IBS (irritable bowel syndrome) is a gastrointestinal disorder
which is amongst the most commonly encountered in primary care. It is approximately
twice as prevalent in women as it is in men and presents as a collection of symptoms
which vary between patients and, within a single patient, over time. Symptoms always
include lower abdominal pain which may be associated with constipation and/or diarrhoea.
Diagnosis of IBS is performed by excluding other diseases,
followed by positive symptomatic diagnosis which is carried out according to the
Manning or Rome criteria. A recent review by experts in the field has led to publication
of revised diagnostic criteria (Rome 2), and updated diagnosis and treatment recommendations,
based on research results. Recent approaches to the treatment of IBS have been based
around the finding that levels of the neurotransmitter 5-hydroxytryptamine (5-HT)
and it's metabolites are raised in the plasma of patients with IBS. 5-HT has a number
of different receptor subtypes found in the human body.
Observations in animal studies have shown that both 5-HT3
and 5-HT4 receptors are present on myenteric neurones and mediate the
release of excitatory neurotransmitters which cause contraction of gastrointestinal
smooth muscle. This has led to the development of both 5-HT4 agonists
and antagonists, and 5-HT3 antagonists. However, studies in man have
not revealed the presence of either 5-HT4 or 5-HT3 receptors
on myenteric neurones, so the specific receptors exerting neuronal control of gastrointestinal
function are unknown.
The prior art has also speculated that a 5-HT2B
antagonist may have use in the treatment of irritable bowel disorders these speculations
are in the public domain. However, the basis for credible development of such an
approach has not previously been determined. The supposition is based on observations
in rat, where the receptor is present in the stomach fundus, and mediates contraction.
Similarly, the authors of
US patent US-5457101
) base their proposal for the use of 5-HT2B antagonists on the observation
of 5-HT2B mediated contraction of rat colon smooth muscle.
also proposes a role for 5-HT in the pathology of IBS. However, the claim
that 5-HT2B receptors might be beneficial in the treatment of IBS is
based on three indirect observations:
5-HT2B receptors are present in human intestine.
However, the two references cited in
report the presence of 5-HT2B receptors which contract small
intestinal smooth muscle. There is no evidence, either experimental or clinical,
that this mechanism of action has any relevance to IBS. In addition, there is no
evidence that these actions in human intestinal tissue are accompanied by production
of nitric oxide, leading to the sensitization of sensory nerve fibres.
Non-selective 5-HT2B receptor antagonists are
clinically effective in reducing the pain associated with IBS. The first of these
studies investigated the use of mianserin in the treatment of IBS and non-ulcer
dyspepsia, and showed the treatment to offer alleviation of symptoms. Mianserin
is a non-selective alpha-2 adrenoceptor, 5-HT1, 2 and 3, and histamine H1 and H2
receptor antagonist, and it's beneficial effects can not therefore be attributed
solely to 5-HT2B receptor stimulation. In the second reference, the disease
under investigation was "abdominal migraine" which shares little similarity to IBS.
Entry criteria for the trial included at least bimonthly attacks, abdominal pain
with facial pallor, and at least one first-degree (or two second degree) relatives
with a history of migraine or recurrent headaches. These authors themselves stated
that there is no reason to believe that the treatment [pizotifen] would be effective
in patients with abdominal pain who did not meet these criteria, and it is therefore
difficult to find evidence from this trial to support the use of pizotifen in IBS.
In addition, pizotifen is a non-selective 5-HT2 receptor, which is non-surmountable
at 5-HT2A receptors (Prins et al., 1997), and additionally shows significant
affinity for 5-HT3 receptors (Schmidt & Peroutka, 1989). The evidence
(from these clinical studies) to support the use of 5-HT2B receptor antagonists
in IBS is therefore not credible.
These separate and independent pieces of evidence are claimed
as support for the use of 5-HT2B receptor antagonists in the treatment
of IBS. However,
provides no experimental evidence to' support this claim.
The prior art contains several publications that provide
examples of compounds that are capable of binding to 5-HT receptors.
discloses compounds that comprise covalently connected ligands that are
capable of binding to a 5-HT receptor. The interaction of the 5-HT receptor with
the ligand may inhibit or initiate an effect of the 5-HT receptor, which may be
used to provide a therapeutic effect.
discloses several classes of compounds that are capable of binding to
the 5-HT2B receptor in mammals and which may be used for treating or
preventing a variety of 5-HT2B related conditions. Agents that target
5-hydroxytryptamine receptor subtypes, particularly the 5-HT3 or 5-HT4
subtypes, for the treatment of IBS have also been reviewed (De Ponti et al, 2001).
Disclosure of the invention
The present inventors have investigated the location and
action of 5-HT2B receptors in human colon tissue. The inventors have
demonstrated, however, that 5-HT2B receptors in human colon have no significant
effect on basal tone in human colon (i.e. no effect on resting colon), but that
their activation potentiates contractile responses to neuronal stimulation. In other
words, it is believed that in the human colon, 5-HT acts on 5-HT2B receptors
to make the colon hypersensitive to neuronally-mediated stimulation. It is this
mechanism which it is believed to be the key to the involvement of 5-HT2B
receptors in the pathogenesis of IBS, rather than merely by causing direct smooth
Thus for the first time, it is shown herein that 5-HT2B
receptors are present on both nerves and smooth muscle of human colon, and that
their activation potentiates neuronally-mediated responses, and are thus causative
in the abnormal motility and pain associated with IBS. For this reason, there is
provided direct experimental evidence that 5-HT2B receptors will be useful
in the treatment of IBS.
The demonstration of 5-HT2B receptors localised
on neuronal elements within the human GI tract is not in the public domain, nor
is the potentiation of neuronally-induced contractions induced by activation of
5-HT2B receptors. Thus the present findings allow the development of
a rational approach to the development of an IBS treatment.
The present invention thus relates to a rational approach
to the treatment of IBS via 5HT2B antagonists, as well as a novel means for the
development of novel compounds useful in the treatment of IBS.
The present invention provides an assay to determine whether
or not a compound is capable of antagonising 5-HT2B receptor activity
in the human colon, which assay comprises:
- placing a section obtained from a human colon; under tension;
- electrically stimulating said section in the presence of a potential 5-HT2B
receptor antagonist; and
- observing whether said potential 5-HT2B receptor antagonist is capable
of antagonising the 5-HT induced potentiation of electrically stimulated contractions
of said human colon.
The present findings provide a novel means in the development
of compounds for use in the treatment of GI disorders, the means being the use of
human colon smooth muscle preparation as an assay for the detection of compounds
which may be beneficial in the treatment of GI disorder, and particularly IBS. The
colon smooth muscle preparation may be used in an assay in which it is electrically-stimulated,
so as to contract in the presence of such stimulation, and compounds assayed for
the ability to inhibit 5-HT-induced potentiation of such contraction. Such an assay
provides a means of detecting compounds with 5-HT2B activity.
Description of the drawings.
Detailed description of the invention.
5-HT2B Receptor Antagonist.
- Figure 1 shows the effect of 5-HT on electrically-induced contractions of longitudinal
muscle strips from human colon. The transient contractile response to EFS, and the
potent potentiation of this neurally-mediated response by increasing concentrations
of 5-HT (-9 to -5M) is illustrated.
- Figure 2 shows the effect of 5-HT in colon smooth muscle. Figure shows mean
concentration-effect curve to 5-HT, with data expressed as increase in contractility
(mN) over basal EFS-induced contractions. Data are given as mean±s.e.mean for
n=49 donors, and have been fitted to the Hill equation according to a 3 parameter
- Figure 3 shows 5-HT2B receptor immunoreactivity in the human colon.
In the muscularis externa, immunoreactivity was predominantly localised to the longitudinal
(L) and circular (C) muscle cells (MC) and the myenteric nerve plexuses (NP).
- Figure 4 shows immunoreactivity of adjacent human colonic sections to (A) 5-HT2B
(B) Neurofilament 68 and (C) control IgG1 antibodies.
A large number of 5-HT2B receptor antagonists
are known per se in the art. These include spirotetrahydro-beta-carboline derivatives
(eg as described in
), aryl pyrimidine derivatives (eg
), indole derivatives (
), fused indoles (
), heterocyclic urea derivatives (
), condensed indole derivatives (
), benzocondensed five membered heterocycle carboxamides (
), tricyclic derivatives (
), thieno-indole derivatives (
), indoline derivatives (
), carboline derivatives (
) and benzodiazepines (
). The disclosures of such compounds are incorporated herein by reference.
These compounds may be used in the present invention.
It is preferred that the 5-HT2B receptor antagonist
is a selective 5-HT2B receptor antagonist. Selective means fulfilling
one or more of the following parameters:
- 1. Affinity (KD) at the human 5-HT2B receptor (measured
at the cloned human receptor transfected in a mammalian cell line) ≥ 7.0,
more preferably ≥8.
- 2. Antagonist potency (pKB at the human 5-HT2B receptor
in colon ≥ 7.0 more preferably ≥8.
- 3. Ratio of binding at the 5-HT2B vs 5-HT2C receptor (measured
at the cloned human receptors transfected in a mammalian cell line) ≥ 10,
more preferably ≥100.
- 4. Ratio of binding over other members of 5-HT family of (measured at the cloned
human receptors transfected in a mammalian cell line) ≥30, more preferably
- 5. MW of compound ≤500.
The values KD and pKB may be determined
as described herein below. Criteria 1-4.are expressed without an upper limit, since
the skilled person will appreciate that it is not conventional when defining criteria
of this type to do so. However, in practice compounds which fulfil these criteria
will be in a range which is from the specified values to 3-4 log higher (e.g. for
(1) above from ≥7.0 to 10.0 to 11.0.
Preferably, compounds meet at least two, preferably at
least three of the above criteria. For example, compounds which meet criteria 1+2,
1+3, 1+4, 1+5, 2+3, 2+4, 2+5 are preferred groups of two criteria. Groups of three
criteria include 1+2+3, 1+2+4, 1+2+5, 2+3+4., 1+3+4, 2+3+5, 3+4+5 and the like.
Compounds which meet one of more of the above criteria
and which may be absorbed orally with no significant effect on hepatocyte function
and of low or non-toxicity are particularly preferred.
Specific compounds useful in the practice of the present
- RS-127445 (2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine) or a salt
or N-oxide thereof;
- LY-23728 (N-(1-methyl-1H-indol-5-yl)N'-(-benzyl)-2,3,4,9-tetrahydro-1H-beta-carboline).
This has a 100-fold selectivity for 5-HT2B over 5-HT2C and
5-HT2A. It has not been tested in the prior art in human colon.;
- SB-204741 (N-(1-methyl-5-indolyl)-N'-(3-methyl-5-isothiazolyl) urea). Active
in colon, potency of 6.8, at least 10-fold selectivity over other 5-HT2 receptors;
- SB-200646 (N-(1-methyl-5-indolyl)-N'-(3-pyridyl)urea hydrochloride). Non-selective
5-HT2B/2C receptor antagonist;
- SB-206553 (5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f]indole).
This is a non-selective 5-HT2B/2C receptor antagonist with >100-fold
selectivity over 5-HT2A receptors and is a potent antagonist of 5-HT
response in human colon (8.5); and
- SB-215505 (6-chloro-5-methyl-N-(5-quinolyl)-2,3-dihydro-1H-indole-1-carboxamide).
Potent, selective 5-HT2B receptor antagonist, potency of 9.9 and 100-fold
selectivity over 5-HT2A and 5-HT2C receptors.
It will be appreciated that the precise therapeutic dose
of a 5-HT2B receptor antagonist, expressed in the form of its free base,
will depend on the age and condition of the patient and the nature of the IBS to
be treated, and the affinity of the 5-HT2B receptor antagonist for the
human 5-HT2B receptor, and will be at the discretion of the attendant
However, in general, effective doses for the treatment
of IBS patients will lie in the range of 0.001 to 1000mg, such as 0.01 to 500mg,
preferably 0.1 to 250mg, for example 0.5, 10, 20, or 50mg of a 5-HT2B
receptor antagonist per unit dose, which could be administered in single or divided
doses, for example 1 to 4 times per day.
The 5-HT2B receptor antagonist, or a pharmaceutically
acceptable derivative thereof, may be formulated in conventional manner using one
or more acceptable carriers or excipients. Thus a 5-HT2B receptor antagonist,
or a pharmaceutically acceptable derivative thereof, may for example be formulated
for oral, sub-lingual, buccal, parenteral, rectal or intranasal administration,
or in a form suitable for administration by inhalation or insufflation (either through
the nose or mouth), or in a form suitable for topical administration.
For oral administration the pharmaceutical compositions
may take the form of, for example, tablets or capsules prepared by conventional
means with pharmaceutically acceptable excipients such as binding agents (eg pregelatinised
maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); filters (eg
lactose, microcrystalline cellulose or calcium phosphate); lubricants (eg magnesium
stearate, talc or silica); disintegrates (eg potato starch or sodium starch glycollate);
or wetting agents (eg sodium lauryl sulphate). The tablets may be coated by methods
well known in the art. Liquid preparations for oral administration may take the
form of, for example, solutions, syrups or suspensions, or they may be presented
as a dry product for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with pharmaceutically
acceptable additives such as suspending agents (eg sorbitol syrup, methyl cellulose
or hydrogenated edible fats); emulsifying agents (eg lecithin or acacia); non-aqueous
vehicles (eg almond oil, oily esters or ethyl alcohol); and preservatives (eg methyl
or propyl-p-hydroxybenzoates or sorbic acid).
For buccal administration the compositions may take the
form of tablets or lozenges formulated in conventional manner.
For parenteral administration the compositions may take
the form of injections, conveniently intravenous, intramuscular or subcutaneous
injections, for example bolus injections or continuous intravenous infusions. Formulations
for injection may be presented in unit dosage form eg in ampoules or in multi-dose
containers, optionally with an added preservative.
The compositions for parenteral administration may take
such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and
may contain formulatory agents such as suspending, stabilising and/or dispersing
agents. Alternatively, the compositions may be in dry form such as a powder, crystalline
or freeze-dried solid for constitution with a suitable vehicle, eg sterile pyrogen-free
water or isotonic saline before use. They may be presented, for example, in sterile
ampoules or vials.
Tablets for sub-lingual administration may be formulated
in a conventional manner.
For intranasal administration, or administration by inhalation
or insufflation, conventional formulations may be employed. For topical administration
the pharmaceutical compositions may be liquids, for example solutions, suspensions
or emulsions presented in the form of creams or gels.
Compositions may also be formulated as depot preparations
or delayed release formulations. Depot preparations may be administered by implantation
(for example subcutaneously, transcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compositions may be formulated with suitable polymeric
or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion
exchange resins, or as sparingly soluble derivatives, for example, as a sparingly
Compositions adapted for release of the 5-HT2B
receptor antagonist in the colon may be used. These include compositions in the
form of suppositories or retention enemas for rectal administration.
Other such compositions included delayed release formulations,
in which the 5-HT2B receptor antagonist is formulated in a manner which
allows specific delivery to the small and large intestine, and more preferably specifically
to the colon. This can be achieved by using delayed release formulations which are
There are numerous teachings in the art of means to provide
delayed release formulations of compounds such that the active ingredient is released
primarily in the colon. Generally, such means provide for protection of the active
ingredient from the environment of the stomach and intestines by means of one or
more coatings. For example,
, describes a delayed release composition comprising a core of active ingredient
optionally combined with one or more excipients, coated with an intermediate layer
which delays release of the ingredient independently of pH, and an outer layer which
dissolved in a pH dependent manner.
The outer layer may be a polymer such as cellulose acetophthalate,
cellulose acetate terephthalate, cellulose acetate trimellitate, hydroxypropyl-methyl
cellulose phthalate, polyvinyl alcohol phthalate, polyacrylate or a polymethacrylate.
The coating will have little or no solubility at a pH lower than 5, but will dissolve
at a more neutral or alkaline pH, for example of 7.5 or above. This coating protects
the active ingredient during its passage through the stomach and intestines.
The intermediate layer will be a material such as a hydrophobic
gelling polymer such as methylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose,
polyvinyl alcohols, polysaccharides and the like.
An alternative approach is described in
, which also uses two protecting layers. In this reference, the intermediate
layer is an amorphous amylose, such as glassy or rubbery amylose. This layer is
broken down by the action of enzymes of the microflora which are found in the colon.
An outer layer of a film forming cellulose material or acrylic polymer material
is then added to provide protection to the amylose layer. This outer layer dissolves
in a pH-independent manner. The amylose is preferably glassy amylose with a glass
transition temperature, Tg, of no less than 17°C, preferably no less than 30°C,
and a molecular weight of at least 20,000, e.g. at least 100,000 daltons.
The film forming cellulose may be, for example, sodium
carboxymethyl cellulose, sodiumcarboxymethyl 1-hydroxyethyl cellulose, 2-hydroxycellulose,
2-hydroxypropyl cellulose, methyl cellulose, and the like, preferably with a molecular
weight range of from 42,0000 to 280,000 daltons. Acrylic polymer materials include
acrylate and methacrylate polymers and copolymers thereof, preferably in the molecular
weight range of from 15,000 to 250,000 daltons.
, the disclosure of which is incorporated herein by reference, the amylose
coating may be from 5 to 50µM in thickness around a core of active ingredient
of, for example a 1mm sphere, and the outer coating may also be of a similar size
range. Coatings comprising a mixture of the amylose and film forming material are
Delayed release compositions of the invention, including
those as described above, will allow the substantial bulk of the active ingredient
(for example at least 50% of each unit dosage form) released in the body of a subject
to be released in the colon.
For example, a delayed release composition will show less
than 20%, preferably less than 10% release of active ingredient after 3 hours in
0.1N HCl at 20°C, but more than 50%, preferably more than 75% release of active
ingredient within 24 hours when at a pH of 7.0 or above (e.g. 7.5) and/or exposed
to anaerobic microbial digestion by faecal microflora (for example using digestive
conditions described in Example 4(d) of
The invention also provides a means to assay and verify
the effectiveness of compounds as candidates for the treatment of IBS.
The assay of the invention comprises:
- placing a section obtained from a human colon under tension;
- electrically stimulating said section in the presence of a potential 5-HT2B
receptor antagonist; and
- observing whether said potential 5-HT2B receptor antagonist is capable
of antagonising the 5-HT induced potentiation of electrically stimulated contractions
of said human colon.
The section of human colon will be from fresh tissue, for
example recovered from patients undergoing surgery. The section is provided in a
manner in which it can be conveniently placed under tension in suitable apparatus
for the purposes of the assay. For example, a longitudinal section comprising muscle
strips of the human colon may be used, as described in the accompanying examples.
In the accompanying examples, the strips of colon are about
2 mm wide by 20 mm long, so that they fit the apparatus used by the present inventors.
The size of the strips may be varied according to experimental preference.
The strips may be placed in an oxygenated chamber containing
a physiologically balanced salt solution, such as the Kreb's solution described
in the accompanying examples. The strips of tissue are then placed under a tension
(for example between hooks or other attachment means) and left to equilibrate. A
tension of from 5 to 25, such as 10 to 15 mN is suitable, but may be varied according
to the particular apparatus used. Once the tissue has equilibrated, it may be used
to assay potential 5-HT2B receptor antagonists.
In the assay, the colon tissue is stimulated by electrical
pulses (for example a 10 second pulse every 60 seconds) so that contraction of the
tissue is observed upon pulsing, and relaxation of the tissue back to the resting
tension is achieved between pulses. Under these conditions, we have observed that
upon increasing concentrations of 5-HT, the contraction is markedly increased, though
the basal level without stimulation is unaffected - see Figure 1.
Compounds which are being assayed will be provided to the
tissue at a range of concentrations (for example from 10-6M to 10-10M,
preferably from 10-7M to 10-9M) and their ability to inhibit
the 5-HT-induced EFS contractions determined. Compounds which have this ability
will produce a rightward shift in the response curve of the type shown in Figure
The alteration in the response curve can be calculated
by a standard method, according to the method of
Arunlakshana, A. & Schild, H.O. ((1959). Some quantitative uses of drug antagonists.
Br. J. Pharmacol. Chemother., 14, 48-58
). This is a well known piece of work, which details the classical Schild
Equation, providing the pKB. The equation is: pKB = log(concentration
ratio - 1) - log(antagonist concentration), where the concentration ratio is the
concentration of agonist producing a defined response (such as 50% of the maximum)
in the presence of an antagonist, divided by the concentration producing the same
response in the absence of antagonist, and the antagonist concentration is the concentration
of antagonist producing that displacement in the agonist response.
The binding affinity, KD, may be calculated
by standard methods, for example by reference to
Kenakin, T, ((1993), Pharmacologic analysis of drug-receptor interaction,
2nd Edition, Raven Press
Compounds which demonstrate antagonism may be selected
as candidates for the treatment of IBS.
The compounds which may be used in the assay of the invention
include compounds which have been proposed in the art as having 5-HT receptor binding
activity, particularly 5-HT2B receptor binding activity, but whose activity
in colon tissue in particular has not been investigated. In such circumstances,
the assay of the invention provides a validation means in the development of IBS
Furthermore, the assay provides a means for the testing
and developments of compounds whose 5-HT2B activity is unknown, including
novel compounds produced as part of a drug development program. Libraries of compounds
are commercially available from a variety of sources and these may be used in the
assay of the present invention.
Compounds which show antagonism at the 5-HT2B
receptors in colon tissue may be formulated and used in accordance with the other
aspects of the invention described herein, and such compounds and their use form
a further aspect of the invention.
The following Examples illustrate the invention.
Gene Expression Data.
The present applicants have developed protocols for quantitative
analysis of mRNA expression using the ABI prism 7700 Sequence Detection System (Perkin
Elmer). The system uses fluorogenic PROBES to generate sequence specific fluorescent
signals during PCR. The probes are oligonucleotides with fluorescent reporter and
quencher dyes attached. While a probe is intact, the intensity of reporter fluorescence
is suppressed by a quencher. When a probe forms part of a replication complex during
the PCR process, the quencher is separated from the reporter dye resulting in an
increase in fluorescence which is then picked up by the 7700 sequence detector.
The ABI 7700 has a built in thermal cycler, and a laser
directed at each of the 96 sample wells via bi-directional fibre optic cables. Emitted
fluorescence travels through the cables to a detector where emissions which fall
between 520nm and 660nm are collected every few seconds. The system software analyses
the contribution of each component dye to the experimental spectrum, and normalises
the signal to an internal reference dye. The peaks of these normalised 'reporter'
values (Rn) are then plotted against thermal cycle number to produce an amplification
plot - to allow visualisation of the extent of PCR product generation.
The starting copy number of a target sequence (Cn) is established
by determining the fractional PCR cycle number (Ct) at which a PCR product is first
detected - the point at which the fluorescence signal passes above a threshold baseline.
Quantification of the amount of target mRNA in each sample is established through
comparison of experimental Ct values with standard curves for the target sequence
which are constructed during each experiment.
Total RNA was isolated from 72 human tissue samples, each
from three different donors. Samples of RNA were used in this study only if intact
18s and 28s ribosomal RNA were detected by gel electrophoresis, if actin mRNA could
be detected using the ABI 7700 sequence detector, and if genomic DNA formed less
than 10% of the total nucleic acid sample. Total RNA samples were annealed to PGN-826R
plus a GAPDH primer and reverse transcribed using MuLV reverse transcriptase. Quantitative
sequence detection was carried out on the resulting cDNA.
A DNase treatment step is carried out on all RNA samples
prior to mapping, the effectiveness of the treatment is monitored for each set of
map samples by running a control PCR plate which does not undergo the reverse transcription
A primer/probe set has been designed to span an intron/exon
boundary in the sequence of the ubiquitously expressed GAPDH gene. The probe in
this reagent set is labelled with a fluorophore which is spectrally distinct from
that attached to the specific probe for the target of interest. A determination
of the presence of GAPDH mRNA can therefore be carried out in the same well as that
used to examine the copy number of the target of interest. Absence of a GAPDH signal
within a well indicates that the RNA within that sample has been degraded.
Primer probe design.
Primer / probe sets for detection of 5-HT2B
mRNA were designed by the applicants and positioned to amplify target sequences
encoding amino acids in the intracellular loop between putative transmembrane domains
V and VI. Off-line homology searches revealed no significant matches with gene sequences
logged at Genbank.
The structure of the PRIMER/PROBE set used to amplify the 5-HT2B receptor
was as follows:
- Forward Primer: PGN-826F
5'- ACGCCTAACATGGTTGACTGTGTC-3' (SEQ ID NO:1)
- Reverse Primer: PGN-1024R
5'-TGAGGCTCTCTGTTCGTTGGAA -3' (SEQ ID NO:2)
- Taqman Probe: PGN-894T
5'-AGGTGGCAATGCTGGATGGTTCTCGA -3' (SEQ ID NO:3)
Reaction conditions were optimised using genomic DNA as
a template and a primer concentration grid followed by a probe concentration gradient
experiment. Primer concentrations were selected to give the most efficient amplification
of gene product i.e. those which generated a low threshold cycle and a relatively
high accumulation of fluorescence (300 nM forward, 50 nM reverse). These optimal
primer concentrations were then used to select the optimum probe concentration.
A probe concentration of 200 nM gave the maximum fluorescent signal and minimum
threshold cycle value.
Of the 72 tissues taken from the 3 donors tested, mRNA
for the human 5-HT2B receptor was found in at least one donor of all
tissues tested, showing that the receptor gene is ubiquitously expressed. Expression
was found to be highest in the reproductive system, with particularly high expression
being seen in uterus. Expression of 5-HT2B receptor RNA was also high
in all regions of the alimentary tract. In addition, high levels of 5-HT were found
in liver parenchyma, kidney medulla, and the adrenal and thyroid glands.
In the alimentary system, 5-HT2B receptor expression
was found in all regions of the gastrointestinal tract. In colon smooth muscle,
there was a large degree of variation in the levels of 5-HT2B receptor
mRNA between the three donors tested, with values ranging from 10 to approximately
13,000 copies of mRNA per 100ng protein. There is no obvious reason for this high
degree of variance; the levels of GAPDH was relatively comparable between the three
donors. In summary, mRNA for the human 5-HT2B receptor is expressed in
all tissues tested. Particularly high levels were found in the GI tract, and in
particular oesophagus and colon.
A monoclonal anti-5-HT2B receptor antibody was
purchased from Pharmingen, Inc, USA (Catalog Number 60531A, Lot no. M017437). The
antibody was raised against a recombinant fusion protein between glutathione-S transferase
and the human 5-HT2B receptor. The antibody has been shown to recognise
both the immunogen and rat 5-HT2B receptor.
Preparation of human tissue extracts.
Snap frozen human tissue (approx. 1 g) was added to 10
volumes of ice-cold 50 mM HEPES (pH 7.4), 1 mM EDTA, 1 mM EGTA, 250 mM sucrose,
0.2 mM PMSF. The tissue was homogenised using an Ultra-Turrax homogeniser on full
speed for approx. 10 seconds. Sodium dodecyl sulphate (SDS) was added at a final
concentration of 1%, for 10 min at room temperature, to solubilise proteins. Insoluble
material was removed by centrifugation at 1,000 xg for 10 min at room temperature.
The supernatant was decanted and re-centrifuged 40,000 xg for 15 min at room temperature.
Protein was determined by the BCA method using BSA as a standard. The final protein
extracts were stored at -20°C until use.
Dot-blotting of 5-HT
-like receptor protein.
The level of 5-HT2B-like receptor protein expression
was determined in protein extracts of 82 human tissues and 2 stable human cell lines
(HEK293 and 132N1). In brief, protein extracts were diluted to 0.4 mg protein/ml
in 62.5 mM Tris-HCl (pH 6.8); 1% (w/v) SDS. Immunoblotting was performed using a
96-well perspex blotting manifold (Life Technologies). Before use, the manifold
was washed in detergent, and rinsed in distilled water. A nitrocellulose sheet,
pre-soaked with distilled water and a buffer containing 25 mM Tris (pH 8.3), 192
mM glycine, 20% methanol, was loaded into the manifold and clamped in placed by
vacuum pressure. No sample vacuum was applied. Protein samples (100 µl/well)
were added to wells and incubated for 1 hour at room temperature.
The resultant protein blot was removed from the manifold
and rinsed in phosphate-buffered saline (PBS) for 5 min. The blot was stained with
Ponceau S for 15 min to visualise the protein bound and to inactivate endogenous
alkaline phosphatase enzyme. Destaining was achieved using PBS before blocking in
PBS containing 1% (w/v) Marvel, 1% (w/v) BSA, 1% (v/v) sheep serum for 1 hour at
room temperature. Anti-5-HT2B antibody diluted to 0.1 µg/ml in PBS,
1% Marvel, 0.25% BSA (reagent dilutent) was added to the blot and incubated for
90 min at 37°C. After thorough washing, bound antibody was localised using
an anti-mouse fluorescein-linked secondary antibody (1:600) (Amersham) and an anti-fluorescein
alkaline phosphatase conjugate (diluted in 1:2500 in Tris-buffered saline, 1% Marvel,
0.25% BSA). Finally, bound conjugate was localised using enhanced chemifluorescence
and imaged using a fluorimager (STORM ; Molecular Dynamics). The resultant image
was quantified using ImageQuant software. Control values obtained using non-immune
mouse IgG1 as the primary antibody were subtracted from the results for the anti-5-HT2B
antibody to correct for endogenous fluorescence or residual alkaline phosphatase
activity in protein extracts.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE).
SDS PAGE was performed according to a modified method of
Laemmli (1970). Samples for electrophoresis were solubilised in SDS sample buffer
(25 mM Tris-HCl (pH 6.5), 4% (w/v) SDS, 10% (v/v) glycerol, 5% (v/v) &bgr;-mercaptoethanol,
0.001% (w/v) bromophenol blue) and incubated for 1h at room temperature. Electrophoresis
was performed in a vertical gel apparatus (Flowgen) equipped with 12 x 10 cm plates.
Samples (40 µg protein) were resolved using a 4% stacking gel and a 7.5% resolving
gel at a constant voltage of 200 V.
Western Blotting of Resolved Proteins.
Following SDS PAGE, resolved proteins were transferred
to a 0.2 µm nitrocellulose membrane (Flowgen) according to Towbin et al. (1979)
using a mini-blotting tank (Flowgen). Efficient transfer was achieved at 100 V for
1 hour in 25 mM Tris-HCl (pH 8.3), 192 mM glycine, 20% methanol, 0.1% SDS. 5-HT2B-like
receptor protein was localised using a selective antibody as detailed in section
2.2.2. molecular weights of proteins were determined from FITC-labelled marker proteins
(Sigma) using Fragment Analysis software (Molecular Dynamics).
Fresh frozen (10 mm) or formalin-fixed paraffin-embedded
(5-7 mm) sections of human tissue including colon and ileum, were mounted onto silane-coated
slides. Frozen sections were stored at -80°C until use, while paraffin-embedded
sections were stored at room temperature (RT).
Frozen sections were brought to RT and air-dried for 1hr,
when required. These sections were then fixed in acetone for 30 min and air-dried
for 1 h. Paraffin-embedded sections were rehydrated in graded alcohol. Endogenous
peroxidase activity in frozen and paraffin-embedded sections was quenched by incubation
in either 1% H2O2/0.1% NaN3 in PBS for frozen sections,
or 3% H2O2 in distilled water for paraffin-embedded sections
(30 min). (All incubations and washes were carried out on an orbital shaker at room
temperature unless otherwise stated.) At this stage, an antigen retrieval step was
performed on paraffin-embedded sections. These sections were microwaved in 0.01M
citrate buffer for 20 min followed by cooling in water. All sections were then incubated
for 30 min, with 10% normal serum from the species that the secondary antibody was
raised in. Subsequently, sections were incubated with the 5-HT2B receptor
antibody (1-2 mg/ml in PBS) for 16 h - 72 h, at 4°C. Control sections were
incubated with PBS alone or mouse IgG1 at 1-2 mg/ml. Unless otherwise
stated, sections were then washed (2 x 5 min) and incubated with biotinylated anti-mouse
IgG (1:300 dilution; Amersham) for 30 min, followed by washing (2 x 5 min) and incubation
with streptavidin-biotin conjugated to horse-radish peroxidase (1:600 dilution;
Amersham) for 30 min. Subsequently, sections were incubated with 3',3-diaminobenzidine
tetrachloride (0.025% w/v) / H2O2 (0.02% v/v) in 0.05M Tris
buffer (pH 7.6) for 5 min, followed by washing in distilled water. Some sections
were counterstained in Mayer's haematoxylin (1 min). Sections were then dehydrated
with xylene and coverslipped with DPX mountant (BDH Laboratories). Immunostained
sections were viewed with a Zeiss Axioplan2 microscope.
Dot blotting: The 5-HT2B-like protein was detected
in most of the 82 human tissue samples, but was highly abundant in extracts from
both the urinary and gastrointestinal tracts.
Western Blotting: To confirm the presence and abundance
of 5-HT2B-like receptor protein in the samples as determined by dot-blotting,
all 84 protein extracts were resolved by traditional SDS polyacrylamide gel electrophoresis
and analysed by Western Blotting. The techniques allowed confirmation of the presence
and abundance of 5-HT2B-like protein, and also determination of the molecular
weight of the receptor protein. There was a highly significant correlation (P<0.001;
r=0.721) between protein quantification using the two blotting methods. The Western
blotting confirmed the presence or absence of 5-HT2B-like receptor protein
in all samples. This included extracts from both the urinary and gastrointestinal
The molecular mass of the of 5-HT2B-like receptor
was found to be about 80,000 Da using information gained from FITC-labelled standard
protein run alongside the protein extracts. In some samples, a lowered molecular
mass protein species was present with a molecular weight of approximately 60,000
Da. This is either degraded 5-HT2B-like receptor species or a related
protein recognised by the antibody.
In the gastrointestinal tract, 5-HT2B receptor
localisation was studied in human colon. In sections of human colon, 5-HT2B
receptor-like immunoreactivity (5-HT2BR-ir) was observed in several cell
types. In the muscularis externa, moderate 5-HT2BR-ir was seen in the
circular (C) and longitudinal (L) muscle layers, while moderate to strong staining
was observed in putative myenteric nerve plexuses (MP) lying between the two muscle
layers (Figure 3). Localisation of 5-HT2HR-ir to the myenteric plexus
was confirmed by immunolabelling of adjacent sections with the 5-HT2B
receptor antibody (Figure 4A), a neuronal marker antibody, neurofilament 68 (Figure
4B), or the control IgG1 antibody (Figure 4C).
Dot blot and Western Blot analysis has provided a detailed
protein expression profile of the - 80KDa 5-HT-2B receptor protein throughout
the human body. These data have demonstrated highly abundant expression of the 5-HT2B
receptor protein expression within the urinary and gastrointestinal tracts, and
within the lung parenchyma, cerebral artery, stomach fundus smooth muscle, uterus
and prostate. In detailed immunocytochemical studies of the human colon and ileum,
5-HT2B receptor immunoreactivity (5-HT2B-ir) was observed
in both the longitudinal and circular smooth muscle layers within the muscularis
externa, and in the myenteric nerve plexuses lying between these two muscle layers.
Sections of human colon were cut open along its longitudinal
axis. The section was pinned out flat and the mucosa carefully removed using sharp
dissecting scissors. Once the mucosa was removed, the section was turned over to
reveal the three taenia coli (taenia mesencolica, taenia omentalis and taenia libera)
and the muscle bands that lie between them. Longitudinal muscle strips (2mm wide
by 20mm long) were then cut from the tissue between the taenia coli and suspended
between stainless steel hooks in organ chambers containing oxygenated (95% O2/5%
CO2) Krebs solution at 37°C. The composition of the Krebs solution
was as follows: NaCl (118.2mM), KCl (4.69mM), MgSO4.7H2O (1.18mM),
KH2PO4 (1.19mM), glucose (11.1mM), NaHCO3 (25.0mM),
CaCl2.6H2O (2.5mM). Tissues were placed under a tension equivalent
to 10mN and left to equilibrate for a period of at least 60 minutes.
Responses were recorded using isometric transducers coupled
to an Apple Macintosh computer via a MacLab interface. After 60 minutes, the longitudinal
muscle sections of the human colon were stimulated electrically (sub-maximal voltage
and frequency with 60s between successive stimulations) using parallel platinum
wire electrodes and a Multistim D330 pulse stimulator. Upon electrical stimulation,
the strips of human colon longitudinal smooth muscle responded with a rapid contraction.
Once the response to electrical stimulation had stabilised
(stimulated responses differed by no more than 10%), the strips were exposed to
increasing concentrations of 5-HT (or 5-HT receptor agonists), in the absence or
presence of selective receptor antagonists.
Electrical stimulation (15V, 1ms pulse width, at sub-maximal
frequency for 10s every 60s) causes highly reproducible, transient, contractile
responses of isolated preparations of colon smooth muscle. the responses are inhibited
by either tetrodotoxin or by atropine, indicating that they are neuronal in nature,
and they involve (at least in part) cholinergic neurotransmission.
Application of 5-HT, and a selection of 5-HT receptor agonists,
has been shown to produce a concentration-dependent potentiation of the contractile
response to electrical stimulation (Figures 1 and 2). The order of agonist potency
would appear to implicate a receptor of the 5-HT2 family (Table 1), since
the profile for the various agonists in colon matches most closely that observed
for 5-HT2B receptors.
The effects of various selective 5-HT receptor antagonists
have also been tested, the antagonists caused a rightward shift of the concentration-response
curve to 5-HT, and the profile generated corresponds to a receptor of the 5-HT2B
receptor class (Table 2).
Table 1. Potencies of some selective receptor agonists at the 5-HT receptor
in human colon longitudinal muscle. Table 1 shows the mean pEC50 for
agonists at the 5-HT receptor in colon (mean±s.e.mean for at least 3 donors)
and also at human 5-HT2A, 2B, 2C and 5-HT4 receptors, whereby
all data are for human 5-HT receptors. NA indicates data are not available at the
Table 2. Affinities of some selective receptor antagonists at the 5-HT receptor
in human colon longitudinal muscle.
NSA at -9M
Table 2 shows the mean pKB or pA2
for antagonists* or from binding data#, at the 5-HT receptor in colon and also at
human 5-HT2A, 2B, 2C and 5-HT4 receptors, whereby all data
are for human 5-HT receptors. In colon, values given are pKB estimates,
where antagonists were tested over at least 3 concentrations, except methysergide
and methiothepin which were each tested at a single concentration each. All data
were obtained in at least 3 donors. NA indicates data are not available at the human
receptor, NSA indicates non-surmountable antagonism.
In summary, the agonist and antagonist profile of the receptor
in human colon which mediates 5-HT-induced potentiation of the neuronal response
to electrical stimulation corresponds to a receptor of the 5-HT2B receptor
sub-type. This shows for the first time that an antagonist of this receptor would
counteract the effects of 5-HT in the human colon and would represent an effective
treatment for IBS. pKB is calculated as described above; pA2
is defined as the negative logarithm of the molar concentration of antagonist which
would produce a 2-fold shift of the concentration-response curve for an agonist.
At equilibrium, and assuming the antagonist is competitive, pA2 and pKB should be
Physiological and pharmacological studies have indicated
that 5-hydroxytryptamine (5-HT) may play a pivotal role in mediating sensory and
reflex responses in the gastrointestinal tract of various species, including man
(Read & Gwee, 1994). Administration of the 5-HT precursor, 5-hydroxytryptophan,
has been shown to mimic the symptoms of IBS, and increased levels of 5-HT and its
metabolites have been detected in plasma of patients suffering from IBS.
The nature of neuronal control of human intestinal motility
is only partially elucidated. 5-HT has been reported to induce both excitatory and
inhibitory responses throughout the human gastrointestinal tract (Misiewicz et al.,
1966; Costal & Naylor, 1990). The nature of the receptors mediating these responses
have not been well characterised. Extrapolating from animal data, we would expect
5-HT3 and/or 5-HT4 receptors to be implicated in these effects.
However, little evidence has been found to support a role for such receptors in
human gastrointestinal reflexes.
In the present invention, a receptor site (5-HT2B)
has been identified which is located in two key areas of the human gastrointestinal
tract - 1. neurones within the sub-mucous & myenteric plexus, and 2. smooth muscle.
These data, along with certain functional evidence, have led the present inventors
to develop a hypothesis in which the 5-HT2B receptor plays a key role
in the initiation and maintenance of the symptomatically complex phenotype of irritable
bowel syndrome (IBS).
Thus it is believed that the 5-HT2B receptor
is located on neurones of the myenteric plexus and modulates the activity of these
neurones, and that pathological increases in the levels of endogenous 5-HT causes
'misfiring' of the enteric reflexes which are essential for co-ordinated gastrointestinal
motility and peristalsis. Finally, immunocytochemical and functional data indicate
the presence of 5-HT2B receptors mediating gastrointestinal smooth muscle
spasm in response to elevated levels of endogenous 5-HT.
The use of a 5-HT2B receptor antagonist will
thus increase the activation threshold for neuronal and muscular 5-HT2B
receptors, by endogenous 5-HT. This would result in a reduction of neuronal misfiring
and muscular spasm, allowing 'normal', co-ordinated peristaltic activity to be restored.
In a novel, in vitro preparation of human colon, 5-HT2B receptors
are activated by low (nM) concentrations of 5-HT which mediates a substantial potentiation
in the magnitude of electrically stimulated contractions. It is believed that activation
of these 5-HT2B receptors is upstream of a chain of events which leads
to the stimulation of sensory afferents leading to the pain of IBS.
Thus the present data show for the first time direct evidence
to support the use of 5-HT2B antagonists in the treatment of IBS.
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- <110> Pharmagene Laboratories Ltd
Borman, Richard A
Tilford, Nicholas S
Baxter, Gordon S
- <120> Methods for the Treatment of IBS
- <130> AHBCP5904602
- <160> 3
- <170> PatentIn Ver. 2.1
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<223> Description of Artificial Sequence: Taqman Probe:
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