Controlled Release Gel Formulations

and

Preclinical Screening of Drug Candidates

Tofeeq Ur-Rehman

Department of Chemistry Umeå University, Sweden Doctoral Thesis 2011

Copyright © Tofeeq Ur-Rehman, 2011 ISBN: 978-91-7459-161-3 Elektronisk version tillgänglig på http://umu.diva-portal.org/ Printed by: VMC-KBC, Umeå University Umeå, Sweden 2011

i

To My parents

ii

Author Tofeeq Ur-Rehman

Title

Controlled Release Gel Formulations and Preclinical Screening of Drug Candidates

Abstract Simple gel formulations may be applied to enhance the systemic and local exposure of potential compounds. The aim of this thesis is the development and characterization of controlled release formulations based on thermo-reversible poloxamer gels, which are suitable for novel drug delivery applications. In particular co-solvents (DMSO, ethanol), mucoadhesive polymers (chitosan, alginate) and salts (sodium tripolyphosphate, CaCl2) have been used to enhance the applications of poloxamer 407 (P407) formulations in preclinical animal studies. The impact of these additives on the micellization and gelation properties of P407 aqueous solutions was studied by calorimetric methods, nuclear magnetic resonance spectroscopy (NMR) and “tube inversion” experiments. The drug release behavior of hydrophobic and hydrophilic drugs was characterized by using a membrane/membrane-free experimental setup. Finally, preliminary pharmacokinetic studies using a mouse model were conducted for screening of selected inhibitors of bacterial type III secretion and for evaluation of different formulations including P407 gel.

All additives, used here, reduced the CMTs (critical micelle temperature) of dilute P407 solutions, with the exception of ethanol. The gelation temperature of concentrated P407 solutions was lowered in the presence of CaCl2, DMSO, TPP and alginate. 1H MAS (Magic Angle Spinning) NMR studies revealed that DMSO influences the hydrophobicity of the PPO segment of P407 polymers. Low concentrations of DMSO did not show any major effect on the drug release from P407 gels and may be used to improve the exposure of lead compounds in poloxamer gels. A newly developed in situ ionotropic gelation of chitosan in combination with TPP in P407 gels showed an enhanced resistance to water and reduced the release rates of model drugs. From preliminary pharmacokinetic studies in mice it was revealed that poloxamer formulations resulted in an increased plasma half-life of the lead compound.

Keywords

Poloxamer, drug delivery, micellization, DMSO, calorimetry, NMR, in situ gelation, chitosan, preclinical pharmacokinetics, type III secretion inhibitors

iii

Table of Contents 

 

Abstract ii Table of Contents iii Preface v List of the Papers vi List of Abbreviations vii Introduction 1 

Drug Discovery and Formulation 1 Controlled Release Formulations and Preclinical Screening 3 In Situ Gels as the Pharmaceutical Dosage Form 4 Poloxamer Gels 5 Polysaccharide Gels 6 Type III Secretion Inhibitors 7 Preclinical Pharmacokinetics 8 

Aim of this Thesis 9 Materials and Methods 10 

Polymers 10 Drugs and Compounds 10 Other Materials 11 Preparation of Poloxamer Solutions 11 Differential Scanning Calorimetry 11 Isothermal Titration Calorimetry 13 Nuclear Magnetic Resonance Spectroscopy 14 Gelation of P407 Solutions 16 Dissolution/Erosion of a Gel 18 In Vitro Drug Release Studies 18 Drug Analysis 19 Solubilization of Salicylidene Acylhydrazides 19 Preparation of Capsules for Rodents 20 Polysaccharide Particles 21 Preclinical Animal Studies 22 

Results and Discussion 23 Characterization of Poloxamer 407 Solutions 23 Co-solvents to Enhance the Loading Capacity of P407 Gels 26 In Situ Gelation to Enhance Residence Time 29 Controlled Release from P407 Gel Formulations 32 In Situ Particle Formation 33 Formulation for Screening of Drug Candidates 35 Parenteral Administration 35 Oral Formulations for Rodents 36 

iv

Preliminary Pharmacokinetics 37 Summary and Outlook 39 Acknowledgements 41 References 43 

v

Preface

This thesis is the result of work carried out at the Department of Chemistry, Umeå University, Sweden. The work consists of two parts: 1) characterization of the sustained release poloxamer formulation and 2) formulation development of potential drug candidates for preclinical animal studies.

Poloxamer gels have the potential of sustained delivery of drugs in multiple applications from local to systemic administration. They are ideal for being loaded – by simple procedures - with drugs of different nature, ranging from small molecules to proteins, and from water soluble to hydrophobic ones. Initially, the main focus was on the biophysical characterization of polymer gels by NMR spectroscopy and calorimetric studies. The main aim of this part was to generate a molecular understanding of the release mechanism and a correct description of the results obtained from the release trials. In the second part of my work, the focus was shifted towards the development of formulations suitable for novel drugs in combination with preclinical studies. Therefore, the impact of different co-solvents, salts and polymers on micellization, gelation and drug release properties of poloxamer gels were investigated. This way valuable information was obtained, suitable to improve drug loading, gel strength and controlled release properties of these gels in a way which extends their delivery application with lead compounds.

As a part of an ongoing interdisciplinary research project at Umeå University, I have been responsible in the development of suitable formulations of newly synthesized lead compounds targeting bacterial type III secretion systems. The screening of potential compounds on the basis of pharmacokinetic profiling and proof of concept efficacy studies in small animals was the aim of the project. The parenteral formulations of selected compounds were optimized for preliminary pharmacokinetic studies. In addition, gel formulations were developed and characterized which would be suitable for ocular, vaginal and oral administration of lead compounds. This work also includes preliminary work on a colon targeted oral capsule for small laboratory animals.

vi

List of the Papers

This thesis is based on the following papers, which are referred to in the text by Roman numerals (I-V). The published papers have been added as reprint at the end of this thesis, with kind permission of the publisher.

I. Effect of DMSO on micellization, gelation and drug release profile of

Poloxamer 407. Ur-Rehman, T., Tavelin, S., Gröbner, G. International Journal of Pharmaceutics (2010) 394, 92-98

II. Chitosan in situ gelation for improved drug loading and retention in

poloxamer 407 gels. Ur-Rehman, T., Tavelin, S., Gröbner, G. International Journal of Pharmaceutics (2011) in press

III. Effect of alginate, ethanol and CaCl2 on micellization and gelation of P407 aqueous solutions. Ur-Rehman, T., Tavelin, S., Gröbner, G. Manuscript

IV. Inhibitors of bacterial type III secretion, evaluation of anti-chlamydial activity and preliminary pharmacokinetics. Ur-Rehman, T., Nordfelth, R., Blomgren, A., Elofsson, M., Gylfe, Å. Manuscript

V. Preliminary pharmacokinetics of the bacterial virulence inhibitor

3,5-dibromo-2-hydroxy-benzylidene nicotinic acid hydrazide. Ur-Rehman, T., Nordfelth, R., Blomgren, A., Elofsson, M., Gylfe, Å. Manuscript

vii

List of Abbreviations

AUC Area under the plasma concentration time curve

AUMC Area under first moment of plasma concentration time curve

CGC Critical gelation concentration

CGT Critical gelation temperature

Cmax Maximum (peak) plasma drug concentration

CMC Critical micellization concentration

CMT Critical micellization temperature

CR Controlled release

CRO Contract research organization

DSC Differential scanning calorimetry

DMSO Dimethylsulfoxide

DMEM Dulbecco’s modified eagle medium

HTS High throughput screening

ICG Indocynine green

IND Investigational new drug

ITC Isothermal titration calorimetry

LC-MS Liquid chromatograpgy-mass spectrometry

LC-MS/MS Liquid chromatography tandem mass spectrometry

MAS Magic angle spinning

MEC Minimum effective concentration

MIC Minimum inhibitory concentration

MTC Minimimum toxic concentration

NCA Noncompartmental analysis

NDA New drug application

NMR Nuclear magnetic resonance spectroscopy

P407 Poloxamer 407

PEO Poly (ethyleneoxide)

PK Pharmacokinetics

ppm Parts per million

PPO Poly (propyleneoxide)

R & D Research & development

RPMI Roswell Park Memorial Institute

t ½ Elimination half-life

T3S Type III secretion

viii

TEM Transmission electron microscopy

TPP Sodium tripolyphosphate

UV-VIS Ultraviolet-visible spectroscopy

1

Introduction

Drug Discovery and Formulation

During the last century, a majority of drugs have been developed by the pharmaceutical industry where new drug development may be divided into four distinctive phases: i) Drug discovery  

ii) Preclinical development  

iii) Clinical development 

iv) Manufacturing and marketing 

The selection of a suitable lead compound (drug candidate) is the goal of the discovery phase. In the context of the drug approval process investigational new drug (IND) and new drug application (NDA) are the goals of developmental stages. It was envisioned that during the 21st century, various organizations would significantly contribute to the pharma industry during the discovery and development of new drugs (Figure 1) [1]. Academic research units are now actively involved in the different stages of drug discovery, including areas such as target identification, hit identification, lead identification and lead optimization.

Traditionally, the formulation studies were conducted during the last three stages of drug development for animal testing, human trials, shelf life stability and product extension. Now, the “pre-preclinical paradigm” is under consideration in the industry where preliminary physicochemical characterization, formulation, pharmacokinetics and toxicity are emphasized for lead selection in parallel to preclinical development [2]. The early in vivo studies aim at the identification of lead structures with the necessary features to select the best lead structures from a range of compounds as early as possible. In both cases, precious resources can be used directly on the candidates which have a better chance as a therapeutic agent in the development phase [3].

High throughput screening (HTS), combinatorial chemistry, and advancements in analytical techniques in the last three decades have made it possible to synthesize large numbers of compounds using limited resources. Current drug discovery methodologies are biased towards more lipophilic compounds which possess low potency [4, 5]. For the accurate assessment of compounds in the drug discovery phase, it is essential to ascertain their aqueous solubility and to identify the appropriate formulations suitable for early animal as well as for in vitro testing [6]. Therefore, the development of

2

safe and tolerable formulations for preclinical pharmacokinetic and efficacy studies can maximize the potential of the vast numbers of novel “hit” compounds generated by these new screening and synthesis approaches.

Figure 1: Drug development strategies in 21st century, adapted from [1].

The limited availability of compounds, limited drug characterization data and the need for rapid results are the main challenges for the formulation development during the discovery phase. Mostly, compounds are formulated rapidly for in vivo testing, without considering the commercial viability of the formulation. Routine formulation screening methods [7], are optimized for large pharmaceutical companies with well resourced R&D organizations. In the work presented here, two concepts - the immediate and

3

sustained/controlled release formulation - are suggested for testing of lead compounds in the discovery phase. Both strategies are suitable for smaller research groups with limited resources.

Controlled Release Formulations and Preclinical Screening

A drug delivery system is a formulation or device that safely brings a therapeutic agent to the specific body site at a certain rate to achieve an effective concentration at the site of drug action. The release of drug in a predesigned manner is termed controlled drug release (CR), which is used to promote therapeutic benefits while minimizing toxic side-effects [8]. Some examples of hypothetical plasma concentration profiles obtained by CR formulations are given in Figure 2. Generally CR formulations have been used for drug candidates during the clinical trials or to extend the life cycle of marketed drugs. Often, a compound with a short half-life, Cmax related toxicity, poor solubility and a narrow therapeutic index is not developed despite its superior potency, selectivity and/or efficacy. Therefore, testing of feasibility and application of controlled release formulations at an earlier stage would not only offer the use of a much wider range of promising lead compounds, but may also accelerate the conversion of certain drug candidates into successful CR products [9, 10].

Figure 2: Hypothetical blood plasma concentration of a drug displaying a sawtooth type profile after repeated dosing (□) a constant controlled release over long period (○) and controlled release triggered by environment (▲) (adapted from [8]). The concentration between minimum effective concentration (MEC) and minimum toxic concentration (MTC) is termed as therapeutic range. During the in vivo studies, the application of CR formulations can reduce animal stress caused by restraints, handling and repeated dosing. Moreover

Level of toxicity

Dru

g le

vel

Time

Subtherapeutic level

4

it can save money and time. A precise dose of compounds may be administered in a convenient way with CR formulations as compared to repeated administration via water or food [11]. For compounds with a short half-life, repeated dosing is required to maintain the plasma concentration above the MEC / MIC and in this process Cmax may fluctuate above MTC. CR formulations would be beneficial to flatten the plasma drug concentration curve of such compounds in the case of systemic administration or to enhance the contact time in the case of topical administration.

In Situ Gels as the Pharmaceutical Dosage Form

A gel is a soft, solid or solid-like material which consists of at least two components, one of them being a liquid, present in substantial quantity [12]. Gels combine the cohesive properties of solids and the diffusive transport characteristics of liquids. Hydrogels are three dimensional, cross-linked networks of water soluble polymers which can retain large amounts of water. In gels, the polymer network is formed by the cross-linking of polymer chains either by the formation of covalent bonds (chemical cross-linking) or non-covalent bonds (physical cross-linking). Due to the toxicity concerns, there is increasing interest in physically crosslinked gels. Non-covalent bonds may be formed by hydrogen bridge linkage, counter ion interaction, crystallization or physical entanglements. [13, 14]. A gel can prolong the drug contact at the site of administration due to its rheological and mucoadhesive properties as compared to an aqueous solution. The gels also possess a broad application spectrum and can be applied in almost every route of administration. Oral, ophthalmic, rectal, transdermal, subcutaneous and vaginal gels are available for different pharmaceutical applications. In the discovery phase, the gel formulations can be used to enhance the local and systemic exposure of potential lead compounds; which is ideal to establish animal models for various conditions quickly and cost efficiently.

Despite the huge diversity of gels, a special class of gels namely smart polymer gels are in the focus of pharmaceutical research during the last decades. These smart polymers change their physicochemical properties in response to an altered environment. In situ gel formation of drug delivery systems can be defined as a liquid formulation generating a solid or semi-solid depot after administration. The impact of external stimuli such as temperature, pH and ionic strength, on the cross-linking of polymer chains have been studied to improve the gel strength or to induce in situ gelation. Poly (N-isopropyl acrylamide) and poloxamer solutions undergo sol-gel phase transition at critical temperature. Poly (acrylic acid), Poly (methacrylic acid) and chitosan form pH-responsive gels [15-17].

5

Poloxamer Gels

Poloxamers also known as Pluronics® are a family of ABA block copolymers, where a hydrophobic poly (propyleneoxide) (PPO) block is sandwiched between two hydrophilic poly (ethyleneoxide) (PEO) blocks. The general structural formula of a poloxamer is ExPyEx, where x and y denote the number of ethylene oxide and propylene oxide monomers per block. In general, poloxamers behave like nonionic surfactants due to the nature of their block units. In certain solvents like water, these polymers form various structures, ranging from micellar to gel-like features, depending upon the length of the polymer subunits, concentration, and the temperature (Figure 3). Solubility of PPO block in aqueous solution is reduced at elevated temperatures and the core-shell type of micelles are formed when the concentration and temperature rise above the CMC (critical micellization concentration) and CMT (critical micellization temperature) [18]. The relatively dehydrated core of the micelle facilitates the incorporation of hydrophobic drugs in aqueous poloxamer solutions. The concentrated aqueous solutions of poloxamer form gels above the CGC (critical gelation concentration), when the temperature is raised above the CGT (critical gelation concentration). The gelation is due to physical entanglement and packing of the micellar structures [19, 20]. This is a reversible process and below the CGT these gels remain fluid, a behaviour which can be advantageously exploited for administration to the smaller orifices.

Poloxamers have been studied for drug and gene delivery applications due to their biocompatibility and low toxicity [21-23]. Poloxamer 407 (P407) is an important member with nominal weight of 12600, and 70% of a hydrophilic block (Figure 3). P407 has been approved as a formulation adjuvant in oral solutions, ophthalmic solutions, periodontal gels and topical emulsions. P407 gels have also been evaluated for ocular periodontal, dermal vaginal, subcutaneous, nasal, rectal and intratumoral delivery [24-30]. In this work, poloxamer based formulations are characterized for preclinical applications. These are cheap and can be produced without expensive laboratory equipment.

6

Figure 3: A Schematic representation of micellization and gelation of aqueous P407 solutions.

Polysaccharide Gels

Polysaccharides are polymers of monosaccharides, which have large number of reactive groups. Chitosan is a positively charged polysaccharide, while alginate and pectins represent negatively charged polysaccharides. The general molecular structures of these polymers are shown in Figure 4. Chitosan, alginate and pectin are polysaccharides which are frequently used in different drug delivery applications due to the mild preparatory conditions and their biocompatible nature. As a natural biomaterial, these are highly stable, safe, non-toxic, hydrophilic and biodegradable [31]. Gels based on such polyelectrolytes may be cross-linked using ionic interactions and are called ionotropic gels. Alginate and pectin form ionotropic gels with multivalent cations such as Ca2+, whereas chitosan may be crosslinked with poly-anions such as sodium tripolyphosphate (TPP). By tuning cross linking conditions, either the gel or the particulate formulation (from nanoparticle to bead) can be produced [31]. These polymers are frequently used as a matrix for the encapsulation of living cells and the controlled release of drugs and proteins. Most of the ionotropic gels degrade under physiological conditions e.g., calcium alginate gel can be destabilized by the extraction of Ca2+ ions using chelating agents and pectin is enzymatically degraded by the microflora in the colon [13]. Chitosan and alginate are mucoadhesive and

7

their in situ gelation has been proposed to enhance the residence time of poloxamer gels (Paper II and III). The polysaccharides were also used to develop colon specific oral formulations (capsules and microparticles) for rodents.

Figure 4: Molecular structures of polysaccharides used in the study.

Type III Secretion Inhibitors

Antibacterial drugs with novel mechanisms of action are needed since bacterial resistance mechanisms rapidly adapt to cover novel compounds in old antibiotic classes. Antibiotic resistance is a growing global problem limiting the alternatives for infection treatment and causing increased mortality in bacterial infections. Type III secretion (T3S) is a virulence system found in several pathogenic Gram- negative bacteria such as Yersinia spp., Pseudomonas aeruginosa, Salmonella Typhimurium, Chlamydia spp., enterohemorrhagic Escherichia coli (EHEC) and Shigella spp. [32]. T3S enables these bacteria to secrete and inject pathogenic proteins from their cytoplasm into the cytosol of eukaryotic host cells. These proteins are capable of interfering with the eukaryotic signaling pathway which results in the disarmament of the host immune response. By inhibiting T3S and suppressing the virulence of pathogenic bacteria, the commensal flora is left unaffected which would reduce the risk of the development of antimicrobial resistance [33, 34].

Inhibitors of bacterial Type III secretion (T3S) are promising drug candidates for the treatment of bacterial infections. Salicylidene acylhydrazides are the most thoroughly studied T3S inhibitors which were identified using as screening based strategy (Figure 6). This compound class has been shown to inhibit type III secretion in Yersinia, Salmonella, Shigella, enterohemorrhagic Eschericha coli, and Chlamydia [35-40]. This work also includes formulations for preclinical testing of these compounds against different bacteria in animal models.

8

Preclinical Pharmacokinetics

Pharmacokinetics is a branch of pharmacology which deals with the study of the time course of drug concentration in different body media such as plasma, blood, urine, cerebrospinal fluid and tissues [41]. More specifically it can be defined as the study of the kinetics of absorption, distribution, metabolism and excretion (ADME) of a drug (Figure 5). In vitro cultures of cells/enzymes systems and in vivo animal models are used to define the pharmacological profile of a compound. During the development phase animal studies are used to evaluate the pharmacologic effects of the agent on specific organ system. These studies are followed by studies using animal models of the human diseases for which the compound is considered to be active. Mostly preclinical animal testing is done on small animals (usually rodents) and pharmacokinetics/pharmacodynamics relationship is used to support drug discovery and to interpret toxicokinetic experiments.

Figure 5: A schematic representation showing the entrance and elimination of a drug from the central compartment (adapted from [41]).

It is of particular interest to estimate the PK behaviour of a drug candidate as early as possible so that the most promising compounds may be selected for further development. In vivo animal pharmacokinetic screening is necessary to assess the clearance and half-life in preclinical species. Pharmacokinetics allows the identification of the limiting features of a drug candidate in relation to the desired route of administration. Combining with in vitro data, pharmacokinetic data enable the identification of compound liabilities, which will provide the basis of structural modification, or change in development strategy [42]. Pharmacokinetic studies of selected compounds require the identification of formulation for small animals, quantification of the compounds in plasma or whole blood, and the determination of pharmacokinetic parameters.

BloodExtravascular dosee.g. oral, IP, SC, IM

AbsorptionDistribution Tissue

IV dose

9

Aim of this Thesis

The overall aim of this work is the development, characterization and application of simple and economical formulations (for both immediate and controlled release), suitable in the selection and optimization process of lead compounds. The research conducted in this thesis can be divided into two main parts.

In the first part, the overall objective was to improve the use and application range of poloxamer based drug delivery systems by using specific additives and to study the effect of these additives on the physicochemical and drug release properties of poloxamer solutions. The specific objectives were:

To study the effect of co-solvents such as DMSO and ethanol on the

micellization, gelation and release properties of P407 based drug delivery systems (Paper I, III).

To study the effect of mucoadhesive polymers (chitosan and alginate) and specific salts (TPP and CaCl2) on the CMT and CGT of poloxamer based formulations (Papers II and III).

To investigate the effect of in situ gelation of chitosan and alginate on the dissolution and drug release profile of P407 gels (Papers II).

To study in situ particle formation properties of chitosan and alginate in P407 gels (Papers II).

In the second part, the general objective was the identification of formulation approaches and concepts, suitable for testing of potential lead compounds on small animals. The specific objectives were:

Improving solubilization of compounds for animal studies. In vitro characterization of simple controlled release formulations,

suitable for screening of potential drug candidates in early animal studies (Papers I, II and III).

Identifying the tolerance level of poloxamer gels in rodents after chronic dosing (Paper V).

Preclinical screening of lead compounds on basis of PK parameters like elimination half-life (Paper IV).

Preparation of colon targeting oral capsules for rodents.

10

Materials and Methods

Polymers

P407 (Pluronic F127, culture-tested, manufactured by BASF, USA) with an average molecular weight of 12600 Da, low molecular weight chitosan (50000-190000 Da, 75-85% deacetylated) and sodium alginate (80000-12000 Da, medium viscosity) were purchased from Sigma (Sweden). Classic (AU 901, AM 901, AU 701) and amidated Pectin (AF020, AU020, CF 020) were generously provided by Herbstreith & Fox (Germany).

Drugs and Compounds

Flufenamic acid and doxycycline hyclate were purchased from Sigma (Sweden); metoprolol tartrate was a gift from Astra Zeneca (Sweden). Type III secretion inhibitors, shown in Figure 6, were synthesized in the laboratory of Mikael Elofsson (Department of Chemistry, Umeå University, Sweden) [40, 43], and INP0341 was provided by Creative Antibiotics, Sweden.

Figure 6: Molecular structures of salicylene acylhydrazides, a class of type III secretion inhibitors (from M. Elofsson).

11

Other Materials

TPP, CaCl2, glycerol, DMSO, acetic acid, citric acid, ethanol, propylene glycol, NaOH, NaH2PO4, glycerol and Na2HPO4 were all of analytical grade. DMSO (99.5%) was from Fluka (Sweden), D2O (99.9%) from Sigma (USA), DMSO-d6 (99.8%) from Euriso-top (UK). Hydroxy propyl β-cyclodextrin and Indocynine green (ICG) and RPMI (cell culture media) were purchase from Sigma (Sweden), whereas cell culture media, DMEM (Dulbecco’s modified eagle medium) was purchase from Invitrogen. The water used in all the experiments was deionized and filtered (Millipore, USA).

Preparation of Poloxamer Solutions

Poloxamer solutions were prepared according to the “cold method” first described by Schmolka [44]. The main advantage of this “cold method” compared to procedures using elevated temperatures, is the suitability for thermo-labile drugs, which can be incorporated at any preparation stage. Briefly, a weighed amount of P407 was added to water or an aqueous solution which contained additives. For micellization studies, P407 solutions of low concentrations were prepared by diluting a stock solution with defined amounts of an additive directly or an aqueous solution containing a specific additive. For gelation, dissolution and release studies, gels formulations were prepared on a weight by weight basis (w/w).

Differential Scanning Calorimetry (Papers I, II and III)

A differential scanning calorimeter measures the apparent specific heat of a system as a function of temperature. Differential scanning calorimetry (DSC) is used to examine a heat induced phase transition or conformational change of a system [45]. As seen in Figure 7, a DSC instrument contains two identical cells (named the reference and the sample cell) suspended in an adiabatic jacket and connected by various heating and temperature sensing circuits. The sample cell is filled with the solution (buffer or water) containing the polymeric substances (P407 in our case) and additives of interest while the reference cell usually is filled with the solution without additives. Normally, the temperature in these cells is gradually increased or decreased (between 0 and 110 °C for the aqueous solution). If there is a heat induced endothermic transition in the sample during this change in temperature, the temperature of the sample cell will fall below that of the reference cell, which is detected. An additional electric power is supplied to the sample cell in order to compensate, which is proportional to the energy associated with the thermally induced transition. An opposite power control is maintained in case of exothermic transitions in the sample cell. The

12

temperature at which the excess heat capacity is at maximum defines the transition temperature of the sample. With this setup DSC is a non-invasive method.

Figure 7: A schematic representation of differential scanning calorimetry.

The temperature at which micelles appear in a poloxamer solution of given concentration is termed CMT. Due to the strong dependence of the CMC on the temperature, both CMC and CMT are used to characterize the micellization in poloxamer solution and DSC has been previously for this purpose [46].

Calorimetric experiments were performed on a Microcal VP-DSC microcalorimeter (MicroCal USA) equipped with VP Viewer software. For analyses of all samples the reference cell was filled with pure water, the pressure adjusted to 15 psi and thermograms acquired by applying heating and cooling cycles between 2 and 65 °C at temperature scan rates of 90, 60, 30, 15 or 5 °C/h. The specific chosen rate was depending on the used concentration of the test solution, except for precise determinations of the gelation temperature of highly concentrated solutions (when a rate of 0.5 °C/hour was applied using a very narrow temperature interval). The pre/post scan equilibration time was set to 5 minutes. All the data were analyzed using Microcal Origin software which was supplied with the DSC equipment.

13

Isothermal Titration Calorimetry (Paper III)

Most physical and chemical processes may be accompanied by a gain or release of heat. Isothermal titration calorimetry (ITC) directly measures the heat evolved or absorbed as a result of mixing of two solutions at constant temperature. It can provide the thermodynamic parameter for non-covalent interactions of two molecules, such as enzyme-ligand, protein-protein, and surfactant-membrane interactions [47, 48].

Figure 8: A schematic diagram of isothermal titration calorimetry device.

Like DSC a reference and a sample cell are enclosed in an adiabatic jacket and the temperature difference between reference and sample cell is measured and calibrated to differential power (DP) or feedback power (Figure 8). The reference cell is filled with water or the buffer, while the sample cell contains one of the compounds to be tested for interactions. Repeated injection of the second component (3-15 µL) by a syringe into the sample cell produces heat effects that are due to stirring and dilution of the compounds, dilution and the heat of occurring interactions. The amount of power that must be applied to actively compensate for heat produced in the sample cell, after an injection of the second compound is measured directly. ITC is used mostly for binding/affinity assays; however this technique is also used to determine the CMC of surfactants and polymers [49, 50]. In this work ITC was used to investigate the CMC of P407 aqueous solution at different temperatures and the effect of additive (such as CaCl2) on this property. P407 solution (with or without CaCl2) was loaded in the syringe and the sample cell was filled with water/Cacl2 solution. After injection of micellar P407 solution, ITC produced an exothermic thermogram which is

Reference cell  Sample cell 

Adiabatic jacket

Injectorwith stirrer

HeaterHeater

F.B

14

due to stirring, demicellization and dilution (micelle and monomer). By integrating the raw data and fitting to a sigmoidal curve is used to determine the CMC of small surfactans, which is also applied for P407 solutions (Figure 9).

Figure 9: Typical ITC data with repeated injection of P407 solution into water at 30○C, raw data (upper panel) and integrated heat data (lower panel).

Nuclear Magnetic Resonance Spectroscopy (Paper I)

In recent decades, nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful tool to provide not only structural and dynamic information of molecules at atomic level but also to provide unique diagnostic information in medicine ranging from images to metabolic profiling of intact tissues and body fluid [51, 52].

The technique relies on the property of various nuclei which have a nuclear spin quantum number (I) greater than 0. 1H, 13C, 19F and 31P, possess a spin I=1/2, and are the most common used nuclei for applications of NMR in life science. If a sample is inserted into an outer magnetic field (B0) the magnetic

0 10 0 200 3 00 40 0 500 6 00 7 002

4

6

8

1 0

Tim e (m in )

DP

(µ

ca

l/s

ec

)

0 . 0 0 0 . 0 5 0 . 1 0 0 . 1 5 0 . 2 0 0 . 2 5

- 5 5

- 5 0

- 4 5

- 4 0

- 3 5

- 3 0

- 2 5

- 2 0

- 1 5

M o l a r R a t i o

dH

(

kc

al

/m

ol

e)

15

moments of these nuclei will possess different, but discrete orientations. These different orientations are characterized by different discrete energy levels of the nucleus. Therefore a nucleus such as a proton which has a spin of ½, can adopt two possible magnetic quantum states; m=1/2 (low energy state, α) and m=-1/2 (high energy state, β) with the corresponding energy expressed as:

E = γ ћB0mI

Where γ is the gyromagnetic ratio, ћ is the Planck constant/2л and m1 is the spin quantum number of a specific nucleus. This process is accompanied by a precession of the magnetic moments around the outer magnetic field. The frequency of this precession is called the Larmor frequency (), whose size depends on B0 and the γ of the specific nucleus, and reflects the energy difference between the two states as follows:

E= νh and ν/2=-γBo

Due to this energy difference between the states, there will be a population difference causing a macroscopic magnetization of the sample in the magnet. In an NMR experiment, by applying radio frequency pulses at this frequency (), an absorption of energy occurs. Applying the pulse along the x-axis (the outer magnetic field parallel to z-axis) will cause the macroscopic magnetization vector to move away from its z-direction and a transverse magnetization (maximum upon application of a 900 pulse) can be detected in a typical NMR experiment. This transverse magnetization induces an electric current in a detection coil and can be registered as Free Induction Decay (FID) signal. Fourier-Transformation will convert this signal into an NMR spectrum, where intensities are plotted as a function of frequencies [53, 54].

In a typical liquid state NMR spectrum these resonances have line shapes which are very sharp due to the averaging of all magnetic interactions except the isotropic chemical shift and the J-coupling. However, due to the missing averaging processes, a solid state NMR spectrum consists of very broad and overlapping line shapes which make it difficult to extract information. Andrew and Lowe developed a method to suppress dipole-dipole coupling in solid state NMR which is known as magic angle spinning (MAS) NMR [55-57]. All the anisotropic features of a solid state NMR spectrum can be removed by spinning the sample around an axis where axis of rotation is tilted to a specific angle (54.70) with reference to the external magnetic field and solution-like spectra with sharp resonances at their isotropic chemical

16

shift values is obtained (indicating the location of a nucleus in a specific chemical environment). When a solid sample is placed in special ceramic rotor, spinning frequencies up to over 100 kHz are feasible.

Different chemical environments (and therefore different electronic environments), produce a type of shielding effect against the outer magnetic field which cause the protons to have slightly different Larmor frequencies and therefore different chemical shift values. Chemical shifts are a function of the nucleus (such as hydrogen) and its local chemical environment, which provides detailed information about the molecular segment, where this specific nucleus is located. Therefore it is possible to obtain structural information about a molecule. The frequency shifts are extremely small in comparison to the fundamental NMR frequency and is represented in ppm (relative to a reference).

However, NMR spectra often do not only contain resonances at different frequencies but also a phenomenon called J-coupling which describes the splitting up of a specific resonance in multiple lines. This phenomenon also called spin-spin coupling is based on the coupling of the intrinsic angular moments of different nuclei in a molecule via chemical bonds, causing a splitting in their respective energy levels. The number of generated energy levels depends on the type and number of chemically bonded neighboring nuclei. The size of splitting of the resonance lines is described by a coupling constant J, and is independent of the outer magnetic field and is measured in Hertz units. The aggregation of poloxamers in aqueous solutions has been studied by using different techniques of NMR spectroscopy such as self diffusion, chemical shift and spin relaxation [58-60]. Here, temperature dependent 1H MAS NMR experiments were performed using a CMX 100 instrument (Varian, USA) operating at 100.13 MHz (Paper I). The MAS rate was kept at ca. 1700-1800 Hz and all NMR acquisitions started after sample equilibration for at least 10 minutes at the required temperature (± 0.5 °C). The acquired data were processed using the Spinsight software (Varian, USA). The water peak at 294 K was used as an external reference (4.796 ppm) for the chemical shift determinations of the NMR peaks in all samples.

Gelation of P407 Solutions

The determination of CGT is important in the development of the gel formulations, which otherwise are characterized by rheological studies. The gelation temperatures of the formulations of P407 used here were determined by the “Visual Tube Inversion Method” with minor

17

modifications [61, 62]. Briefly, two glass vials of 13 mm diameter, one containing 1 g of sample and the other 1 ml of water, were placed in a water bath. In the water filled vial a thermo-couple of a digital thermometer (Fluke, USA) was placed. The temperature was slowly increased and the temperature at which the solution in the sample containing vial stopped flowing upon tilting was noted as the gelation temperature (t1). Similarly, the temperature of the water bath was lowered and the temperature, at which the gel started flowing, was noted as the gelation temperature (t2). The average of both temperatures as mean±sd of t1 and t2, is then defined as the critical gelation temperature (CGT). The CGT of different poloxamer solutions is shown in Figure 10.

Figure 10: The critical gelation temperature of aqueous P407 solutions in the presence of DMSO and DMEM media.

The viscosity of a gel is an important parameter in the physicochemical characterization of gel formulations and is normally studied by flow or oscillatory measurements [63]. It is recommended that the gel formulations intended for parenteral administration should be characterized in term of syringeability and injectability [64]. Syringeability and injectability of poloxamer formulations at room temperature was assured using 23-27G needles. The gelation time (at 36-37°C), gelation temperature and injectability and of poloxamer solution was assured prior to animal study (Paper II).

18

Dissolution/Erosion of a Gel

The interaction of the administered poloxamer gel with body fluids like tears, extracellular fluid, vaginal fluid etc. will result in gel dissolution. Dissolution profiles of the different P407 gels in aqueous environments were determined by a gravimetric method [65]. For this purpose water and a pre-weighed glass vial of 13 mm diameter containing gel was equilibrated at 37 °C. The water was layered over the gel and at pre-determined time intervals the liquid medium was removed, the vial was re-weighed and the weight of the dissolved gel was calculated from the difference in the weight of the vial.

In Vitro Drug Release Studies (Papers I, II and III)

In vitro drug release studies serve as a comparative tool during the formulation development. Drug release from poloxamer gel into the release medium is regulated by the diffusion of the drug and/or dissolution of the poloxamer gel, depending on the experimental conditions. The diffusion of the drug from the gel into the release media is the predominant mechanism of drug release in an experimental setup where the poloxamer gel is confined within a permeable membrane (with pores blocking the poloxamer molecules) or in the cases where a non-aqueous release medium (e.g isopropyl myristate) is used instead of an aqueous release medium. The diffusion experiments are usually done when gels are intended for transdermal, subcutaneous or intramuscular applications [22]. Polycarbonate membrane models are commonly used in studies of drug permeability across epithelial cell monolayers [66]. The experimental setup based on polycarbonate membrane inserts was applied with the intention to simulate the conditions where release media have restricted access to the gel (Figure 11). A membrane-free experimental setup is preferred for in vitro release studies when gels are intended for ocular, rectal or vaginal applications. In this case, release of drug is predominantly controlled by erosion/dissolution of the gel. In both models the conditions do not match the in vivo conditions perfectly. Nevertheless, the use of suitable release media (like phosphate buffer, water), maintaining the physiological temperature (37 ○C) and agitation (20 rpm) was assured to simulate the in vivo environments. Metoprolol tartrate, doxycycline and flufenamic acid were used as model drugs which possess different physicochemical and pharmacological spectra.

19

Figure 11: The experimental setup with permeable membrane inserts for in vitro drug release studies.

Drug Analysis

UV-VIS absorption spectroscopy was used for the determination of the concentration of a drug in the release medium. The linear calibration equations were obtained with standard solution using Cary 5000 UV-VIS spectrophotometer (Varian). LC-MS/MS (Liquid chromatography tandem mass spectrometry) analysis was conducted for determination of compounds in plasma/whole blood.

Solubilization of Salicylidene Acylhydrazides (Papers IV, V)

During the discovery phase, the main purpose of a formulation is to solubilize the maximum amount of compound in a buffer suitable for animal studies [67]. We were also aiming on maintaining the compound solubilized at a 15 µM to 20 mM concentration range for in vivo testing. Some solubilization strategies have been reported in the literature with pH adjustment, preparation of salts, use of co-solvent/ surfactant, or by reducing the particle size. For compounds with polar groups pH adjustment is a very simple and biologically suitable one [68, 69]. Under the restriction of limited resources and time, we truncated and modified the flow chart of the solubilization as shown in Figure 12. If pH adjustment did not produce the required solubilization, co-solvents (ethanol, propylene glycol, PEG 400 along with DMSO), surfactants (Tween 80, Tween 20, P407) or cyclodextrin (hydroxypropyl β-cyclodextrin) were used. The solubility of ME0052 in different buffers was also determined by shaking methods and the stability

20

of the compounds in the formulations was assured by measuring the molar mass by LCMS (liquid chromatography-mass spectrometry).

Figure 12: A Solubilization enhancement scheme for lead compounds in drug discovery.

Preparation of Capsules for Rodents

Oral administration to small animals (mice and rats) is mostly done in liquid formulations through oral gavages. Solid dosages for oral administration to rodents are scarce and only size 9 capsules are available for preclinical studies in rats (e.g., Torpac® and Capsugel®). The gelatin capsules are also under trial for preclinical studies in mice (Torpac®). In this work a novel cone-cup design is introduced instead of the conventional body-cap design and we have prepared small capsules of pectin, chitosan using the dipping - quenching method [70, 71]. A scheme for preparation of pectin capsules is shown in Figure 13. Briefly, a home-made mold was dipped in pectin/alginate/chitosan solution while rotating, after few seconds it was dipped in CaCl2/TPP solution and air dried. The dried cone was removed and cut to size. To check the fate of capsule in the gastrointestinal tract (GIT), the capsules were placed in a stirring HCl solution (pH 1.5) for 2 hours.

P

0.1MNaOH

P

S

1MNaOH

P

S

Dissolve in PBS

Formulation

P

P

Stock solutionin DMSO

Dilute with PBS

PP

S

CPB (10)

S

CPB (5) P

S

CPB (8) P

S

CPB (10)CS 20%

S

AS  (2) 

S

P AS (5)

S

P

CPB (3) 

S

P

Dilute with PBS and adjust pH

Compound

S

CPB (3)CS 20%

S

P

AS (2‐5)+CS 20%

S

P

Dilute with PBS and adjust pH

BS (9‐12)+CS20%

S

S:     solubleP: Precipitate CS: co‐solventCD:  cyclodextrinsAS:  acidic solution (pH)BS:  Basic solution (pH)PBS: Phosphate buffer  salineCPB: citrate‐phosphate buffer (pH)

CS1 5%, CS2 10%, CS3 40%  CS + Surf +PBS /CS+ CD and PBS

21

Figure 13: Scheme for preparation of a polysaccharide capsule (upper panel) and beads (lower panel).

Polysaccharide Particles (Papers II and III)

Polysaccharide particles are commonly propagated for colon specific delivery of drugs. Pectin, alginate and chitosan are not digested in the upper GIT and normal flora present in the large intestine take part in their enzymatic degradation. In this work particles of pectin and alginate were prepared by ionotropic gelation using Ca2+ ions [72]. Briefly, a pectin/alginate solution with/without dye (ICG) was dropped through a needle into to CaCl2 solution, where polymer droplets turn into beads (Figure 13). The beads were air dried/freeze dried. A modification of the previously published method [73] was employed for chitosan nanoparticle formation, and both, simple mixing and in situ double syringe mixing were applied. The size distribution of the particles was determined by a Zeta sizer, nano-S (Malvern industries); Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to characterize the nanoparticle/microparticles.

22

Preclinical Animal Studies (Papers IV and V)

Preclinical PK studies with different formulations of selective compounds were conducted in mice. The tolerance of ME0052 and P407 gels was also monitored after chronic dosing. BALB/c mice (9-12 week old) were kept on normal diet before/during study, and were held according to permission by the departmental ethical committee. Drug formulations were administered parenterally (intraperitoneal, intravenous and subcutaneous) and at a predetermined time interval blood samples (20 µL) were collected from the tail vein into heparinized capillary tubes. After the last sample the mice were euthanized by exsanguinations under anesthesia. Blood samples were fortified with EDTA and centrifuged (1000x g for 10 minutes) to get plasma. For whole blood analysis, the samples were diluted 3 times with hearin containing water. All plasma and whole blood samples were stored at -20 ◦C before analysis.

Cassette dosing involves the simultaneous administration of several compounds to a single animal followed by rapid sample analysis by LC-MS/MS. The cassette dosing approach was applied for screening of seven compounds. The important PK parameters are elimination half-life, area under the plasma concentration curve, volume of distribution, maximum residence time, clearance and bioavailability. Non compartment analysis (NCA) of plasma/whole blood concentration was performed using PKSolver a Microsoft Excel add-in program [74]. AUC0-t and AUMC0-t (area under the zero and first moment curves from 0 to a last sampling time) were calculated using linear trapezoidal method. The terminal elimination slope (λz) was estimated using regression with largest R2 on the last points (3-4) but prior to Cmax. The compounds which showed better PK profile such as t½ and Cmax

after cassette dosing were also administered individually at high dose.

23

Results and Discussion

Characterization of Poloxamer 407 Solutions

In this study the poloxamer P407 was used as the core component in the development of aqueous thermo-reversible gel formulations. Depending on the application and route of administration, these formulations need specific additional substances to provide the optimal conditions (e.g., solubility, isotonicity and pH). In the work presented here, we therefore studied the impact of various promising additives on the basic physicochemical properties of P407 based aqueous formulations and their drug release behavior by using physicochemical and pharmaceutical methods.

Most drug delivery applications using P407 solutions are merely based on their ability to form gels at physiological temperatures; a process which depends on the packing of micelles (Figure 3). Before describing the effect of additives on P407 systems, a short description will be given on the effect of P407 concentration on the micellization, gelation and release characteristics of P407 solutions. Micellization of these systems is usually characterized by CMC, CMT, aggregation number and size of micelles. These parameters can be determined by different techniques including surface tension measurement, spectroscopy, light scattering or calorimetry analysis [18, 20, 75-77]. DSC provides a non-invasive approach for the precise determination of the CMT of P407 solutions. Dehydration of the PPO block of P407 polymer occurs at elevated temperatures and is the driving force behind the micelle formation of the PEO-PPO-PEO copolymers; a process visible as an endothermic peak in the DSC thermogram [46, 78]. Characteristic DSC profiles of 1.26% and 22% P407 solutions are displayed in Figure 14. The Tonset indicates the temperature at which micelles start to form and Tendset is the temperature at which the micellization process is completed. Tpeak is the temperature which denotes the maximum heat to be adsorbed by the system and the area under the peak reflects the enthalpy change [18]. In general, the micellization can be characterized by Tonset, Tendset, Tpeak or the area under the peak [62] [79], Tpeak is used as the definition of the CMT of P407 solutions in this work here. As shown in Figure 13, the CMT is moving towards lower values for higher poloxamer concentrations, while the corresponding peak area in the DSC thermogram increases; an observation in good agreement with previous reports [78, 80].

For concentrated P407 solutions a second small peak is also observed at the high temperature shoulder of the micellization peak (21.7-22.8°C in case of a 22% P407 solution) as seen in Figure 14. This peak can be seen as an

24

indication of a transition from a micellar to a gel-like system [79]. To distinguish this small peak from the micellization peak, samples were equilibrated just prior to the onset of this peak and scanned by DSC at a very slow scanning rate of 0.5°C/h (inset in Figure 12). It was, however, difficult to study the effect of additives on this minor peak by DSC methods due to difficulties in filling the sample cell using higher P407 concentrations. Therefore, the CGTs of different poloxamer solutions were measured by the tube inversion method. In general, the poloxamer solution forms gel above the CGC of around 16% with the CGT of P407 solutions being reduced upon increasing concentrations of poloxamer [60]. A similar trend was observed during this work (Figure 15). The CGT of a highly concentrated solution (> 20%) drops at around room temperature, which restricts its use due to the difficulty in syringeability and injectability. To understand the mechanism of the gelation of a poloxamer solution different mechanisms have been proposed such as ordering of micelles to form a cubic crystalline structure, dehydration of PPO core, or micellar entanglements [19, 20]

Figure 14: Differential Scanning Calorimetry profiles of aqueous P407 solutions: Thermograms are shown for 22% (black line) and 1.26% aqueous solutions (red line). Temperature points used for the determination of CMT are indicated. The small endothermic peak (blue line) is indicative of a gelation process (adapted from Paper I)

0 20 40 60

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Tonset

Tendset

1.26%

22%

Tpeak

Tgelation

Cp

(cal

/oC

)

Temperature (oC)

25

Figure 15: The effect of P407 concentration on the critical micellization temperature (○) and the critical gelation temperature (□) of P407 aqueous solutions, as determined by differential scanning calorimetry and tube inversion method.

The CMC of P407 has been reported previously at different temperatures but there is lack of CMC data at physiological temperatures. We aimed to investigate the effect of additives on CMC of P407 at 37°C by applying ITC. We spent a considerable time with ITC studies, but despite repeated attempts results were not reproducible. Different values of CMC of P407 (at 37°C) were obtained due to poor fitting. Many ITC curves could not be fitted using a sigmoidal curve, whereas reasonable sigmoidal pattern was observed in few (Figure 9). The calculated CMC of P407 solutions at at 30°C was 51 µM which dropped to 0.4 µM at 37°C. Recently Bouchemal et al. have reported the same problem in estimation of CMC of P407 solutions at higher temperatures [81]. Instead of sigmoid fitting, they have applied another approach, wherein start transition (ST) and end transition (ET) are noted manually from the curve. The effect of CaCl2 on CMC of P407 at 30°C was studied and it was observed that it reduces to 11 µM in presence of 0.2 M CaCl2 (Figure 3c Paper III).

0 5 10 15 20 2512

16

20

24

28

Tem

per

atu

re 0 C

P407 Conc (wt%)

CGT CMT

26

Co-solvents to Enhance the Loading Capacity of P407 Gels

During the discovery phase often only little data is available regarding the basic properties of newly synthesized compounds with the majority of them having poor aqueous solubility. Although micelle formation in P407 solutions supports the solubilizing process of hydrophobic compounds, this process is mostly not sufficient for loading of the required high dose of drugs into P407 solutions. However, most of the compounds discovered during the screening process have good solubility in DMSO, which is used as a kind of “universal solvent” for compounds, and also in vitro cell culture assays. Ethanol and propylene glycol are other mild co-solvents which have low toxicity and can be used in parenteral, oral formulations [68]. DMSO, ethanol and propylene glycol are therefore commonly used as co-solvents to enhance the solubility of lead compounds during the formulation development. The phase diagram of P407 aqueous solutions and different co-solvents has been reported previously [82]. Here, we found that the increasing concentration of ethanol resulted in a slightly increased CMT value, accompanied by a dramatic reduction in the area under the micellization peak of 1mM P407 solution (Figure 16); in agreement with previous studies [61]. Kwon et al. have studied the effect of methanol, ethanol, propanol and butanol on the gelation properties of P407 solutions and reported that gelation temperature of 20% P407 solutions is shifted to higher temperatures in the presence of methanol and ethanol. They further reported that higher concentration of ethanol (>30%) strongly affect the gel formation in poloxamer solution [61].

Figure 16: Thermograms of 1mM poloxamer 407 aqueous solutions containing increasing amounts of a) DMSO or b) ethanol. The decrease in CMT correlates with the presence of DMSO. In the presence of ethanol this pronounced correlation does not exist.

27

This effect may be exploited for preparation of gels at room temperature while loading a high dose of the compounds which have higher solubility in ethanol [76]. The effect of ethanol in presence of CaCl2 on CMT (Figure 19) and dissolution of P407 aqueous solution (Figure 5 Paper III)

The Effect of DMSO on micellization, gelation and drug release properties of P407 solution was not reported earlier, and therefore studied in detail as discussed in Paper I. From the DSC studies it was revealed that DMSO reduces the Tonset, CMT, Tendset and the area under the peak of dilute P407 solutions (s. Figure 16).

NMR spectroscopy has been used for the characterization of block polymers [83]. To obtain any insight into the possible behaviour of P407 solutions prior and upon the presence of DMSO as co-solvent, temperature dependent 1H MAS NMR experiments were carried out. A typical 1H MAS NMR spectrum of 1mM (1.26%) P407 solution is shown in Figure 4 in paper I, which was recorded at 294 K, below the CMT (300K). In general, the fast intra- and intermolecular dynamics of this system is reflected in sharp NMR resonances for the methylene and methyl groups, with only the CH2-CH units giving broadened NMR lines, in agreement with earlier studies [84]. The methyl group of the PPO segments resonates at 1.1 ppm and displays a J-coupling of 5.6 Hz due to the coupling with an adjacent proton. Interestingly, these methyl proton resonances remain unchanged at all temperatures below the CMT for 1.26% P407 solution.

But upon increase in temperature above the CMT, the methyl NMR lines broaden and the J-coupling diminishes. Both observations indicate a type of exchange process at the kHz time scale or the occurrence of a reduced motional freedom due to micelle formation. Similar observations were also made for P407 solutions at various concentrations (see Figure 17 for 20% solutions), where changes in the NMR spectra occurred upon passing the concentration dependent CMT temperature. The NMR spectra clearly indicate, that high concentrations of DMSO induce a broadening of the methyl NMR resonances originating from P407 at room temperature; a behaviour similar to the one observed upon increase in the temperature of DMSO-free aqueous polymer solutions (see Figure 4, Paper I). Presumably, this behaviour reflects the ability of DMSO to disrupt water solvation structures around the PPO block as it occurs upon an increase in temperature [85-88]. This NMR approach also allows for the study of the behavior of the different P407 molecular segments during temperature-induced phase changes, at a molecular level.

28

Another objective of the work presented here, was to investigate the gelation phenomenon in concentrated solutions (20%) as a function of co-additives such as DMSO, ethanol, CaCl2 and the model drug flufenamic acid. The 1H MAS NMR spectra of 20% P407 solution at different temperatures in D2O prior and upon incorporation of flufenamic acid are displayed in Figure 17. It is apparent that there is no effect on the lineshape of the methyl protons around CGT. We did not observe any transitional change in the lineshape, chemical shift of resonance of the PEO group at this concentration.

Figure 17: 1H MAS NMR spectra of Poloxamer 407 aqueous solution, effect of increasing temperature on methyl NMR signal of 20% P407 in a) D2O and b) flufenamic acid containing solution. The CMT of 20% P407 solutions was determined as 14.1 0C.

In general, P407 solutions form gel above the CGC (ca. 16%) with the CGT of P407 solutions being reduced upon increasing concentrations of poloxamer [60]. As shown by us, the presence of 5% DMSO, lowered the CGT values compared to DMSO free solutions of different P407 concentrations (Figure 5, Paper I). Moreover, P407 aqueous solution (20%) containing 15% of DMSO had a CGT of around 12oC. But 15% P407 solution did not undergo any gelation under the same conditions. From our gelation experiments one could reveal that 5% of DMSO may safely be added to P407 solutions intended for parenteral formulation while higher concentrations of DMSO surely hamper the injectability and syringeability of the formulation specifically for small animals, besides the DMSO induced toxicity. However for local treatment of burns and in transdermal applications higher concentration of DMSO may be used in P407 solutions for drug solubilization and maintaining the CGT below room temperature.

29

Apart from the influence of DMSC on micellization and gelation, it also has an impact on the dissolution and release profile of P407 gels. Here, we found that low concentrations of DMSO have minor effects on the dissolution of gels and the release of model drugs (see also Paper I for more details).

In Situ Gelation to Enhance Residence Time

As the gelation in poloxamer solutions is due to weak cross-links (physical entanglements or crystallization of micelles), interactions with physiological fluids result in the reduction of the poloxamer concentration below the CGC (at physiological temperature). As the viscosity of the poloxamer solutions is directly proportional to the concentration, the dilution leads to erosion and dissolution of the gel. There have been numerous attempts made to enhance the strength of the poloxamer gel either by increasing the viscosity of the solution or by introducing covalent cross-linking. The residence time of the formulation at the site of administration may also be enhanced by incorporating mucoadhesive polymers like carbophil, sodium alginate, polycarbophil, carageenan, or chitosan [89-96]. Chitosan and alginate are two of the most studied mucoadhesive polymers which due to their attractive properties are often used in oral delivery formulation. Due to the cationic nature of chitosan, it is able to form ionotropic gels in the presence of anions (such as TPP). The viscosity of chitosan solutions can also be enhanced by higher pH values due to a reduction in solubility. In a similar way, alginates are anionic in nature and form gel structures with divalent cations (such as Ca2+). A schematic representation of the ionotropic gelation process for chitosan and alginate is shown in Figure 18. In this work, our goal was to enhance the residence of P407 gels by the incorporation of chitosan (alginate) by an in situ gelation process by using a double syringe system (Paper II and III). However it is also important to ascertain the effect of both polymers (chitosan and alginate) and salts (TPP and CaCl2) on the micellization, gelation and dissolution properties of P407 solutions produced this way. DSC studies of dilute solutions of P407 containing chitosan and alginate revealed that P407 gels undergo micellization (which is the initial step in gel formation) at lower temperatures (see Paper II, III).

The effect of an increasing chitosan concentration on CGTs of 17, 18 and 20% P407 solutions is presented in Figure 3 of Paper 2. The addition of 0.2% chitosan caused a slight increase in the CGT relative to that observed for pure aqueous P407 solutions. Most likely, this moderate increase is caused by the acidic nature of the formulation, as previously mentioned [30, 97]. The addition of increasing amounts of chitosan to this acidic P407 solution resulted in a slight decrease in CGT. Most practically, up to 1% of chitosan and sodium alginate may be used in formulations, while higher

30

concentrations would make the proper handling of these formulations difficult at room temperature.

Figure 18: In situ gelation of mucoadhesive polymers for improvement of the residence time of P407 gels. a) Cross-linking in chitosan solution with anion, reprinted with permission from ref [98], b) Schematic representation of in situ gelation of polysaccharide and P407 with a double syringe system (adapted from Paper II).

Figure 19: The effect of different additives on CMT of P407 solutions.

The effect of both TPP and CaCl2 on the CMT of P407 solutions was also investigated along with NaCl (Paper II and III). In the DSC thermograms a typical endothermic peak of P407 solutions appeared at lower CMTs in presence of all the salts. It has been reported previously that salts like NaCl and CaCl2 induce micellization at a lower temperature (Figure 19) due to the slating out effect, and the reduction in the solubility of the PPO block [46, 99]. Both TPP and CaCl2 also showed the CGT lowering effect on P407

0 1 2 316

17

22

24

26

28

CaCl2

EtOH EtOH+CaCl

2

DMSO

CM

T (

0 C)

Additive [M]

31

solutions. The influence of different salts (such as NaCl, MgSO4, Al2(SO4)3,NaHPO4, Na2SO4 and Na3PO4) on the gel forming temperature and gel melting temperature of P407 solutions has been reported. This sometimes leads to the loss of gelation ability of P407 solutions [60, 100, 101].

During the formulation development the concept of syringeability and injectability is a recommended test for controlling applicability of these gel-based systems [64]. Small laboratory animals (mostly rodents) are used during the preclinical studies who require the use of fine needles and small volumes [102, 103]. Therefore gel formulations were classified with respect to their syringeability and injectability via fine needles (23G and 27G) prior to any animal testing (see Fig. 4 Paper II). Proper syringeability of concentrated poloxamer solutions was difficult at animal house temperature; therefore we suggest the use of prefilled syringes and applicators kept at refrigerator temperatures. For subcutaneous administration, 17-19% P407 with some salt may be used more easily, however the use of 20% P407 gel (including salts) may not be feasible due to its enhanced viscosity and low gelation temperature. Injectability of these formulations using a double syringe system was also ascertained prior to any in vitro drug release studies.

When P407 gels come in contact at their application site with aqueous physiological fluids (e.g., tears, vaginal fluids, and extracellular fluids) both drugs and P407 undergo a process of dissolution. For understanding the role of additives on this dissolution and erosion process of P407 gels, gravimetric dissolution tests were conducted. Although mucoadhesive polymers like chitosan are considered to enhance the gel retention at the site of action due to their interaction with the mucus membrane, surface erosion of the gels (e.g., in ocular applications) also has to be considered and characterized. We observed that the presence of chitosan (alone) increased the dissolution of both 18% and 20% P407 gel (Fig. 5 Paper II). This may be due to the acidic nature of the formulation (chitosan is solubilized at acidic pH) which can be neutralize by chitosan-TPP complex formed in situ (Paper II). In paper II we could show that chitosan-TPP-poloxamer gel formed has more resistance to an aqueous environment than the individual chitosan-poloxamer and TPP-poloxamer gels. Like chitosan, the in situ gelation of mucoadhesive alginate and pectin with Ca2+ should presumably also diminish the surface erosion rates of P407 gels.

32

Controlled Release from P407 Gel Formulations

In preclinical trials, the gel based formulations as studied here, may be applied to prolong the local or systemic exposure of lead compounds. Therefore, three model drugs with different pharmacological and solubility profile were selected for drug release experiments. Metoprolol tartrate itself is an antihypertensive drug, while doxycycline hyclate has antibacterial properties, both are water soluble. Flufenamic acid is an anti-inflammatory drug with a very low aqueous solubility. Unlike covalently crosslinked gels which may control the release of the drug over a period of months, P407 gels are intended for shorter time periods. Their main advantage is that neither special insertion instrument nor surgical removal of the gel is required, since they dissolve without any toxic effect. To study the in vitro release of drugs in the case of ocular, rectal and parenteral applications, we used a membrane-less experimental setup, as described previously [22]. From the obtained results it is quite clear that the release of a hydrophilic drug is restricted at least for 6 hours due to the sustained release properties of P407 gels which is further controlled in the presence of chitosan-TPP complex formed in situ (Fig. 6 in Paper II). DMSO has a minor effect on release of both the hydrophilic and the hydrophobic drug (Figure 6 in Paper I). Both ethanol and CaCl2 increased the dissolution of the gel and the release of metoprolol (Figure 5 in Paper II). The compounds with short elimination half-life need to be administered repeatedly. Such compounds in P407 gels may be administered using a convenient dosing schedule. Normally, membrane-based systems are used in experimental setups to study the diffusion characteristics of drugs independent of the gel dissolution process (s. material and methods). This may be good for topical administration, but in some applications a restrictive dissolution (membranes with larger pore size) of the gel may be better suited for in vivo simulations (such as vaginal applications or burn wounds treatment). In this work, therefore a permeable polycarbonate membrane (3 µm pore size) was used (see Figure 11). Doxycycline was used as a control drug during the testing of antibacterial agents like T3S inhibitors, which have been proven to be active against a number of pathogens. In the next step we were going to establish our formulations for testing these compounds in rodents (Paper V). As our formulation development in Paper II clearly reveals, sustained release of doxycycline is evident from chitosan-poloxamer and chitosan-TPP-poloxamer gels (Figure 7a Paper II). The drug flufenamic acid with low solubility (like most of recently developed compounds) in aqueous media at neutral and acidic pH values (supplementary table 1 Paper II). As a prerequisite prior any animal testing the release profile was measured for flufenamic acid containing formulations into a release medium of pH 7 and

33

4, respectively (Figure 7b, c in Paper II). In both cases the release of the flufenamic acid from PCT gels were slower than for simple P407 gels. These results demonstrate that the combination of chitosan-TPP and P407 poloxamer can have major advantages for the loading of hydrophobic compounds as well as their controlled release in different application environments. Especially, these mucoadhesive formulations based on the combination of chitosan-TPP and poloxamer gels would be suitable for ocular, buccal and vaginal delivery applications.

In Situ Particle Formation

The interactions of chitosan with TPP can induce the spontaneous formation of the nanoparticles, under the optimum conditions of concentration, pH and stirring [73, 104, 105]. The efficiency of potent compounds can be enhanced due to the ability of nanocarriers to penetrate the mucosal barrier [26, 106-109].

Figure 20: TEM images of nanoparticle prepared by mixing in a double syringe a) 0.2% chitosan with 0.07% TPP in P407 gels and b) 0.2% of chitosan with 0.08% TPP in water (4:1).

34

We also checked the possibility of in situ nanoparticle formation in aqueous solution and in P407 gels. TEM images of nanoparticles obtained by mixing of chitosan and TPP solution in dual syringe are shown in Figure 20. Here, we observed that the nanoparticles are not of uniform shape and size, formed either by simple mixing or with a dual syringe system (Fig. 8 Paper II). Nanoparticles dispersed in poloxamer gels would be beneficial in mucosal delivery as the particles are likely to exhibit enhanced penetration and would facilitate the delivery of different types of drugs. However this method needs further evaluation regarding the amount of drug loaded and the drug release profile from nanoparticle loaded poloxamer gels.

35

Formulation for Screening of Drug Candidates

Parenteral Administration

For in vivo testing of specific drugs in small animals, these compounds need to be solubilized in multiples of their MEC or MIC values. Unfortunately, the majority of the salicylidene acylhydrazides compounds have relatively low potency (MIC 12.5-50 µM) and poor aqueous solubility. The aqueous solubility of compounds can be enhanced by converting it to a more soluble salt form or by an adjustment of the pH of solvent if sufficient polar groups are present in the compound. The scheme as shown in Figure 12 is used for most compounds used in this work. Some of the salicylidene acylhydrazides showed higher solubilization in alkaline solutions (e.g., ME0164, ME0177, ME0184, INP 0341 and ME0264,), whereas one was soluble in acidic solutions. However pH adjustment alone was not sufficient for solubilizing the following compounds (ME0053, ME0190 and ME0192) (Paper IV). The compound ME0052 is an important member of salicylidene acylhydrazides family of T3s inhibitors which has been evaluated for its in vitro activity [33, 110]. The solubility of ME052 is negligible in water and PBS. In 50 mMNa2HPO4 solution just 16 µg/ml of ME0052 was soluble, however in 1MNaOH more than 90 mg/ml could be solubilized. The stability of compound in alkaline solutions was checked by LCMS and no fragmentation of mass was observed. Different approaches were tested to administer large doses of ME0052 while maintaining the pH of the final formulation in the range of 7-9 (see table I, Paper V).

Controlled release formulations of ME0052 were prepared in P407 aqueous gels, with some formulations including co-solvents. To ascertain the controlled release of ME0052 from gels, a simple test was performed as described in section 3.2 of Paper V. First, the effect of culture media on the gelation temperature of different P407 solutions was characterized. With this information one was able to select the suitable P407 concentrations for further tests (for DMEM see Figure 9). In a second step, the in vitro activity of ME0052 against Chlamydia trachomatis was tested by layering the formulations (solution and P407 gel) on top of cell cultures of infected HeLa cells. The comparison revealed that the double amount of ME0052 is required to be loaded into the gel formulation compared to a free solution for obtaining a similar inhibitory action.

36

Oral Formulations for Rodents

Generally, liquid formulations are administered to rodents by oral gavages for preclinical testing of lead compounds and vaccines. The gastric environment of rodents is not identical to the one of humans or large animals. It has been recently reported that the pH in different segments of gastrointestinal tract of mice varies between three to five [111]. Most of the salicylidene acylhydrazides have very low solubility in this pH range, and some of them even loose their activity. By loading the drug flufenamic acid (low solubility at pH 4) in P407 gels we have shown that the activity and the solubility of such compounds may be retained in mouse GIT despite the hostile pH (Figure 7C Paper II). We have further demonstrated that the controlled release properties of P407 can be enhanced by in situ ionotropic gelation using chitosan (Paper II). Here, we speculate that we can exploit a similar effect for these novel compounds by using the polymers alginate and pectin in P407 gels (some basic characterization has been done in Paper III).

Yersinia pseudotuberculosis is a pathogen which resides in the cecum/colon before entering into the blood circulation. Here, our aim was to prepare colon targeted oral formulations for testing of T3S inhibitors. Enteric coating of solid dosage form is a popular approach but may not be applicable for mice due to the small pH gradient present. Due to enzymatic degradation of chitosan, pectin and alginate in the large intestine, these polysaccharides are used in colon specific drug delivery systems [112]. To determine the correct size and administration of a solid dosage carrier for mice is a big challenge. The conventional capsules have a body–cap design, for which special administrating tools are required. In the work here, a novel cone-capsule approach has been introduced to prepare oral capsule by simple methods. This shape of capsules will allow simple filling and administration as it may be mounted on an oral gavage needle, where a flush with water will take down the capsule to the stomach of the mouse. The pectin capsules have more strength as compared to chitosan containing ones. The oral capsules for rats having a length of 8.6 mm and 2.65 mm of outer diameter are available on the market. The outer diameter of ball-tip needle used for gastric administration of liquid to mouse is 1.25 to 2.25 mm depending on the size of the mouse. Here, we prepared capsules with dimensions in these ranges. Some of the capsules are shown in Figure 21. The minimum diameter of the capsules prepared from pectin, and chitosan gel is 1.45 mm and we were able to load them with 2.54-4.5 mg of powder depending on the length of the specific capsule.

37

Figure 21: A photograph of the few cone type polysaccharides capsules.

Preliminary Pharmacokinetics

During the discovery stage, the pharmacokinetic studies are conducted in a way that compounds with better in vivo properties are selected for further evaluation and development stages. First, a cassette dosing approach was followed to compare the Cmax and t½ of seven compounds which were selected on the basis of MIC (table 1, Paper IV). From the plasma profile and PK parameters, ME0177 and ME0192 were chosen for an individual administration, one having very high Cmax and the other having a long half-life (Figure 2, table 2 in Paper IV). ME0177 showed a better PK profile than ME0192 after a higher individual dosing (Figure 3, table 3 in Paper IV). ME0192 may not be suitable for systemic administration due to difficulties with the formulation and very low plasma concentrations, whereas ME0177 may be further evaluated for systemic administration.

In a second study the pharmacokinetic parameters of ME0052 were compared after administration of different formulations. It was observed that Cmax after administration of P407 gel formulations was reduced in comparison to normal solution. But t ½ and AUC of ME0052 was increased (Figure 1 and table 2, Paper V). The results indicate that the controlled release of ME0052 had compensated the rapid elimination. By looking at the PK profile of ME0052, administered in P407 gels, it is apparent that each dose should be repeated three times a day or a very high dose should be administered to maintain the plasma concentration above the MIC. Therefore, the tolerance of ME0052 and P407 gels in mice was checked for repeated and for high dosing. The formulation with a pH of 9.0 did not induce any visible discomfort in the animals neither during administration nor afterwards. However, there were side effects such as weight loss, rugged fur and reduced motor activity after repeated administration of P407

38

formulations (three times a day); possibly due to hyperlipidemia and nephrotoxicity [22]. This observation revealed that P407 gel may not be suitable for parenteral administration to mice in a used dose of 1-2g/kg during a multiday trial and that P407 gels may be more suitable for topical applications (e,g vaginal). Recently INP 0341 has shown anti-chlamydial activity after vaginal administration in mice [113]. By using hydroxypropyl β-cyclodextrin we were able to incorporate a higher dose in the formulation. For this formulation Cmax of 33 µg/ml was achieved using the highest dose (Figure 2, Paper V). Repeated administration of the highest dose (three times a day) for four days did not cause accumulation of the compound (Fig. 3, Paper V). ME0052 inhibits T3S in vitro at 4-20 µg/ml [39, 43, 114] and we have observed a plasma concentration of 33 µg/ml in this study. The evaluation of ME0052 may lead to the discovery of successful inhibitor of bacterial T3S.

39

Summary and Outlook

Concentrated micellar solutions of poloxamers can transform from being a mobile fluid at room temperature to immobile gel at body temperature. This reversible process can be exploited for use as an in situ gelling drug delivery system. Most pharmaceutical formulations do not include a single ingredient or carrier only, but require additives to enhance their drug-carrier compatibility and carrier suitability for the body. Therefore, the main aim of this work was to generate thorough understanding of the influence of additives on carrier and overall formulation. In particular, we studied the impact of co-solvents (DMSO, ethanol), salts (CaCl2, TPP) and mucoadhesive polymers (chitosan, alginate) on P407 based gel formulations and their suitability in drug delivery applications.

Here, we could show that DMSO stimulates the micellization and gelation of P407 aqueous solution at lower temperatures. However, if it is present in higher concentrations (10-15%) in P407 solutions, the system was non-administrable due to very low gelation temperatures. In general, DMSO at low concentration (≤ 5%) had minor effects on the CMT, CGT and the dissolution of P407 gels and can safely be used as a co-solvent for hydrophobic drugs (Paper-I). Whereas the co-solvent ethanol suppresses both micellization and gelation in poloxamer solution and elevated concentration of ethanol nearly abandon these phenomenons. Ethanol may be used for solubilization of high concentration of drugs as well as P407 at room temperature.

As compared to chemically cross-linked gels, poloxamers gels are easily diluted by the body fluids. We have explored the possibility of ionotropic gelation of mucoadhesive polymers in P407 gels to overcome its fast dissolution in body. The use of double syringe system facilitated drug loading and handling during administration and in situ ionotropic gelation of chitosan enhanced the strength and restricted dissolution and release of drug (Paper II). In next step we intend to apply this approach for alginate-CaCl2 and pectin-CaCl2 based ionotropic gelation in P407 gels (Paper III). Dual syringe approach may also be adapted for the in situ formation of nanoparticles dispersed in P407 gels, and preliminary experiments have shown a high potential for this concept (Paper II). In situ formed nano-particles, dispersed in P407 gels would facilitate the sustained delivery of potent drugs. Further work in this area is required to estimate the drug entrapment efficiency and drug release properties from these formulations.

40

In the second part, we have focused on development of formulations for preclinical animal studies and a strategy for solubilization of lead compounds belonging to a class of antibacterial salicylidene acylhydrazides has been developed. Out of 58 active compounds, two compounds were selected on the basis of their in vitro activities and pharmacokinetic profiles (Paper IV). Formulations for smaller animals need to be fluid enough during the administration (whether parenteral or local). We observed that a poloxamer formulations having concentration exceeding 20% has excellent drug controlling behaviour at body temperatures (in vitro). However the difficulty in administeration makes these solutions unacceptable for rodents, especially mice. For single dose studies, P407 gel can be applied to extend the plasma half-life (as shown in Paper V), but repeated parenteral administration was not suitable in mice models. Therefore the focus shifted towards the application of P407 based gel formulations in vaginal applications to enhance the local exposure of drug.

In a similar way in situ forming gels (poloxamer-chitosan-TPP and poloxamer- alginate-CaCl2) have a potential for use in oral formulations for small animals. Moreover, we have also developed a new design of capsule which will be convenient in filling (small quantity) and in administration to small animals. For successful oral capsule, continued optimization is needed which includes concentration of polymer, dipping time, drying time, thickness and film coating with enteric polymer. For these tests the surface appearance of the capsule films can be studied by scanning electron microscopy. The microparticles based on polysaccharides (such as alginate, chitosan) have been prepared to target the colon specific delivery. After the in vitro characterization, ICG loaded powder/microparticles will be administered in the capsule to mouse for in vivo characterization. Multilayered and coated capsules will be an interesting extension of this work.

41

Acknowledgements

The journey from scholarship application to dissertation is filled with joys, surprises and need to discover new dimensions of patience. There are many people who inspired, encouraged, helped, supported and guided me during this period, some of them will be acknowledge here. I would like to express my gratitude to:

Gerhard Gröbner, my supervisor, who came forward to support me in the state of uncertainty, when Staffan moved from the department. Gerhard! Thank you so much for sharing your knowledge of NMR and calorimetry and inculcating in me the basics of research and presentation (especially English and figure work). Thanks a lot for your patience and generosity; I will miss this entire social working environment.

Staffan Tavelin, my first supervisor, who reoriented my initial thoughts in the field of pharmaceutical technology and continued to guide me despite his workload at the new place. Thank you Staffan for fruitful discussions and neverending ideas in the field of pharmaceutics (ceramics, gels, diffusion NMR, protein stability are a few which we explored during the first year).

Mikael Elofsson & Åsa Gylfe, my co-supervisors, who introduced me to the world of type III secretion, and guided me for animal experiments. Thank you Mikael (and Cecilia) for arranging all the social activities and for hosting typical Swedish dinners at home. Åsa, thanks a lot for providing necessary support in presentation and writeup. Both Roland Nordfelth & Hans Wolf-Watz are greatly acknowledged for their fruitful collaboration and kind support. Thanks are due to Anders Blomgren (at Astra Zeneca), who saved us from “sample eating machine”.

Prof. Atta Ur-Rehman, former chairman HEC, who managed to divert substantial amount of government fundings towards the tertiary education in Pakistan. Thank you so much Dr. Atta for enlightening the hundreds of students with research culture.

Johana Jeppson & Therese Nydahl (Svenska Institute), Ishfaq Anwar and administration of HEC for assuring bread and butter to all HEC scholars. Johana Tusen tusen tack! for all of your guidance and support during this period.

Barbro, Anita, and Rosita, for their kind help and guidance in all the possible administrative issues at the Department of Chemistry.

Magnus & Pernilla for arranging GMP group meetings; it was a wonderful experience during the last two years, research, shooting, games, dinners, and surströmming, Tack så mycket!

42

Göran & Leif (for lending me your books), Greger & Andrey (diffusion experts), Per-Olof & Lennart (calm mentors), Solomon, Bertil, Britta, Ida, Ulrika & Ximena (Fika organizers), Jörgen, Istvan, Moritz, Maria, Alexander2(the great smilling Germans), Barbara, Miguel, & Maribel (the social biochemists), Liudmila, Anna, Helder, Tania, Michael (innebandy magician), Oleg (for original help), Therese, Radek, Matteus & Nils (the geneious) and Christoph (witty American, God bless you). I am proud of enjoying your company.

Marcus & Caroline (for all of you sincere help, wish you a same smile for whole life), Johan (thank you for fika calls), Marco, Martin, Linh, Hagiang, Roberth, Christopher2, Markus (for synthesizing T3S inhibitors), Andres, Sara, Micke, Remi, Per Anders, Julia. All the members (in both groups) were so nice to me during this stay and group meetings.

Tobias, one and only social Sparrman, our innebandy and bränball guru, he never said no, whenever I approached him for my computer, equipment or other problems. (Tusen tack! Må Gud välsigna honom i belöning). Lenore & Per for their help in SEM amd TEM analysis and Lenous for DLS analysis.

Erik, initially a roommate and now a best Swedish friend, with whom I may have discussed every topic (Swedish culture, religion, politics, sports, movies, comedies and off course shi…). Erik thanks for all of your help in translation, spectroscopy, review and many more. Good luck with new job and home assignment (Love to Benjmin).

Dr. Abid Riaz, Jamil2, Dr. Bashir, Dr. Jamshaid, Dr. Ikram, Qadeer and particularly Abdus Salam Mufti for encouragement and guidance. Saleem, Qaiser, Tanvir, Yasir, Sardar and Kamran who continued feeding me with jokes, links, news, poetry despite all of my “susties”.

Dr. Hamyon, Israr, Naeem, Khanzeb, Khuram, Ghazanfar, Farooq, Shafique, Waseem, Zohaib, Tayyab, Shahid, Zubair and Pak-Umeå families for cozy dinners and friendly environment which make this stay pleasant. Thanks are also due to Amanda Johansson for her hospitality, guidance and arranging summer trips for Fari, Nabeed and Touseeq. God bless you.

Family members here and in Pakistan, you all are so caring and nice to me. Farukh, for all of your patience and taking care of children (and also me) during the last months. Nabeed & Touseeq, thank you boys for allowing papa for all these activities. I love your questions.

43

References

1. Ratti, E. and D. Trist, The continuing evolution of the drug discovery process in the

pharmaceutical industry. Farmaco, 2001. 56(1-2): p. 13-19.

2. Caldwell, G.W., et al., The new pre-preclinical paradigm: compound optimization in early and

late phase drug discovery. Current topics in medicinal chemistry, 2001. 1(5): p. 353-66.

3. Lakings, D.B., PROFILE: Developability: a science for lead candidate selection. Pharmaceutical

Science & Technology Today, 1998. 1(6): p. 280-282.

4. Chaubal, M.V., Application of formulation technologies in lead candidate selection and

optimization. Drug Discovery Today, 2004. 9(14): p. 603-609.

5. Lipinski, C.A., Drug-like properties and the causes of poor solubility and poor permeability. J

Pharmacol Toxicol Methods, 2000. 44(1): p. 235-49.

6. Li, P. and L. Zhao, Developing early formulations: Practice and perspective. International

Journal of Pharmaceutics, 2007. 341(1-2): p. 1-19.

7. Mansky, P., et al., Screening method to identify preclinical liquid and semi-solid formulations for

low solubility compounds: Miniaturization and automation of solvent casting and dissolution

testing. Journal of Pharmaceutical Sciences, 2007. 96(6): p. 1548-1563.

8. Minko, T., Drug delivery systems, in Martin's Physical pharmacy and pharmaceutical

sciences:physical chemical and biopharmaceutical principles in the pharmaceutical sciences,

P.J. Sinko, Editor. 2006, Lippincott Williams & Wilkins: Philadelphia.

9. Eichenbaum, G., et al., Preclinical assessment of the feasibility of applying controlled release

oral drug delivery to a lead series of atypical antipsychotics. Journal of Pharmaceutical Sciences,

2006. 95(4): p. 883-895.

10. Thombre, A.G., Assessment of the feasibility of oral controlled release in an exploratory

development setting. Drug Discovery Today, 2005. 10(17): p. 1159-1166.

11. Rothen-Weinhold, A., R. Gurny, and M. Dahn, Formulation and technology aspects of conrolled

drug delivery in animals. Pharmaceutical Science & Technology Today, 2000. 3(7): p. 222-231.

12. Almdal, K., et al., WHAT IS A GEL. Makromolekulare Chemie-Macromolecular Symposia, 1993.

76: p. 49-51.

13. Hennink, W.E. and C.F. van Nostrum, Novel crosslinking methods to design hydrogels. Advanced

Drug Delivery Reviews, 2002. 54(1): p. 13-36.

44

14. Peppas, N.A., et al., Hydrogels in pharmaceutical formulations. European Journal of

Pharmaceutics and Biopharmaceutics, 2000. 50(1): p. 27-46.

15. He, C.L., S.W. Kim, and D.S. Lee, In situ gelling stimuli-sensitive block copolymer hydrogels for

drug delivery. Journal of Controlled Release, 2008. 127(3): p. 189-207.

16. Gupta, P., K. Vermani, and S. Garg, Hydrogels: from controlled release to pH-responsive drug

delivery. Drug Discovery Today, 2002. 7(10): p. 569-579.

17. Chaterji, S., I.K. Kwon, and K. Park, Smart polymeric gels: Redefining the limits of biomedical

devices. Progress in Polymer Science. 32(8-9): p. 1083-1122.

18. Alexandridis, P. and T.A. Hatton, Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene

oxide) block-copolymer surfactants in aqueous-solutions and at interfaces - thermodynamics,

structure, dynamics, and modeling. Colloids and Surfaces a-Physicochemical and Engineering

Aspects, 1995. 96(1-2): p. 1-46.

19. Mortensen, K. and Y. Talmon, Cryo-tem and sans microstructural study of pluronic polymer

solutions. Macromolecules, 1995. 28(26): p. 8829-8834.

20. Cabana, A., A. AitKadi, and J. Juhasz, Study of the gelation process of polyethylene oxide(a)

polypropylene oxide(b) polyethylene oxide(a) copolymer (Poloxamer 407) aqueous solutions.

Journal of Colloid and Interface Science, 1997. 190(2): p. 307-312.

21. Singh-Joy, S.D. and V.C. McLain, Safety assessment of poloxamers 101, 105, 108, 122, 123, 124,

181, 182, 183, 184, 185, 188, 212, 215, 217, 231, 234, 235, 237, 238, 282, 284, 288, 331, 333, 334,

335, 338, 401, 402, 403, and 407, poloxamer 105 benzoate, and poloxamer 182 dibenzoate as

used in cosmetics. International Journal of Toxicology, 2008. 27: p. 93-128.

22. Dumortier, G., et al., A review of poloxamer 407 pharmaceutical and pharmacological

characteristics. Pharmaceutical Research, 2006. 23(12): p. 2709-2728.

23. Kabanov, A.V., E.V. Batrakova, and V.Y. Alakhov, Pluronic® block copolymers as novel polymer

therapeutics for drug and gene delivery. Journal of Controlled Release, 2002. 82(2-3): p. 189-

212.

24. Desai, S.D. and J. Blanchard, In vitro evaluation of pluronic F127-based controlled-release ocular

delivery systems for pilocarpine. Journal of Pharmaceutical Sciences, 1998. 87(2): p. 226-230.

25. Amiji, M.M., et al., Intratumoral administration of paclitaxel in an in situ gelling poloxamer 407

formulation. Pharmaceutical Development and Technology, 2002. 7(2): p. 195-202.

26. Barichello, J.M., et al., Absorption of insulin from Pluronic F-127 gels following subcutaneous

administration in rats. International Journal of Pharmaceutics, 1999. 184(2): p. 189-198.

45

27. Charrueau, C., et al., Poloxamer 407 as a thermogelling and adhesive polymer for rectal

administration of short-chain fatty acids. Drug Development and Industrial Pharmacy, 2001.

27(4): p. 351-357.

28. Liu, Y., et al., Controlled delivery of recombinant hirudin based on thermo-sensitive Pluronic (R)

F127 hydrogel for subcutaneous administration: In vitro and in vivo characterization. Journal of

Controlled Release, 2007. 117(3): p. 387-395.

29. Pillai, O. and R. Panchagnula, Transdermal delivery of insulin from poloxamer gel: ex vivo and

in vivo skin permeation studies in rat using iontophoresis and chemical enhancers. Journal of

Controlled Release, 2003. 89(1): p. 127-140.

30. Scherlund, M., et al., Thermosetting microemulsions and mixed micellar solutions as drug

delivery systems for periodontal anesthesia. International Journal of Pharmaceutics, 2000.

194(1): p. 103-116.

31. Liu, Z., et al., Polysaccharides-based nanoparticles as drug delivery systems. Advanced Drug

Delivery Reviews, 2008. 60(15): p. 1650-1662.

32. Hueck, C.J., Type III protein secretion systems in bacterial pathogens of animals and plants.

Microbiol. Mol. Biol. Rev., 1998. 62(2): p. 379-433.

33. Keyser, P., et al., Virulence blockers as alternatives to antibiotics: type III secretion inhibitors

against Gram-negative bacteria. Journal of Internal Medicine, 2008. 264(1): p. 17-29.

34. Levy, S.B. and B. Marshall, Antibacterial resistance worldwide: causes, challenges and

responses. Nat Med, 2004.

35. Kauppi, A.M., et al., Salicylanilides are potent inhibitors of type III secretion in Yersinia. Adv Exp

Med Biol, 2003. 529: p. 97-100.

36. Nordfelth, R., et al., Small-molecule inhibitors specifically targeting type III secretion. Infect

Immun, 2005. 73(5): p. 3104-14.

37. Negrea, A., et al., Salicylidene acylhydrazides that affect type III protein secretion in Salmonella

enterica serovar typhimurium. Antimicrob Agents Chemother, 2007. 51(8): p. 2867-76.

38. Tree, J.J., et al., Characterization of the effects of salicylidene acylhydrazide compounds on type

iii secretion in Escherichia coli O157:H7. Infection and Immunity, 2009. 77(10): p. 4209-4220.

39. Muschiol, S., et al., A small-molecule inhibitor of type III secretion inhibits different stages of the

infectious cycle of Chlamydia trachomatis. Proceedings of the National Academy of Sciences of

the United States of America, 2006. 103(39): p. 14566-14571.

46

40. Dahlgren, M.K., et al., Statistical molecular design of a focused salicylidene acylhydrazide

library and multivariate QSAR of inhibition of type III secretion in the Gram-negative

bacterium Yersinia. Bioorganic & Medicinal Chemistry, 2010. 18(7): p. 2686-2703.

41. Gabrielsson, J., Weiner, D., Pharmacokinetic & pharmacodynamic data analysis. 4th ed. 2000,

Stockholm: Swedish Pharmaceutical Society.

42. Theil, F.-P., et al., Utility of physiologically based pharmacokinetic models to drug development

and rational drug discovery candidate selection. Toxicology Letters, 2003. 138(1-2): p. 29-49.

43. Nordfelth, R., et al., Small-molecule inhibitors specifically targeting type III secretion. Infection

and Immunity, 2005. 73(5): p. 3104-3114.

44. Irving, R.S., Artificial skin I. Preparation and properties of pluronic F-127 gels for treatment of

burns. Journal of Biomedical Materials Research, 1972. 6(6): p. 571-582.

45. O'Brien, R., Haq, I., Applications of Biocalorimetry: Binding, stability and enzyme kinetics, in

Biocalorimetry 2: Applications of calorimetery in the biological sciences, J.E. Ladbury, Doyle,

M.L., Editor. 2004, John Wiley & Sons Ltd.: West sussex.

46. Alexandridis, P. and J.F. Holzwarth, Differential scanning calorimetry investigation of the effect

of salts on aqueous solution properties of an amphiphilic block copolymer (poloxamer).

Langmuir, 1997. 13(23): p. 6074-6082.

47. Heerklotz, H. and J. Seelig, Titration calorimetry of surfactant-membrane partitioning and

membrane solubilization. Biochimica Et Biophysica Acta-Biomembranes, 2000. 1508(1-2): p. 69-

85.

48. Seelig, J., Titration calorimetry of lipid-peptide interactions. Biochimica Et Biophysica Acta-

Reviews on Biomembranes, 1997. 1331(1): p. 103-116.

49. Paula, S., et al., Thermodynamics of micelle formation as a function of temperature: a high

sensitivity titration calorimetry study. The Journal of Physical Chemistry, 1995. 99(30): p.

11742-11751.

50. Couderc, S., et al., Interaction between the nonionic surfactant hexaethylene glycol mono-n-

dodecyl ether (c12eo6) and the surface active nonionic aba block copolymer pluronic f127

(eo97po69eo97)formation of mixed micelles studied using isothermal titration calorimetry and

differential scanning calorimetry. Langmuir, 2001. 17(16): p. 4818-4824.

51. Freeman, R., Magnetic resonance in chemistry and medicine. 2003, New York: Oxford University

Press Inc.

52. Beckonert, O., et al., High-resolution magic-angle-spinning NMR spectroscopy for metabolic

profiling of intact tissues. Nature Protocols, 2010. 5(6): p. 1019-1032.

47

53. Ernst, R.R., Bodenhausen, G., Wokaun, A., Priniciples of nuclear magnetic resonance in one and

two dimensions, in The international series of monographs on chemistry R.B. Bernstein,

Breslow, R., Green, M.L.H., Helpren, J., Rowlinson, J.S., Editor. 1990, Oxford University Press

Oxford.

54. Levitt, M.H., Spin dynamics:basics of nuclear magnetic resonance. 2nd ed. 2008, West sussex:

John Wiley & Sons Ltd.

55. Andrew, E.R., A. Bradbury, and R.G. Eades, Nuclear magnetic resonance spectra from a crystal

rotated at high speed. Nature, 1958. 182(4650): p. 1659-1659.

56. Duer M.J., Introduction to solid state NMR spectroscopy. 2004, Oxford: Blackwell Publishing

Ltd. .

57. Lowe, I.J., Free induction decays of rotating solids. Physical Review Letters, 1959. 2(7): p. 285-

287.

58. Walderhaug, H. and O. Soderman, NMR studies of block copolymer micelles. Current Opinion in

Colloid & Interface Science, 2009. 14(3): p. 171-177.

59. Ma, J.-h., et al., 1H NMR Spectroscopic Investigations on the Micellization and Gelation of

PEO−PPO−PEO Block Copolymers in Aqueous Solutions. Langmuir, 2007. 23(19): p. 9596-9605.

60. Malmsten, M. and B. Lindman, Self-assembly in aqueous block copolymer solutions.

Macromolecules, 1992. 25(20): p. 5440-5445.

61. Kwon, K.W., et al., Effects of alcohol addition on gelation in aqueous solution of poly(ethylene

oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer. Polymer Journal, 2001.

33(5): p. 404-410.

62. Yu, G.E., et al., Micellization and gelation of triblock copoly(oxyethylene oxypropylene

oxyethylene), f127. Journal of the Chemical Society-Faraday Transactions, 1992. 88(17): p. 2537-

2544.

63. Edsman, K., J. Carlfors, and R. Petersson, Rheological evaluation of poloxamer as an in situ gel

for ophthalmic use. European Journal of Pharmaceutical Sciences, 1998. 6(2): p. 105-112.

64. Burgess, D., et al., Assuring quality and performance of sustained and controlled release

parenterals: Workshop report. The AAPS Journal, 2002. 4(2): p. 13-23.

65. Zhang, L., et al., Development and in-vitro evaluation of sustained release Poloxamer 407 (P407)

gel formulations of ceftiofur. Journal of Controlled Release, 2002. 85(1-3): p. 73-81.

48

66. Artursson, P. and J. Karlsson, Correlation between oral-drug absorption in humans and

apparent drug permeability coefficients in human intestinal epithelial (caco-2) cells. Biochemical

and Biophysical Research Communications, 1991. 175(3): p. 880-885.

67. Lipinski, C.A., et al., Experimental and computational approaches to estimate solubility and

permeability in drug discovery and development settings. Advanced Drug Delivery Reviews,

1997. 23(1-3): p. 3-25.

68. Strickley, R.G., Solubilizing excipients in oral and injectable formulations. Pharmaceutical

Research, 2004. 21(2): p. 201-230.

69. Lee, Y.-C., P.D. Zocharski, and B. Samas, An intravenous formulation decision tree for discovery

compound formulation development. International Journal of Pharmaceutics, 2003. 253(1-2): p.

111-119.

70. Wang, G.M., et al., Novel design of osmotic chitosan capsules characterized by asymmetric

membrane structure for in situ formation of delivery orifice. International Journal of

Pharmaceutics, 2006. 319(1-2): p. 71-81.

71. Xu, C., et al., Calcium pectinate capsules for colon-specific drug delivery. Drug Development and

Industrial Pharmacy, 2005. 31(2): p. 127-134.

72. Maestrelli, F., et al., Development of enteric-coated calcium pectinate microspheres intended for

colonic drug delivery. European Journal of Pharmaceutics and Biopharmaceutics, 2008. 69(2): p.

508-518.

73. Calvo, P., et al., Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers.

Journal of Applied Polymer Science, 1997. 63(1): p. 125-132.

74. Zhang, Y., et al., PKSolver: An add-in program for pharmacokinetic and pharmacodynamic

data analysis in Microsoft Excel. Computer Methods and Programs in Biomedicine, 2010. 99(3):

p. 306-314.

75. Nakashima, K. and P. Bahadur, Aggregation of water-soluble block copolymers in aqueous

solutions: Recent trends. Advances in Colloid and Interface Science, 2006. 123-126: p. 75-96.

76. Wanka, G., H. Hoffmann, and W. Ulbricht, The aggregation behavior of poly-(oxyethylene)-poly-

(oxypropylene)-poly-(oxyethylene)-block-copolymers in aqueous solution. Colloid &

Polymer Science, 1990. 268(2): p. 101-117.

77. Linse, P. and M. Malmsten, Temperature-dependent micellization in aqueous block copolymer

solutions. Macromolecules, 1992. 25(20): p. 5434-5439.

49

78. Alexandridis, P., J.F. Holzwarth, and T.A. Hatton, Micellization of poly(ethylene oxide)-

poly(propylene oxide)-poly(ethylene oxide) triblock copolymers in aqueous solutions:

thermodynamics of copolymer association. Macromolecules, 1994. 27(9): p. 2414-2425.

79. Trong, L.C.P., M. Djabourov, and A. Ponton, Mechanisms of micellization and rheology of PEO-

PPO-PEO triblock copolymers with various architectures. Journal of Colloid and Interface

Science, 2008. 328(2): p. 278-287.

80. Scherlund, M., A. Brodin, and M. Malmsten, Micellization and gelation in block copolymer

systems containing local anesthetics. International Journal of Pharmaceutics, 2000. 211(1-2): p.

37-49.

81. Bouchemal, K., et al., A concise analysis of the effect of temperature and propanediol-1, 2 on

Pluronic F127 micellization using isothermal titration microcalorimetry. Journal of Colloid and

Interface Science, 2009. 338(1): p. 169-176.

82. Ivanova, R., P. Alexandridis, and B. Lindman, Interaction of poloxamer block copolymers with

cosolvents and surfactants. Colloids and Surfaces A: Physicochemical and Engineering Aspects,

2001. 183-185: p. 41-53.

83. Nilsson, M., et al., Influence of polydispersity on the micellization of triblock copolymers

investigated by pulsed field gradient nuclear magnetic resonance. Macromolecules, 2007.

40(23): p. 8250-8258.

84. Wanka, G., H. Hoffmann, and W. Ulbricht, Phase-diagrams and aggregation behavior of

poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) triblock copolymers in aqueous-

solutions. Macromolecules, 1994. 27(15): p. 4145-4159.

85. Cowie, J.M. and P.M. Toporowski, Association in binary liquid system dimethyl sulphoxide-

water. Canadian Journal of Chemistry-Revue Canadienne De Chimie, 1961. 39(11): p. 2240-&.

86. Qian, X.Q., et al., Vapor pressure of dimethyl sulfoxide and water binary system. Journal of

Solution Chemistry, 1995. 24(11): p. 1183-1189.

87. Catalan, J., C. Diaz, and F. Garcia-Blanco, Characterization of binary solvent mixtures of DMSO

with water and other cosolvents. Journal of Organic Chemistry, 2001. 66(17): p. 5846-5852.

88. Lu, Z.J., et al., Dielectric relaxation in dimethyl sulfoxide/water mixtures studied by microwave

dielectric relaxation spectroscopy. Journal of Physical Chemistry A, 2009. 113(44): p. 12207-

12214.

89. Liu, Y., et al., Effect of carrageenan on poloxamer-based in situ gel for vaginal use: Improved in

vitro and in vivo sustained-release properties. European Journal of Pharmaceutical Sciences,

2009. 37(3-4): p. 306-312.

50

90. Cho, K.Y., et al., Release of ciprofloxacin from poloxamer-graft-hyaluronic acid hydrogels in

vitro. International Journal of Pharmaceutics, 2003. 260(1): p. 83-91.

91. Gratieri, T., et al., A poloxamer/chitosan in situ forming gel with prolonged retention time for

ocular delivery. European Journal of Pharmaceutics and Biopharmaceutics. 75(2): p. 186-193.

92. Kim, I.Y., et al., Evaluation of semi-interpenetrating polymer networks composed of chitosan

and poloxamer for wound dressing application. International Journal of Pharmaceutics, 2007.

341(1-2): p. 35-43.

93. Niu, G., et al., Synthesis and characterization of reactive poloxamer 407s for biomedical

applications. Journal of Controlled Release, 2009. 138(1): p. 49-56.

94. Ryu, J.-M., et al., Increased bioavailability of propranolol in rats by retaining thermally gelling

liquid suppositories in the rectum. Journal of Controlled Release, 1999. 59(2): p. 163-172.

95. Shin, S.-C., J.-Y. Kim, and I.-J. Oh, Mucoadhesive and physicochemical characterization of

carbopol-poloxamer gels containing triamcinolone acetonide. Drug Development & Industrial

Pharmacy, 2000. 26(3): p. 307.

96. Sosnik, A., et al., Crosslinkable PEO-PPO-PEO-based reverse thermo-responsive gels as

potentially injectable materials. Journal of Biomaterials Science-Polymer Edition, 2003. 14(3): p.

227-239.

97. Choi, H.-G., et al., Effect of additives on the physicochemical properties of liquid suppository

bases. International Journal of Pharmaceutics, 1999. 190(1): p. 13-19.

98. Berger, J., et al., Structure and interactions in covalently and ionically crosslinked chitosan

hydrogels for biomedical applications. European Journal of Pharmaceutics and

Biopharmaceutics, 2004. 57(1): p. 19-34.

99. Anderson, B.C., et al., The effect of salts on the micellization temperature of aqueous

poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) solutions and the

dissolution rate and water diffusion coefficient in their corresponding gels. Journal of

Pharmaceutical Sciences, 2002. 91(1): p. 180-188.

100. Gilbert, J.C., et al., The effect of solutes and polymers on the gelation properties of pluronic F-127

solutions for controlled drug delivery. Journal of Controlled Release, 1987. 5(2): p. 113-118.

101. Pandit, N.K. and J. Kisaka, Loss of gelation ability of Pluronic® F127 in the presence of some

salts. International Journal of Pharmaceutics, 1996. 145(1-2): p. 129-136.

102. Baumans, V., Remie, R., Hackbarth, H.J., Timmerman, A., , Experimental procedures, in

Priniciples of laboratory animal science, V. Zutphen, Baumans, V., Beynen, A.C.,, Editor. 2001,

Elsevier Science Amsterdam. p. 313-333.

51

103. Shimizu, S., Routes of Administration, in The laboratory mouse, H. Hedrich, Bullock, G., Petruz,

P., Editor. 2004, Elsevier Academic Press: Amsterdan. p. 527-542.

104. Gan, Q., et al., Modulation of surface charge, particle size and morphological properties of

chitosan-TPP nanoparticles intended for gene delivery. Colloids and Surfaces B: Biointerfaces,

2005. 44(2-3): p. 65-73.

105. Hasanovic, A., et al., Chitosan-tripolyphosphate nanoparticles as a possible skin drug delivery

system for aciclovir with enhanced stability. Journal of Pharmacy and Pharmacology, 2009.

61(12): p. 1609-1616.

106. Gou, M., et al., A novel injectable local hydrophobic drug delivery system: Biodegradable

nanoparticles in thermo-sensitive hydrogel. International Journal of Pharmaceutics, 2008.

359(1-2): p. 228-233.

107. Zhang, X.-Z., P. Jo Lewis, and C.-C. Chu, Fabrication and characterization of a smart drug

delivery system: microsphere in hydrogel. Biomaterials, 2005. 26(16): p. 3299-3309.

108. Lai, S.K., Y.-Y. Wang, and J. Hanes, Mucus-penetrating nanoparticles for drug and gene delivery

to mucosal tissues. Advanced Drug Delivery Reviews, 2009. 61(2): p. 158-171.

109. Moghimi, S.M., A.C. Hunter, and J.C. Murray, Nanomedicine: current status and future

prospects. FASEB J., 2005. 19(3): p. 311-330.

110. Baron, C., Antivirulence drugs to target bacterial secretion systems. Current Opinion in

Microbiology, 2010. 13(1): p. 100-105.

111. McConnell, E.L., A.W. Basit, and S. Murdan, Measurements of rat and mouse gastrointestinal pH

fluid and lymphoid tissue, and implications for in-vivo experiments. Journal of Pharmacy and

Pharmacology, 2008. 60(1): p. 63-70.

112. Vandamme, T.F., et al., The use of polysaccharides to target drugs to the colon. Carbohydrate

Polymers, 2002. 48(3): p. 219-231.

113. Chu, H., et al., Candidate vaginal microbicides with activity against Chlamydia trachomatis and

Neisseria gonorrhoeae. International Journal of Antimicrobial Agents, 2010. 36(2): p. 145-150.

114. Bailey, L., et al., Small molecule inhibitors of type III secretion in Yersinia block the Chlamydia

pneumoniae infection cycle. Febs Letters, 2007. 581(4): p. 587-595.