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Introduction |
Pharmacological
identification of the histamine (HA) H3 receptor
and subsequent disclosure of possible HA H3
receptor subtypes has led to increased efforts toward the development
of selective HA H3 ligands and keen interest into
the potential existence of HA H3 receptor
subtypes (West et al., 1990
). Several notable imidazole-derived HA
H3 antagonists such as amides (GT-2016),
thioureas (thioperamide), isothioureas (clobenpropit), and ethers
(iodoproxyfan) have been described previously (Arrang et al., 1987; Van
der Goot et al., 1992; Tedford et al., 1995; Stark et al., 1996).
Representative examples are shown in Fig.
1. In addition, the natural product verongamine (Fig. 1) has been described with moderate HA
H3 receptor affinity (Mierzwa et al., 1994), and
has provided our group with conceptual insight into the development of
new HA H3 antagonists with distinct structural
features. Using the verongamine pharmacophore, we have recently
established, from a series of 1H-4-substituted imidazole HA
H3 antagonists, several novel structure-activity relationships (SARs) important for HA H3
receptor-ligand interaction (Yates et al., 1999).
A reduced verongamine analog (Fig. 2)
provided us with the template to synthesize several structurally
distinct compounds and provide the tools necessary for an in-depth
analysis of HA H3 receptor-ligand interactions.
For example, none of the existing potent HA H3
receptor antagonists exhibit any stereochemical presentations. However,
the observation that the HA H3 receptor maintains
a distinct stereochemical bias has been demonstrated previously with
the potent and selective HA H3 receptor agonists
(R)-
-methylhistamine and
(R)-,(S)-
-dimethyhistamine,
as well as in our development of (L)- and
(D)- histamine-amide antagonist derivatives
(Yates et al., 1999).
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Fig. 2.
Model template for HA H3 receptor
antagonist development is shown with a reduced analog of verogamine.
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Moreover, many of the current HA H3 antagonists
including verongamine, clobenpropit, and iodoproxyfan, as well as our
recent 1H-4-substituted imidazoles, provide compounds that
contain a flexible ethylene side chain (Fig. 2B). This limits detailed
investigations on the spatial and/or conformational orientation of the
imidazole head (Fig. 2A) and terminal portions of the HA
H3 ligands (Fig. 2, B-E). Only GT-2016 and
thioperamide utilize cyclic ring presentations, which would impart
conformational restriction and provide insight into the optimal spatial
orientation for ligand-receptor interactions. In the present studies,
we have utilized replacement of the ethylene bridge (Fig. 2B) with an
enantiomerically pure cyclopropane nucleus to investigate both
stereochemical and conformational preferences of the HA
H3 receptor and to build on the previous SAR
findings surrounding the terminal portion of the molecules (Fig. 2,
C-E).
To date, studies by West et al. (1990) have suggested the potential
existence of HA H3 receptor subtypes
(H3A and H3B). More recent
studies by Clark and Hill (1995, 1996) have established that the HA
H3 receptor is
Gi/Go coupled, and that the
classic HA H3 antagonist thioperamide displays
heterogeneity in binding isotherms which are characteristic of a
constitutively active receptor system. Moreover, evidence has suggested
that many G protein-coupled receptor systems including opioid,
dopaminergic, serotonergic, adrenergic, and histaminergic
(H2) receptors may be constitutively active, and
great interest has been generated in the understanding of these novel G
protein-coupled receptor-ligand interactions (reviewed by Milligan et
al., 1995; Milligan and Bond, 1997). The series of compounds
described herein provide further insight into the structural features
of HA H3 receptor antagonists which can
differentiate between receptor subtypes and/or receptor activation
states. Moreover, the present research describes the identification and
initial pharmacological characterization of one of the most potent HA
H3 receptor antagonists to date.
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Materials and Methods |
Chemicals.
GT compounds and thioperamide were synthesized by
Gliatech chemists. Other reagents were purchased as indicated:
triprolidine (Research Biochemicals International, Natick, MA),
aminopotentidine (Tocris Cookson, Bristol, UK),
[3H]N
-methylhistamine
([3H]NAMHA; 81.5 Ci/mmol),
[3H]pyrilamine (>20 Ci/mmol; DuPont NEN
Research Products, Boston, MA), and
[125I]iodoaminopotentidine (2000 Ci/mmol;
Amersham, Arlington Heights, IL or provided by Drs. Leurs and Timmerman
at the Leiden/Amsterdam Center for Drug Research, the Netherlands).
Clobenpropit was kindly provided by Dr. Timmerman.
Animals.
Male Sprague-Dawley rats were purchased from Harlan
Laboratories (Indianapolis, IN) and housed two per cage on a 12-h
light/dark schedule with ad libitum access to Teklad Mouse/Rat Diet
7012 (Harlan Laboratories) and water in accordance with the
Animal Welfare Act of 1994 and amendments. Animals were acclimated to laboratory conditions for a minimum of 1 week before tissue harvesting.
Tissue Harvesting and Plasma Collection.
Animals were
euthanized at 0.5, 1, 2, 4, 8, 16, and 24 h postadministration
(n = 4/group). GT-2331 was administered in a final volume of 1 ml/kg. The animals were euthanized by a lethal injection of
pentobarbital (150 mg/kg i.p., Nembutal; Abbott Laboratories, North
Chicago, IL) at the indicated times postadministration. The thoracic
cavity was opened and blood was drawn from the heart using a 5-ml
syringe fitted with an 18-gauge needle. The blood was transferred to a
Vacutainer containing EDTA and stored at room temperature before
centrifugation and plasma collection. Following the collection of
blood, the upper torso was transcardially perfused through the aortic
arch with 60 ml of 0.9% saline to remove potential vascular drug
contamination. Brains were removed, dissected into various brain
regions, and frozen on dry ice. All of the tissues and plasma were
stored at
80°C before conducting the ex vivo binding and
HPLC-electrochemical detection analysis.
In Vitro HA H3 Receptor-Binding Analysis.
HA
H3 receptor affinity was determined in rat
cortical membranes with [3H]NAMHA as described
previously (Tedford et al., 1995). Animals were euthanized by rapid
decapitation and cortical tissues were harvested and frozen on dry ice.
Cortical membranes were prepared in 50 mM sodium-PBS (pH 7.5 at 4°C)
containing EDTA (10 mM), phenylmethylsulfonyl fluoride (0.1 mM), and
chymostatin and leupeptin (each 0.2 mg/50 ml). The final membrane
pellets were resuspended in water and stored frozen at 80°C before
use. Protein concentrations were determined using the Coomassie Plus
Protein Assay (Pierce, Rockford, IL).
Competition binding was carried out in a total volume of 0.2 ml of 50 mM sodium-phosphate buffer (pH 7.4) using ~1 nM
[3H]NAMHA and 0.01 to 10,000 nM concentrations
of the test compounds. Nonspecific binding was determined using 10 µM
thioperamide. Samples were incubated for 40 min at 25°C and
subsequently filtered through Whatman GF/C glass fiber filters
presoaked in binding buffer with 0.3% polyethyleneimine using an
Inotech cell harvester (Inotech Biosystems International, Lansing, MI).
The filters were rapidly washed three times with Tris-NaCl buffer (25 and 145 mM, respectively, pH 7.4, 4°C). Samples were quantitated
using Ecolume scintillation cocktail (ICN Biomedicals, Costa Mesa, CA)
and a Packard model 1900TR liquid scintillation analyzer (Packard
Instrument Co., Downers Grove, IL). IC50 values
were extraploated from a plot of receptor occupancy (i.e., percent
bound) versus log [competitor]. Inhibition constants
(Kis) were determined using the equation: Ki = IC50/(1 + [ligand]/[Kd]), where
Kd = 0.4 nM for
[3H]NAMHA.
Alternatively, rat cortical membranes were prepared in 25 mM Tris
buffer (pH 7.5 at 4°C) containing the protease inhibitors listed
above. The final membrane pellets were resuspended in water and stored
frozen at 80°C before use. Competition binding was performed as
above using 50 mM Tris buffer (pH 7.4) in place of the sodium-phosphate
buffer. Studies were performed in the absence or presence of 150 mM
NaCl or 30 µM GTP
S (Boehringer Mannheim, Indianapolis, IN).
Binding was terminated and samples were quantitated as described above.
Ex Vivo HA H3 Receptor-Binding Analysis.
Ex vivo
HA H3 receptor occupancy was determined in rat
cortical membranes with [3H]NAMHA as described
previously (Taylor et al., 1992; Tedford et al., 1995). On the day of
binding experiments, the tissue was homogenized using a motor-driven
tissue grinder (Omni 1000) in 9 volumes (w/v) of 50 mM sodium-phosphate
buffer. Ex vivo binding was carried out in a total volume of 0.4 ml of
50 mM sodium-phosphate buffer (pH 7.4) containing ~1 nM
[3H]NAMHA and 0.15 to 1 mg of protein.
Nonspecific binding was determined using 10 µM thioperamide. Samples
were treated as described above for the in vitro binding.
ED50 values (doses which produced 50% inhibition
of [3H]NAMHA binding) in milligrams per
kilograms were determined by linear regression analysis of the data on
a log-linear plot.
In Vitro HA H1 and H2
Receptor-Binding Analysis.
Before binding, HA
H1 or H2
receptor-expressing cells were harvested, washed, and stored as a
pellet at 80°C. In preparation for binding, cell homogenates were
prepared by sonication in distilled water and the protein content was
adjusted as necessary (see below). HA H1 receptor
affinity was determined using membranes prepared from Chinese hamster
ovary (CHO) cells transfected with the human HA
H1 receptor and
[3H]pyrilamine. Stable human HA
H1 receptor-expressing CHO cells were prepared
and provided by Drs. Leurs and Timmerman (Leiden/Amsterdam Center for
Drug Research, the Netherlands). The HA H1
receptor was expressed at ~1 pmol/mg protein in these cells.
Membranes were prepared as described above. Binding was performed in a
total volume of 0.4 ml of 50 mM Na/K-phosphate buffer containing 1 mM MgCl2 (pH 7.4, 25°C). Nonspecific binding was
determined in the presence of 10 µM triprolidine. For competition
studies, 4 nM [3H]pyrilamine was incubated with
~8 µg of membrane protein for 30 min at 25°C in polypropylene
tubes with increasing concentrations of test compounds. Binding was
terminated and samples were quantitated as described for HA
H3 receptor binding.
HA H2 receptor affinity was determined using
membranes prepared from CHO cells transfected with the human HA
H2 receptor and 125[I]iodoaminopotentidine. One HA
H2 clone expressing ~2 pmol/mg protein was
expanded for use in receptor-binding assays. Membranes were prepared as
described above. Binding was performed in a total volume of 0.1 ml of
50 mM Na/K-phosphate buffer (pH 7.4, 25°C). Nonspecific binding was
determined in the presence of 10 µM tiotidine. For competition
studies, 0.25 nM 125[I]iodoaminopotentidine was
incubated with ~7.5 µg of membrane protein for 2.5 h at 25°C
in polypropylene tubes with increasing concentrations of test
compounds. Binding was terminated and samples were quantitated as
described for HA H3 receptor binding.
GT-2331 was also evaluated in a general receptor profile screen
(Novascreen, Hanover, MD) to determine affinity for 59 separate neurotransmitter, neuropeptide, ion channel, hormone, and enzyme systems. A single concentration (1.0 µM) of GT-2331 was tested in
triplicate and the percentage of inhibition was determined.
HPLC-Electrochemical Detection Assay for GT-2331.
Analysis
was performed by utilizing a Prodigy (Phenomenex, Torrance, CA) 5 µm
ODS(2) guard column (30 × 3.2 mm i.d.) in series with a Prodigy 5 µm ODS(2) analytical column (150 × 3.2 mm i.d.) as described by
Handley et al. (1998). The temperature of the column was maintained at
40°C. The BAS (Bioanalytical Systems, Inc., West Lafayette, IN) LC-4C
amperometric detector in conjunction with a BAS CC-5 dual glassy-carbon
electrode flow cell was set with a potential of +1.1 V, with a range of
50.0 nA, and a filter setting of 0.1 Hz. The reference electrode
consisted of a Ag/AgCl electrode. Mobile phase consisted of an
isocratic system of 88:12 acetonitrile to 50 mM sodium acetate buffer
solution (adjusted to pH 6.7 with 1 M acetic acid). All mobile phase
components were filtered through a 0.45-µm filter and sparged with
helium before and through all analytical runs. Mobile phase flow was
maintained at 1.0 ml/min. The injection volume was maintained at 50 µl/run.
Plasma samples were thawed and allowed to come to room
temperature and spiked with a known amount of internal standard,
GT-2260. GT-2331 and GT-2260 were selectively extracted from the plasma by filtration with an Oasis HLB Extraction Cartridge (Waters
Corporation, Milford, MA) preconditioned with 1 ml of methanol and 1 ml
of deionized water. Five hundred-microliter plasma samples were drawn through the cartridge via a vacuum manifold using 5 mm Hg of pressure. Extraction cartridges were washed with 1 ml of a 5% methanol solution. Elution of compounds was accomplished by a 1-ml wash of a 100% acetone
solution into a 5-ml Wheaton reaction vial.
Immediately after extraction, 25 µl of a 50 mM
Na2
CO3-NaHCO2 buffer solution
(pH 9) was added to the eluted acetone solution. Then 200 µl of a 3.5 mM (1.13 mg/ml in acetone) dabsyl chloride solution was added to a vial
containing the 1-ml acetone-buffer solution. The vial was then sealed
and placed into an 80°C oven for 40 min. After 40 min, the vials were
opened and placed back in the oven until completely dry. Once dry, the
vial was allowed to cool to room temperature and then 100 µl of
acetone was added and the sample was vortexed. Then 300 µl of
acetonitrile was added and the sample was vortexed again. The 400 µl
of dabsyl solution was filtered through a 0.45-µm nylon syringe
filter and then loaded onto the autosampler for injection.
Pharmacokinetic Data Analysis.
Individual plasma samples
collected from the various time points were used to determine the mean
GT-2331 plasma levels at various time points. A composite time course
of GT-2331 plasma levels was generated and pharmacokinetic parameters
were determined from the elimination phase using the WinNonlin
program (version 1.1; Scientific Consulting Inc., Cary, NC). The
total area under the curve (AUCall), area under
the curve extrapolated out to infinity (AUC
)
and total area under the moment curve (AUMCall)
were calculated using the linear-log trapezoidal rule from
Cmax to the last time point with
plasma concentrations above the lower limit of quantitation of 100 pg/ml. The plasma clearance (Cl) of GT-2331 was calculated by dividing
the dose administered to the rats by the AUCall.
The mean residence time (MRT) was calculated from the ratio of the
AUMCall to AUCall. The
Vd was calculated by multiplying the
MRT by the plasma Cl rate.
Statistical Analyses.
The effects of NaCl and GTPS on the
inhibition-binding profiles for thioperamide, GT-2016, GT-2212, and
GT-2331 were evaluated by a one-tailed Student's t test. A
p < 0.05 was used to establish statistical differences
between groups (n = 3).
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Results |
In Vitro HA H3 Receptor-Binding and
SAR Studies.
Previously, the coupling of HA with
(L)-phenylalanine provided a compound, GT-2130,
with moderate HA H3 receptor affinity (Ki = 104 nM) and a stereoselective
preference over the ligand containing unnatural
(D)-phenylalanine (IC50 > 10,000 nM) (Yates et al., 1999). The present studies now demonstrate
that incorporation of the racemic trans-cyclopropane ring
into structurally similar analogs imparts high affinity for the HA
H3 receptor (GT-2157, Ki = 4.3 nM; Table
1). Synthesis of the individual
trans-cyclopropane enantiomers of GT-2157 demonstrated a
stereopreference of the HA H3 receptor for the
(1R,2R)-enantiomer GT-2163
(Ki = 1.8) over the
(1S,2S)-enantiomer GT-2164
(Ki = 22 nM). Moreover, the
(1R,2R)-trans-cyclopropane analog GT-2163 had
considerably higher affinity than the corresponding ethylene analog
GT-2130 (1.8 and 30 nM, respectively; Yates et al., 1999). Therefore,
it is evident that the trans-cyclopropane ring, which
imparts conformational restriction as well as a stereoorientation, can
be utilized to improve HA H3 receptor affinity.
Identification of GT-2163 as a potent HA H3
ligand containing the (1R,2R)-cyclopropane ring
system allowed further SAR exploration into regions C-E of the model
template (Fig. 2). In the current studies, a series of
trans-cyclopropane compounds is described containing either
carbamate or retroamide functional groups with various hydrophobic
substitutions (Tables 1 and 2). Several (±)-trans-cyclopropyl carbamate derivatives were developed
with high affinity (~10 nM) for the HA H3
receptor including GT-2263 and GT-2304. A series of amide derivatives
is summarized in Table 2, in which the amide linkage is reversed in
orientation versus GT-2163. Generally, the trans-cyclopropyl
amides displayed lower affinity for the HA H3
receptor. The trans-cyclopropane enantiomers GT-2201/GT-2202
and GT-2211/GT-2212 were synthesized and suggested a preference for the
(1S,2S)-enantiomers contrasting with the high-affinity
(1R,2R)-histamine-amide derivatives.
Finally, cyclopropyl derivatives containing the nonpolar and planar
olefin and acetylene equivalents instead of the polar and planar amide
and amide-oxime spacer in verongamine were made (Table
3). Following the results of a
Topliss operational scheme (Topliss, 1972) described for the
1H-4-substituted imidazoles (Yates et al., 1999, Ali et al.,
1998, 1999), several cyclopropyl acetylene derivatives (GT-2314,
GT-2343, and GT-2344), were made and determined to have high affinity
for the HA H3 receptor (Table 3). Particularly,
the racemic derivative GT-2314 had a
Ki of 0.33 nM. The corresponding
(1R,2R)-trans-cyclopropane enantiomer GT-2331
had a Ki of 0.12 nM, whereas the
(1S,2S)-trans-cyclopropane enantiomer
GT-2342 had substantially lower affinity (~20×) for the HA
H3 receptor (Ki = 5.3 nM). These results illustrate the optimization of several
dependent features for HA H3 receptor binding and
provides one of the most potent HA H3 ligands
described.
In addition, all four trans-cyclopropyl olefins were made to
further evaluate the geometric isomers that are provided within the
olefin series. Minimal differences were seen between GT-2208 and
GT-2209, the corresponding (1R,2R)-trans- and
cis-olefins. Moreover, the
(1R,2R)-trans-cyclopropyl
(cis)- and (trans)-olefin isomers demonstrated marked stereoselectivity over the corresponding (1S,2S)-isomers (GT-2208 versus GT-2207, and GT-2209 versus
GT-2210). These findings as well as those seen with the acetylene
series suggest that a planar alkyl portion of the template molecule (C and D) is the minimal structural requirement needed, provided it
conveys the appropriate overall shape and orientation of the molecule
for the HA H3 receptor. Finally, introduction of
the (L)-amino substitution (Fig 2D), analogous to the
(L)-amino acid derivatives described previously, completely
altered the apparent HA H3 receptor affinity for
the olefin analogs. GT-2252, the cis-olefin derivative was
substantially less active (65×), whereas GT-2232, the
trans-olefin derivative was 10× more potent than their
corresponding unsubstituted analogs. These results demonstrate an
additional site for ligand-HA H3 receptor interaction.
In Vitro HA H3 Receptor-Binding Profiles under Varying
Ionic Buffer Conditions.
Studies in the literature with
thioperamide have shown differential apparent HA
H3 receptor-binding affinities with the HA H3 agonist [3H]NAMHA
dependent on the binding assay buffer conditions (West et al., 1990).
Clark and Hill (1995) recently showed, using Tris buffer and
[3H]NAMHA, that the IC50
for thioperamide could be shifted based on the presence or absence of
50 to 100 mM NaCl. To compare the binding profiles of our new HA
H3 antagonists with that of thioperamide, studies
were performed using Tris buffer and [3H]NAMHA
in the absence or presence of NaCl. We found that like thioperamide
(Fig. 3; Table
4; Clark and Hill, 1995), the apparent HA
H3 receptor affinities for GT-2016 and GT-2212,
both amides from the piperdine and cyclopropane series, respectively,
shifted to a lower affinity state in the absence of NaCl (5-fold and
3.5-fold, respectively). In contrast, the apparent affinity for
GT-2331, a cyclopropane-acetylene derivative, was minimally shifted in the absence of NaCl ions (Fig. 3). Similar findings were seen with
GT-2208, a cyclopropane-olefin derivative (data not shown). These
findings indicate that HA H3 receptor
heterogeneity is seen in selected cyclopropane analogs. More
importantly, the specific isostere moiety (Fig. 2, C and D) may provide
the critical structural modality for establishing HA
H3 receptor subtype selectivity and/or HA
H3 receptor conformational changes.
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Fig. 3.
GT-2016, GT-2212, and GT-2331 displacement curves for
[3H]NAMHA using rat cortical membranes under various
ionic conditions. Membranes were incubated with ~1 nM
[3H]NAMHA in Tris buffer in the absence ( ) or presence
( ) of 150 mM NaCl with the indicated concentrations of GT-2016 (A),
GT-2212 (B), or GT-2331 (C). These data are from a single experiment
and are representative of typical inhibition isotherms, which were
performed in triplicate.
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The studies of Clark and Hill (1995) also showed that GTPS (a
nonhydrolyzable analog of GTP) had an effect similar to that of NaCl on
the HA H3 receptor-binding profile of
thioperamide in Tris buffer. We also found that the addition of GTPS
to the Tris buffer conveyed a dramatic increase in apparent
H3 receptor affinity for GT-2016 and GT-2212
(Table 4) analogous to addition of 100 mM NaCl. However, the addition
of GTPS imparted minimal shifts in the apparent affinity for GT-2331
(Table 4) and GT-2227 (data not shown) consistent with previous findings.
HA Receptor Selectivity Profile.
The HA
H1and H2 receptor
affinities were also evaluated for all of the novel HA
H3 ligands using membranes from cells expressing the cloned human HA H1 and
H2 receptors. HA H3
receptor selectivity profiles were determined and compared for the
different compounds and chemical classes. Overall, the compounds
demonstrated highly selective profiles for the HA
H3 receptor versus the HA
H1 and H2 receptors. These
findings are illustrated with representative candidates from the amide
(GT-2163, GT-2212, and GT-2263), olefin (GT-2208), and acetylene
(GT-2331) series. GT-2331, the most potent HA H3
receptor antagonist, had a selectivity ratio of over ~75,000-fold versus the HA H2 receptor and even better versus
the HA H1 receptor (Table
5). In addition, GT-2331 was further
tested in 59 different assay systems and <50% inhibition was seen at
1.0 µM for all but the 2,
m1, and 
receptors. In those receptor systems,
the percentage of inhibition ranged from 52 to 67%, indicating 1,000 to 10,000-fold selectivity of GT-2331 for the HA
H3 receptor.
Ex Vivo HA H3 Receptor-Binding Studies.
The
central nervous system (CNS) penetration for several of the most potent
HA H3 antagonists was evaluated using the ex vivo binding technique (Table 6). Animals were
dosed with 0.03 to 30 mg/kg (i.p.) HA H3
antagonist, and after 1 h the rats were euthanized and the
penetration of compound into the CNS was evaluated. The most potent
compounds, GT-2208, GT-2212, and GT-2331, produced a dose-dependent
inhibition of [3H]NAMHA binding in rat cortical
homogenates from drug- versus vehicle-treated animals. This is
illustrated for GT-2331, which had an exceptionally low
ED50 consistent with the apparent high affinity
seen in vitro (Fig. 4). Log-linear
regression analysis of these data provided ED50
values of 1.5, 20.7, and 0.08 mg/kg for GT-2208, GT-2212, and
GT-2331, respectively.
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Fig. 4.
Ex vivo HA H3 receptor-binding profile
for GT-2331 in rat brain cortex. GT-2331 (0.03, 0.1, 0.3, and 1 mg/kg)
and vehicle were administered i.p. 1 h before the animals were
euthanized. Ex vivo H3 receptor binding was determined for
each animal (n = 3-4/group) in femtomoles per
milligram of protein and expressed as a percentage of values from
vehicle-treated animals. Absolute values for vehicle-dosed animals were
37.7 ± 1.5 fmol/mg protein (mean ± S.E.,
n = 4).
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The 24-h time course of CNS penetration and HA H3
receptor occupancy were subsequently evaluated for GT-2331 (1 mg/kg
i.p.). GT-2331 produced near maximal inhibition of
[3H]NAMHA binding within 1 h of i.p.
administration (Fig. 5). High levels of
receptor occupancy were maintained out to 8 h for GT-2331. HA
H3 receptor occupancy returned to normal within
16 to 24 h after the single acute administration of GT-2331.
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Fig. 5.
Twenty-four-hour time course ex vivo H3
receptor-binding profiles for GT-2331 in rat brain cortex. GT-2331 (1 mg/kg) and vehicle were administered i.p. Animals were euthanized at
0.5, 1, 2, 4, 8, 16, or 24 h after administration of drug. Ex vivo
H3 receptor binding was determined for each animal
(n = 4/group) in femtomoles per milligram of
protein and expressed as percentage of vehicle-treated values.
Absolute values for vehicle-dosed animals were 40.6 ± 3.8 fmol/mg
protein (mean ± S.E., n = 4).
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Pharmacokinetic Study of GT-2331.
Plasma samples used for
pharmacokinetic analysis were obtained from the animals used in the
GT-2331 ex vivo receptor-binding study (1 mg/kg i.p.). A composite
GT-2331 plasma profile was generated and initial pharmacokinetic
parameters were established for comparison to ex vivo
H3 receptor-binding profiles. Analysis of the
plasma samples indicated that GT-2331 was readily absorbed into the
blood stream with maximal plasma concentrations
(Cmax) of 810.0 ng/ml being seen at
the first 30-min time point. This was in agreement with the rapid
appearance in cortical H3 receptor blockade seen in these animals. Using the linear-log trapezoidal rule, the
AUCall for all observed time points was
calculated to be 2437 ng·h/ml, whereas the
AUC was slightly higher at 2505 ng·h/ml
(Table 7). Subsequent elimination of
GT-2331 from the plasma provided an observed terminal elimination
half-life (T1/2) in excess of 4 h
(Table 7) and paralleled the time course for cortical
H3 receptor blockade.
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Discussion |
The present series of compounds focused on the development of
conformationally restricted and stereoselective apparent high affinity
HA H3 receptor antagonists. Several chiral HA
H3 agonists [i.e.,
(R)--methylhistamine and
(R)-,(S)--dimethyhistamine) have previously been described; however, the incorporation of such
chiral centers has not been exploited in the development of HA
H3 antagonists. In addition, the utilization of a
cyclopropane ring system in the present series provided a number of
conformationally restricted analogs in which orientation of the
imidazole head and terminal tailpiece could be evaluated. Finally, the
use of a template which systematically explored the various structural components required for HA H3 receptor activity
has provided the opportunity to develop diverse compounds with very
high HA H3 receptor affinity. The results of
these studies have lead to the development of novel HA
H3 receptor antagonists like GT-2331 which has a
Ki of 0.12 nM. Recent studies by
Tedford et al. (1998) have characterized the HA
H3 antagonist activity of GT-2331 using the isolated guinea pig jejunum and cardiac synaptosomes and discuss further H3 receptor binding versus functional results.
In the present studies, GT-2331 was evaluated for HA receptor
selectivity and CNS penetration. The compound provides an exceptional selectivity ratio versus the other known HA receptor subtypes. Furthermore, GT-2331 was extensively tested against a number of neurotransmitter, neuropeptide, ion channel, hormone, and enzyme systems and showed minimal interaction. These studies demonstrate the
apparent high affinity and selectivity of GT-2331 for the HA
H3 receptor. Moreover, the ex vivo binding
studies indicate that GT-2331 can penetrate the blood-brain barrier
very readily. The duration of action is very long and amenable for
further development. Pharmacokinetic studies support a good duration of
action for GT-2331 and parallel the CNS HA H3
receptor occupancy profiles.
The functional and pharmacological existence of the HA
H3 receptor, as well as potential HA
H3 receptor subtypes, has been demonstrated by
several laboratories using a variety of structurally diverse compounds
(West et al., 1990; Clapham and Kilpatrick, 1992; Schwörer et
al., 1994; Leurs et al., 1996; Schlicker et al., 1996). However, the
true nature of this receptor has been difficult to prove since the HA
H3 receptor has yet to be cloned. Previously,
thioperamide was shown to produce differential binding affinities using
[3H]NAMHA dependent on the binding buffer
conditions. For example, Clark and Hill (1995) showed using Tris
buffers that the IC50 for thioperamide could be
shifted based on the presence or absence of 50 to 100 mM NaCl. In the
absence of added NaCl, the overall IC50 for
thioperamide was 65 nM and the binding profile was best described by a
two-site model. However, in the presence of NaCl (or in
sodium-phosphate buffer) the overall IC50 was
shifted to 8 nM and was best described by a single-site model. Their
studies also showed that GTPS (a nonhydrolyzable analog of GTP) had
an effect similar to that of NaCl on the binding profile of
thioperamide in Tris buffer. Although it has been generally accepted
that the binding of histaminergic antagonists is independent of the
apparent affinity state of the receptor, such data would suggest
otherwise. Clark and Hill (1995) concluded that the binding of
thioperamide is sensitive to the G protein-coupling state of the
receptor, a phenomenon that is referred to as "negative" antagonism
or "inverse" agonism (Costa et al., 1992; Lefkowitz et
al., 1993). Similar findings have recently been demonstrated for the HA
H2 receptor, and data suggest that the HA
H2 antagonist cimetidine is an inverse agonist
(Smit et al., 1996).
We have also demonstrated that some of our HA H3
receptor antagonists were sensitive to the ionic conditions of the
binding buffer. For selected compounds, lower apparent affinity binding was seen in the ion-free Tris buffer condition with a 4- to 5-fold increase in affinity with the addition of NaCl. The apparent affinities of GT-2016 and GT-2212 in the Tris-NaCl buffer were similar to what we
have previously observed in sodium-phosphate buffer. Moreover, GTPS
produced a similar shift in apparent affinity for both compounds in the
ion-free Tris buffer conditions. These data suggest that GT-2016 and
GT-2212 as well as thioperamide may function as inverse agonists.
However, these studies do not preclude the possible existence of
distinct HA H3 receptor subtypes in which one
subtype may show greater sensitivity to the ionic conditions of the
binding buffer.
Although the binding of GT-2016 and GT-2212 was clearly sensitive to
the ionic conditions of the binding buffer, the binding of
GT-2331, an acetylene, as well as other olefin derivatives was not
dramatically affected by the presence or absence of NaCl. Furthermore,
GTPS minimally altered the binding of GT-2331 under ion-free Tris
buffer conditions. Thus, GT-2331 might display low efficacy inverse
agonism in some systems but may be better characterized as a neutral antagonist.
It appears that there may be important differences in the in vitro
binding profiles exhibited by the different subclasses of
cyclopropane-containing HA H3 receptor ligands.
The different structural features in these new series are of particular
interest and may provide further insight into HA
H3 ligand-receptor interactions. In the case of
the acetylene and olefin derivatives, all heteroatoms have been removed
distal to the imidazole head, thus limiting interactions (e.g.,
hydrogen, ionic, etc.) within the receptor-binding domain. Such
interactions may be critical for the initiation of conformational
changes and/or G protein coupling. Clearly, additional studies are
required to fully characterize the functional activity of these
compounds and their neutral antagonist or inverse agonist nature. Such
studies, however, may not be feasible until the HA H3 receptor is functionally cloned. Moreover,
these studies do not preclude the possibility of HA
H3 receptor subtypes in which one subtype may
show greater sensitivity to the ionic conditions of the buffer.
Finally, constitutive receptor activity has been recently
described as coupling of the receptor-G protein in the activated state
in the absence of an agonist (Lefkowitz et al., 1993). This phenomenon
has been observed in several native and mutant receptor systems
including HA H2, opioid, -adrenergic, and
leptin (Costa et al., 1990; Samama et al., 1993; Smit et al., 1996;
White et al., 1997). The broader implications are that inverse agonists and neutral antagonists might provide different pharmacological effects
in vivo and provide unique opportunities for drug development (Costa et
al., 1992). These recent developments in the understanding of G
protein-coupled receptor interactions and constitutive receptor activity may have dramatic implications on drug development.
In summary, the current studies, as well as those by Yates et al.
(1999), have disclosed two new broad series of potent HA H3 receptor ligands. Insight into the
understanding of the SARs associated with ligand-HA
H3 receptor interactions have been garnered with
the development of these new classes of HA H3
receptor ligands. Finally, several potent and selective HA
H3 antagonists have been identified which will
provide for further biological assessment and potential clinical development.
We gratefully acknowledge the efforts of Dr. Loyd Burgess in the
preparation of the stably transfected HA H2
receptor-expressing Chinese hamster ovary cells and the cell culture
expertise of Ms. June Kocsis Angle.
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Abstract
Introduction
