Design and synthesis of long acting inhaled corticosteroids for the treatment of asthma

Article abstract of DOI:10.1016/j.bmcl.2011.07.106

In this Letter we present data for a novel series of ICS for the treatment of asthma. 'Inhalation by design' principles have been applied to a series of highly potent steroidal GR agonists, with a focus on optimising the potential therapeutic index in human. Pharmacokinetic properties were tuned with high intrinsic clearance and low oral bioavailability in mind, to minimise systemic exposure and reduce systemically driven adverse events. High CYP mediated clearance as well as glucuronidation were targeted to achieve high intrinsic clearance coupled with multiple routes of clearance to minimise drug-drug interactions. Furthermore, pharmaceutical properties such as stability, crystallinity and solubility were considered to ensure compatibility with a dry powder inhaler. This work culminated in the identification of the clinical candidate 15, which demonstrates preclinically the desired efficacy and safety profiles confirming its potential as an inhaled agent for the treatment of asthma.

Full text of DOI:10.1016/j.bmcl.2011.07.106

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5826–5830
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of long acting inhaled corticosteroids for the treatment
of asthma
David S. Millan a, , Stephen A. Ballard b, Sara Chunn a, Joseph A. Dybowski d, Craig K. Fulton e,
⇑
Paul A. Glossop a, Edouard Guillabert e, Christopher A. Hewson b, Rhys M. Jones c, David J. Lamb b,
Carolyn M. Napier d, Toby A. Payne-Cook e, Emmanuelle R. Renery e, Matthew D. Selby a, Michelle F. Tutt b,
Michael Yeadon b
a Worldwide Medicinal Chemistry, Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
b Allergy and Respiratory Research Unit, Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
c Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
d Department of Drug Safety, Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
e Department of Pharmaceutical Sciences, Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
In this Letter we present data for a novel series of ICS for the treatment of asthma. ‘Inhalation by design’
principles have been applied to a series of highly potent steroidal GR agonists, with a focus on optimising
the potential therapeutic index in human. Pharmacokinetic properties were tuned with high intrinsic
clearance and low oral bioavailability in mind, to minimise systemic exposure and reduce systemically
driven adverse events. High CYP mediated clearance as well as glucuronidation were targeted to achieve
high intrinsic clearance coupled with multiple routes of clearance to minimise drug-drug interactions.
Furthermore, pharmaceutical properties such as stability, crystallinity and solubility were considered
to ensure compatibility with a dry powder inhaler. This work culminated in the identification of the clin-
ical candidate 15, which demonstrates preclinically the desired efficacy and safety profiles confirming its
potential as an inhaled agent for the treatment of asthma.
Received 29 June 2011
Revised 26 July 2011
Accepted 27 July 2011
Available online 11 August 2011
Keywords:
Inhaled
Corticosteroid
Asthma
Ó 2011 Elsevier Ltd. All rights reserved.
Inhaled corticosteroids (ICS) as stand alone agents, or in combi-
nation with long acting b2-agonists (LABA) are highly effective
treatments for asthma. The worldwide prevalence of this chronic
disease is substantial with estimates of over 300 million people af-
fected, with reports of this number rising.1 The commercial market
for ICS is therefore extremely large, with inhaled agents such as
fluticasone propionate 1 and budesonide 2 (Fig. 1) achieving mul-
ti-billion dollar sales annually, either as stand alone agents, or as
part of LABA combinations. The next generation ICS fluticasone
furoate 3, which is in phase 3 clinical trials as a combination with
the LABA vilanterol, is also expected to capture a substantial sec-
tion of the market.2 ICS and ICS/LABA combinations are also exten-
sively used in chronic obstructive pulmonary disease (COPD), but
with steroid resistance believed to limit the efficacy of ICS in most
patients.3 The lack of significant clinical progession of novel anti-
inflammatory mechansims in recent years suggests the market
dominance of either ICS or ICS/LABA will not be significantly
challenged in the next 10 years, and therefore ICS will remain as
a necessary treatment for chronic respiratory diseases.
While ICS are generally regarded as offering a good risk/benefit
profile, there exists a general discomfort with steroid safety, partic-
ularly in paediatric use, and at the higher doses generally used in
COPD, for long term systemic safety. Corticosteroid efficacy in asth-
ma is dose limited due to mechanism related adverse events such as
bone metabolism, growth retardation in children, skin thinning and
adrenal suppression.4 There is therefore remaining scope for
improvement by offering an ICS with a better safety/tolerability pro-
file, and hence a greater therapeutic index (TI). Moreover, achieving
once daily dosing across all severities of asthma could lead to greater
patient compliance, and hence better pharmacotherapeutic control
of the disease (all ICS are dosed twice daily in more severe patients).1
All currently available ICS have some bioavailability to the sys-
temic circulation, whether it be through absorption through the
lung, or via oral absorption from a swallowed component of the in-
haled dose.1 If the systemic exposure could be further reduced
through improved pharmacokinetics, alongside greater potency
and a longer duration of action (DoA), then an ICS could be pro-
⇑
Corresponding author. Tel.: +44 1304 643497; fax: +44 1304 651987.
E-mail addresses: dave.millan@gmail.com, david.millan@pfizer.com (D.S. Mill-
an).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.106

D. S. Millan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5826–5830
5827
F
F
O
H
S
OH
O
O
H
S
O
HO
O
HO
O
O
H
HO
O
H
O
O
H
F
F
F
F
H
H
O
O
O
1: Fluticasone propionate
2: Budesonide
3: Fluticasone furoate
duced with a superior TI. A greater TI creates the opportunity to de-
velop a safer ICS at the same level of efficacy as current agents, or
alternatively to dose escalate and generate greater efficacy without
compromising safety in comparison with current agents. This pa-
per will detail our efforts to design a potent, long acting ICS with
a superior safety profile, through improved pharmacokinetics, rel-
ative to the marketed agents.
somewhat, relative to the starting points in 4 and 5, and therefore
we did not pursue biaryl analogues further. 7 demonstrated that
the terminal ring system could be saturated in the form of a mor-
pholine ring system, albeit with weaker activity in our GR reporter
assay. The highly lipophilic 8 also retained excellent potency sug-
gesting that physicochemical properties could be modulated with-
out loss of significant potency.
We decided to restrict the design of a novel ICS series to the
conventional steroid scaffold and utilise the D ring of the structure
to engineer the desired pharmacokinetic and pharmaceutical prop-
erties required for inhalation. In order to design a successful ICS
series we focussed on the ‘inhalation by design’ principles that
have been detailed in a previous publication.5 These include (1)
high potency to achieve a low inhaled dose (61 mg drug product);
(2) duration of action (DoA) for a therapeutically relevant period;
(3) high clearance and low oral absorption of any swallowed dose
to minimise systemic exposure and therefore potential for adverse
events; (4) multiple routes of clearance to minimise drug-drug
interactions (DDIs); (5) physical form characteristics suitable for
a dry powder inhaler including high crystallinity and compatability
with lactose which is the commonly used excipient.
These design principles suggest maintaining or actually increas-
ing the lipophilicity of a novel series, relative to marketed agents,
could potentially deliver a compound with higher clearance, as
well as a slower rate of absorption of drug from the lung to the sys-
temic circulation, through a reduction in permeability and solubil-
ity. The latter concept could conceivably lead to increasing the DoA
of the inhibitor at the site of action, by retaining the compound in
the lung. We also hypothesised that by simply increasing affinity
for the glucocorticoid receptor may also increase residency time
We also measured the potency of several analogues against
inhibition of TNF
a release from human whole blood to assess the
impact of blood binding on the potency, relative to the isolated hu-
man PBMC assay. Fluticase propionate 1 and fluticasone furoate 2
showed a 93 and 169 fold shift in IC50, respectively, from the PBMC
to the human whole blood assay which gave an indication of the
extent of binding. Compounds 4 and 8 showed an even greater
shift of 252 and 474 fold, respectively, from isolated cell to whole
blood suggesting even higher binding. We considered high blood
binding, in combination with high unbound clearance, as another
way to minimise unbound systemic exposure.
It became apparent that achieving potency in this series would
not be difficult and therefore we focussed our attention on opti-
mising the series for inhalation. As mentioned previously we were
keen to follow our ‘inhalation by design’ principles to achieve an
inhaled agent with minimal systemic exposure and hence reduce
systemically driven adverse events and therefore achieving high
metabolic clearance was important.5 Furthermore, we were also
keen to identify compounds that would be cleared by multiple
routes so that becoming a victim to cytochrome P450 (CYP) medi-
ated DDIs would be less likely to occur. To this end we investigated
the introduction of a phenol group onto our inhibitor series to see
if phase II mediated metabolism through glucuronidation could
provide a second route of metabolism. Glucuronidation is well doc-
umented as a phase II metabolic pathway that has high capacity
and is a major route of drug inactivation.9
Gratifyingly, the introduction of a phenol in 9 was well toler-
ated from a potency standpoint. However, the introduction of a
more polar heterocycle into the terminal aromatic ring of the
acetal in 10 led to a significant loss of potency in the GR reporter
assay but interestingly was still potent in the PBMC assay. The
reasons for this are not clear. Compounds 11 and 12 suggested that
introduction of a second linking atom between the aromatic rings
of the acetal was tolerated in terms of potency and therefore the
phenols 13 and 14 were designed and synthesised which were also
highly potent. Chloro phenol 15 was also highly potent in the GR
reporter assay, as well as the PBMC assay. Furthermore, the shift
from PBMC to whole blood of 291 fold suggested that 15 was much
more highly bound relative to fluticasone propionate 1. With a ser-
ies of potent phenols now in hand, we sought to understand the
in vitro and in vivo pharmacokinetic profile of lead compounds
to investigate their potential as optimal inhaled agents with low
systemic expoure, and reduced potential for CYP mediated DDI.
To understand the in vitro intrinsic clearance for CYP mediated
metabolism we utilised a human liver microsome system, which
could also be set up to measure the intrinsic clearance of glucuron-
on the receptor through a slower koff
.
Towards these goals, we began working on an acetal series of
ICS compounds 4–15 which were prepared from a common steroi-
dal scaffold 16 (Table 1). The commercially available acetonide 17
was first reacted with tetrafluoroboric acid to reveal the precursor
16, which was then reacted with various aromatic aldehydes 18
under acidic dehydrating conditions to afford target compounds
4–15 (Scheme 1).6
Many of the aromatic aldehydes 18 were not commercially
available and were therefore synthesised. These were produced
by either a suzuki cross coupling reaction to produce the biaryl
aldehydes leading to 4, 6 and 9 (X = direct bond, or CH2), or by
nucleophlic aromatic substitution to afford aldehydes which went
on to produce 5, 8 and 10 (X = O, S). Aldehydes that produced 11–
15 (X = SCH2, or CH2S) were synthesised by nucleophilic displace-
ment of a benzylic halide with a thiophenol.
Generally this series of ICS compounds demonstrated excellent
in vitro potency, whether it be in our GR agonist reporter assay or
in a whole cell PBMC assay whereby the inhibition of TNFa release
was measured. Initial starting points in 4 and 5 were highly potent
in both the GR agonist reporter assay as well as the PBMC assay,
with similar potency to the marketed agent fluticasone propionate
1, and more potent than budesonide 2. 6 dropped off in potency

5828
D. S. Millan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5826–5830
Table 1
GR agonist activitya, inhibition of TNF
a
release from human PBMCb, and whole bloodc for target compounds 4–15
X
OH
R
O
H
O
HO
O
H
F
F
4-15
O
Compound
X
R
c Log P
GR agonista EC50 (nM)
PBMCb IC50 (nM)
Whole bloodc IC50 (nM)
9.3
1, Fluticasone propionate
2, Budesonide
3, Fluticasone furoate
3.8
2.9
4.3
1.09
12.40
0.49
0.10
0.96
0.05
8.46
43
4
5
6
7
CH2
4.8
4.9
4.7
2.5
0.78
0.43
8.78
0.71
0.17
0.09
0.41
1.81
*
*
*
*
O
Direct bond
CH2
O
N
SMe
8
S
5.7
3.93
0.10
47.4
*
*
OH
9
CH2
O
4.2
2.9
0.38
0.04
0.12
OH
10
21.10
N
N
*
*
*
11
12
SCH2
CH2S
4.9
4.9
7.35
1.75
0.16
0.22
OH
13
14
15
CH2S
CH2S
CH2S
4.2
4.2
4.9
1.87
2.82
2.97
0.06
0.15
0.03
*
*
OH
OH
8.73
Cl
*
a
b
c
Glucocorticoid receptor (GR) agonist activity was determined using a reporter assay, see ref. 7 for details. Differences of <2-fold should not be considered significant.
Inhibition of TNF
a
release from human PBMC was determined by an ELISA assay, see ref. 8 for details. Differences of <2-fold should not be considered significant.
release from human whole blood was determined by an ELISA assay, see ref. 8 for details. Differences of <2-fold should not be considered significant.
Inhibition of TNF
a
idation. Both fluticasone propionate 1, and lead 8, showed high
in vitro intrinsic clearance, as well as high in vivo clearance
(unbound) in rat, leading to a short half life and negligible oral
bioavailability. However, both showed little if any glucuronidation
in the HLM UGT experiment, suggesting a potential for a CYP
mediated DDI. Little glucuronidation would be expected as both

D. S. Millan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5826–5830
5829
X
OH
OH
O
OH
R
O
H
O
H
O
O
OH
O
HO
HO
HO
b
O
a
OH
O
H
H
D
H
+
R
F
F
X
H
F
F
F
F
O
O
O
17
18
16
4-15
Scheme 1. General synthesis of target compounds 4–15. Reagents and conditions: (a) 48% aq HBF4, rt, 7 h; (b) CF3SO3H, MgSO4, 1,4-dioxane, rt, 24 h.
fluticasone propionate 1 and 8 do not contain a phenol, although
incorporation of a phenol does not always lead to high levels of
glucuronidation. Phenol containing 15, however, demonstrated
high levels of glucuronidation, alongside liver blood flow clearance
in the rat and therefore this compound immediately became a very
interesting lead (Table 2). Metabolite identification studies from
human and rat hepatocytes suggested that the major metabolite
for compound 15 was indeed a glucuronide, which is likely to have
much weaker agonist potency against the GR receptor. Gratifyingly
when hepatocyte incubations were carried out in the absence or
presence of ketoconazole (CYP 3A4 inhibitor) there was not a sig-
nificant difference in intrinsic clearance observed, suggesting a re-
duced potential for a CYP mediated DDI. Importantly, the unbound
clearance was significantly higher than that for fluticasone propio-
nate 1 suggesting that 15 has the potential to have significantly
lower unbound drug concentrations in the human systemic circu-
lation, relative to fluticasone propionate and therefore may offer an
improved TI.
With lead compound 15 suggesting it had favourable pharma-
cokinetic properties we were interested to further investigate the
in vitro pharmacology and selectivity in relation to other GR ago-
nists, such as fluticasone propionate 1. Compound 15 was profiled
against a number of other human nuclear hormone receptors
including mineralocorticoid (MR), progesterone (PR), estrogen
(ER) and androgen (AR) receptors in both agonist and antagonist
format to determine selectivity as a GR agonist. 15 was more than
10 fold selective for GR agonism over PR agonism and was superior
to fluticasone propionate 1 (4.1-fold). Compound 15 was a partial
agonist for MR with selectivity of 4.1 fold over MR antagonism
which was comparable to fluticasone propionate 1 (3.7-fold). 15
did not function as an agonist or antagonist of AR or ER. As men-
tioned above we were also interested in identifying a compound
with slow dissociation kinetics from GR to enable a long DoA,
and therefore we sought to investigate the dissociation half-life
from GR of 15 relative to fluticasone propionate 1 using a compe-
tition kinetic binding methodology. 15 exhibited a slower dissoci-
ation half-life from the human receptor (1284 min) in comparison
to fluticasone propionate 1 (544 min) suggesting a potential for a
longer DoA in vivo.10
The intriguing pharmacological and pharmacokinetic properties
of 15 prompted us to evaluate the in vivo potency and DoA of the
compound. The PBMC potency of 15 and fluticasone propionate 1
were similar in rat and human and therefore experiments in rat
were deemed suitable. Bacterial lipopolysaccharide (LPS) challenge
to the lungs of rats results in acute pulmonary neutrophilia. The
use of this model has been extensively cited in the literature to
characterise anti-inflammatory compounds, including ICS.11 Effi-
cacy and DoA studies were dosed via the inhaled route to replicate
the intended human delivery route. In two independent studies, 15
was dosed as an aqueous solution (containing 0.5% Tween-20) in-
tra-tracheally 4 h prior to stimulus, which dose dependently inhib-
ited LPS induced lung neutrophilia with an ED50 of 3
lg and 4.7 lg
(Fig. 1). The same experiment was conducted for fluticasone propi-
onate 1 resulting in an ED50 of 36 lg, suggesting 15 was indeed
significantly more potent in vivo.
To investigate the in vivo DoA, 15 and fluticasone propionate 1
were dosed by intratracheal administration at various times before
LPS challenge. The dose levels were chosen using ED50 values de-
rived from studies dosed 1 h pre-LPS challenge for fluticasone pro-
1.5
1.0
0.5
0.0
Figure 1. Dose dependent inhibition of LPS induced neutrophilia by 15.
Table 2
In vitro and in vivo pharmacokinetic properties for compounds 1, 8, and 15
a
Compound
HLM Clint (Clintu)
(
lL/min/mg)
HLM UGTb
(lL/min/mg)
Rat IV Clb (Clu)c (mL/min/Kg)
Rat PO Fd F (%)
T1/2 (h)
1 Fluticasone propionate
8
15
51 (300)
154 (>3080)
122 (121000)
<3.5
<3.5
100
56 (1800)
26 (>18000)
84 (>59000)
<2
<0.5
<1
1.1
1.9
2.8
a
Compounds were assessed for metabolic stability in human liver microsomes (HLM), and the unbound Clint (Clintu) also determined by first determining the microsomal
binding by equilibrium dialysis.
b
Compounds were assessed for phase II glucuronidation in HLM by including the co-factor UDPGA, and excluding the cytochrome P450 dependent co-factor NADPH.
Intravenous (IV) pharmacokinetics were determined in rats from a 0.5 mg/Kg dose. The unbound clearance was calculated after measuring the plasma protein binding,
c
and blood binding.
d
Oral (PO) pharmacokinetics were determined from a 2 mg/Kg dose.

5830
D. S. Millan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5826–5830
and finally the compound has demonstrated a preclinical safety
Duration of Action at ED50 Dose
profile suitable to move forward into human trials. This promising
overall profile has led to the nomination of compound 15 as a
development candidate for the treatment of asthma. Further infor-
mation relating to the research and development of compound 15
will be reported in due course.
80
60
40
20
0
Fluticasone Proprionate, 1
15
Acknowledgments
We thank Mark Lansdell, Samantha Hughes and David Price for
contributions to the chemistry. We also thank Karl Campany,
Stephen Bradley, Nicole Parker, and William Hood for contribu-
tions to the biology.
NS
NS
NS
References and notes
0
4
8
12
24
1. Barnes, P. J. Trends Pharm. Sci. 2010, 3, 335.
2. Available from: http://www.gsk.com (accessed 6 June 2011).
3. Barnes, P. J.; Adcock, I. M. Lancet 2009, 373, 1905.
Pre-dose time (hours)
Figure 2. Duration of action of 15, and fluticasone propionate
neutrophilia model (NS-not statistically significant from vehicle).
1 in rat LPS
4. Lipworth, B. J. Arch. Intern. Med. 1999, 159, 941.
5. Glossop, P. A.; Lane, C. A. L.; Price, D. A.; Bunnage, M. E.; Lewthwaite, R. A.;
James, K.; Brown, A. D.; Yeadon, M.; Perros-Huguet, C.; Trevethick, M. A.;
Clarke, N. P.; Webster, R.; Jones, R. M.; Burrows, J. L.; Feeder, N.; Taylor, S. C. J.;
Spence, F. J. J. Med. Chem. 2010, 53, 6640.
6. For detailed experimental procedures for the preparation and
characterisationof 15, see: Glossop, P.A, Millan, D.S., Price, D.A., WO Patent
Application 069032, 2009.
7. The potency of marketed agents 1–3 and test compounds 4–15 as a GR agonist
was assessed using a human cell-line (SW1353, a human bone carcinoma cell-
line) expressing endogenous GR and stably expressing an MMTV-luciferase
reporter construct. GR agonism was assessed by the ability of marketed agents
1–3 and test compounds 4–15 to bind the endogenous GR, transactivate the
MMTV promoter and drive expression of luciferase. At least n = 2 determinants
pionate 1 (10 lg) and 4 h pre-LPS challenge for 15 (4.3 lg). Flutica-
sone propionate 1 inhibited neutrophilia by 29% at 1 h, but de-
clined to 11%, 8% and 10% at 2, 4 and 8 h, respectively. 15,
however, inhibited neutrophilia by 52% at 1 h, 59% at 4 h, 46% at
8 h, and 24% at 24 h suggesting that 15 has the potential to have
a significantly increased DoA relative to fluticasone propionate 1
(Fig. 2).
Compound 15 has also demonstrated suitable pharmaceutical
properties, such as crystallinity, stability on lactose (commonly
used inhaled excipient) and solubility. The simulated lung fluid sol-
were used to quote an EC50
8. The potency and efficacy of marketed agents 1–3 and test compounds 4–15
was assessed by measuring the inhibition of TNF release from primary human
.
a
ubility12 of 15 is 0.36
propionate 1 (0.32
l
g/ml, which is comparable to fluticasone
peripheral blood mononuclear cells (PBMC) and human whole blood (WB)
following LPS stimulation, where stated. The cellular composition of human
PBMC preparations was typically: lymphocytes (79.3% 1.3), monocytes
(19.2% 1.2), neutrophils (2.1% 0.3) and basophils (0.4% 0.0). The potency
and efficacy of marketed agents 1–3 and test compounds 4–15 was also
assessed in rat PBMC and WB to support translation to in vivo models, where
l
g/ml). The preclinical safety profile also sug-
gests 15 is suitable to take forward to human trials.
In summary, we have described our approaches toward the
identification of a novel series of GR agonists, which were opti-
mised for potency, and pharmacokinetic properties suitable for
an inhaled drug. Compound 15 was subsequently identified and
shown to possess high potency for the GR receptor, both in vitro
and in vivo, and non-inferior selectivity against a panel of human
nuclear hormone receptors, relative to the marketed agent flutica-
sone propionate 1. The slow in vitro dissociation kinetics against
the human receptor was shown to translate into a relatively long
DoA in an in vivo experiment, when compared to fluticasone pro-
pionate 1. The in vitro and in vivo pharmacokinetic profile suggests
the compound will display low oral bioavailability in humans fol-
lowing any swallowed dose, coupled with very high intrinsic clear-
ance such that any lung absorbed drug will be rapidly metabolised
to less potent metabolites. Moreover, 15 is metabolised by both
phase I and II pathways and therefore should not be a victim or a
perpetrator of any DDIs. The pharmaceutical properties of 15 are
suitable for combination with lactose in a dry powder inhaler,
stated. At least n = 3 determinants were used to quote an IC50
.
9. Fisher, M. B.; Paine, M. F.; Strelevitz, T. J.; Wrighton, S. A. Drug Metab. Rev. 2001,
33, 273.
10. Binding kinetics were determined by a competition assay with radio-labelled
ligand. [3H]-Dexamethasone at 5 nM was incubated together with
recombinant GR ligand-binding domain and three different concentrations of
test compound for various time points. The association of the radio-ligand was
examined over time in the presence and absence of the test compound.
Experiments were performed at 4 °C to maintain protein stability and carried
out over 3 days. The altered profile of the ligand association in the presence of
the test compound allowed the kinetics (kon and koff) of the test compound to
be determined. The T for each compound was then calculated as ln (2)/koff
.
½
Because of assay to assay variation in determining kon and koff for [3H]-
dexamethasone, non-labelled dexamethasone was always tested as a reference
standard so that ratios for T could be calculated, thus standardising data. All
½
statistical tests were performed on this ratio data.
11. Chiang, P.-C.; Hu, Y.; Thurston, A.; Sommers, C. D.; Guzova, J. A.; Kahn, L. E.; Lai,
Y.; Blom, J. D. J. Pharm. Sci. 2009, 98, 4354.
12. The simulated lung fluid solubility is determined using an aqueous solution
containing 0.02%w/v DPPC and 5% v/v dog plasma protein and assessed by the
dialysis bag method.