ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2018

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 262

Improved Molecular Understandingof Lipid-Based Formulations

for Enabling Oral Delivery of Poorly Water-SolubleDrugs

LINDA C. ALSKÄR

ISSN 1651-6192ISBN 978-91-513-0509-7urn:nbn:se:uu:diva-366586

Dissertation presented at Uppsala University to be publicly examined in B:42, BiomedicalCenter, Husargatan 3, Uppsala, Friday, 18 January 2019 at 09:15 for the degree of Doctor ofPhilosophy (Faculty of Pharmacy). The examination will be conducted in English. Facultyexaminer: Senior Lecturer Brendan Griffin ( School of Pharmacy, University College Cork).

AbstractAlskär, L. C. 2018. Improved Molecular Understanding of Lipid-Based Formulations. forEnabling Oral Delivery of Poorly Water-Soluble Drugs. Digital Comprehensive Summaries ofUppsala Dissertations from the Faculty of Pharmacy 262. 68 pp. Uppsala: Acta UniversitatisUpsaliensis. ISBN 978-91-513-0509-7.

The majority of emerging drug candidates are not suited for conventional oral dosage forms, asthey do not dissolve in the aqueous environment of the gastrointestinal (GI) tract. Consequently,a large number of enabling formulation strategies have emerged. One such strategy is todeliver the drug pre-dissolved in a lipid-based formulation (LBF), thereby bypassing the rate-limiting dissolution step. To date, only about 4% of the marketed oral drugs are deliveredas LBFs. The limited use of this strategy is a result of the incomplete understanding of drugsolubility in lipid vehicles, the reduced chemical stability of pre-dissolved drug, and the complexinterplay between drug and formulation undergoing intestinal lipid processing. Hence, thisthesis targeted an improved molecular understanding of lipid-based drug delivery to makean informed formulation development. In the first part of the thesis, drug solubility in LBFexcipients and composed formulations was assessed. Through experimental studies of nearlyforty compounds in nine excipients drug physicochemical properties related to solubility inthese excipients were identified. The obtained data was used to develop in silico tools forprediction of drug solubility in excipients and formulations. The second part of the thesisfocused on LBF performance in vitro and in vivo. Factors associated with the type of solid formthat is precipitating during digestions was revealed, which provides an initial framework forunderstanding drug precipitation behaviour under physiological conditions. It was also shownthat clinically relevant doses of LBF significantly increases intestinal drug solubilization as aresult of GI lipid processing and bile secretion. Moreover, simultaneous assessment of digestionand absorption in vitro provided the same rank order of absorbed drug as the in vivo studies.Coadministration of LBF and drug was shown to be a promising alternative to pre-dissolveddrug in the LBF. In summary, this thesis has improved the molecular understanding of factorsthat govern drug solubility in lipid vehicles and solid form of precipitated drug under digestiveconditions. It was also proved that clinically relevant doses of LBFs significantly increase theintestinal drug solubilization, and proof-of-concept was shown for coadministration of LBF withsolid drug as an alternative to drug-loaded LBF.

Keywords: lipid-based formulation, poorly water-soluble drug, solubility prediction,molecular properties, lipid digestion, precipitation, solid state, intestinal solubilization, invitro in vivo correlation (IVIVC), coadministration

Linda C. Alskär, Department of Pharmacy, Box 580, Uppsala University, SE-75123 Uppsala,Sweden.

© Linda C. Alskär 2018

ISSN 1651-6192ISBN 978-91-513-0509-7urn:nbn:se:uu:diva-366586 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-366586)

Till Edith och Ruth

“And above all, watch with glittering eyes the wholeworld around you because the greatest secrets are

always hidden in the most unlikely places. Those who don't believe in magic will never find it.”

Roald Dahl

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I PerssonI L.C., Porter C.J.H, Charman W.N., Bergström C.A.S. (2013) Computational Prediction of Drug Solubility in Lipid Based Formulation Excipients. Pharmaceutical Research, 30(12):3225-3237.

II Alskär L.C., Porter C.J.H, Bergström C.A.S. (2016) Tools for Early Prediction of Drug Loading in Lipid-Based Formulations. Molecular Pharmaceutics, 13(1):251-261.

III Alskär L.C., Keemink J., Johannesson J., Porter C.J.H.,

Bergström C.A.S. (2018) Impact of Drug Physicochemical Properties on Lipolysis-Triggered Drug Supersaturation and Precipitation from Lipid-Based Formulations. Molecular Pharmaceutics, 15(10):4733-4744.

IV Alskär L.C., Parrow A., Keemink J., Johansson P.,

Abrahamsson B., Bergström C.A.S. Effect of Lipids on Absorp-tion of Carvedilol in Dogs: Is Coadministration of Lipids as Efficient as a Lipid-Based Formulation? Submitted.

I Birth name.

Reprints were made with permission from the respective publishers.

Also contributed to: i Alskär L.C., Bergström C.A.S. (2015) Models for Predicting

Drug Absorption From Oral Lipid-Based Formulations. Current Molecular Biology Reports, 1(4):141-147.

ii Larsson P., Alskär L.C., Bergström C.A.S. (2017) Molecular Structuring and Phase Transition of Lipid-Based Formulations upon Water Dispersion: A Coarse-Grained Molecular Dynamics Simulation Approach. Molecular Pharmaceutics, 14(12): 4145-4153.

Contents

Introduction ................................................................................................... 11 Oral lipid-based formulations ................................................................... 11 

Intestinal lipid digestion and colloidal structuring .............................. 12 Intestinal drug and lipid absorption ..................................................... 13 Classification of lipid-based formulations ........................................... 15 Nomenclature of lipid-based formulations .......................................... 16 Marketed lipid-based formulations ...................................................... 16 Drug candidates suitable for oral lipid-based formulations ................. 16 

Formulation design and development ...................................................... 18 Drug loading capacity .......................................................................... 18 Equilibrium solubility and dissolution rate .......................................... 18 Solubility screening assays .................................................................. 19 In silico prediction of drug solubility in lipids .................................... 20 LBF performance evaluation ............................................................... 21 Drug supersaturation and precipitation ................................................ 21 In vitro lipolysis assay ......................................................................... 22 In vitro in vivo correlation ................................................................... 23 

Current limitations and potentials for LBFs ............................................. 24 

Aims of the thesis.......................................................................................... 26 

Methods ........................................................................................................ 27 Model compounds .................................................................................... 27 Studied excipients and formulations ........................................................ 27 In vitro methods ....................................................................................... 29 

Solubility determinations ..................................................................... 29 In vitro lipolysis assay ......................................................................... 30 In vitro lipolysis permeation assay ...................................................... 30 Cell culture .......................................................................................... 31 Solid state characterization techniques ................................................ 31 

In vivo experiments .................................................................................. 32 In silico methods ...................................................................................... 33 

Quantitative structure–property relationships ...................................... 33 Statistical analysis .................................................................................... 35 

Results and Discussion ................................................................................. 36 Data sets (Paper I, II, III and IV) .............................................................. 36 Drug solubility in lipid vehicles (Paper I and II) ...................................... 36 

Effect of excipient water sorption (Paper I and II) .............................. 38 Excipient solubility predictions (Paper I and II) ...................................... 39 LBF loading capacity predictions (Paper II) ............................................ 41 Physicochemical properties related to drug solubility in LBF excipients (Paper I and II) ......................................................................................... 42 LBF in vitro performance (Paper III and IV) ........................................... 44 

Drug solubility in lipolysis medium (Paper III and IV) ....................... 44 Lipolysis-triggered supersaturation and precipitation (Paper III) ........ 45 Solid state of precipitated drug (Paper III) .......................................... 45 Physicochemical properties related to drug precipitation behavior (Paper III) ............................................................................................ 46 

In vivo dog study (Paper IV) .................................................................... 47 LBF-mediated intestinal drug solubilization ....................................... 47 Coadministration of LBF and drug (Paper IV) .................................... 48 In vitro in vivo correlation ................................................................... 49 

Conclusions ................................................................................................... 50 

Future perspectives ....................................................................................... 52 

Populärvetenskaplig sammanfattning ........................................................... 54 

Acknowledgements ....................................................................................... 56 

References ..................................................................................................... 58 

Abbreviations

ABL Aqueous boundary layer API Active pharmaceutical ingredient BCS Biopharmaceutics classification system BS Bile salt DG Diglyceride DIF Dog intestinal fluid DSC Differential scanning calorimetry FA Fatty acid FFA Free fatty acid GF Glass-former GI Gastrointestinal HBSS Hank's balanced salt solution HLB Hydrophilic-lipophilic balance IVIVC In vitro in vivo correlation LBF Lipid-based formulation LCMIX Long-chain mixed mono-, di-, triglyceride LCTG Long-chain triglyceride LFCS Lipid formulation classification system MCMIX Medium-chain mixed mono-, di-, triglyceride MCTG Medium-chain triglyceride MG Monoglyceride MLR Multiple linear regression MVA Multivariate data analysis Mw Molecular weight nGF Nonglass-former PCA Principal component analysis PLM Polarized light microscopy PLS Projection to latent structure PLS-DA Projections to latent structures discriminant analysis PWSD Poorly water-soluble drug QSPR Quantitative structure–property relationship RMSEE Root-mean square error of the estimate SEDDS Self-emulsifying drug delivery systems SIF Simulated intestinal fluid SMEDDS Self-microemulsifying drug delivery systems SNEDDS Self-nanoemulsifying drug delivery systems

TG Triglyceride Tm Melting point VIP Variable importance to projection

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Introduction

Oral drug products are convenient, cost-effective, and painless to administer. Therefore, it remains the most attractive delivery technique for drugs intended for systemic circulation. To reach their systemic target, drugs need to be sufficiently soluble in the gastrointestinal (GI) fluid, allowing for absorption through the gut wall. However, to date many new drug candidates suffer from poor water solubility, often caused by high lipophilicity. The increasing number of drugs that are highly lipophilic is related to the nature of currently explored therapeutic targets, in combination with the biological screening methods used, where a positive association between lipophilicity and pharmacological activity is well-known.1-3 Depending on the therapeutic area, between 75-95% of all small molecular discovery compounds (Mw < 1500 Da) are in fact displaying poor water solubility.4, 5 This makes new drug candidates difficult to formulate and challenging to design as suitable oral drug products. Consequently, a large number of formulation strategies have emerged to overcome dissolution rate-limited, and solubility limited absorption of lipophilic drug compounds.6-9 One such successful strategy is the delivery of the drug in a lipid-based formulation (LBF).10

Oral lipid-based formulations LBFs consist of mixtures of oils, surfactants, and organic solvents in different proportions. In contrast to conventional oral dosage forms (e.g. tablets) the drug is typically delivered in a pre-dissolved state. Thus, for poorly water-soluble drugs (PWSD), the major advantages associated with LBFs are avoidance of rate-limiting dissolution, and improved intestinal solubility, caused by increased solubilization when the formulation components mix with the GI fluids (Figure 1). Additionally, lipids in the formulation may trigger the endogenous digestion process and lead to release of bile salts (BS), phospholipids, and cholesterol, which contributes to maintaining the drug dissolved in the intestine.11, 12 LBFs may also facilitate lymphatic uptake of highly lipophilic drugs (logP > 5) through fusion with evolving lipoproteins. The lymphatic pathway has the benefit of reducing first-pass metabolism.13, 14

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Intestinal lipid digestion and colloidal structuring The fat we ingest through food is subjected to mechanical mixing and enzymatic lipid digestion.15-17 Formulation lipids are processed in the same way in the GI tract. The main site for lipid digestion within the GI tract is the small intestine (Figure 1). However, the process starts in the mouth and continues in the stomach before reaching the small intestine. Lingual lipase is secreted with the saliva, although, for LBFs delivered in capsules the digestion process is considered to be initiated by gastric lipase in the stomach.11, 18 During the lipid digestion process, triglycerides (TG) are hydrolyzed into diglycerides (DG), and free fatty acid (FFA) molecules. The DGs are then further digested into monoglycerides (MG) and FFAs (Figure 2).

Figure 1. After oral intake of an LBF capsule the formulation excipients are dis-persed and digested in the GI tract. In the small intestine, TGs and DGs are digested by pancreatic lipase and colipase into MGs and FFAs. The presence of exogenous formulation excipients may stimulate bile secretion from the gallbladder. The diges-tion products, BS, phospholipids, and cholesterol are incorporated into different colloidal structures, including multi- and unilamellar vesicles, mixed micelles and micelles. Together, these colloidal species expand the solubilization capacity of the small intestine for lipid-digestion products and PWSD (D).

Between 10-30% of the total lipid digestion is performed by gastric lipase in the stomach. The magnitude of the gastric digestion is related to residence time and formulation dispersion properties.11, 19-21 The stomach further contributes to the lipid processing by mechanical mixing, which assists formation of a crude emulsion, and thus promotes accessibility for the pancreatic lipases. Following transit into the small intestine, where the major part of the lipid breakdown occurs, the digestion progresses by the pancreatic

13

lipase/colipase complex at the oil-water interface (Figure 1).22-24 The presence of exogenous lipids and lipid-digestion products in the small intestine may stimulate endogenous secretion of bile. Secreted bile components, such as BS, phospholipids, and cholesterol are incorporated into different colloidal structures together with digested formulation components (Figure 1).25, 26 The formed colloidal structures increase the solubilization capacity of the small intestine for both poorly water-soluble lipid-digestion products and drug compounds.

Figure 2. Enzymatic lipolysis of triglycerides by digestive lipases in the GI tract. The digestion process starts by gastric lipase in the stomach and continues in the intestine by pancreatic lipases. The final digestion products, monoglycerides and free fatty acids, are incorporated into mixed colloidal structures together with BS, phospholipids and cholesterol. The FFA is exemplified by capric acid (C10:0).

Intestinal drug and lipid absorption Orally administered drugs intended for systemic effect need to be absorbed from the intestine into the blood circulation to reach their target. In the intestine, drug molecules in solution diffuse through the aqueous boundary layer (ABL), and are subsequently absorbed over the gut wall (Figure 3). However, following intestinal digestion of lipid formulations, administered PWSD is largely localized within the formed colloidal species. These colloids

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diffuse across the ABL to the brush border membrane of enterocytes. At the apical membrane the drug molecules dissociate from the colloidal structures, and are absorbed by passive diffusion or carrier-mediated transport. The colloidal drug transport can essentially enhance the mass-transfer across the ABL.27, 28 Diffusion across the ABL depends on the relative size of the colloidal structures or the drug molecule in solution. An inverse relationship between size of the colloid or drug and diffusion coefficient is frequently observed.29, 30 Moreover, it has been suggested that the acidic microclimate at the ABL aids dissociation of the colloidal structures.31 Exposure to the acidic milieu leads to protonation of FFAs, which promotes dissociation from the colloidal structures, and absorption across the apical membrane. Protonated FFAs are expected to partition more readily across the lipophilic intestinal membrane, than non-protonated FFAs.31, 32 Subsequently, the absorption of FFAs and MGs across the intestinal membrane occur through several active and passive transport mechanisms.33, 34

Figure 3. Schematic overview of the absorption of drug (D) from an orally delivered LBF. Following digestion of the formulation glycerides the drug molecules are ei-ther present as free drug in solution, or solubilized in mixed colloidal species. Drug molecules in solution can diffuse through the ABL and subsequently permeate the cells in the gut wall. The drug can also reach the intestinal membrane solubilized in lipid colloids, which considerably enhances the mass-transport of drug.

Additionally, LBF excipients may interact with apical membrane transporters leading to altered absorption of PWSD. For example, surfactants (Kolli-phor/Cremophor, Polysorbate 80, Polysorbate 20, and Labrasol), and cosolvents (PEG400 and Pluronics) commonly used in LBFs have been shown to nonspecifically inhibit the P-glycoprotein (P-gp) efflux transporter.35, 36 Administration of LBFs containing P-gp inhibiting excipients could therefore potentially enhance oral bioavailability of P-gp substrate drugs. However, the

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effect has mainly been studied in vitro, and only a limited number of studies have demonstrated enhanced drug absorption in vivo related to excipient- mediated P-gp inhibition.11

Classification of lipid-based formulations Pouton introduced the lipid formulation classification system (LFCS) to systematically categorize LBFs, as presented in Table 1.37, 38 This system classifies lipid formulations into five different groups; type I, II, IIIA/IIIB, and IV, based on the composition of excipients in the formulation. The LFCS ranges from lipid-rich type I formulations to more hydrophilic type IV formulations. The simplest formulations are type I, which are comprised of only digestible TGs, or TG in mixture with DGs and MGs. Type II LBFs contain glycerides in combination with lipophilic surfactants (HLB <12), for example Tween 85. Type III formulations are combinations of mixed glyceride lipids and more hydrophilic surfactants (HLB > 12). Commonly used surfactants are Kolliphor EL and Kolliphor RH 40. Type III LBFs may also include cosolvents (e.g. PEG400, Transcutol/Carbitol or ethanol). The type III formulations are further classified into type IIIA and IIIB, where the proportion of lipids are larger in a type IIIA than IIIB. Instead, IIIB contain a larger proportion of surfactants and cosolvents. The most hydrophilic class, type IV formulations, does not include any oils, but merely surfactants and cosolvents.

Table 1. Composition of type I-IV lipid-based formulations (% w/w), according to the lipid formulation classification system.37, 38

Excipient Type I

Type II

Type IIIA

Type IIIB

Type IV

Oils: TG or mix of DG/MG 100 40-80 40-80 < 20 - Lipophilic surfactant (HLB<12)

- 20-60 - - 0-20

Hydrophilic surfactant (HLB>12)

- - 20-40 20-50 30-80

Hydrophilic cosolvent - - 0-40 20-50 0-50 Particle size of dispersion (nm) Coarse 100-250 100-250 50-100 <50

Although the LFCS classifies LBFs based on composition, the ingredients reflect particle size upon dispersion in aqueous media (Table 1), as well as formulation digestibility. In general, lipophilic type I formulations require digestion to mix with the GI fluids and promote drug absorption. Whereas type II-IV formulations contain sufficient proportions of surfactant to obtain spontaneous dispersion in aqueous fluids. Additionally, the type IV LBFs without pure oil components are not likely to be affected by lipase digestion.39

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Nomenclature of lipid-based formulations Lipid formulations have been attributed several different names in the scientific literature. Firstly, the most simple name; lipid-based formulations, which is used synonymously with lipid-based drug delivery systems (LbDDS). Later, the term self-emulsifying drug delivery systems (SEDDS) was introduced,40 followed by self-microemulsifying drug delivery systems (SMEDDS), and self-nanoemulsifying drug delivery systems (SNEDDS). The naming of the SEDDS, SMEDDS and SNEDDS refers to their self- emulsification ability and droplet size upon dilution in aqueous media, such as GI fluids. In general, SEDDS are characteristic type II formulations, while SMEDDS and SNEDDS are classified as either type IIIA or IIIB.41 However, due to their thermodynamical instability, many pharmaceutical microemul-sions may be re-defined as nanoemulsions, which has prompted a debate about the use of the SMEDDS/SNEDDS terminology.42-44 Moreover, an increasing number of studies have indicated that the in vivo performance of lipid formulations is poorly correlated with the physical state of the initial dispersion (i.e. emulsion droplet size).45-48 For formulations that undergo bile dilution and continued digestion along the intestine (resulting in dynamically changing colloidal structures), the relation to drug absorption is complex, and cannot simply be linked to initial colloidal structures. Hence, throughout this thesis the term lipid-based formulations is used, since the dispersion properties are not always known, and more importantly, might not be relevant for drug absorption. In vitro in vivo correlations (IVIVC) of LBFs are further deliberated in the following sections.

Marketed lipid-based formulations LBFs have been explored as a strategy for increasing oral drug absorption for approximately 50 years.10 Although lipid formulations cannot be considered a recent pharmaceutical innovation, there are as of now a limited number of oral drugs formulated as LBFs. In total, only about 2-4% of marketed oral drug products use this delivery strategy.12 Some successful examples are; Sandimmune© and Sandimmune Neoral© (cyclosporin A), Norvir® (ritonavir), and Fortovase® (saquinavir).49 Comprehensive summaries of commercially available LBFs and included excipients are available in several review articles.12, 49-52 Likely reasons for the limited use of this delivery strategy are discussed in the following sections.

Drug candidates suitable for oral lipid-based formulations This thesis has explored LBFs for enabling the delivery of PWSD, and as such, the focus has been on compounds that display solubility and dissolution limited absorption. PWSD are typically categorized as class 2 (low solubility,

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high permeability), or class 4 (low solubility and permeability) in the biopharmaceutics classification system (BCS). This classification system was introduced in 1995 to classify the absorption behavior of drugs, with the main purpose to identify compounds for which biowaivers could be granted.53 It is used by both the European Medicines Agency (EMA), and the Food and Drug Administration (FDA) for regulatory purposes,54 and has found extensive application during the drug discovery and development process.55, 56 Indeed, a recent analysis of marketed drugs, formulated as orally delivered LBFs, showed that the majority of the compounds fall into BCS class 2 or 4 (19 out of 25).52 All compounds categorized as BCS class 2 or 4 were extracted from this analysis, and Figure 4A-C displays selected physicochemical properties of the nineteen drugs. Most of the compounds have low molecular weight (Mw), with a median of 417 g/mol, and cyclosporine being a strong outlier (1203 g/mol). The logP values range from 1.9 – 7.4, with a median of 4.9. Melting point (Tm) varies from 59 °C – 350 °C, with a median of 150 °C.

Figure 4. Physicochemical properties of BCS class 2 and 4 drugs marketed as orally delivered LBFs. (A) Molecular weight (Mw), (B) logP, and (C) Melting point (Tm). The black lines display the median with the interquartile range. Data adapted from Salva et al.52

The physicochemical data of PWSD marketed as LBFs follow the general conviction that drugs with a logP > 2 are suitable candidates for LBFs.37 logP reflects the lipophilicity of a drug, and according to the concept “like dissolves like” a lipophilic drug should be more soluble in lipids than water. Although this is true, the drug crystal lattice structure (as reflected by the melting point) will also have an impact on the resulting solubility in lipids. Compounds with high logP and Tm values can be seen as ‘anything-phobic’, and their interaction with both water and lipids is limited by strong intermolecular forces. In Figure 4C, this is reflected by the melting point of the marketed BCS class 2 and 4 drugs, where only two of the compounds have a Tm above 250 °C. Thus, potential candidates for LBFs are likely to be of lipophilic character,

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but also have relatively low crystal packing energy. Moreover, as an experimental guideline, it has been suggested that drug solubility above 25 mg/g in lipid excipients is desired to support the development of an LBF.57

However, these molecular properties should be considered as a guideline for when an LBF could be a successful delivery system. Physicochemical properties, such as logP and Tm, cannot be used alone to guide the formulation work, since they do not predict in vivo performance. For such evaluations in vitro and in silico tools are needed.

Formulation design and development LBF performance depends on the nature of the included excipients and the physicochemical properties of the drug compound. Ideally, the formulation should be able to solubilize the entire drug dose in a unit dosage form, keep the drug in a solubilized state during intestinal dispersion and digestion, and hence, prevent drug precipitation. These criteria require careful excipient selection, both with regard to drug loading capacity of the LBF, and maintenance of solubilized drug throughout the GI tract. To accurately evaluate the performance of drug-loaded LBFs, both predictive in silico and in vitro models are warranted. However, currently the development of LBFs is largely empirical, and therefore experimentally demanding.57-59 In the following sections the main steps and techniques, together with basic concepts applied in lipid formulation development will be introduced, and their shortcomings and potentials are discussed.

Drug loading capacity Throughout this thesis, the term drug loading capacity is used to refer to maximum drug solubility in composed formulations (containing several excipients), while the word solubility is used to refer to the equilibrium solubility in single excipients. Thus, the same methodologies can be applied to determine drug solubility in a single excipient and drug loading capacity of an LBF.

Equilibrium solubility and dissolution rate Equilibrium (thermodynamic) solubility is the saturated solubility of a substance in the end of the dissolution process; at the point when there is an equilibrium between dissolved and undissolved substance. Thermodynamic solubility is often regarded as the ‘true’ solubility of a drug compound. Therefore it is an essential parameter during formulation development. Yet, it is important to understand that the equilibrium solubility is influenced by a

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number of factors, such as; temperature, pH, polymorphism, compound purity, compound ionization states, and stability in solution.

Dissolution rate of a drug is closely related to thermodynamic solubility, because the crystal lattice must be disrupted during the process. The dissolution of any material includes removal of single molecules from the crystal lattice, formation of a cavity in the solvent, and solvation of the molecule. Hence, the solubility is dictated by the sum of the changes in free energy (ΔG) during the dissolution steps.60 Consequently, drug dissolution has been extensively investigated in pharmaceutical research,61 and the process was described by Noyes and Whitney more than a century ago.62 The relationship between drug solubility and dissolution was further evolved by Nernst and Brunner,63 and is today commonly referred to as the Noyes- Whitney/Nernst-Brunner equation (Equation 1):

Eq. 1

where dW/dt is the amount of drug released over time, D the diffusion coefficient of drug in solution, A the surface area of drug in contact with the solvent, h the diffusion layer thickness, Cs the saturated solubility, and Ct the amount of drug in solution at time t.

Solubility screening assays LBF development often starts with experimental screening of drug solubility in a large number of commonly used oils, surfactants and cosolvents.57 The excipient solubility screening is typically performed in a 96 well format (total volume 40-200 µl), and can either be of kinetic or thermodynamic character (Figure 5). In a kinetic solubility assay, the investigated compound is pre-dissolved in an organic solvent, and a few microliters of the concentrated drug solution is added into each well of the plate (using micrograms of drug per well). The excipients are added into to the wells, either diluted in an aqueous buffer or in concentrated form. Aqueous dilution of excipients is performed because of the complexity of pipetting viscous liquids. UV absorbance is measured after incubation, and solubility is determined as the value when drug precipitation occurs.64 As an extension to kinetic solubility assays, solvent evaporation methods have been introduced, i.e. the organic solvent is first evaporated, followed by addition of excipient or medium of interest. Thereafter, the plate is incubated, the samples filtered, and the drug concentration in the filtrate is determined with a plate reader or HPLC.65-67 In a thermodynamic solubility assay the solid compound is dispensed (10-15 mg) directly into the wells containing excipients, and drug concentration is determined by HPLC after incubation and filtration.68

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The described approaches are suitable at different stages during the drug development process. The kinetic assay gives qualitative measurement that classifies the drug as “poorly” or “highly” soluble in an excipient, rather than providing exact solubility values. On the other hand, the assay consumes a small amount of drug, making it suitable for late discovery and early drug development stages. A thermodynamic solubility screening can be performed later in the drug development process, when slightly more compound is available (milligrams). Through this approach, the formulator is informed about how much of the drug dose that can be dissolved in an excipient or formulation. The thermodynamic solubility assay can also be combined with solid state analysis of the residual drug pellet to evaluate the solid form.

Figure 5. Schematic representation of thermodynamic and kinetic drug solubility. In a thermodynamic solubility assay, the solid drug is add in excess into the excipi-ent and the drug concentration at equilibrium determined. In a kinetic solubility assay, the drug is pre-dissolved in an organic solvent, and a few microliters of the concentrated stock-solution added into the excipient. The solubility is determined as the value when drug precipitation occurs. In a kinetic assay, solubility of the stable crystalline polymorph is reached over time.

In silico prediction of drug solubility in lipids Prediction of drug solubility in lipid vehicles is complex because it is governed by molecular interactions between solute and solvent, the lipid microstructure, and physicochemical properties of included excipients and drug. Early solubility theories that do not account for molecular interactions (e.g. ideal solubility theory, regular solution theory, and Flory–Huggins theory) are therefore unable to accurately predict drug solubility in polar organic lipid solvents.69 Considerable efforts have been made to develop models for solubility in binary mixtures of cosolvent and water.70 One of the most renowned models for prediction of drug solubilization in cosolvent/water mixtures is the log-linear model by Yalkowsky and co-workers.71, 72 This relationship was later applied to calculate solubility in lipid formulations.73-75

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The weakness of such methodologies is that it requires experimental efforts, which limits the applicability for rapid estimation of LBF drug loading capacity early in the development process. Other applied approaches to predict drug solubility in cosolvent/water mixtures are linear regression analysis76 and quantitative structure–property relationships (QSPR).77 Although methods for calculating solubility in cosolvents/water mixtures exist, at the start of this thesis work no models yet existed for prediction of de novo drug solubility in lipid excipients or composed LBFs based on physicochemical properties of the drug compound.

LBF performance evaluation One key design criteria for developing LBFs is the maintenance of intestinal drug solubilization, and hence prevention of drug precipitation.11, 12, 57 The main purpose of biopharmaceutical evaluation of LBFs is therefore to assess whether the drug remains in solution during formulation dispersion and digestion. Intestinal absorption may be affected by drug supersaturation and precipitation, and these parameters are similarly important determinants during in vitro evaluation of LBFs.

Drug supersaturation and precipitation Intestinal drug supersaturation occurs when the concentration of drug exceeds equilibrium solubility of the GI fluid. By definition, the supersaturated state is thermodynamically unstable, and hence provides the driving force for drug precipitation. Intestinal drug precipitation may lead to reduced absorption due to the lower concentration of dissolved drug. Supersaturation, in the absence of precipitation, has the potential to enhance drug absorption as compared to saturated solutions. Drug supersaturation increases the free concentration of drug in equilibrium with the solubilized reservoir, and thus the flux across the intestinal membrane is favored because of higher thermodynamic activity. Indeed, many enabling drug delivery systems, e.g. amorphous solid dispersions and mesoporous materials, make use of this absorption enhancement strategy.78-80

The supersaturated state can be induced through several mechanisms in the GI tract. For weak bases, transit from the low pH environment of the stomach to the higher pH of the small intestine may result in supersaturation through the pH shift mechanism.81, 82 More specifically, for drugs delivered in LBFs the supersaturated state can be induced by the loss of solubilizing capacity during intestinal dispersion83, 84 and digestion.85, 86 LBF-mediated supersaturation may also occur as a result of bile dilution of lipid colloids87 and absorption of lipid-digestion products.28 However, supersaturation through bile dilution may be drug dependent, and most pronounced for basic drugs.88 The augmented awareness that drug supersaturation might be a key

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factor for absorption has resulted in strategies to maintain supersaturation during intestinal formulation processing, for example by addition of polymeric precipitation inhibitors.85, 89

The drug molecules are prone to precipitation once supersaturation has been induced, since transition from the supersaturated high-energy state to the thermodynamically stable state is energetically favored. Traditionally, intestinal precipitation is undesired because it lowers the absorbable drug dose and may lead to unpredictable and erratic absorption. However, recent studies have shown that precipitation is not always detrimental to absorption.90-92 Rather the absorption is dependent on the dissolution capacity of the precipitated solid form. If the precipitate is of amorphous character, it may easily re-dissolve and not hamper absorption as much as if the thermodynamically stable crystalline polymorph precipitates.93, 94 Recognizing the potential impact of solid form on absorption has led to an evolving interest in solid state characterization of the drug precipitate upon in vitro lipolysis. Commonly used techniques for determination of solid form (amorphous or crystalline) of precipitated drug are introduced in the next section.

In vitro lipolysis assay The in vitro performance of an LBF is commonly assessed through lipolysis studies.11, 95 At present, several versions of the experimental setup exist, but in general, it involves a temperature-controlled (37 °C) vessel which contain simulated intestinal fluid (SIF) (e.g. buffer containing BS and phospholipids with a pH of 6.5), a pH meter, and a titration unit (Figure 6).96, 97 The drug-loaded LBF is added into the vessel and, after a period of dispersion (to mimic gastric emptying prior to bile secretion), digestion is initiated by addition of digesting enzymes, commonly pancreatic lipase. Lipid digestion results in liberation of FFAs (Figure 2), which causes the pH to drop in the SIF. The digestion process is pH-sensitive and requires compensation for the liberated FFAs. Compensation of pH is attained by addition of sodium hydroxide (NaOH). An estimate of the rate and extent of digestion is obtained by monitoring pH and addition of equimolar NaOH. Samples are taken throughout the assay and centrifuged to separate the water, oil and pellet phase; the latter being composed of the enzymes and potentially precipitated drug. Drug concentration is analyzed to determine the distribution between the different phases, and thus obtain an estimate of the extent of drug that has remained solubilized.98 Additionally, the pellet phase that contains any precipitated drug can be characterized with various solid state techniques to determine the solid form of the precipitate.99 Commonly adapted characterization methods for lipolysis pellet samples are polarized light microscopy (PLM),93 X-ray diffraction (XRD),85 Fourier Transform Infrared Spectroscopy (FTIR),100 and Raman spectroscopy.101

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Figure 6. General setup of the in vitro lipolysis model used for assessment of drug solubilization, supersaturation and precipitation. Drug-loaded LBF and digestive enzyme are added into the lipolysis vessel, and samples taken throughout the assay. After centrifugation drug distribution between oil, aqueous and pellet phase can be determined.

Lately, the importance of gastric lipolysis has been realized, and as an extension to the standard intestinal in vitro lipolysis model an additional gastric step has been proposed.102, 103 However, because it is not a commercially available product, the use of gastric lipase is currently restricted, and in the in vitro assay, the low gastric pH leads to technical difficulties with the automatic titration due to unionized FFAs.104 Nevertheless, the combined model provides an improved reflection of the total GI lipid processing, which might be essential for improved IVIVC. Recently, it was shown that the emulsion structures, as a result of gastric lipolysis, impacts the intestinal lipolysis rate.105

In vitro in vivo correlation The complex relationship between intestinal formulation processing and absorption of drug and lipid products complicates the establishment of robust IVIVC for LBFs. The possibility to perform predictive in vitro tests is a prerequisite for rational formulation development, and to this end several studies have investigated the IVIVC of LBF performance. In some cases, a rank order correlation was established between in vitro lipolysis performance and in vivo drug exposure.47, 106-108 However, no correlation was apparent in several other studies.103, 109-112 These findings suggests that there is a need for improved understanding of the dynamic digestion, as well as a refinement of the in vitro lipolysis assay. The standard in vitro assay consists of a one-compartment setup (Figure 6), and thus lacks an absorptive sink.

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The absence of such an absorptive compartment has been proposed to be one of the main reasons for the poor in vivo predictions.10, 111, 113 In a one- compartment model, free drug is not transported away from solution, as would be the case for highly lipophilic, freely permeating compounds in vivo. This may cause much higher supersaturation levels in the in vitro lipolysis experiment, resulting in drug precipitation that would not occur in vivo.

Beyond the lack of an absorptive sink, there are other factors which may limit the predictability of in vivo lipid formulation performance. As mentioned above, inclusion of a gastric step may be necessary to provide predictive in vitro testing. If drug precipitation is occurring during in vitro lipolysis, additional solid state characterization of the pellet phase may help convey whether drug precipitation will have an impact on absorption or not. Yet another consideration is the selection of animal model to study IVIVC. GI physiology, bile secretion, and enzyme activity are substantially different between species,114-116 and thus species adaptions might be required.117 It should be mentioned that simpler in vitro dispersion assays have also been evaluated as an approach to obtain sound IVIVC of LBFs. The results are scattered, and to date, this approach cannot be considered to provide more reliable results than the dynamic in vitro lipolysis.10

Current limitations and potentials for LBFs LBF development involves a trade-off between maximizing the drug load in the formulation and promoting moderate supersaturation to drive absorption, while at the same time avoiding drug precipitation. Thus, essential factors for the limited use of this delivery strategy are related to both LBF properties and performance. Firstly, the complexity of LBFs with numerous possible combinations of excipients, makes formulation development resource- and time-consuming, and also require significant amount of active pharmaceutical ingredient (API) to be synthesized.57 Secondly, the reduced chemical stability of pre-dissolved drug in LBFs is problematic, specifically for compounds prone to oxidation.118 Finally, the limited understanding of the dynamic interactions between drug, formulation and GI fluid, including drug solubilization, supersaturation, and precipitation results in poor predictions of in vivo performance.59, 111, 119As such, the current formulation approach may lead to development of non-optimized LBFs, which fail to provide improved drug exposure compared to standard formulations.

In view of this, refined in vitro and in silico tools are required for assessing both drug loading capacity and formulation performance. Currently, the majority of research has repeatedly used the same lipophilic model compounds, e.g. danazol, fenofibrate, halofantrine and cinnarizine. For the field to progress, the same limited number of model compounds cannot be used over and over again. The molecular understanding of drug–LBF

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interactions could be increased by expanding the database, which may reveal trends and facilitate establishment of guidelines to identify the suitability of formulating a drug candidate as an LBF. To summarize; to improve the application of LBFs, the existing knowledge gaps must be filled, and concrete guidance for the formulation scientists provided. Hence, an improved molecular understanding of lipid-based drug delivery is warranted at several levels to make an informed formulation development. This has been the central emphasis of this thesis.

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Aims of the thesis

The overall aim of this thesis was to identify molecular characteristics of PWSD that define the potential benefit of LBFs, and through this knowledge develop experimental and computational tools able to assist the formulation development. In the first part of the thesis, drug solubility in lipid vehicles was studied (Paper I-II). Thereafter, biorelevant performance (solubilization, supersaturation and precipitation) of LBFs was studied through in vitro lipolysis experiments (Paper III). The last project focused on LBF-mediated intestinal solubilization, and evaluation of the effect on in vitro and in vivo absorption (Paper IV). The specific aims were to:

Identify physicochemical properties and molecular descriptors of PWSD

linked to solubility in LBF excipients and composed formulations.

Develop experimental and computational tools to enable rational experimental screening and computational prediction of drug loading capacity in LBFs.

Study supersaturation and precipitation behavior of drugs formulated in

LBFs when subjected to in vitro lipolysis to explore the relationship between the solid form and drug inherent properties.

Assess the intestinal drug solubilization effect, and the resulting effect on

absorption, of a weak base from a low dose of LBF. Explore whether drug pre-dissolved in LBF and drug coadministered with

lipids produce comparable absorption in vitro and in vivo.

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Methods

Model compounds The compound data sets in Paper I-IV were selected to include potential candidates for LBFs. As such, the studies include orally delivered BCS class 2 compounds with a diverse range in structure and physicochemical properties. To specifically target lipophilic compounds, which could benefit from being formulated in an LBF, a specific inclusion criterion was also applied: all studied compounds have a logP > 2. Additionally, only compounds in their free form were included, i.e. no salt forms. In Paper I and II comprehensive drug data sets were studied to gather sufficient data for in silico QSPR development of drug solubility in excipients and drug loading capacity in LBFs. For Paper III a smaller number of compounds were selected to study in vitro formulation performance. In Paper IV, one model compound was evaluated for the impact of LBFs on absorption in an in vivo dog study.

Studied excipients and formulations All studied excipients were selected because they are commonly used in LBFs, and include examples of the major classes of oils, surfactants and cosolvents used in the LFCS.38, 98 An overview of the excipient properties are provided in Table 2, and their molecular structures are depicted in Figure 7. In Paper I Soybean oil, Captex 355, Polysorbate 80, and PEG400 were investigated for their solvation capacity, and in Paper II the excipient data set was extended with Maisine 35:1, Capmul MCM EP, and Carbitol. These excipients were then used to compose different types of LBFs, representative of the LFCS, studied in Paper II-IV.

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Table 2. Overview of the oils, surfactants and cosolvents in Paper I-IV.* Name Excipient

type Mw

(g/mol) Description

Soybean oil LCTG 873 FAs: 10% palmitic acid (C16:0), 7% linolenic acid (C18:3), 53% linoleic acid (C18:2), 25% oleic acid (18:1), 5% steric acid (18:0)

Maisine 35:1 LCMIX 490

Blend of: 45% MG, 46% DG, 9% TG. FAs: 11% palmitic acid (C16:0), 1% linolenic acid (C18:3), 55% linoleic acid (C18:2), 31% oleic acid (18:1), 2% steric acid (18:0)

Captex 355 MCTG 505 FAs: 59% caprylic acid (C8:0) and 41% capric acid (C10:0)

Capmul MCM EP MCMIX 248 Blend of: 60% MG, 35% DG, 5% TG. FAs: 84% caprylic acid (C8:0), 16% capric acid (C10:0)

Cremophor EL/ELP Surfactant 2424 Polyethoxylated castor oil, HLB 12–14 Polysorbate 80 Surfactant 1310 Polyoxyethylenesorbitan monooleate, HLB 15

Carbitol Cosolvent 134 Diethylene glycol monoethyl ether

PEG400 Cosolvent 400 Poly(ethylene glycol) *Long-chain triglyceride (LCTG), long-chain mixed mono-, di-, triglyceride (LCMIX), medium-chain triglyceride (MCTG), medium-chain mixed mono-, di-, triglyceride (MCMIX). Molecular weight (Mw) is the average weight as stated in the product specification, or calculated from specified constituents. Source of excipients, Sigma: Soybean oil, Cremophor EL/ELP, Poly-sorbate 80, Carbitol and PEG400, Gattefossé: Maisine 35:1, Abitec: Captex 355 and Capmul MCM EP.

Cremophor EL and ELP changed trade name during the course of this thesis work, and are currently called Kolliphor EL and ELP. Carbitol is also referred to as Transcutol in the scientific literature, since that is the tradename for diethylene glycol monoethyl ether by another producer. The same batches of excipients were used for all solubility determinations in Paper I-II, and all excipients were stored under argon gas to minimize water uptake. Prior to any experiments, excipients and LBFs were preheated to 37 °C, except for Maisine 35:1 which was heated to 70 °C (as recommended by manufacturer). This was done to facilitate handling of the viscous excipients and to obtain homogenous mixing. After heating, the excipients LBFs were prepared by weighing the ingredients into vials in predefined fractions (% w/w).

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Figure 7. Molecular structures of LBF excipients studied in Paper I-IV. The oils are represented by the glycerol backbone, and the FA composition for each oil is pro-vided in Table 2.

In vitro methods

Solubility determinations The experimental solubility values in excipients and LBFs were determined by a small-scale shake-flask method. Briefly, an excess of API was added into 300-700 mg of excipient or LBF in a test tube, and placed on a plate shaker in an incubator at 37 °C. After 24, 48, and 72 h (or longer if required) the vials were centrifuged at 37 °C, 2800 g for 30 min, to separate solid drug from the solution. The supernatant was diluted with methanol (MC glycerides, surfactants, cosolvents) or isopropanol (LC glycerides), and analyzed with UV-plate reader, HPLC-UV, or LC-MS/MS, depending on drug concentration and excipient UV interference. The same solubility assay with minor adjustments was applied to determine drug solubility in SIF media and dog intestinal fluid (DIF). Excess of drug was added into 300-900 ul of medium. After ~2 h of incubation (37 °C) the pH of the SIF samples was measured and adjusted back to 6.5 (pH used at in vitro lipolysis) if needed. For the DIF samples, the pH was measured after ~20 h of incubation (37 °C). At predetermined time points, the samples were centrifuged, supernatant was diluted in organic solvent and analyzed with HPLC-UV or LC-MS/MS,

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depending on the sensitivity needed. Analytical details for each analysis technique and drug are specified in the respective paper.

In vitro lipolysis assay The in vitro performance of selected model compounds and LBFs were studied in Paper III using the in vitro lipolysis assay, as described in the introduction. In vitro lipolysis was performed with both drug-loaded LBF and placebo-LBF (no drug added). The SIF media from the lipolysis of placebo-LBF was used to determine drug concentration (as described in Solubility determinations), with the purpose of evaluating time-dependent supersaturation potential and the probability of drug precipitation.86, 120 The measured drug concentration was applied to calculate dose number (Do) (Equation 2):

/ Eq. 2

where M0 is the dose of the compound (mg), V0 is the volume (ml), and Cs is the equilibrium solubility in the medium used (mg/ml).121 In Paper III, a Do > 5 was targeted to increase the probability of drug supersaturation and precipitation during lipid digestion. This level was selected because previous studies have observed lipolysis-triggered precipitation at concentrations around 3-fold greater than the solubility.86, 120 The pellet samples were characterized for solid form of the drug precipitate with PLM and Raman spectroscopy, as described in the coming section.

In vitro lipolysis permeation assay The in vitro lipolysis with an absorption compartment was carried out in a newly developed lipolysis-permeation setup at 37 °C (Figure 8).122 The device consists of two compartments separated by a Caco-2 cell monolayer, where the upper chamber is used to perform digestion studies and the lower chamber is for determining drug permeation. The digestion experiment was performed similarly to the regular in vitro lipolysis assay, with minor modifications. The LBFs were digested with immobilized lipase, which has been proven compatible with the Caco-2 cell monolayer.123 The receiver chamber contained HBSS supplemented with 4% bovine serum albumin (235.4 ml, pH 7.4). Samples were withdrawn from both chambers at predefined time points, centrifuged, diluted, and analyzed with HPLC-UV (donor samples) and LC-MS/MS (receiver samples). Loss of integrity results in mixing of the digestion (pH 6.5) and receiver media (pH 7.4), and hence, a stable pH in the digestion chamber (pH 6.5) was used as an in situ marker for membrane

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integrity. Samples withdrawn 15 min prior to a pH increase in the digestion chamber were discarded, to be certain that the flux was measured over an intact cell monolayer.122

Cell culture Caco-2 cells (American Type Culture Collection, VA, USA) were cultivated in an atmosphere of 90% air and 10% CO2, as described previously.124 For the permeation membrane, Caco-2 cells (passage 95 to 105) were seeded on permeable, polycarbonate filter supports (0.45 µm pore size, 75-mm diameter; Transwell Costar, Sigma-Aldrich) at a density of 170,000 cells/cm2. Monolayers were used for experiments between day 21 and 24 after seeding.

Figure 8. Schematic drawing of the two-compartment in vitro lipolysis-permeation setup, which enables evaluation of drug absorption in the presence of LBF. In this study, a Caco-2 cell monolayer was used. However, other types of membranes, artificial or cell-based, could be applied.

Solid state characterization techniques

Differential scanning calorimetry Differential scanning calorimetry (DSC) (TA Instrument Co., USA) was applied to verify crystallinity and purity, as well as to determine melting point (Tm) and enthalpy of fusion (ΔHf) for all drug compounds (as received by manufacturer). In Paper III, DSC was also applied to verify the conversion from the crystalline to the amorphous form. Samples of 1-5 mg were weighed into an aluminium pan and sealed with an aluminium lid containing pin holes.

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Conventional DSC was applied for the analysis,125 and the thermal analysis started with equilibration at 0 °C, after which the sample was heated 10 °C/min to 20–30 °C above the expected Tm. From the resulting thermogram the onset of melting was used to report the Tm.

Polarized light microscopy In Paper III, the solid form of the precipitated drug after 60 min of in vitro lipolysis was characterized using PLM (Olympus BX51, Olympus, Japan), equipped with crossed polarizing filters. At termination of the lipolysis experiment, the pellet was transferred to a microscope slide and images recorded. The microscopy technique uses birefringence to indicate crystalline material and visualize the crystal structure; crystals are visible as glowing particles, whereas amorphous material is non-visible.126

Raman spectroscopy In addition to the PLM analysis in Paper III, the precipitated drug was investigated with Raman spectroscopy (Rxn-2 Hybrid, Kaiser Optical System Inc., USA). A fiber-optic PhAT probe with a laser wavelength of 785 nm, and laser power of 400 mW was used. All spectra were monitored in the range 100–1890 cm–1, baseline corrected by standard normal variate, and a reference spectra without drug was used to correct for the background of the pellet samples (e.g., pancreatic enzyme). The vibrational Raman spectra provides a fingerprint that is unique to each compound, and differences can be seen between hydrate forms, crystal forms, or between the crystalline and amorphous form.127

In vivo experiments A Labrador dog model was used to evaluate the effect of a small amount of coadministred LBF on drug absorption and intestinal solubilization (Paper IV). The study was conducted at AstraZeneca R&D (Mölndal, Sweden), and approved by the local ethics committee for animal research (no: 34-2015). The weak base carvedilol was chosen as the model compound (Figure 9A), and the selected LBF was a long-chain IIIA (IIIA-LC) (Figure 9B). The in vivo dog study was divided into part I and II. In the first part, DIF was sampled through permanent duodenal stomas,128 after oral administration of placebo LBF (1 g and 2 g, respectively) and water. The sampled DIF was analyzed for digestion products (total concentration of FFA), BA, and carvedilol solubility (as described in Solubility determinations). The carvedilol DIF solubility was compared to carvedilol SIF solubility after lipolysis with the standard porcine pancreatic enzyme98 and the immobilized enzyme.129 In Part II, 25 mg carvedilol was formulated in four different ways: (F1) pre-dissolved in 1 g LBF, (F2) coadministred with 1 g LBF, (F3) coadministred with 0.5 g LBF,

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and (F4) as a micronized powder. F1-F4 were orally administered in hard gelatin capsules to the dogs at four separate occasions. Plasma samples were withdrawn between 0-24 hours post administration. The area under the curve (AUC) from the concentration-time profile, maximum concentration (Cmax), and the relative bioavailability (Frel) as compared to formulation F4 (i.e. the micronized powder) were calculated. The acquired data was used to evaluate the IVIVC between the in vitro lipolysis-permeation and dog plasma exposure of carvedilol.

Figure 9. (A) The model compound carvedilol and (B) composition of the type IIIA-LC LBF used in the in vivo dog study (Paper IV).

In silico methods

Quantitative structure–property relationships Multivariate data analysis (MVA) was applied to demonstrate diversity of the data set, and to relate measured physicochemical properties and calculated molecular descriptors to solubility in LBF excipients. All statistical modeling was carried out with Simca (Umetrics, Sweden), version 13.0 (Paper I and II) and version 15.0 (Paper III).

Molecular descriptors To calculate molecular descriptors, SMILES strings were retrieved for all compounds. Corina (Molecular networks, Erlangen, Germany) was applied to convert the molecules into three-dimensional structures. The 3D molecules were used as input for calculation of molecular descriptors with DragonX (Talete, Italy) for the data sets in Paper I (version 1.4) and Paper II (version 6.0.16). The generated molecular descriptors give 1D, 2D and 3D information about the drug molecules, such as size-correlated properties, charge characteristics, topological information, and lipophilicity. To improve the accuracy of the excipient models, a number of experimentally determined solid state related properties commonly available during early drug development were added to the descriptor matrix. These were Tm (Paper I and II), enthalpy and entropy of fusion, and ideal solubility at 37 °C (Paper II).

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The ideal solubility has previously been identified to be closely related to solubility of crystalline organic nonelectrolytes in aqueous systems,130 and was therefore included as a descriptor.

Model development All molecular descriptors were blinded to avoid selection bias, and skewed descriptors (skewness > |1.5|) were removed to avoid weighting the model. In Paper II, an additional step to reduce the number of strongly linearly correlated (≥ |0.9|) descriptors prior to variable selection was performed with an R-script (R, 3.2.0, Vienna, Austria). The remaining descriptors were mean centered and scaled to unity of variance prior to further analysis. Principal component analysis (PCA) was performed to assess the diversity of the studied data sets. In Paper I and II, the score plot of the PCA was also applied to validate that the selected test sets covered the drug space of the training sets. Outliers identified in the PCA and the distance-to-the-model-of-X (DModX) plot were moved to the test set to avoid distortions of the model. The solubility in the logarithm form of mol compound/mol excipient was used as the response value. The average molecular weights used for calculations of mol excipient are provided in Table 2.

In Paper I and II, projection to latent structure (PLS) models were developed to link physicochemical properties and structural features to solubility in excipients. The variable selection procedure followed a previously published protocol,131, 132 with successive variable removal to reduce the model complexity, facilitate model interpretability and robustness. The first exclusion step removed all variables except the 100 most important for the response. Thereafter, additional variables were removed based on the variable importance to projection (VIP) and the loading plot, and monitored by the leave-one-out (using 7 groups), and cross-validated R2 (Q2). If the exclusion of a variable had no effect or increased Q2, it was permanently excluded from the model. The variable selection procedure continued until no further descriptors could be removed without a drop in Q2. The accuracy of the models was assessed by R2 and root-mean square error of the estimate (RMSEE). Furthermore, based on the obtained PLS results for Soybean oil and Captex 355 (Paper I) the applicability of multiple linear regression (MLR) (Microsoft Office Professional Plus 2010) based on a few easily calculated and transparent molecular descriptors (e.g. number of double bounds, hydrogen acceptors/donors, double bonds) was investigated.

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In Paper II, the predicted excipient solubility from the PLS models was applied to calculate loading capacity of the LBFs by multiplying the predicted solubility with the fraction of each excipient in the final formulation (Equation 3).

Eq. 3

where SLBF is the total drug loading in the LBF, which is equal to the sum of the solubility in the pure excipient (Se), multiplied by the weight fraction of that excipient in the formulation (We). This approach was adapted after the log-linear model developed for water/cosolvent systems.71, 72 However, here the weighted mean is used instead of the weighted geometric mean of solubil-ity. An overview of the methodology in Paper II is visualized in Figure 10.

Figure 10. Schematic drawing of the applied methodology for prediction of drug loading capacity in LBFs. PLS models were developed based on experimental drug solubility in excipients and molecular descriptors. The predicted drug excipient solubility was then applied to calculate the drug loading capacity of the LBFs.

Additionally, in Paper III, projections to latent structures discriminant analysis (PLS-DA) was used to identify trends between solid form of the drug precipitate and physicochemical properties. The response variables were categorical, and all compounds classified as either amorphous (A) or crystalline (C). The X-variables were solely drug physicochemical properties previously related to solid form of drug precipitate99 and/or glass-forming ability,133, 134, e.g. ionization, melting point, and molecular weight.

Statistical analysis The experimentally determined data in this thesis is determined in at least triplicates. Statistically significant differences were assessed by unpaired t-test, one-way or two-way ANOVA with post-hoc test as appropriate. Corrections were made for multiple comparisons. Results were deemed significant at p < 0.05.

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Results and Discussion

Data sets (Paper I, II, III and IV) All studied compounds in Paper I-IV were selected due to their potentials as LBF candidates. However, since the projects had different purposes, the selection criteria and number of compounds varied. In Paper I and II, larger data sets were desired for in silico model development, while in Paper III the extensive in vitro experimental work limited the number of compounds. For the in vivo study in Paper IV, a single model compound was selected. The PCA score plot shows the distribution of the studied drug data sets (Paper I-IV), as well as BCS class 2 and 4 compounds marketed as orally delivered LBFs in the chemical space (Figure 11).

Figure 11. PCA score plot of the studied model compounds in Paper I-IV, and BCS class 2 and 4 drugs marketed as orally delivered LBFs. Data for marketed LBFs were adapted from Salva et al.52 The strong outlier is cyclosporine.

Drug solubility in lipid vehicles (Paper I and II) In the first part of this thesis, solubility in lipid vehicles was studied to identify physicochemical properties and molecular descriptors of PWSD linked to solubility in LBF excipients (Paper I) and composed formulations (Paper II). At the start of these projects no comprehensive studies of drug solubility in lipid vehicles was openly available. Thus, the experimental data retrieved during the course of this work for nearly forty compounds in nine different

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excipients represents a unique database of solubility in lipid vehicles. When the experimental work for Paper I started, the determined solubility values for three compounds in Soybean oil and Captex 355 were compared to literature data to verify the accuracy of the solubility assay with minimal amount of excipient (300-700 mg) (Figure 12).

Figure 12. Comparison between the established small-scale method and literature values using larger volumes of excipients. The downscaled method was in agree-ment with literature data and still allowed at least six consecutive samples to be taken for slowly dissolving compounds. Literature data were extracted from Kauko-nen et al.135 The solubility data (37 °C) represent cinnarizine, danazol and halofan-trine solubility in Soybean oil and Captex 355, ranging from low to high glyceride solubility.

From studying a larger drug data set, general trends and experimental tools could be revealed. For example, the overall rank order of drug solubility in lipid excipients was confirmed to be: LCTG < MCTG < surfactant < cosolvent. Additionally, the drug solubility was found to be higher for 32 of the 35 compounds in the mixed mono-, di-, triglycerides (Maisine 35:1 and Capmul MCM EP) than the corresponding triglycerides (Soybean oil and Captex 355, respectively). It was also observed that the drug solubility was frequently twice as high (mg drug/g excipient) in the TGMC (Captex 355) compared to the TCLC (Soybean oil). Their solvation capacity proved to be close to equal when the solubility values were converted into mol drug/mol excipient (R2 0.99). Further investigation into excipient solvation capacity showed a similar relationship between the two mixed glycerides (Maisine 35:1 and Capmul MCM EP, R2 0.89). Recently, drug solubility in different FFAs (C6-C18) was investigated, and in line with the results obtained within this work, the solubility decreased with increasing chain length of the FFAs.136 However, in the same study, it was also shown that the hydrogen bonding ability of the FFAs was important for drug solubilization. Similarly, TG ester concentration (reflecting hydrogen bonding ability) has been related to increased solubility of small molecules,137 rather than, for example, TG chain length.

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Moreover, the solvation capacity of the surfactants and cosolvents were also related (Carbitol compared to: Cremophor EL (R2 0.93), Polysorbate 80 (R2 0.90), PEG400 (R2 0.85)), which is suggested to be linked to the ethoxylation of these excipients. Knowledge about the solubility relationships presented here could aid in reducing the need for solubility screening experiments during LBF formulation development; measured values can be used to estimate the solubility in other related excipients.

Effect of excipient water sorption (Paper I and II) One concern with LBF excipients is water sorption that can affect drug solubility.137, 138 To minimize the water uptake all excipients were stored under argon gas, and the work was carried out using small aliquots from the original bottles. Yet, despite these precautions, water uptake may occur. This was addressed in several different ways throughout the work. First, the water content in PEG400 was determined by Karl Fischer titration (KF Coulometer 831, Metrohm). The amount of water in the PEG400 used in the original study was 0.32% w/w, which can be compared to a freshly opened container that contained 0.11% w/w water.

Figure 13. Assessment of the effect of excipient water sorption on drug solubility. (A) Drug solubility of four model compounds in PEG400 under ambient and dry conditions. For itraconzole, danazol, and indomethacin, no significant difference in solubility between the two conditions was observed, while for candesartan a slight decrease was observed under ambient conditions compared to dry conditions (* significant difference p < 0.05). (B) Correlation between drug solubility for 25 compounds in Cremophor EL (water ≤ 3% w/w) versus Cremophor ELP (water ≤ 0.5% w/w).

Secondly, to address the impact of water sorption on drug solubility, four compounds (itraconazol, candesartan, danazol and indomethacin) were selected for complementary studies under dry conditions. The solubility of the four drugs ranged from low to high in PEG400, and represents neutral, basic

39

and acidic drugs. To obtain an anhydrous milieu, the solubility samples were placed in a desiccator containing phosphopentaoxide (relative humidity ≤ 1.8%), and only sampled once (72 h). The solubility determinations of itraconazol, danazol and indomethacin confirmed the original results (Figure 13A); no significant difference was seen between ambient and dry conditions. The solubility of candesartan was of the same magnitude in both conditions; 13.8 ± 1.5 mg/g in ambient milieu, and 16.0 ± 0.6 mg/g in dry milieu, although still significantly different (p = 0.042). Moreover, drug solubility for a larger number of compounds was studied in Cremophor ELP to evaluate the difference compared to Cremophor EL. Cremophor ELP is a refined version of Cremophor EL that contains less water (Cremophor EL ≤ 3% w/w, Cremo-phor ELP ≤ 0.5% w/w). Here, 14 out of 25 compounds had significantly different solubility (p < 0.05), while still following a similar rank order (Figure 13B). For most compounds the solubility was slightly higher in the excipient containing more water, although some compounds displayed lower solubility with higher water concentration. Similar deviations in drug solubility determined under ambient and dry conditions have been observed in glyceride excipients.138, 139 Drug solubility may increase or decrease with water content in the excipients. As an example, the solubility of progesterone slightly increased in hydrated Soybean oil from 23.5 mg/g to 24.1 mg/g, while in Olive oil the solubility slightly decreased from 23.9 mg/g to 20.9 mg/g.139 Thus, the impact of a small amount of water is both excipient and compound dependent, and careful considerations need to be made when working with such excipients under standard laboratory conditions. With regards to the model development marginal water sorption and solubility deviations, in the same magnitude as observed for candesartan and progesterone, is not expected to distort model development, because it does not shift a low solubility compound into becoming highly soluble and vice versa.

Another concern with the lipid excipients is the batch to batch variability.140, 141 Slight deviations in the FA composition and the concentration of FFAs can have large impacts on the resulting drug solubility, specifically for weak bases.142 To avoid unnecessary variations in the solubility data due to batch variations, the same batches of excipients were used for all solubility determinations in Paper I and II.

Excipient solubility predictions (Paper I and II) In silico predictions allow evaluation of compounds even before they have been synthesized. Thus, they are attractive methods in early drug development and the preformulation stage, when minimal amount of compound is available. Predictive tools for aqueous solubility,143, 144 SIF media,145 and human intestinal fluid3, 145 are available. However, the models developed in this thesis (Paper I and II) are the first to predict drug solubility in a range of

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different LBF excipients. Prior to this work, one QSPR model for prediction of drug solubility in pure PEG400 based on 92 compounds existed, which showed modest accuracy, with an R2 0.71 and RMSETr 0.55.76

The models developed in Paper I and II provided insights into molecular features associated with increased drug solubility in oils, surfactants, and cosolvents. All developed models were based on 2-3 principal components and contained 4-6 molecular descriptors, resulting in statistically strong models (R2 0.81-0.94 and Q2 0.78-0.91) (Paper II). The accuracy of the models improved as descriptors related to the solid state were incorporated. However, no lipophilicity descriptor was found to be important for the predictions in any of the studied vehicles. The PLS models for PS80 and PEG400 in Paper I only allowed qualitative predictions (R2 < 0.62, both vehicles). Thus, for Paper II the following alterations were made; the calculated descriptor matrix was expanded (from ~1000 to ~5000) by using a newer version of DragonX, the drug data set was extended with a few more compounds to further increase structural diversity, and ideal solubility (log Xi

c) was incorporated as an experimental variable. These efforts resulted in PLS models for PS80 and PEG400 with a higher accuracy (R2 0.86, RMSETr 0.22 and R2 0.81, RMSETr 0.27, respectively). The herein developed excipient models have comparable predictive power as similar models for prediction of drug water solubility.146,

147 In addition to the developed PLS models, MLR was applied to allow for

transparent estimations of triglyceride solubility. The equations found to best predict solubility in Soybean oil and Captex 355 were (Equation 4 and 5):

0.19 0.01 0.26 0.21 Eq. 4

0.15 0.01 0.27 0.18 Eq. 5

where Tm is the melting point, nN the number of nitrogens and nDB the number of double bonds (non-aromatic) of the drug compound. These equations have the possibility to be used as a simple first estimation of solvation capacity in triglycerides (Figure 14).

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Figure 14. The relation between experimental triglyceride solubility and calculated solubility through suggested equations (Equation 4 and 5) applied on the full data set (n=37, Paper I and II) in Soybean oil and Captex 355.

LBF loading capacity predictions (Paper II) Predicted excipient solubility from the PLS models was applied to calculate the loading capacity (Equation 3) for nine model compounds in four types of LBFs. Overall, the predicted loading capacity was in agreement with experimental loading capacities of the formulations (R2 of 0.79 all LBFs) (Figure 15). However, the predictions were slightly better for the lipid-rich LBFs (II and IIIA) (R2 0.82) than the formulations with higher quantities of surfactants and cosolvents (IIIB and IV) (R2 0.79). The improved accuracy of the predictions in the lipid-rich formulations was not unexpected, given the performance of the computational lipid models compared to the surfactant and cosolvent models.

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Figure 15. Experimentally determined loading capacity plotted against predicted loading capacity for nine model compounds in four different formulations. Predic-tions were made through the developed excipient PLS models, and the predicted solubility values used to calculate the LBF loading capacity through Equation 3.

Physicochemical properties related to drug solubility in LBF excipients (Paper I and II) The link between LBF excipient solubility and drug physicochemical properties was examined both in Paper I and II. Even though drug candidates suitable for LBFs in general are rather lipophilic (Figure 4), the data gathered herein does not linearly correlate drug solubility in LBF excipients to lipophilicity (represented by logP). This is visualized in Figure 16A and B, where the solubility data in four glycerides, and four ethoxylated surfactants and cosolvents are plotted against logP. However, for compounds with a logP value greater than 5 the solubility drastically drops in oils, surfactants and cosolvents.

The relation between Tm and drug solubility in the studied excipients was also assessed (Figure 16C and D). In all glycerides, the drug solubility decreases above a Tm of 150 °C, but a similar threshold value was not observed for the surfactant and cosolvents for the data set used. Moreover, it was observed that acidic compounds in general displayed lower solubility in the glycerides than bases and neutral compounds. Acidic compounds may be less prone to dissolve in glycerides because of the low fraction FFAs inherently present in such excipients. However, for this particular data set, a further explanation may be that the acids had relatively high Tm.

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Figure 16. Drug solubility in glycerides and ethoxylated excipients compared to logP and Tm. The dotted lines are inserted to visualize the solubility decrease for compounds with logP >5 (glycerides and ethoyxlated excipients) and Tm > 150°C (glycerides). (A) Drug solubility in glycerides versus logP. (B) Drug solubility in ethoyxlated surfactants and cosolvents versus logP. (C) Drug solubility in glycerides versus Tm. (D) Drug solubility in ethoyxlated surfactants and cosolvents versus Tm.

In summary, from the physicochemical analysis of this data set and the most important variables in the model development, it was found that; suitable candidates for lipid-rich LBFs (I-IIIA) would be lipophilic, low-melting, have few polar groups, and have a capacity to distribute charges. Examples meeting these criteria are fenofibrate and perphenazine. In contrast, more “hydrophilic” compounds (lower end of the logP scale investigated) could benefit from formulation in less lipophilic LBFs (IIIB-IV), in which the polar descriptors were unimportant. For solubility in these systems, important descriptors were related to ionization state, electronegativity, size, and shape. Hence, suitable candidates for these formulations are polar but still lipophilic, and low-to-high melting, e.g., diflunisal and niclosamide.

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LBF in vitro performance (Paper III and IV)

Drug solubility in lipolysis medium (Paper III and IV) Maximum drug solubility in the aqueous dispersion and digestion phases was determined after digestion of placebo-LBF for nine compounds; eight compounds in Paper III, and the model compound carvedilol in Paper IV. Overall, the neutral and acidic compounds had higher solubility in the dispersion compared to the digestion medium, regardless of LBF type, whereas an opposite pattern was observed for the weak bases. For the latter, the digestion of the formulations into FFAs significantly boosted the aqueous phase solubility of the basic drugs (cinnarizine, haloperidol, ketoconazole and carvedilol). The rank order of the solubility increase between dispersion and digestion medium reflects the extent of ionization of the weak bases, and thus their attraction to FFAs. In Figure 17A the rank order of the solubility fold-increase in IIIA-LC dispersion and digestion medium is visualized; ketoconazole (×2) < cinnarizine (×6) < carvedilol (x7) < haloperidol (×11). The extent of ionization at pH 6.5 (pH used during in vitro lipolysis) follows the same order for these compounds: ketoconazole (42%) < cinnarizine (90%) < carvedilol (98%) haloperidol (99%). Drug solubilization is known to decrease as the formulation is dispersed and digested in the GI tract.11 However, these results shows that this is not always true for weak bases, for which the solubilization in SIF media is boosted as the formulation is digested. Based on our findings, a formulation strategy for weak bases would be to incorporate specific lipid species (e.g., long-chain lipids) in the formulation, which will boost solubility upon digestion. Thus, the solubilization effect in the GI tract can be prolonged and the risk of drug precipitation minimized.

Figure 17. (A) Solubility fold-increase for the weak bases between IIIA-LC disper-sion and digestion media. The solubility increase is linked to the attraction for released FFAs, and thus follows the extent of ionization of the drug at pH 6.5 (pH during in vitro lipolysis). (B) Liberated ionized FFAs during 60 min of in vitro lipolysis (37 °C) from LBFs; IIIA-LC, IIIB-MC and IV.

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Lipolysis-triggered supersaturation and precipitation (Paper III) In vivo processing of the formulation and the resulting effects on drug solubilization and precipitation are critical for absorption of drugs formulated as LBFs.57 If the drug precipitates during the GI transit, the form of the precipitated drug may impact the potential of the drug to redissolve, where an amorphous precipitate is expected to redissolve at a faster rate than a crystalline material.93 103 Thus, in Paper III, lipolysis-triggered supersaturation and precipitation was investigated to explore the relationship between precipitated solid form and inherent physicochemical properties of the drug.

Formulation IIIB-MC was chosen as the model formulation. This selection was made because among those studied in Paper III, this LBF is digested to the greatest extent (Figure 17B). From the original data set of eight drug compounds, the five compounds displaying a Do >1 (Equation 2) were investigated. However, as suspected from previous studies,85, 86, 120 high levels of supersaturation were necessary for lipolysis-triggered drug precipitation to occur. Therefore the lipolysis medium was spiked with a highly concentrated stock-solution for two of the compounds (indomethacin and griseofulvin), to increase the apparent Do closer to the initially targeted level of ~5. The formulations dispersed readily, and no evidence of drug precipitation was apparent during the initial 10 min of dispersion, despite high supersaturation levels, for all drugs. During the following digestion phase, drug precipitation occurred in all lipolysis experiments. Between 2%-67% of the drug dose had precipitated at 60 min of digestion. However, none of the drugs reached the level of equilibrium solubility, and hence, the drugs were still in a supersaturated state throughout the lipolysis despite significant precipitation. This demonstrates one of the main advantages of delivery of molecularly dispersed drugs in LBFs; attainment of the solvation capacity throughout lipid digestion, previously described as “LBF-mediated prolonged metastable supersaturation”.10 In Figure 18A and B felodipine distribution between aqueous and pellet phase is shown as an example.

Solid state of precipitated drug (Paper III) At the termination of the lipolysis experiment, the isolated pellet material was examined with PLM and Raman spectroscopy. Both techniques could be used to verify the presence of drug crystals, despite minimal amount of precipitated drug in some cases. In Figure 18C and D the solid state characterization of felodipine is shown as an example. For all drugs, crystals were present in the microscopy images. Analysis of the Raman spectra identified that danazol and griseofulvin precipitated in their crystalline form, niclosamide precipitated in a hydrate form, and felodipine and indomethacin precipitates were mixtures of the crystalline and amorphous forms.

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Figure 18. In vitro lipolysis of felodipine with solid state characterization of the precipitated drug after 60 min of digestion. (A) Lipolysis profile of loaded type IIIB-MC. The black dotted line shows the thermodynamic drug solubility at 10 min of dispersion and 60 min of digestion. (B) Felodipine distribution between aqueous and pellet phase. (C) Raman spectra, and (D) PLM graph of the pellet phase contain-ing precipitated felodipine. Pure crystalline and amorphous drug are shown as reference in the Raman spectra.

Physicochemical properties related to drug precipitation behavior (Paper III) The results in our study was combined with data collected under digestive conditions from previous studies.84, 86, 93, 107, 148 MVA, in the form of PLS-DA was applied to elucidate the relationship between solid form of precipitated drug to physicochemical properties and glass-forming ability (as defined by melt quenching).133, 134 This analysis revealed that precipitation in a crystalline form is more likely to occur for compounds with low Mw (<350 g/mol) and high Tm (>200 °C), whereas amorphous precipitate is found for drugs with high Mw and low Tm (Figure 19A and B). All of the drugs that had previously been classified as nonglass-formers (nGF) precipitated in a crystalline form. In the group of compounds classified as glass-formers (GF), five of the

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compounds precipitated in an amorphous form and three in a crystalline form (Figure 19C). Furthermore, as hypothesized in previous works, our analysis strengthened the finding that charged weak bases favor amorphous precipitation.84, 100

It should be noted that this analysis was made on a limited data set (12 compounds), and the analysis therefore provides initial trends of lipolysis-triggered precipitation behavior. Yet, by considering molecular properties and performing a glass-forming classification, an evaluation of the probable solid form (amorphous or crystalline) of the precipitate can be made.

Figure 19. Drug physicochemical properties related to lipolysis-triggered precipita-tion behavior. (A) Molecular weight (Mw), and (B) melting point (Tm) of drugs precipitating in amorphous and crystalline form, respectively. (C) Observed relation between glass-forming classification,133, 134 and lipolysis-triggered precipitation behavior. For the three drugs classified as nonglass-formers (nGF) all precipitated in a crystalline form, while in the group classified as glass-formers (GF), most drugs precipitated in an amorphous form. Halofantrine had not been classified (GF/nGF) in previous studies; thus, the total number of compounds is 11.

In vivo dog study (Paper IV)

LBF-mediated intestinal drug solubilization Although lipid intake is generally thought to trigger BS release, only one study has previously investigated the effect of clinically relevant doses of LBF on bile secretion.149 No study prior to this work had investigated the effect on intestinal drug solubilization from low-dose LBF. The dog model used herein allows continued sampling of intestinal fluid trough duodenal stomas.128 Therefore, it was possible to determine BS concentration, digestion products and drug solubility in the intestinal fluid after oral administration of LBF.

Administration of 2 g LBF significantly increased carvedilol DIF solubility, as compared to administration of only water or 1 g LBF (Figure 20A). The solubility differences observed in the DIF fluid was compared to the levels of FFA (Figure 20B), and BS (Figure 20C). Carvedilol DIF

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solubility followed the same rank order (water ˂ 1 g LBF ˂ 2 g LBF) as both the FFA and BS concentrations. Hence, these data indicate that a clinically relevant dose of LBF (2 g) is enough to trigger bile secretion. As a result of the elevated BS concentration and presence of formulation digestion products, the intestinal solubility of the weak base increased significantly.

Figure 20. After oral administration of water, 1 and 2 g of placebo LBF: (A) Carve-dilol solubility in DIF, (B) total concentration of FFA and (C) total concentration of BS in DIF. The graphs show average concentrations in DIF from three dogs 5-10 min after administration. Administration of 2 g LBF resulted in significantly higher carvedilol DIF solubility compared to 1 g LBF and water, respectively. The increase in carvedilol intestinal solubilization is linked to the increase in FFA and BS concentrations after administration of 2 g LBF. *represents significant difference. ****p < 0.0001; ***p < 0.001, **p < 0.01, and *p < 0.05.

Moreover, it was found that the average carvedilol DIF solubility correlates better with the in vitro lipolysis solubility when the immobilized enzyme was used for the digestion, compared to the standard pancreatic enzyme. Hence, the relatively low digestion capacity of the immobilized enzyme, as compared to the pancreatic extract, more accurately predicted the in vivo DIF solubility of carvedilol in this study.

Coadministration of LBF and drug (Paper IV) One limiting factor with LBFs is the chemical instability of pre-dissolved drug.118 In the dog model this problem was addressed by evaluating coadministration of drug and LBF, compared to pre-dissolved drug in the formulation. Both formulation approaches produced comparable plasma exposure, and there was no difference in relative bioavailability of carvedilol plasma exposure between F1 (coadministred carvedilol with 1 g LBF) and F2 (pre-dissolved carvedilol in 1 g LBF). These results support the use of coadministration of LBF as a potential dosing regimen in cases where it is

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beneficial for the drug to be in a solid form, e.g. due to chemical instability in the lipid vehicle.

In vitro in vivo correlation A newly developed lipolysis-permeation device (Figure 8),122 that enables simultaneous study of digestion and absorption was assessed to evaluate IVIVC of lipid formulations. As opposed to solubilization profiles, drug permeation across the Caco-2 monolayers into the receiver compartment accurately predicted similar in vivo absorption of carvedilol for formulation F1 and F2 in Labrador dogs. These results highlight the importance of capturing the lipid digestion simultaneously with drug absorption to obtain IVIVC for LBFs. Moreover, the lipolysis-permeation device has the potential to aid the current sub-optimal in vitro evaluation, and thus to broaden the understanding of LBF performance.

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Conclusions

This thesis presents an improved molecular understanding of lipid-based drug delivery at several levels. By means of in silico, in vitro and in vivo methods, molecular properties were linked to; increased drug solubility in LBF excipients, solubilization by lipid-digestion products, and precipitation behavior under digestive conditions. This led to experimental and computational tools that can assist in the development of LBF. The possibility for coadministration of LBF instead of pre-dissolved drug was demonstrated, and a promising in vitro lipolysis-permeation device was assessed for its potential to generate IVIVC. The specific conclusions of this thesis are: Melting point is as an important molecular property for drug solubility in

glycerides. This property can provide a first guidance of the dose (high/low) possible to dissolve in LBFs containing large amounts of oils. Drugs with higher melting point might be well solubilized in LBFs containing large quantities of cosolvents and surfactants. The number of double bonds and the number of nitrogens were additional molecular properties with strong impact on solubility in glycerides.

Solubility in one vehicle can be used to estimate solubility in similar excipients. This was demonstrated by the correlation between glycerides and ethoxylated excipients, respectively. It was confirmed that the general rank order of drug solubility in LBF excipients are: long-chain triglyceride < medium-chain triglyceride < surfactant < cosolvent.

Predictive in silico models (PLS) of drug solubility in LBF excipients

were developed. Performance of the models improved by the inclusion of experimental solid state properties. This allowed calculation of loading capacity in composed formulations by multiplying the predicted excipient solubility value with the fraction of the excipient in the LBF.

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Molecular weight, melting point, glass-forming classification, and ioniza-tion are factors associated with the type of solid form that precipitates during lipolysis. These properties provide an initial framework for understanding drug precipitation behavior under digestive conditions.

Solubilization in intestinal fluid of weak bases may significantly increase

when, small but clinically relevant doses, of LBFs are administered. This is a result of the in vivo processing of the LBF excipients and triggered bile secretion.

Coadministration of LBF is a promising alternative to pre-dissolved drug

in LBF. The potential use of this principle could enable use of lipid formulations for drugs that are prone to chemical degradation.

A recently developed device which provides simultaneous assessment of

in vitro lipolysis and absorption showed the same rank order of absorbed drug as observed in the in vivo studies.

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Future perspectives

In this thesis, the first computational models for prediction of drug solubility in lipid excipients and drug loading in lipid-based formulations were developed. Solubility predictions based on molecular descriptors generally performs within 5-10 fold accuracy. Thus, the excipient solubility models presented in this thesis can be used as a first guidance in the LBF development, to assess if a compound will display low, moderate or high solubility in lipid excipients. While these models are developed on a considerably larger number of compounds than are typically included in LBF studies in the scientific literature, the data set is still of medium size, and more data is needed for validation. Additionally, this work has provided improved insight into which molecular properties govern solubility in lipid vehicles and hence, formulations. To further progress the understanding, in silico methods that can better consider specific as well as non-specific interactions between the API and the LBF excipients, and capture the colloidal restructuring upon dispersion and digestion, hold considerable promise. One such computational tool is molecular dynamic (MD) simulations. This type of simulation can be used to study the complex interplay on a molecular level and improve mechanistic insights of lipid-based drug delivery.

The commonly applied in vitro methods makes it difficult to predict the tendency for in vivo supersaturation to occur. In the GI tract, the interplay between dosage form, API and intestinal physiology (fluid composition, enzymatic capacity, permeation through the gut wall, gastric emptying, and transit times) determine the in vivo supersaturation and precipitation. This dynamic environment is difficult to capture through the in vitro methods currently in use. The two-compartment lipolysis-permeation device assessed in this thesis work, allows for digestion and permeation to be studied simultaneously. Thus, this is one step closer to mimicking the complex interplay and provide better IVIVC for drugs delivered in LBFs. More studies are needed to confirm the applicability domain of the lipolysis-permeation chamber. However, this type of in vitro assay still requires a significant amount of API to be available. Hence, scaled-down approaches, using for example a transwell format, could be an alternative approach.

Potential chemical instability of pre-dissolved API in LBF makes this delivery system less attractive to the pharmaceutical industry, because it requires significant resources spent on stability studies. The study herein opens up the potential for coadministration of lipid and drug in separate

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capsules, and thus avoids API-LBF stability issues, while minimizing formulation optimizing efforts. Other promising strategies for improving chemical stability of API in lipid formulations are solidification through adsorption onto mesoporous material or the use of high melting lipids to produce solid formulations that can be processed to form lipid-based multiparticulates. Moreover, the use of LBFs is typically limited to rather lipophilic and low melting drugs, for which the solubility in lipid vehicles is sufficiently high. To address this problem, ionic liquids or lipophilic prodrugs provide new approaches to improve the solubility in lipid systems, and thus expand the applicability of LBFs.

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Populärvetenskaplig sammanfattning

De flesta patienter föredrar att ta läkemedel i tablettform eftersom det är enkelt och smidigt i jämförelse med till exempel en injektion. En förutsättning för att få bra effekt från tabletter är att läkemedlet löser upp sig i tarmen och därmed kan tas upp genom tarmväggen. Tyvärr är många nya läkemedel svårlösliga i tarmvätskan som till största delen består av vatten. Enligt konceptet ”lika löser lika” så löser sig dessa läkemedel ofta bättre i olja. Ett sätt att öka lösligheten och således möjligheten att utveckla dessa läkemedel till fungerande produkter är därför att använda oljebaserade hjälpämnen (lipider). Istället för att göra en tablett av läkemedlet så löses det in i lipider (t.ex. olivolja) och fylls i en kapsel, en så kallad lipidbaserad läkemedelsformulering. För att kunna utveckla lipidbaserade formuleringar krävs det både kunskap om läkemedels löslighet i lipider, samt vad som händer när formulering och läkemedel blandar sig med tarmvätskan. Förståelsen för dessa processer är ännu begränsad och hämmar därför utvecklingen av lipidbaserade formuleringar inom läkemedelsindustrin. Detta avhandlingsarbete har fokuserat på att öka kunskapen om vilka läkemedel som är bäst lämpade för lipidbaserade formuleringar, samt att öka förståelsen för hur dessa beter sig i tarmen.

Ett stort antal kombinationer av läkemedel och hjälpämnen har undersökts för att utröna vilka egenskaper eller funktionella grupper hos läkemedelsmolekylerna som är kopplade till hög respektive låg löslighet i lipider. De experimentella resultaten har använts för att utveckla statistiska beräkningsmodeller som kan förutsäga nya läkemedels löslighet i lipider utifrån läkemedelsmolekylernas struktur. Modellerna kan användas för att uppskatta läkemedels löslighet i lipider redan i ett tidigt utvecklingsskede då de inte är kemiskt framställda. De framtagna modellerna ökar därför utvecklingsbarheten av lipidbaserade formuleringar eftersom de väsentligt minskar de experimentella löslighetsbestämningarna.

För att utvärdera hur lipidformulering och läkemedel beter sig i magtarm-kanalen görs in vitro försök. Det innebär att försöken inte görs i levande djur utan i laboratoriet. I avhandlingsarbetet användes en in vitro modell med vätska som efterliknar tarmvätskan, samt de ämnen som bryter ned lipider till mindre beståndsdelar. In vitro försöken utfördes för att studera om läkemedlen faller ut som fast material när lipidformuleringen bryts ned. Resultaten visade att trots nedbrytning av lipidformuleringen så är läkemedlen till hög grad fortfarande lösta i tarmvätskan. Molekylära funktioner hos

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läkemedelsmolekylerna analyserades och kopplades till utfällningsegenskaper av läkemedelssubstans från lösning. Detta är betydelsefull information för att förstå om den lipidbaserade formuleringen kommer leda till bra absorption av läkemedel i tarmen eller ej.

I det sista projektet utvärderades effekten en liten mängd lipid för absorptionen av läkemedel. Resultatet visade att två kapslar med lipidformulering triggade gallutsöndringen hos hundar vilket ledde till ökad löslighet av läkemedel i tarmvätskan. Vidare undersöktes möjligheten att dosera läkemedel och lipider i separata kapslar. Då visades det att läkemedlet absorberas i lika hög grad om det är inlöst i lipidformuleringen som om det administreras i en egen kapsel. Detta ökar möjligheten att använda lipidbaserade formuleringar för läkemedel som bryts ner vid långtidsförvaring i kapslar med lipider.

Sammantaget har denna avhandling ökat den grundläggande förståelsen för vilka typer av läkemedel som passar för administrering i lipidbaserade formuleringar. De metoder och modeller som presenterats i det här avhandlingsarbetet kan användas för att underlätta beslut under läkemedelsutvecklingsprocessen. Det kan i sin tur leda till effektivare och mindre kostsam utveckling av nya läkemedel.

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Acknowledgements

The work presented in this thesis was carried out at the Department of Pharmacy, Faculty of Pharmacy, Uppsala University. The studies were financially supported by Swedish Research Council and the European research council. The Wallenberg foundation, Letterstedts resestipendium, Anna Maria Lundin’s, and IF’s stiftelse are gratefully acknowledged for the additional financial support, which has given me the opportunity to participate in courses, collaborate abroad and visit conferences. I would like to express my sincere gratitude to all who have contributed, directly or indirectly, to this thesis and especially to: Min handledare Docent Christel Bergström. Först och främst, tack för att du som unik förebild i forskningsvärlden inspirerade mig till att ta mig an doktorandstudierna! I både med- och motgång har du en enastående förmåga att entusiasmera och peppa. Det har alltid fått mig att (emellanåt med ett visst mått av frustration) ge lite mer och försöka en gång till. Jag är tacksam för att du guidat mig till att bli en självständig forskare, i allt från rent praktisk hand-ledning på labbet i hur man gör löslighetsförsök till att slutföra avhandlingen. My co-supervisor professor Christopher Porter, for hosting me in your laboratory at MIPS twice, and thus allowing me learn from your extensive knowledge in lipid-based drug delivery, get to know your research group, and taking part in the Friday research seminars (aka Fat Club). I would also like to thank all my collaborators and co-authors who contributed with data and knowledge. Bertil Abrahamsson for making the dog study pos-sible. Pernilla Johansson and Petra Delavaux, for your experimental help and expertize, and because I appreciated working and chatting with you. Albin and Janneke, for extremely efficient and skilful lab work under a tight deadline. Former and current colleagues in the Drug Delivery group. Thank you for an inspiring and challenging research environment, but most of all for discussions, laughs and pranks in the (often extremely crowded) lab. You have made the work during these years much more fun! Anna and André, I missed your ambitious minds and the Friday experiments during my last years.

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The Bergström group. My first PhD colleagues; Sara and Jonas, for the kind and warm atmosphere in the slightly smaller research group. Then there was an exponential growth of the group, and I have truly enjoyed the benefit of getting so many new colleagues that share my lipid research interest. Thanks to all and every one of you for fruitful discussions, brilliant ideas, and many laughs! Special thanks to Khadijah, Caroline, and Janneke, for sharing my structured and organized side in the lab. Alexandra, for understanding the huge amount of coffee needed to combine research and family. Jamal, for proof reading this thesis. Mina kontorskamrater: Jenny och Magnus, för att ni stått ut med mitt snackande och peppat när det behövts som mest. Laura, som fick dela ett minimalt kontor med en aningen stressad och förvirrad avhandlingsskrivan-desmåbarnsförälder. Emelie och Ilse, hjärtligt tack för att ert kontor alltid stått öppet för samtal om stort som smått. En kopp kaffe och en pratstund med er har gjort många jobbdagar betydligt mycket bättre. Jonas för det artistiska fotot till framsidan och Oliver som är modellen. Tack!

Colleagues and PhD students at MIPS. Special thanks to Orlagh, for being an A-Maisine scientist, and making me realize exactly how much time that should be spent on colouring pretty pictures. Joanne, for sharing my adventure in Melbourne, and literally taking me in under your roof. I miss you! Master students: Angelica Friberg, Elena Diesch, Jenny Johannesson, Daniel Brundel and Paulina Jakubiak, for skillful experimental work. I would also like to thank all PhD-students, post-docs, administrators, teachers past and present not specifically named. Stort tack till mina föräldrar för att ni alltid uppmuntrat och stöttat mig i de val jag gjort och för att ni är världens bästa mormor & morfar. Mina syskon, Daniel och Malin, tack för att ni följde efter mig till Uppsala, det har varit fantastiskt att ha er så nära. Till sist, min underbara och tålmodiga familj. Ni är det finaste jag vet ❤ Oskar, utan ditt enastående lugn hade detta aldrig varit möjligt. Edith, Ruth och Majken för att ni skänker glädje och sätter perspektiv på tillvaron.

Linda Alskär, December, 2018

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 262

Editor: The Dean of the Faculty of Pharmacy

A doctoral dissertation from the Faculty of Pharmacy, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofPharmacy. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Pharmacy”.)

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