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
To
My parents
i
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 thermoreversible 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 halflife of the lead compound.
Keywords
Poloxamer, drug delivery, micellization, DMSO, calorimetry, NMR, in situ
gelation, chitosan, preclinical pharmacokinetics, type III secretion inhibitors
ii
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
Materials and Methods
Polymers
9 10 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
Results and Discussion
Characterization of Poloxamer 407 Solutions
22 23 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 iii
Preliminary Pharmacokinetics
37 Summary and Outlook
39 Acknowledgements
41 References
43 iv
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.
v
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 antichlamydial 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
vi
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
t½
Elimination half-life
T3S
Type III secretion
vii
TEM
Transmission electron microscopy
TPP
Sodium tripolyphosphate
UV-VIS
Ultraviolet-visible spectroscopy
viii
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)
ii)
iii)
iv)
Drug discovery Preclinical development Clinical development 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
1
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
2
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].
Level of toxicity
Drug level
Subtherapeutic level
Time
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
3
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 semisolid 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].
4
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.
5
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
6
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.
7
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.
Extravascular dose
e.g. oral, IP, SC, IM
IV dose
Absorption
Blood
Distribution
Tissue
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.
8
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.
9
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 (8000012000 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).
10
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
11
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.
12
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].
Adiabatic jacket
Injector
with stirrer
Heater
Heater
F.B
Reference cell Sample cell 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
13
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).
D P (µc al/s ec)
10
8
6
4
2
0
10 0
200
3 00
40 0
500
6 00
7 00
(k c a l/m
o le )
T im e (m in )
-1 5
-2 0
-2 5
-3 0
d H
-3 5
-4 0
-4 5
-5 0
-5 5
0 . 0 0
0 .0 5
0 . 1 0
M
o l a r
0 .1 5
0 .2 0
0 . 2 5
R a tio
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
14
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 [5557]. 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
15
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
16
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).
17
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 preweighed 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.
18
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
19
of the compounds in the formulations was assured by measuring the molar
mass by LCMS (liquid chromatography-mass spectrometry).
Stock solution
in DMSO
P
CPB (3)
CS 20%
P
S
AS (2‐5)
+CS 20%
S
CPB (5) P
P
Dilute with PBS
S
P
P
CPB (3) P
AS (2) S
P
S
AS (5)
Compound
S
S
CPB (8) P
S
CPB (10)
P
P
CPB (10)
CS 20%
S
0.1 M
NaOH
S
P
1M
NaOH
S
S
P
BS (9‐12)
+CS 20%
P
S
Dissolve in PBS
Dilute with PBS and adjust pH
S: soluble
P: Precipitate CS1 5%, CS2 10%, CS3 40% CS: co‐solvent
CD: cyclodextrins
AS: acidic solution (pH)
BS: Basic solution (pH)
PBS: Phosphate buffer saline
CPB: citrate‐phosphate buffer (pH)
S
Dilute with PBS and adjust pH
CS + Surf +PBS /CS+ CD and PBS
Formulation
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.
20
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.
21
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 LCMS/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.
22
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
23
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]
0.12
22%
0.10
o
Cp(cal/ C)
0.08
Tpeak
0.06
0.04
Tgelation
0.02
1.26%
Tonset
Tendset
0.00
0
20
40
60
o
Temperature ( C)
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)
24
24
0
Temperature C
28
20
CGT
CMT
16
12
0
5
10
15
20
25
P407 Conc (wt%)
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).
25
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.
26
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 temperatureinduced phase changes, at a molecular level.
27
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.
28
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
29
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).
28
0
CMT ( C)
26
CaCl2
EtOH
EtOH+CaCl2
DMSO
24
22
17
16
0
1
2
3
Additive [M]
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
30
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 gelbased 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 TPPpoloxamer 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.
31
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-TPPpoloxamer 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
32
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).
33
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.
34
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.
35
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.
36
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 halflife (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
37
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 βcyclodextrin 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.
38
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 nonadministrable 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 alginateCaCl2 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 nanoparticles, 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.
39
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.
40
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!
41
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.
42
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