid-Based Formulation. J. Pharm. Sci. 2015.



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33. Arnold, Y. E.; Imanidis, G.; Kuentz, M. T. Advancing in-vitro drug precipitation testing: new process monitoring tools and a kinetic nucleation and growth model. J. Pharm. Pharmacol. 2011, 63, (3), 333-341. 34. Kaukonen, A. M.; Boyd, B. J.; Charman, W. N.; Porter, C. J. H. Drug solubilization behavior during in vitro digestion of suspension formulations of poorly water-soluble drugs in triglyceride lipids. Pharm. Res. 2004, 21, (2), 254-260. 35. Williams, H. D.; Sassene, P.; Kleberg, K.; Bakala-N'Goma, J. C.; Calderone, M.; Jannin, V.; Igonin, A.; Partheil, A.; Marchaud, D.; Jule, E.; Vertommen, J.; Maio, M.; Blundell, R.; Benameur, H.; Carriere, F.; Mullertz, A.; Porter, C. J. H.; Pouton, C. W. Toward the establishment of standardized in vitro tests for lipidbased formulations, part 1: method parameterization and comparison of in vitro digestion profiles across a range of representative formulations. J. Pharm. Sci. 2012, 101, (9), 3360-3380. 36. Thomas, N.; Richter, K.; Pedersen, T. B.; Holm, R.; Mullertz, A.; Rades, T. In Vitro Lipolysis Data Does Not Adequately Predict the In Vivo Performance of Lipid-Based Drug Delivery Systems Containing Fenofibrate. AAPS J. 2014, 16, (3), 539-549. 37. Kostewicz, E. S.; Abrahamsson, B.; Brewster, M.; Brouwers, J.; Butler, J.; Carlert, S.; Dickinson, P. A.; Dressman, J.; Holm, R.; Klein, S.; Mann, J.; McAllister, M.; Minekus, M.; Muenster, U.; Müllertz, A.; Verwei, M.; Vertzoni, M.; Weitschies, W.; Augustijns, P. In vitro models for the prediction of in vivo performance of oral dosage forms. Eur J Pharm Sci. 2014, 57, (0), 342-366. 38. Crum, M. F.; Trevaskis, N. L.; Williams, H. D.; Pouton, C. W.; Porter, C. J. H. A new in vitro lipid digestion - in vivo absorption model to evaluate the mechanisms of drug absorption from lipid-based formulations. Pharm. Res. 2016, 33, (4), 970-82. 39. Crum, M. F.; Trevaskis, N. L.; Pouton, C. W.; Porter, C. J. H. Transient Supersaturation Supports Drug Absorption from Lipid-Based Formulations for Short Periods of Time, but Ongoing Solubilization Is Required for Longer Absorption Periods. Mol. Pharmaceutics 2017. 40. Pouton, C. W. Lipid formulations for oral administration of drugs: non-emulsifying, self-emulsifying and 'self-microemulsifying' drug delivery systems. Eur J Pharm Sci 2000, 11, S93-S98. 41. Khoo, S. M.; Humberstone, A. J.; Porter, C. J. H.; Edwards, G. A.; Charman, W. N. Formulation design and bioavailability assessment of lipidic self-emulsifying formulations of halofantrine. Int. J. Pharm. 1998, 167, (1-2), 155-164. 42. Cuine, J. F.; Charman, W. N.; Pouton, C. W.; Edwards, G. A.; Porter, C. J. H. Increasing the proportional content of surfactant (Cremophor EL) relative to lipid in self-emulsifying lipid-based formulations of danazol reduces oral bioavailability in beagle dogs. Pharm. Res. 2007, 24, (4), 748-757. 43. Williams, H. D.; Sassene, P.; Kleberg, K.; Calderone, M.; Igonin, A.; Jule, E.; Vertommen, J.; Blundell, R.; Benameur, H.; Mullertz, A.; Pouton, C. W.; Porter, C. J. H.; Consortium, L. Toward the Establishment of Standardized In Vitro Tests for Lipid-Based Formulations, Part 3: Understanding Supersaturation Versus Precipitation Potential During the In Vitro Digestion of Type I, II, IIIA, IIIB and IV Lipid-Based Formulations. Pharm. Res. 2013, 30, (12), 3059-3076. 44. Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B.; Tinevez, J. Y.; White, D. J.; Hartenstein, V.; Eliceiri, K.; Tomancak, P.; Cardona, A. Fiji: an open-source platform for biological-image analysis. Nature Methods 2012, 9, (7), 676682. 45. Johnson, B. M.; Chen, W. Q.; Borchardt, R. T.; Charman, W. N.; Porter, C. J. H. A kinetic evaluation of the absorption, efflux, and metabolism of verapamil in the autoperfused rat jejunum. J. Pharmacol. Exp. Ther. 2003, 305, (1), 151-158. 46. Stappaerts, J.; Brouwers, J.; Annaert, P.; Augustijns, P. In situ perfusion in rodents to explore intestinal drug absorption: Challenges and opportunities. Int. J. Pharm. 2015, 478, (2), 665-681. 47. Cao, J.; Chen, X.; Liang, J.; Yu, X. Q.; Xu, A. L.; Chan, E.; Duan, W.; Huang, M.; Wen, J. Y.; Yu, X. Y.; Li, X. T.; Sheu, F. S.; Zhou, S. F. Role of P-glycoprotein in the intestinal absorption of glabridin, an active flavonoid from the root of Glycyrrhiza glabra. Drug Metab. Dispos. 2007, 35, (4), 539-553. 48. Trevaskis, N. L.; Nguyen, G.; Scanlon, M. J.; Porter, C. J. H. Fatty acid binding proteins: potential chaperones of cytosolic drug transport in the enterocyte? Pharm. Res. 2011, 28, (9), 2176-90. 49. Singhal, D.; Ho, N. F. H.; Anderson, B. D. Absorption and intestinal metabolism of purine dideoxynucleosides and an adenosine deaminase-activated prodrug of 2 ',3 '-dideoxyinosine in the mesenteric vein cannulated rat ileum. J. Pharm. Sci. 1998, 87, (5), 569-577. 50. Caldwell, J. The Biochemical Pharmacology of Fenofibrate. Cardiology 1989, 76, 33-44. 38 Environment ACS Paragon Plus

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51. Lovin, I.; Albu, F.; Tache, F.; David, V.; Medvedovici, A. Solvent and salting effects on sample preparation for the determination of fenofibric acid in human plasma by HPLC-DAD. Microchem. J. 2003, 75, (3), 179-187. 52. Persson, L. C.; Porter, C. J. H.; Charman, W. N.; Bergstrom, C. A. Computational prediction of drug solubility in lipid based formulation excipients. Pharm. Res. 2013, 30, (12), 3225-37. 53. Williams, H. D.; Anby, M. U.; Sassene, P.; Kleberg, K.; Bakala-N'Goma, J. C.; Calderone, M.; Jannin, V.; Igonin, A.; Partheil, A.; Marchaud, D.; Jule, E.; Vertommen, J.; Maio, M.; Blundell, R.; Benameur, H.; Carriere, F.; Mullertz, A.; Pouton, C. W.; Porter, C. J. H. Toward the Establishment of Standardized in Vitro Tests for Lipid-Based Formulations. 2. The Effect of Bile Salt Concentration and Drug Loading on the Performance of Type I, II, IIIA, IIIB, and IV Formulations during in Vitro Digestion. Mol. Pharmaceutics 2012, 9, (11), 32863300. 54. Guzman, H. R.; Tawa, M.; Zhang, Z.; Ratanabanangkoon, P.; Shaw, P.; Gardner, C. R.; Chen, H.; Moreau, J. P.; Almarsson, O.; Remenar, J. F. Combined use of crystalline salt forms and precipitation inhibitors to improve oral absorption of celecoxib from solid oral formulations. J. Pharm. Sci. 2007, 96, (10), 2686-2702. 55. Augustijns, P.; Brewster, M. E. Supersaturating drug delivery systems: Fast is not necessarily good enough. J. Pharm. Sci. 2012, 101, (1), 7-9. 56. Van Eerdenbrugh, B.; Taylor, L. S. Small Scale Screening To Determine the Ability of Different Polymers To Inhibit Drug Crystallization upon Rapid Solvent Evaporation. Mol. Pharmaceutics 2010, 7, (4), 1328-1337. 57. Yeap, Y. Y.; Trevaskis, N. L.; Porter, C. J. Lipid absorption triggers drug supersaturation at the intestinal unstirred water layer and promotes drug absorption from mixed micelles. Pharm Res 2013, 30, (12), 3045-58. 58. Williams, H. D.; Trevaskis, N. L.; Yeap, Y. Y.; Anby, M. U.; Pouton, C. W.; Porter, C. J. H. Lipid-Based Formulations and Drug Supersaturation: Harnessing the Unique Benefits of the Lipid Digestion/Absorption Pathway. Pharm. Res. 2013, 30, (12), 2976-2992. 59. Anby, M. U.; Nguyen, T. H.; Yeap, Y. Y.; Feeney, O. M.; Williams, H. D.; Benameur, H.; Pouton, C. W.; Porter, C. J. H. An in Vitro Digestion Test That Reflects Rat Intestinal Conditions To Probe the Importance of Formulation Digestion vs First Pass Metabolism in Danazol Bioavailability from Lipid Based Formulations. Mol. Pharmaceutics 2014, 11, (11), 4069-4083. 60. Alonzo, D. E.; Zhang, G. G. Z.; Zhou, D. L.; Gao, Y.; Taylor, L. S. Understanding the Behavior of Amorphous Pharmaceutical Systems during Dissolution. Pharm. Res. 2010, 27, (4), 608-618. 61. Tanno, F.; Nishiyama, Y.; Kokubo, H.; Obara, S. Evaluation of hypromellose acetate succinate (HPMCAS) as a carrier in solid dispersions. Drug. Dev. Ind. Pharm. 2004, 30, (1), 9-17. 62. Gao, Y.; Olsen, K. W. Drug-Polymer Interactions at Water-Crystal Interfaces and Implications for Crystallization Inhibition: Molecular Dynamics Simulations of Amphiphilic Block Copolymer Interactions with Tolazamide Crystals. J. Pharm. Sci. 2015, 104, (7), 2132-2141. 63. Obara, S.; Maruyama, N.; Nishiyama, Y.; Kokubo, H. Dry coating: an innovative enteric coating method using a cellulose derivative. Eur. J. Pharm. Biopharm. 1999, 47, (1), 51-59. 64. Curatolo, W.; Nightingale, J. A.; Herbig, S. M. Utility of hydroxypropylmethylcellulose acetate succinate (HPMCAS) for initiation and maintenance of drug supersaturation in the GI milieu. Pharm. Res. 2009, 26, (6), 1419-31. 65. Ilevbare, G. A.; Liu, H. Y.; Edgar, K. J.; Taylor, L. S. Understanding Polymer Properties Important for Crystal Growth Inhibition-Impact of Chemically Diverse Polymers on Solution Crystal Growth of Ritonavir. Cryst. Growth Des. 2012, 12, (6), 3133-3143. 66. Friesen, D. T.; Shanker, R.; Crew, M.; Smithey, D. T.; Curatolo, W. J.; Nightingale, J. A. S. Hydroxypropyl Methylcellulose Acetate Succinate-Based Spray-Dried Dispersions: An Overview. Mol. Pharmaceutics 2008, 5, (6), 1003-1019. 67. Takemura, S.; Kondo, H.; Watanabe, S.; Sako, K.; Ogawara, K.; Higaki, K. Aminoalkylmethacrylate Copolymer E Improves Oral Bioavailability of YM466 by Suppressing Drug-Bile Interaction. J. Pharm. Sci. 2013, 102, (9), 3128-3135. 68. Mohsin, K. Design of Lipid-Based Formulations for Oral Administration of Poorly Water-Soluble Drug Fenofibrate: Effects of Digestion. AAPS PharmSciTech. 2012, 13, (2), 637-646.



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69. Anby, M. U.; Williams, H. D.; Feeney, O.; Edwards, G. A.; Benameur, H.; Pouton, C. W.; Porter, C. J. H. Non-linear Increases in Danazol Exposure with Dose in Older vs. Younger Beagle Dogs: The Potential Role of Differences in Bile Salt Concentration, Thermodynamic Activity, and Formulation Digestion. Pharm. Res. 2014, 31, (6), 1536-1552. 70. Sassene, P. J.; Knopp, M. M.; Hesselkilde, J. Z.; Koradia, V.; Larsen, A.; Rades, T.; Mullertz, A. Precipitation of a Poorly Soluble Model Drug during In Vitro Lipolysis: Characterization and Dissolution of the Precipitate. J. Pharm. Sci. 2010, 99, (12), 4982-4991. 71. Thomas, N.; Holm, R.; Mullertz, A.; Rades, T. In vitro and in vivo performance of novel supersaturated self-nanoemulsifying drug delivery systems (super-SNEDDS). J. Controlled Release 2012, 160, (1), 25-32. 72. McEvoy, C. L.; Trevaskis, N. L.; Feeney, O. M.; Edwards, G. A.; Perlman, M. E.; Ambler, C. M.; Porter, C. J. H. Correlating in vitro solubilisation and supersaturation profiles with in vivo exposure for lipid based formulations of the CETP inhibitor CP-532,623. Mol. Pharmaceutics 2017. 73. DiNunzio, J. C.; Hughey, J. R.; Brough, C.; Miller, D. A.; Williams, R. O.; McGinity, J. W. Production of advanced solid dispersions for enhanced bioavailability of itraconazole using KinetiSol (R) Dispersing. Drug Dev. Ind. Pharm. 2010, 36, (9), 1064-1078. 74. Alonzo, D. E.; Gao, Y.; Zhou, D. L.; Mo, H. P.; Zhang, G. G. Z.; Taylor, L. S. Dissolution and Precipitation Behavior of Amorphous Solid Dispersions. J. Pharm. Sci. 2011, 100, (8), 3316-3331.

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Figure 1. Schematic representation of the composition of the three LBF types employed during the in vitro digestion experiment (% content is % w/w). The Type IIIA and Type IIIB formulations contained a medium chain lipid phase comprising a mixture of Captex® 300 and Capmul® MCM, based on the LFCS classification of LBFs proposed by Pouton et al. 40 214x47mm (300 x 300 DPI)

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Figure 2. In vitro evaluation of drug solubilization profile of fenofibrate during dispersion and digestion of three LBFs, [mean ± SD (n = 3)] in the presence and absence of Eudragit E100 (EU E100). Fenofibrate was incorporated at 40% (upper row) and 85% (lower row) of the equilibrium solubility in the formulation and in the absence (grey circles) and presence (blue circles) of 1% w/w EU E100. The horizontal (blue) dashed line represents the APMAX and the black circles (dashed black line) represents the apparent equilibrium solubility of fenofibrate in the aqueous colloidal phase, produced during drug-free LBF dispersion (APDISP) and digestion (APDIGEST). The blue shaded section represents the experimental duration of the dispersion phase. 275x147mm (300 x 300 DPI)


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Figure 3 Fenofibrate solubilisation and supersaturation during in vitro dispersion and digestion of LBFs. The AUC of solubilized fenofibrate during in vitro dispersion and digestion, calculated using the linear trapezoidal method is shown in the bars, with data obtained using formulations loaded at 40% of saturation solubility in the open bars and data at 85% saturation in the grey bars (left axis). The degree of fenofibrate supersaturation during dispersion and digestion is shown as the blue squares (data at 40% saturation loading) or blue triangles (data at 85% saturation loading) (right axis). Data expressed as the supersaturation ratio (S) and calculated with AUCDISPandDIGEST of the solubilization profiles, [mean ± SD (n = 3)]. 244x126mm (300 x 300 DPI)



Figure 4. Venn diagram showing the solubility of PPIs in the Type IV LBF (yellow) and micellar solution (blue) respectively. Polymers displayed outside the diagram were not soluble in either of the media, whereas those in the overlapping region were soluble in both media. Polymer solubility was tested at 1% w/w in the formulation and the corresponding mass was used to assess solubility in 40 mL micellar solution. 191x113mm (149 x 149 DPI)


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Figure 5. In vitro evaluation of the impact of the presence of PPIs pre-dispersed in different media on fenofibrate solubilization and supersaturation after dispersion (15 min) and digestion (60 min) in a Type IV formulation at 85% saturated solubility [mean ± SD (n ≥ 3)]. Panel A: Shows the % drug solubilized over the period of formulation dispersion and digestion, calculated by dividing the AUC of the concentration versus time profile obtained in the in vitro test by the AUC of the APMAX over the same time period (representing 100% solubilized). The horizontal black dotted line represents the extent of drug solubilization obtained from the formulation in the absence of polymer (also shown as the black (control) bar). Data above the line represent formulations where polymer addition leads to lower drug precipitation during formulation dispersion and digestion (and therefore greater drug solubilization). The green panel highlights the area where the % solubilized fenofibrate exceeds that of the formulation without polymer (control). Panel B: Mean supersaturation ratio (S) obtained over the period of dispersion and digestion of the formulations, data grouped for polymers with different solubility profiles in the LBF. 276x104mm (300 x 300 DPI)



Figure 6. Impact of PPI concentration (1 and 5% w/w) on the extent of fenofibrate solubilization. Percentage (%) drug solubilized over the period of formulation dispersion and digestion was calculated by dividing the AUC of the concentration versus time profile obtained in the in vitro test by the AUC of the APMAX over the same time period (representing 100% solubilized). PPIs were incorporated at 1 and 5% w/w, pre-dispersed in the LBF (Type IV) [mean ± SD (n = 3)]. 128x73mm (300 x 300 DPI)


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Figure 7. In vitro impact of PPIs on drug solubulization and supersaturation. Panel A: Drug solubilization profiles of fenofibrate in a Type IV formulations at 85% fenofibrate loading pre (orange open symbols) - and post-perfusion of the jejunum during dispersion and digestion in the absence (grey circles) and presence (black circles) of three different polymers pre-dispersed in the LBF [mean ± SD (n = 4-5)]. Polymer concentrations in the LBFs were 1, 5 and 1% w/w for EU RL100, HPMC E4M and PPGAE respectively. Blue panels represent the dispersion stage. The black circles and dashed line shows fenofibrate solubility in the aqueous colloidal phase produced by dispersion (APDISP) and digestion (APDIGEST) of the formulation and the upper horizontal dotted black line represents the APMAX. Panel B: Fenofibrate supersaturation in the absence and presence of polymers, expressed by the supersaturation ratio (S) calculated with AUCDISPandDIGEST of the solubilization profiles in panel A, [mean ± SD]. **** Statistically significant difference (p < 0.0001), compared to the Type IV control (no PPI) LBF. 267x156mm (300 x 300 DPI)



Figure 8. Fenofibric acid absorption assessment using the coupled in vitro digestion-in vivo absorption model for Type IIIB LBF and Type IV LBFs in the absence and presence of PPIs (HPMC E4M, PPGAE, EU RL100) with fenofibrate incorporated at 85% saturated solubility [mean ± SEM (n=4–5)]. Panel A: Overlaid fenofibric acid flux into mesenteric vein blood across a ~ 10 cm segment for the Type IIIB LBF and Type IV LBFs (with and without PPIs). Panel B: Calculated AUC of fenofibric acid flux into the mesenteric vein over time (t = 060 min, data from Panel A). Data represent mean ± SEM (n = 4-5). Panel C: IVIVC of in vivo absorptive drug flux of fenofibric acid and the vitro supersaturation ratio (S), data from in vitro digestion tests in Figure 7. Linear regression for all five formulations was R2 = 0.74, for just the Type IV LBFs was R2 = 0.87 (regression line for fit to Type IV LBFs only shown). * Statistically significant difference (p < 0.05) compared to the control, Type IV LBF (no PPI). 245x183mm (300 x 300 DPI)


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Figure 9. Crossed-polarized light micrographs of precipitated fenofibrate generated during dispersion and digestion of a Type IV formulation loaded at 85% saturation, in the presence of PPIs. Images of the pellet phase were obtained at 0 min (end of dispersion phase) and at 30 min after initiation of digestion. 169x97mm (149 x 149 DPI)


Polymeric Precipitation Inhibitors Promote Fenofibrate Supersaturation

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