Note: Descriptions are shown in the official language in which they were submitted.
CA 02374358 2002-O1-04
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IU
METHODS AND COMPOS1TI(aNS FO.R REGULATING
GUT MOTTL1TY AND FOOD INTAKE
This application claims the benefit ofU.S. Provisional Application No.
60/143,054,
15 filed July 6, 1999.
Field of the Invention
This invention is generally in the field of treating obesity and regulating
food intake.
In particular, this invention relates to compositions and methods of
regulating food intake in
20 which trichothecenes, derivatives and analogs thereof, or purinergic
compounds are
administered to alter gut motility and thereby satiety. The invention also
relates to methods
of screening for derivatives or analogs of trichothecenes and also for
agonists and antagonists
of purinergic receptors that are useful for regulating food intake-
2~ Background of the Invention
Overeating leading to obesity is a major health problem. Obesity increases the
risk of
diabetes, heart disease, cancer and other chronic diseases, in addition to
increasing the
physical or mechanical restrictions imposed on the body. Although such adverse
health
effects of obesity arc scientifically well documented and generally well
understood by the
30 public, the effective control of appetite and overeating on an individual
basis has been a goal
difficult to attain for millions of people- Approximately 25 percent of the
children in North
America are considered overweight or obese. North Americans alone spend
approximately
$4U billion per annum on weight lass treatments, and this amount appears to be
increasing. A
recent study conservatively estimated the annual cost of treating obesity in
Canada at $1.8
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billion, representing 2.4 percent of the total health care expenditures for
all diseases (see,
"Cost of Obesity: $1.8 billion," in Pharmaceutical Manufacturers A.ssocialion
of Canada,
March 1999, page 11 ).
Currently available anti-obesity drugs work for the most part by targeting
central
nervous system (CNS) pathways to induce appetite suppression. However, such
drugs have a
number of CNS-related side effects, such as anxiety, and there is the
potential for chronic
health problems such as hypertension, cardiovascular disease, and diabetes.
Another current
approach to treating obesity is to control appetite by using "bulk" products,
which are
ingested instead of normal food. Such bulk products have the problem of
altering nutritional
status in that the bulk product does not contain the necessary range of
desirable nutrients.
Moreover, the individual who ingests a bulk product may refuse to consume any
Food, even
desirable nutrients.
Drugs that suppress appetite are among the least desirable means to treat
obesity
because weight is usually regained once administration of such drugs is
halted. Furthermore,
serious undesirable side effects, including increased risk of diseases such as
primary
pulmonary hypertension may limit the use of such drugs. For example, the
appetite
suppressants fenfluramine and dexfenfluramine were recently pulled off the
market by their
manufacturers because of a potential for serious adverse effects on the lungs
and heart.
Another type of obesity treatment that has emerged recently is the use of
drugs that
interfere with fat absorption from the small intestine. Such a drug may, for
example, inhibit
pancreatic enyzmes used for fat digestion. Undigested fat is then passed
through the
intestines and excreted Decreasing fat absorption can result in oily stool,
oily spotting of
undergarments, intestinal gas, frequent bowel movements, and decrease
absorption of fat-
soluble nutrients such as vitamins A, D, and E
There is currently no medical approach that cuts weight gain without unhealthy
side
effects or increased risk of disease. Needs remain for effective treatments
for obesity and
methods of controlling weight gain in humans and other animals without
untoward nutritional
and medical side effects.
Summary of the Invention
This invention provides methods oftreating obesity and controlling food intake
in
humans and other animals. The invention is based on the discovery of how
mycotoxin
trichothecenes produce food or feed refusal in humans and other vertebrate
animals and also
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on the elucidation of the neural circuitry regulating patterns of gut motor
activity ("gut
motility"), which propels food through the organs of the gut. The methods of
treatment
described herein involve administering a compound that affects the pattern of
gut motility,
that is, the pattern of contractions, relaxations, and quiescence of the
smooth muscle tissue of
the organs of the gut. Stimulating the "fed pattern' of gut motor activity
signals satiety, that
is, a feeling of fullness, which shortens the time an individual spends eating
or feeding. Thus,
compounds that stimulate the fed pattern of gut motility are useful in methods
of treatment
where the goal is to limit food intake, as in treating obesity. C'.ompounds
that stimulate the
"fasting pattern" or prolong or prevent the onset of the fed pattern of gut
motility will tend to
increase eating or feeding time because satiety is not signaled to the body.
Such compounds
are particularly useful in methods of increasing weight gain in animals, such
as livestock and
poultry raised as commercial sources of meat.
Methods of treating obesity provided by the invention comprise administering
an
effective amount of a trichothecene mycotoxin, or derivative thereof, which
stimulates the fed
pattern of gut motility. In a preferred embodiment of the invention, the
methods of treating
obesity comprise administering to an individual a rrichothecene from the
nivalenol-related
group of structurally related compounds consisting of nivalenol; 4-
deoxynivalenol ("DON",
C,SHzoOb); trichothecolon; trichothecin; 3-acetyldeoxynivalenol ("3-acetyl
DON", C~7H22O7);
7-acetyldeoxynivalenol; 3,15-diacetyldeoxynivalenol; 4-acetylnivalenol
(fusarenon-h7; 4,15-
diacetylnivalenol Other DON-based derivatives are also usefiil in the
preferred methods of
the invention, including DON carbonate (i.e., 3-hydroxy-12,13-epoxy-9-
tricothecin-8-one-
7,15 carbonate, Ci6Hig07); 3-acetyl-DON carbonate (i.e , :i-acetoxy-12,13-
epoxy-9-
tricothecin-8-one-7,15 carbonate, C~RH2oOs); 3-acetyl-DON benzylidene acetal
(i.e., 3-
acetoxy-7,15-benzylidene-12,13-epoxy-9-tricothecin-8-one, (',24HzeOs); L>ON-
benzylidene
acetal (i.e., 3-hydroxy-7,1..5-benzylidene-12,13-epoxy-9-tricothecin-8-one,
Czzfiz407);
isopropylidine DON (i.e., 3-hydroxy-7,15-isopropylidine-12,13-epoxy-9-
tricothecin-8-one,
C,BHzaOs); and isopropylidine-3-acetyl-DON (i.e.. 3-acetoxy-7,15-
isopropylidine-12,13-
epoxy-9-tricothecin-8-one, C2oH2~0?). More preferably, the methods of treating
obesity
comprise administering a trichothecene, such as DON or a DON-based derivative,
to an
individual at a dose which is non-toxic and non-emetic, hut which stimulates
the fed pattern
of gut motility in the individual. The trichothecene, or derivative thereof,
may be
administered by any of a variety of routes, including orally or parenterally.
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Alternatively, methods of treating obesity comprise administering a
trichothecene
analog, which is a compound that functions like a trichothecene to stimulate
the fed pattern of
gut motility. Trichothecene analogs may be structurally related to or
structurally distinct
from trichothecenes. Thus, trichothecene analogs may be derived from a
trichothecene such
as DON or may be any of a variety of compounds, including inorganic compounds,
organic
compounds, amino acids, peptides, polypeptides, proteins, nucleotides, nucleic
acids,
carbohydrates, lipids, and combinations thereof, which have the ability to
stimulate fed
pattern gut motility.
In another embodiment, the invention provides compositions and methods for
I 0 regulating gut motility and treating obesity in an individual. Such
methods of the invention
comprise administering to the individual a compound that binds to and
stimulates Pzx, purine
receptors (purinoceptors}, which are present in smooth muscle of gut tissues
and are directly
involved in regulating the fed pattern of gut motility. In particular, an
agonist of the Pzxl
purine receptor is a purinergic compound that binds the receptor to stimulate
the fed pattern
I S of gut motility. As with tricothecenes, stimulating the find pattern of
the gut motor activity
with a purinergic compound signals satiety and thereby shortens feeding time
and decreases
food intake. Preferably, the agonist of the Pzxl receptor that is useful in
treating obesity
according to the invention is a "non-desensitizing" agonist of the purine
receptor, in that
molecules of the agonist are able to bind the Pzxi receptor and to stimulate
the Pzx~ mediated
20 fed pattern of gut motility, without eventually blocking or inactivating
the receptor. In a
more preferred embodiment, the non-desensitizing agonist of the Pzxl receptor
is a structural
analog of ATP or of 2',3'-O-(2,4,6-trinitrophenyl)-ATP ("TNP-ATP") for use in
methods of
stimulating the fed pattern of gut motility and for treating obesity.
In another embodiment, the invention provides compositions and methods for
25 increasing weight in an individual. Such methods comprise administering to
the individual a
desensitizing agonist or an antagonist, such as TNP-ATP, of the Pzxc receptor.
A
desensitizing agonist or an antagonist compound useful in such methods of the
invention
binds and blocks the P2x, receptor, and thereby inhibits or prevents the fed
pattern of gut
motility and/or prolongs the fasting pattern of gut motility, which in turn
increases feeding
30 time and food intake Such methods are particularly useful in raising
commercial livestock
and poultry for market.
1n yet another aspect of the invention, methods are pravided for identifying a
compound that stimulates or inhibits (prevents) the fed pattern of gut
motility by directly
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recording the pattern of gut motility in vivo in a test subject during
administration and
metabolism of the compound in the subject. Such methods may be used to test
the regulatory
activity of a compound, such as a known or new trichothecene compound, a
trichothecene
derivative compound, a trichothecene analog compound, an agonist of the PZxI
receptor, or an
S antagonist of the P2x, receptor. The ability of a compound to regulate
(stimulate or inhibit)
patterns of gut motility can be measured using an in vitro gut bath assay, an
ex vivo gut organ
assay, or an in vivo assay. Trichothecene and purinergic compounds, and
derivatives or
analogs thereof, that are identified by such screening methods as capable of
stimulating the
fed pattern of gut motility may be used to treat obesity according to the
methods of the
invention, whereas compounds that inhibit fed pattern of gut motility may be
used to increase
food uptake, as in promoting weight gain in livestock and poultry for
commercial markets
Brief Description of the Drawings
Figure 1 shows a schematic diagram of the in vivo set up of Krantis et al.
(Can. J.
Physiol. I'harmacol., 7=l: 894-903 ( 1996)), employed for recording
gastrointestinal motility in
anaesthetized experimental animals, such as a rat ( 1 ). In a detail (A)
magnification of the rat
gut, foil strain gauges (2) are shown attached (e.g., with a glue) to selected
sites of gut organs,
for example, the serosal surface of the gastric antrum (3), proximal duodenum
(4), or distal
ileum (5), along the longitudinal muscle layer. Wire leads are attached to an
IBM computer
data acquisition system (6),
Figure 2 shows a schematic representation of the neural pathways controlling
fed and
fasting patterns of gut motility in the duodenum and ileum. The arrangement
ofcholinergic
(ACh), nitrergic (NO), and purinergic (ATP) neurons is shown together with the
different
receptor targets and/or inputs: mus. (cholinergic muscarinic), 5-HT3
(serotonergic), nic.
(cholinergic nicotinic), P2x (purinergic). A plus sign ("+") indicates a
stimulatory input
between neurons, and stimulation and contraction at smaoth muscle of the gut;
a minus sign
("-") indicates an inhibitory input. DON = deoxynivalenal, stimulator of gut
hyperactivity
(fed pattern) and satiety. NO = nitric oxide, which is a non-adrenergic, non-
cholinergic
(NANC) inhibitory transmitter in the proximal duadenum and which is also an
inhibitory
transmitter of the propagatory P2x-purinergic and cholinergic (muscarinic,
mus.) motor
activity in duodenum and ileum. A circuit pathway of a nitrergic interneurone
(NO) with
cholinergic nicotinic input (nic.) (far right) is not pr-went in the ileum.
A'fP -= adenosine
triphosphate, de-sensitizing agonist of purinergic receptors, such as, P2;c
receptors. ACh =
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acetyl choline, the cholinergic chemical signal that binds at muscarinic
(onus.) receptors to
excite motor neurons. 5-HT = 5-hydroxytryptamine (serotonin), binds to 5-HT;
(serotonergic) receptors on neurons and is the majcar transmitter of enteric
interneurones
mediating neurogenic stimulation of NANC relaxations and cholinergic
contractions of the
smooth muscle of the gut. nic. = cholinergic nicotinic receptor of neurons.
Figures 3A and 3B show diagrammatic chemical structures of 4-deoxynivalenol
(DON) and related derivative compounds. Figure 3 A shows diagrammatic chemical
structures for DON (C,SHzoO6); 3-acetyl-DON (C,7Hzz07); isopropylidine DON
(C,sHz4O~,
designated EN 139491); and isopropylidine-3-acetyl-DON (CzoHz6O7, designated
EN139492).
Figure 3B shows diagrammatic chemical structures for DON carbonate (C,6H,x07,
designated EN 139494); 3-acetyl-DON carbonate (C,gHzoOs, designated EN
139495); 3-
acetyl-DON benzylidene acetal (CZaHz60g, designated EN 139496); and DON-
bcnzylidene
acetal (CzZHz407, designated EN139497). "Ph" represents a phenyl group; "OAc"
represents
an acetyl group.
Figure 4 shows a recording of the spontaneous motor activity of the rat
gastric antrum
in a control animal showing the oscillatory appearance of contractile and
relaxant responses.
Vertical marks indicate time (t) at 0 and 50 minutes after start of recording.
Administration
of DON (first arrow to right of t = 0 minutes) at 10 mg/kg of body weight,
i.v., abruptly
attenuated the motor activity of gastric antrum. Within 40 minutes, the
control motor pattern
recovered, however, a proximal readrninistration of DON (second arrow) was
without effect.
Figure 5 shows an example of a recording ~uf the in vivo motility pattern of
the rat
duodenum control activity. The spontaneous in viva motility pattern of the
duodenum control
activity (no DON} consists of periodic "grouped" (G) and "intergroup" (I}
activity. Vertical
marks indicate tune (t) at 0, 30, 120, and 150 minutes after start of
recording. The first arrow
after t ° 30 minutes, indicates systemically administered DON (arrow)
at 10 mg/kg of body
weight (i.v.), which induced a sustained hyperactivity (46 ~ 1 S min)
Following recovery of
motor activity to control level, readministration of~ DON (arrow after t = 150
minutes) was
without effect.
Figures 6A-6D show data from a quantitative analysis of the effects of L-NAME
and
ex, R-methylene ATP on frequency (Freq) and amplitude (Amp) of DON-induced
relaxations
in the rat duodenum (Figure 6A, frequency, and Figure 6B, amplitude) and ileum
(Figure 6C,
frequency, and Figure 6D, amplitude). Grouped motor activity in the presence
of L-NAME
and DON (broadly spaced diagonal bars) was equivalent to that with DON alone
(closely
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spaced diagonal bars). a, (3-methylene ATP significantly attenuated the
frequency and
amplitude of DON induced relaxations (filled bars) to the level of control
intergroup activity
(no DON, open bars), in the duodenum (n ---- 8) and ileum (n = 4).
Figures 7A-7D show results of a quantitative analysis of the et~ects of the 5-
HT3
receptor antagonist, granisetron, on spontaneous and DON-induced activity in
the rat
duodenum. Granisetron (i.v_ or i.a., broadly spaced diagonal bars) selectively
attenuated the
frequency (Freq) and amplitude (Amp) of "grouped" relaxations (n = 6) (Figure
7A,
frequency, and Figure 7B, amplitude) and contractions (n -= 3) (Figure 7C,
frequency, and
Figure 7D, amplitude), however, it did not alter the stereotypic DON induced
hyperactivity
(compare closely spaced diagonal bars (DON alone) with filled bars (DUN +
granisetron)).
Control "grouped" activity (open bars}.
Figure 8 is a bar graph showing the ef~'ects (as percem of control) of a,~i-
methylene
ATP on DON-enhanced motor activity for contractions and relaxations ofthe
duodenum in
piglets. "Control" represents a group of piglets that received no DON and no
a,p-methylene
ATP. "DON" represents a group of piglets that received DON only ( l mg~kg'').
"a,~i-
methylene ATP -~- DON" represents a group of piglets that received intra-
arterial injection of
a,(3-methylene ATP (300 frg/kg, i.a.) during DON (1 mg/kg) enhanced motor
activity ofthe
duodenum. Control group values were set as 100%. All other values are percent
of control
values. Open bars represent average (4 piglets) amplitude of relaxations.
Filled bars
represent average (5 piglets) frequency of relaxations. Open cross-hatched
bars represent
average (3 piglets) amplitude of contractions. Closely spaced cross-hatched
bars represent
average (2 piglets) frequency of contractions. "~" indicates p ~~ 0.05
compared to control.
"~" indicates.p<0.05 compared to DON enhanced activity.
Figure 9 is a bar graph showing the effects (as percent of control) of a,(3-
methylene
ATP on DON-enhanced motor activity (contractions and relaxations) of the ileum
in piglets.
"Control" represents the group of piglets that received no DON and no a,(3-
methylenc ATP.
"DON" represents the group of piglets that received DO N only (10 mg/kg).
"a,(3-methylene
ATP + DON" represents the group of piglets that received intra-arterial
injection of a,(3-
methylene ATP (300 ug/kg, i.a.) during DON (10 mg/kg) enhanced motor activity
ofthe
ileum. Control group values were set as l00%. A.11 other values are per cent
of control
values. Open bars represent average (4 piglets) amplitude of relaxations
Filled bars
represent average (5 piglets) frequency of relaxations. Open cross-hatched
bars represent
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average (3 piglets) amplitude of contractions. Closely spaced cross-hatched
bars represent
average (2 piglets) frequency of contractions. "~" indicates p < 0.05 compared
to control.
"~" indicates p<0.05 compared to DON enhanced activity.
Figure l0 shows a schematic representation of the arrangement of cholinergic,
nitrergic, GABAergic, purinergic and VIPergic neural elements within the
proposed tonic and
modulatory pathways controlling spontaneous motor activity in the rat duodenum
and ileum
A circuit pathway having GABAergic and nitrergic interneurones (NO) with
cholinergic
nicotinic input (nic.) (far right) is not present in the ileum. VIP =
vasoactive intestinal
peptide, which is an activator of nitrergic prejunctional inhibition of motor
innervations.
Figure I I shows an example of a recording of the contral spontaneous motor
activity
of the rat gastric antrum (site S1) and proximal duodenum (site D1). DON
(lOmg/kg of body
weight ("bw"), administered i.v.) abruptly attenuated the antral motor
activity, and induced a
sustained hyperactivity in the duodenum (DI). Within 60 minutes, the control
motor pattern
recovered.
Figure 12 shows effects of 3-acetyl DON on rat gastrointestinal motor activity
in vivo.
Typical fasting pattern of gut motor activity in the duodenum (recorded at
duodenal site D2)
and the gastric antrum (S 1 ) are shown prior to administration of 3-acetyl
DON treatment.
Following injection (vertical arrow) of 3-acetyl DON (lOmg/kg, i.v.) the motor
activity
changed into a typical fed pattern motor activity lasting approximately 40
minutes "MMC"
is the "grouped" activity portion of fasting pattern of gut motility.
Figure 13 shows the elFects of intravenously administered 3-acetyl DON at 10
mg/kg
of body weight on spontaneous motor activity in the rat gastric antrum (S 1 )
and duodenum
(D2). Within 60 minutes, the control motor pattern recovered (see recording
after 130 min).
"MMC" is the "grouped" activity portion ofthe fasting pattern of gut motility.
Figure 14 shows typical in viva recordings of the motor activity in the rat
duodenum
(D,) and gastric antrum (S 1 ) illustrating the action of the compound EN I
:39491 ( l Omg/kg bw,
i.v.) on "fasting pattern" motor activity. The top panel shows 20 minutes of
normal fasting
pattern motor activity prior to administration of compound. Recording during
this period
shows that the duodenum displayed a typical pattern of low frequency
spontaneous motor
activity together with propagating "grouped" motor activity ("MMCs") and that
the gastric
antrum displayed a typically rhythmic motor activity. The second panel of
recording shows
the time of injection for EN139491 and development within 30 seconds of
injection of a long
lasting (40-60 minutes) hyperactivity in the duodenum and a simultaneous and
parallel
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attenuation of motor activity in the gastric antrum, characterisitic of fed
pattern motor
activity. Recovery of fasting pattern motor activity is shown in the bottom
panel as
evidenced by periods of "grouped" MMC activity and less active "intergroup"
activity.
Figure 15 shows the effect of compound EN139491 on duodenal motor activity
recorded at site D2 (1.5 cm distal to the D1 strain gauge). Abbreviations are
the same as in
previous figures.
Figure 16 shows bar graphs of the efFect on the amplitude of the relaxation
component
of gut motor activity by administration of the EN139491 DON derivative
compound (10
mg~kg t i.v.) during spontaneous fasting pattern motor activity recorded irr
vivo from the
proximal duodenum (D1 ) and gastric antrum of Halothane anaesthetized male
Sprague
Dawley rats. Amplitude of relaxation is expressed as percent of control
"intergroup" activity.
Amplitudes of the relaxation component of motor activity of "grouped" motor
activity prior
to administration of EN139491 (diagonal bar); for control "intergroup" motor
activity prior to
administration of EN139491 (open bar, set as I00%); and for hyperactivity
after intravenous
administration of EN139491 are shown. Each bar graph is the mean t Sf?M of
data compiled
from in vivo recordings obtained from 5-8 Sprague Dawley rats.
Figure l7 shows bar graphs as described in Figure 16, except that the efFect
of
administration of EN139491 on gut motor activity on the frequency of the
relaxation
component of gut motor activity is shown.
Figure 18 shows bar graphs as described in Figure 16, except that the effect
of
administration of EN 139491 on gut motor activity on the amplitude of the
contraction
component of gut motor activity is shown.
Figure 19 shows bar graphs as described in Figure 16, except that the effect
of
administration of EN139491 on gut motor activity on the frequency of the
contraction
component of gut motor activity is shown.
Figure 20 shows a typical irr vivo recording of the motor activity in the rat
duodenum
(D1 and D2 duodenal recording sites) and gastric antrum (S1 antral recording
site) illustrating
the action of the DON derivative compound EN139492 (at lOmg~kg', i v ) on the
fasting
pattern of gut motor activity. The typical fasting pattern motor activity is
evident in the
recording prior to administration of EN139492. During this fasting pattern
period, the
duodenum (D I and D2) displayed a typical pattern of lo~,u frequency
spontaneous motor
activity together with propagating "grouped" motor activity (MMCs), and the
gastric antrum
displayed a typically rhythmic motor activity. Within 30 seconds of
administration of
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EN139492 (vertical arrow) by injection, there developed a long lasting (40-60
minutes)
hyperactivity in the duodenum and a simultaneous attenuation of motor activity
in the gastric
antrum, characteristic of the fed pattern of gut motor activity, though the
effects on antral
motor activity were not as sustained as in duodenal motor activity.
Figure 21 shows a typical in vivo recording showing the effects of
intravenously
injected Pzx~ pur7noceptor antagonist TNP-ATP (3.5 mg~kg'') on spontaneous
motor activity
in the duodenum (at site D1) of a Halothane anaesthetized male Sprague Dawley
rat. oahe
recording shows that TNP-ATP did not evoke any response upon immediate
injection
(vertical arrow), but that, within 1 minute of injection, propagating motor
activity (MMCs)
was reduced. "Intergroup" motor activity was not significantly altered.
Recovery of motor
activity to within 90% of control level occurred within 20 minutes.
Figure 22 shows a typical in vivo recording showing the effects of
intravenously
injected TNP-ATP (3.5 mg/kg) on DON-induced (10 mg/kg bw, i.v , vertical arrow
above
DOI~ fed pattern motor activity in the rat duodenum (site D I ). TNP-ATP
inhibitory effect
on DON-induced fed pattern occurred within 1 minute of injection with "fNP-ATP
(vertical
arrow above T'M'-ATP).
Figure 23 shows the effects of intravenously administered TNP-ATP (3.5 mg/kg)
an
DON-induced fed pattern motor activity in the rat gastric antrum (S 1) and
duodenum (D2).
Within 60 minutes, the control motor pattern recovered. T'he boxed portions of
the recording
2.0 show the initial actions of TNP-ATP at each site.
Figure 24 shows bar graphs of the amplitude ofthe relaxation component of gut
motor
activity recorded in vivo at the.proximal duodenum (site Dl) in Halothane-
anaesthetized male
Sprague Dawley rats. The amplitude ofthe relaxation component ofDON-induced
(1U
mg/kg, i.v.) gut hyperactivity in the absence of TNP-ATP (open bar) and
presence of TNP-
2,5 ATP (3.5 mg/kg, i.v.) (checkered bar) is shown. T'he amplitude of the
relaxation component
of "grouped" MMC (diagonal bar) and the control "intergroup" activity (tilted
bar) is also
shown. The amplitude of the relaxation component is expressed as the percent
of the control
'°intergroup" relaxation amplitude, which was set at 100'Yo. Asterisk
indicates a significant
(p<0.05) difference compared to control "intergroup" motor activity Each bar
graph is the
30 mean ~ SEM of data compiled from in vivo recordings obtained from 5-8
Sprague Dawley
rats. Each animal was its own control. D1 represents the recording obtained
from a strain
gauge positioned at 10 mm distal to the pyloric sphincter.
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Figure 25 shows bar graphs of the frequency of the relaxation component of gut
motor
activity recorded in viva at the proximal duodenum {site D1) in Halothane-
anaesthetized male
Sprague Dawley rats. The frequency ofthe relaxation component of DON-induced
(10
mg/kg, i.v_) hyperactivity in the absence of TNP-ATP (open bar) and presence
of TNP-ATP
(3.5 mg/kg, i.v.) (cross-hatched bar) is shown. The frequency ofthe relaxation
component of
"grouped" MMC (diagonal bar) and the control "intergroup" (filled bar)
activity is also
shown. The frequency of the relaxation component is expressed as the percent
of the control
"intergroup" relaxation frequency, which was set at 100%. Asterisk,
statistics, and recording
conditions of experiment as in Figure 24.
Figure 26 shows bar graphs of the amplitude of the contraction component of
gut
motor activity recorded in vivo at the proximal duodenum (site D 1 ) in
Halothane-
anaesthetized male Sprague Dawley rats. The amplitude ofthe contraction
component of
DON-induced ( 10 mg/kg, i.v.) gut hyperactivity in the absence of TNP-ATP
(open bar) and
presence of TNP-ATP (3.5 mg/kg, i.v.) (cross-hatched bar) is shown. The
amplitude of the
1 S contraction component of "grouped" MMC (diagonal bar) and the control
"intergroup"
activity (filled bar) is also shown. The amplitude of the contraction
component is expressed
as the percent ofthe control "intergroup" contraction amplitude, which was set
at 100°/o.
Asterisk, statistics, and recording conditions of experiment as in Figure 24.
Figure 27 shows bar graphs of the frequency of the contraction component of
gut
motor activity recorded in vivo at the proximal duodenum (site D 1) in
Halothane-
anaesthetized male Sprague Dawley rats. 'rhe frequency of the contraction
component of
DOI~-induced (10 mg/kg, i.v.) hyperactivity in the absence of TNP-ATP (open
bar) and
presence of TNP-ATP (3.5 mg~kg', i.v.) (cross-hatched bar) is shown. The
frequency of the
contraction component of "grouped" MMC (diagonal bar) and the control
"intergroup" (filled
2 S bar) activity is also shown. The frequency of the contraction component is
expressed as the
percent of the control "intergroup" contraction frequency, which was set at
100% Asterisk,
statistics, and recording conditions of experiment as in Figure 24.
Detailed Descr~tion of the Invention
This invention provides compositions and methods for treating obesity and
regulating
food intake by modulating the motor activity of gut organs in humans and other
vertebrate
animals. These methods are based on the discovery that trichothecene
compounds, such as 4-
deoxynivalenol (DOI~, stimulate the pattern of contractions and relaxations in
organs ofthe
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gut that normally occur when food is ingested. Stimulation of this "fed
pattern" of gut
motility signals satiety, that is, the feeling of fullness, which is an
important factor that affects
the time that an individual spends eating. Trichothecenes, such as DON, act at
a site outside
the organs of the gut and send a signal, which is sent down neural pathways
leading to the
smooth muscle of the gut organs. We have discovered that a specific receptor,
the purinergic
receptor PZx~, present on cells of the smooth muscle of the small intestine is
involved in
regulating specific aspects of gut motor activity. Accordingly. compounds that
act as
agonists or antagonists of the PZxr receptor are also useful in regulating gut
motility and to
controlling satiety and time spent sating.
In order to accurately describe the invention the following terms are defined_
As used herein, "gut" refers to the gastrointestinal tract consisting of the
stomach,
small intestine, and large intestine.
As used herein, "gut motility" or "gut motor activity" refers to the motor
behavior of
the smooth muscle in the gastrointestinal organs (stomach, small intestine,
and large
1:5 intestine) of humans and other animals which activity consists of periods
of alternating
muscular contractions and relaxations, as well as periods of quiescence or
relatively little
activity. For example, in normal, healthy humans and other animals, the
liequency and
amplitude of muscular contractions and relaxations of the small intestine
become heightened
when food is ingested in order to propel food aborally (forward) into the
intestines for
2~ nutrient extraction and absorption (see, "fed pattern" of gut motility,
below). Other patterns
of gut motility may occur depending on the presence or absence of food in
various parts of
the gut organs. Furthermore, the proximal portion of a particular gut organ
may exhibit
motor behavior that differs from the activity in a distal portion of the
organ, such as in the
case of the duodenum (the beginning portion of the small intestine) and the
ileum (the
25 terminal portion of the small intestine).
As used herein, the "fed pattern", "fed pattern activity", and "segmentation"
are
synonymous and refer to the continuous pattern of contractions and relaxations
of the small
intestine of the gut in an animal, including humans, that normally occurs as
the result of
ingesting food. The fed pattern of gut motility propels ingested food through
the gut for
30 nutrient extraction and absorption, and eventually excretion of unabsorbed
material as waste.
The fed pattern ofgut motor activity typically begins within minutes of
ingesting food and is
responsible for signaling satiety, that is, the feeling of fullness. Thus,
satiety from the fed
pattern of gut motility normally informs an individual that eating can be
ended. Satiety is
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sensed by an individual via the fed pattern of gut motility long before the
brain has an
opportunity to analyze the nutrient content of the blood (a separate process
that takes place
hours after food has been consumed and that is responsible for signaling
cravings for specific
nutrients, such as, proteins, carbohydrates, salt, and fats, which are
maintained at specific
levels for health).
The fed pattern is both characteristic and different for each organ and even
different
sites in the same organ of the gut. In the small intestine, the fed pattern is
characterized by a
continuous series of contractions and relaxations of the smooth muscle, which
mixes
intestinal contents, propels food aborally into the intestines, and delays
anterograde
propulsion to enhance substrate absorption (Lundgren et al., I~ig. Dis. Sci.,
3~J: 264-283
(1989)) When measured and recorded in vivv by the method of Krantis of al.
(Can. J.
Physiol. Pharmacol., 74: 894-903 (1996)), the fed pattern ofgut motor activity
in the
duodenum is a characteristic intense pattern of hyperactivity whereas
simultaneously in the
gastric antrum, the fed pattern is characteristized as a measurable
suppression or decrease in
recorded tissue motor activity. 'This fed pattern activity replaces the
"fasting pattern" of gut
motor activity (see, below), which occurs after food has been propelled
through the gut for
nutrient extraction. Fed pattern motility is activated primarily by peripheral
autonomic
ganglia via primarily vagal inputs and also, but to a lesser extent, is
controlled by the central
nervous system (CNS) (see, Yoshida et al., .l. 1'harmacol. F,xp. T~rerap.,
256: 272-278 (1991);
Tanaka et al., J Surd. Res., 53: 588-595 {1996); Chung et al., (.'art. .l.
Physiol. Pharmacnl.,
70: 1148-1 153 (1992)), Over-activation of autonomic nerves accelerates the
onset and
increases the duration of the fed pattern, concurrently increasing the
frequency and amplitude
of propagatory motor activity ofthe gut (ftall et al., Am. J. Physiol., 250:
6501-6510 (1986);
Johnson et al., Am. J. S"urg., 167: 80-88 (1994), see also Examples below). As
noted above,
trichothecene mycotoxins, such as DON, can now be used in proper amounts to
stimulate the
fed pattern of gut motility.
"Fasting pattern" or "fasting cyclic motor pattern"' of gut motor activity
refers to the
motor behavior of the gut in the absence of ingested food matter or before
ingesting food,
when no ingested material is present for propulsion from the stomach and into
intestines. In
the duodenum (the start of the small intestine), the fasting pattern of gut
motor activity is
characterized by alternating periods of spontaneous, irregular contractions
and relaxations
("grouped" activity) and relatively quiescent periods ("intergroup" activity).
An example of a
duodenal fasting pattern with its alternating grouped and intergroup
activities is shown in the
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early portion (between t = 0 and t = 30 minutes) of the recording of gut
motility in Fig. 5. In
the ileum (the end region of the small intestine), the fasting pattern is
characterized by
random contractile and/or relaxant motor activity cc a generally quiescent
state. Ingestion of
food matter interrupts the fasting pattern of gut motility and stimulates the
continuous activity
of the fed pattern of gut motility.
Until recently, methods were not available for accurately measuring and
characterizing gut motility in that only one component, either contraction ac
relaxation, could
be measured under experimental conditions. More recently, however, Krantis and
co-
workers have developed a method of simultaneously measuring the contraction
and
relaxation components of gut motility for various organs of the
gastrointea~tinal tract using
miniaturized, flexible, foil strain gauges that can be attached in viva to
various locations on
organs ofthe gut (see, Krantis et al., Can. .l. Physrnl. Pharrnacx~l., 74: 894-
903 (1996)). In
this method, wires from the gauges attached to the organs are connected to a
computerised
data analysis system (see, Fig. 1). The method of Krantis et al. (1996) may be
used for
t 5 pharmacological, neurological, and physiological studies of the gut using
in viva, ex viva
(organs positioned out of the body cavity), and irr vitro (extricated tissue
from gut organs)
procedures (see, Examples, below). The ability to simultaneously record
contractions and
relaxations in gut organs and at multiple sites within an argan provides a
more precise
characterization of gut motility, including distinct patterns of gut motility,
and the effect of
2~3 food and various chemical compounds on such patterns.
In the fasted state, the gut exhibits a cyclic motor behavior known as "MMC",
"migrating motor complex", or "migrating myoelectric complex". MMCs are
associated with
interdigestive propulsion of intestinal contents and involve sequential
activation of excitatory
and inhibitory neurons to propagate cycles of contractions and relaxations
that originate in
25 the stomach and terminate at the ileum. An MMC cycle consists of three
distinct phases:
phase I is a quiescent phase; phase II is a period of irregular spiking of
activity, and phase III
is a short period of rapid spike bursts of activity. MMCs provide a basic
intrinsic motor
pattern, which functions as a "housekeeper" of the small intestine. For
example, the highly
propulsive phase Ill motor activity of each MMC cycle sweeps the intestinal
lumen, clearing
3~0 it of remnants to prevent bacterial overgrowth, back flow, and the
accumulation of intestinal
secretions (Caenepeel et al., Dig. Drs. Sri., 34: 1180-1184 (1989)). Using the
method of
Krantis et al. (1996), it is now clear that gut motility may comprise both
contractions and
relaxations of smooth muscle. In the absence of food, the "grouped" activity
of the fasting
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pattern of intestinal gut motility appears to correspond to the same type of
motor activity
classically ascribed to phase III of MMCs. The presence of food in the
intestinal lumen
induces a switch from the fasting pattern to the fed pattern of gut motor
activity.
The method ofKrantis et al. (1996) has also enabled the discovery ofthe mode
of
action of compounds called trichothecenes or trichothecene mycotoxins on gut
motility. As
shown in Examples 1 and 2 (infra), the trichothecene 4-deoxynivalenol (DON)
acts at sites
outside the gut to stimulate the fed pattern of gut motility, which
characteristically occurs
after ingesting food and which signals satiety, that is, the sensation of
fullness. These
findings provide the mechanism to explain to the well-documented anorectic or
feed refusal
behavior of humans and other animals that have ingested crops contaminated
with fungal
species that produce DON or other trichothecenes. 'This invention provides a
method of
treating obesity that takes advantage of the ability of trichathecene
compounds to induce the
fed pattern of gut motility and satiety. Methods of treating obesity described
herein comprise
administering a trichothecene or similar acting compound, which stimulates the
fed pattern of
gut motility and, thereby, satiety. Sensing fullness, the individual is thus
given a signal to
stop eating. When circulating levels of the administered compound decrease,
satiety will
decline and the individual may continue to eat or feed.
This invention also provides methods of regulating food intake by
administering an
agonist or antagonist of the Pzx, purine receptor (purinoc;eptar), which
mediates grouped
relaxations of gut tissue. According to the invention, a trichothecene, such
as DON, and
derivatives and analogs thereof, which stimulates the fed pattern of gut
motility, actually acts
at a site outside the gut. From that remote site of action, a signal travels
down neural
pathways to smooth muscle cells of the gut that express Pzx, purine receptors,
which are
involved in regulating the fed pattern of gut motility (see, Fig 2). Thus, a
compound that
binds and affects the Pzxl purinoceptor is acting at the terminal portion of
the neural pathway,
whereas DON or other such trichothecenes act upstream ,According to the
invention, one
group of compounds useful in the methods described herein consists of analogs
of adenosine
triphosphate (ATP) which may act as agonists or as antagonists of the Pzx,
purinoceptor. As
explained below, certain types of agonists of the Pzxi purinoceptor bind the
receptor and
stimulate the fed pattern of gut motility. Such agonists of the Pzx~
purinoceptor may be used
in lieu of trichothecenes in methods of treating obesity An antagonist of the
Pzx,
purinoceptor is a compound that binds and blocks the receptor, thereby
switching off or
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attenuating the fed pattern. Such PZxc receptor antagonists suppress or
prevent fed pattern
and satiety and, thus, may be used to prolong eating time and promote weight
gain.
Trichothecenes Useful in the Invention
Historically, trichothecene compounds were identified as one of the toxic
secondary
metabolites produced by various fungi that can contaminate crops, hence the
designation
trichothecene mycotoxins. Animals, including humans, that ingest such
contaminated crops
may experience a variety of pathological symptoms of mycotoxicosis, such as
vomiting,
diarrhea, hemorrhagic lesions in internal organs, alimentary toxic aleukia
(ATA),
agranolocytosis, aplastic anemia, necrotic angina, Inflammation of mucous
membranes,
refusal to eat, convulsions, sepsis, and in some cases, death (see, for
example, Ueno,
"Trichothecene Myeotoxins: Mycology, Chemistry, and 'Toxicology," in Advarrces
in
Nutritional Research 1980, 3; 301-353 ( 1980)).
As used herein, "trichothecene mycotoxin" or "trichothecene" refers to a
member of a
1'.> group of sesquiterpenoid family of chemical compounds based on the non-
olefinic parent or
core compound trichothecane. All trichothecenes are modified sesquiterpenes,
and contain
an olefinic (double) bond (hence, trichothecene) between carbon atoms at
positions 9 and 10
(C-9, C-10), and an epoxy ring formed between carbon atoms at positions l2 and
13 (C-12,
C-13). Thus, trichothecenes are also characterized as 12"13-epoxytrichothecene
compounds.
Ueno classified naturally-occurring trichothecene rnycotoxins into four groups
based on
structural and also fungal characteristics (see, for example, Ueno, 1980,
sy~pra). According to
this classification scheme, members of a group of trichothecenes represented
by nivalenol are
non-macrocyclic compounds that have the carbon-8 (C-8) substituted with a
ketone (oxo-)
group. In addition to nivalenol, the group of "nivalenol-related"
trichotheeenes includes such
2'i naturally-occurring trichothecene mycotoxins as 4~-deoxynivalenol (00N;1,
trichothecolon,
trichothecin, 3-acetyldeoxynivalenol (3-acetyl-DON), 7-acetyideosynivalenol,
3,15-
diaeetyldeoxynivalenol, 4-acetylnivalenol (fusarenon-X), and 4,15-
diacetylnivalenol. As
used herein, "DON", "4-DON", "deoxynivalenol", "4-deoxynivalenol", and
"vomitoxin" all
refer to the same trichothecene compound having the chemical structure shown
in Fig. 3.
Thus, nivalenol differs from DON in that nivalenol contains a hydroxyl group
at C-4,
whereas DON lacks the hydroxyl group ("4-deoxy") at position 4.
Although clearly capable of causing severe and widespread incidences of
toxicosis
when ingested in suf~'ICiently high quantities, DON is nevertheless considered
as one of the
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least potent trichothecenes with respect to sub-lethal toxicosis (see, for
example, Prelusky et
al., Arch. Environ. Contam. Toxicol., 22: 36-40 (1992); Friend et al., Can. J
Anim. Sci., 6Gv
765-775 (1986); Ueno, in Developments in Food Science 1V. Trichothecenes,
chemical.
biological, and toxicological aspects (Elsevier, Amsterdam, 1983), pp. 135-
146).
S DON is also non-mutagenic as determined using a hepatocyte-mediated mutation
assay with V79 Chinese hamster lung cells (Rogers and Heroux-Metcalf, Cancer
Lett., 20
29-35 (1983)) or a skin tumorigenesis Sencar mouse model (Lambert et al., Food
Chem.
7oxicol., 33: 217-222 (1995)). The cellular toxicity is not mediated by
alteration in
deoxyribonucleic acid (DNA) synthesis or repair (Bradlaw et a1_, I«od C.'hem.
Toxicol., 23:
1063-1067 (1985); Robbana-Barnat et al., Tbxicolo~y, 9~: ISS-166 (1988))
DON appears to undergo no extensive liver metabolism and is readily and
predominantly eliminated in the urine. The derivatives deoxynivalenol
glucuranide and de-
epoxide DON have also been found in urine, apparently as the result of
metabolism by
microbes in the gut of animals that have received DON (see, for example,
Worrell et al.,
1 S Xenobiotica, 19: 25-32 ( 1989); Lake et al., Food C'hem. Toxicol., 25: 589-
592 ( 1987)).
Moreover, as we have discovered, DON and other trichothecenes operate by
stimulating
responses outside the gut which have a pronounced effect on the muscular
activity of the gut.
That is, DON does not act directly on the smooth muscle or other structures of
gut tissues and
involves no harmful effects on gut tissues to achieve its effects on gut
motility. Accordingly,
DON and the other nivalenol-related trichothecene compounds are particularly
well suited for
use in the methods described herein for treating obesity. Trichothecenes that
are not
structurally related to DON and the other nivalenol-related compounds, may
also be used in
methods of treating obesity provided they also stimulate the fed pattern of
gut motility and at
doses that do not result in any of the undesirable or severe symptoms of
clinical
mycotoxicosis.
Trichothecenes and derivatives thereof useful in the invention may be produced
biologically from fungal cultures or by chemical synthesis. A variety of soil
fungi that have
been found contaminating and growing on cereal grains and other crops produce
trichothecenes as secondary metabolites. Such fungi include species
off'usarium,
Tricothecium, Trichoderma, Myrothecium, Cylindrocarpon, and StachybotryS (see,
Ueno,
1980). Methods of producing and purifying DON and acetyl esters ofDON (such as
3-acetyl
DON and I S-acetyl DON) from Fusarium cultures have been described (see,
C.'an. J.
Microbiol., 29: 1 171-1178 (1983); Miller and Blackwell. C.'an J. Bot., 64 1-5
(1986);
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Greenhalgh et al., Proceedings of the 6th IUPACInternatioruxl Symposium on
Mycotoxins
and Phycotoxins: 137-152 (Steyn, P.S., ed.) (Elsevier Press, Amsterdam, 1986);
Miller and
Arnison (Can. J. Plant Path., 8: 147-150 (1986)). Thus, various trichothecenes
useful in the
methods described herein may be produced and extracted from fungal cultures
using standard
S culture and production techniques (see, for example, Ehrlich et al.,
Biochim. Biophys. Acta,
932: 206-213 ( 1987); Ueno, 1980 (supra) and references cited therein). As
noted above,
DON is an abundant, natural contaminant of corn and wheat. Thus, DON and other
tricothecenes may also be isolated from contaminated crops. Alternatively,
they can be
isolated from the Brazilian shrubs Baccharis magapotomica and cordfolicx
(Kupchan et al., ,l.
Urg. Chem., 42: 4221-4225 (1977)). In addition, the invention provides new
derivatives of
DON that can be synthesized from DON or 3-acetyl-DON as described in the
Examples
section below.
In addition, fungi that produce trichothecenes may also be used to modify pre-
existing
trichothecenes. Such bio-transformation of DON and its derivatives has been
undertaken in a
variety of laboratories employing bacteria (Shima et al., Appl. Fnviron.
Microbiol., 63: 3825-
3830 (1997)} or strains ofFusarium. For example, I''. roseum maintained in
peptone-
supplemented medium converts 3-acetyldeoxynivalenol to DON (Yoshizawa et al.,
Appl.
Microbiol., 29: 54-58 (1975)). F nivale can acetylate DON at the carbon at
position 3 to
give 3-acetyl-DON. Furthermore, these strains can deacetylate 7,15-diacetyl-
DON to give 7-
acetyl-DON.
The chemistry of the tricothecenes is well known so that various trichothecene
compounds may be synthesized by chemical or biochemical procedures. The
tricothecenes
are sesquiterpene alcohols or esters chemically related by the tetra-cyclic
12,13-
epoxytricothec-9-ene skeleton (Williams, Arch. Environ Contum. lbxicol., 18:
374-387
( 1989)). It has been reported that certain trichothecenes related to 4-DON
can also be
prepared using the trichothecene T-2 toxin (413,15-diacetoxy-3a-hydroxy-8a-[3-
methylbutyryloxy]-12,13 epoxytrichothec-9-ene) as starting material, since it
is produced
abundantly by 1r: tricinctum and is easy to modify at the C-3 and C-8
positions (Ehrlich et al.,
Appl. Environ. Microbiol , 50: 914-919 ( 1985); Udell et al., Z. Naturfarsch,
44: 660-668
( 1989)). Removal of the C-3 hydroxyl of T-2 involves initial conversion of T-
2 to the 3-
phenylthionocarbonate and then reduction of this intermediate with tri-n-
butyltin hydride to
give 3-deoxy-T-2. This approach has been used by others to prepare 3-
deoxyanguidine and
4-deoxyverrucarol (Schuda et al., .l. Nat. Prnd, 4i: S 14-519 ( 1984)).
CA 02374358 2002-O1-04
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It has also been shown that to generate the C-8 oxo-functionality (that is,
trichothecenes related to DON), T-2 and deoxy-T-2 are oxidized with selenium
dioxide
(Bamburg et al., Tetrahedron, 24' 3329-3336 (1968)). Additional derivatives,
such as THP-
7-DON (tetrahydropyranyl-7-DON) and D1DON (3, 7-dideoxynivalenol), have been
produced by introducing a C-8 ketone into the T-2 toxin (Bamburg et al.,
1968). This is
achieved by first preparing 3-THP-T-2 trio! and 3-deoxy-'T-2 trio! and then
oxidizing them
with manganese dioxide (MnOZ) (Warpehoski et al., J. (Jrg. ("hem., 47v 2897-
2900 (1982))
Oxidation is not possible for preparation of 7-DON from T-2 tetraol because of
competing
ring cleavage reactions and the low solubility of T-2 tetraol in methylene
chloride, which is
used as the reaction solvent. For preparation of THP-7-DON from THP-T-2 trio!,
MnOz
oxidation is the only possible method, since the acetic acid used as the
solvent for selenium
dioxide oxidation, would remove the tetrahydropyranyl group. 'The side
products in the
Mn02 oxidations are trichothecene, with l5-carboxaldehyde functionality.
Identification of DON-Related Compounds
DON-related trichothecene compounds can be identified using mass spectra, NMR
(nuclear magnetic resonance) spectroscopy, infra-red spectroscopy,
anisaldehyde staining,
and TLG (thin layer chromatography), to detect the presence of one or more
various
structural features of DON or other trichothecenes
2U All DON-related trichothecenes should have NMR spectra showing the expected
AB
coupling pattern from the C-l3a and C-lab protons, and a proton at 6.5 ppm for
C-10 (Cole
and Cox, Handbook of Toxic Fungal Metabolites (Academic Press, New York,
1981), pp.
152-263)
With anisaldehyde staining, the 8-oxo-substituted (keto) trichothecenes form a
lemon-
yellow adduct, whereas compounds lacking the keto group at position 8 form red
or brown
colored adduct.
In the infrared spectra, the carbonyl group at position 8 absorbs at 1660 -
1680 cm' .
This confirms that the trichothecene retains the alpha, beta-unsaturated
ketone functionality.
The mass spectral data for the acetylated analogs of DON should show the
parent ion
and fragment ions anticipated by loss of acetyl or acetic acid during the
process.
Each trichothecene used in the methods described herein is preferably purified
until it
migrates as a discrete spot on thin layer chromatography (TLC). Homogeneity
can be further
assessed by using high pressure liquid chromatography (HPLC) in which the
particular
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trichothecene should elute as a single peak. GC-MS (gas chromatography and
mass
spectroscopy) analysis has also been useful in assessing purity, for example,
as in showing a
single peak for each type of species of purified acetylated trichothecene
(Cole and Cox, 1981,
supra).
As discussed below, the invention also encompasses compounds in which one or
more of the above-described structural features of DON, or another
trichothecene, has been
modified or even eliminated to make a derivative compound that may also be
used in the
compositions and methods of the invention.
1~0 Trichothecene Derivatives and Analogs
DON may be used in the methods of the invention, such as treating obesity, but
must
be used at a dose that stimulates the fed pattern of gut motility without
causing any of the
other undesirable side effects, including emesis (vomiting), one of the
clinical symptoms of
mycotoxicosis. Although the pharmaceutically acceptable dose of a
trichothecene such as
15 DON can be determined using standard methods, it would be desirable to
produce a
modification in a trichothecene chemical structure to yield a structurally-
related compound,
which is even more benign with respect to possible untoward side effects. Such
a "benign
trichothecene" (for example, a "benign DON") is a derivative of the original
trichothecene
and is expected to be comparable or more potent than the original
trichothecene in
20 stimulating the fed pattern of gut motility, but with fewer or no unwanted
side effects.
Hence, a preferred derived trichothecene (e.g., DON derivative) will exhibit
one or more
improved properties and will be preferred over a known trichothecene, such as
DON, in
methods of treating obesity.
For example, various structural features of DON provide sites on the compound
that are
25 particularly attractive candidates for modification to foam DON
derivatives. Advantageously,
DON is a relatively small molecule and has a limited number of sites available
for
modification to alter activity of this compound. Such sites include the
unsaturated bond
between C-9 and C-10, the presence of the 12,13 epoxy ring, the presence. of
hydroxyl or
other goups on the structural nucleus of the trichothecene, and the occurrence
of hydroxyl or
30 other substituents at C-3, C-4, and C-15 (see, Figure 3A). In addition,
space-filling molecular
models reveal several features of the trichothecene nucleus that provide
additional
information in considering which sites) to modify in providing a useful
derivative ofDON.
Oxygen substituents in the A-ring (C-8 keto and C-7 hydroxvi groups) make this
side of the
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molecule more hydrophilic than when no substituent is present or when, like in
T-2 toxin, an
isovaleroxy side chain is present. The presence of hydroxyl groups at
appropriate positions
on the nucleus modifies biological activity. For example, the difference
between 4-
deoxynivalenol (DON) and nivalenol is the presence of a hydroxyl group at C-4
in DON.
Studies of the relationship between trichothecene stnrcture and the
characteristic
property oftrichothecenes to inhibit protein synthesis have also revealed
several interesting
features that may be considered in making derivatives of DON or other
trichothecenes that
may be employed in the methods ofthe invention (see, Erlich et al., Bioch~m.
Biophys. Acta,
923: 206-213 (1987); Rotter et al., Erm. Health, 48: I-34 (1996)). With
respect to inhibition
of protein synthesis, the most potent trichothecenes lack substitution in the
A-ring containing
the 8-oxo substituent, or have esterified hydroxyls. When a C-7 hydroxyl is
present,
hydrogen (H) bonding to the C-8 keto can occur, but this makes the ring more
sterically
strained. H-bonding can also occur between the C-15 and C-7 hydroxyl groups.
Removal of
the C-7 hydroxyl group exposes the C-L S substituent on the side of the
trichothecene away
from the 12,13-epoxide. In addition, the C-7 hydroxyl group must contribute to
trichothecene potency since nivalenol, which possesses the C-7 hydroxyl group,
is an order of
magnitude more potent than 7-DON, which lacks this hydroxyl group. It is,
thus, understood
that comparable or corresponding sites in DON and other trichothecenes,
especially the
nivalenol-related trichothecenes, may be considered as potential sites for
modification to
2,0 produce a derivative of DON or other trichotheeene, which is useful in the
compositions and
methods of this invention.
Examples of derivatives of the trichothecene DON useful in the compositions
and
methods of the invention include compounds referred herein as 3-acetyl-I)ON
(C,7Hzz07);
isopropylidine DON (3-hydroxy-7,15-isopropylidine-12.13-epoxy-9-tricothecin-8-
one,
CraHza06, designated EN 139491); isopropylidine-3-acetyl-DON (3-acetoxy-7,15-
isopropylidine-12,13-epoxy-9-tricothecin-8-one, CzoHz607, designated EN
139492); BON
carbonate (3-hydroxy-12,13-epoxy-9-tricothecin-8-one-7,15 carbonate, Ct6Hrx07,
designated
EN 139494); 3-acetyl-DON carbonate (3-acetoxy-12,13-epoxy-9-tricothecin-8-one-
7,15
carbonate, CrgHza08, designated EN139495); DON benzylidene acetal (3-hydroxy-
7,15-
benzylidene-12, l3-epoxy-9-tricothecin-8-one, CZZ.Hz4O7, designated EN
139497); and 3-
acetyl-DON benzylidene acetal (3-acetoxy-7,1 S-benzylidene-12,13-epoxy-9-
tricothecin-8-
one, CzaHz60g, designated EN139496) (see, Figures 3A and 3B). These compounds
may also
serve as "parent" compounds or starting materials that may be further modified
to yield
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additional new DON derivative compounds, which, preferably, exhibit one or
more improved
properties that will make the new derivative compound preferred over the
parent compound
or other known trichothecenes for use in compositians and methods for
regulating gut
motility described herein.
It is also understood that an alternative to DON or other trichothecene need
not be a
structurally related, derivative compound, as any compound that regulates gut
motility at a
dose having minimal or no untoward side effects may be useful in the methods
described
herein.
According to the invention, a trichothecene analog is any compound that mimics
one
or more of the characteristic and desirable biochemical activities of a
trichothecene, whether
or not the compound has structural characteristics of a trichothecene. Like
DON,
trichothecene analogs useful in the methods of the invention regulate gut
motility by acting
outside the gut, in the periphery. In particular, trichothecene analogs which
are useful in the
methods described herein for treating obesity, act outside the gut to
stimulate the fed pattern
1 S of gut motility and, thereby, signal satiety to stop eating. Such
trichothecene analogs may be
structurally related or chemically derived from DON or another trichothecene
(see above); an
inorganic molecule; an organic molecule unrelated to the trichothecenes;
biomolecules, such
as nucleotides, nucleic acids, peptides, polypeptides, proteins,
carbohydrates, lipids; or
combinations thereof Whether a particular compound is a trichothecene analog
according to
the invention may be determined using one or more methods of screening for
trichothecene
activities described herein.
A$onists and Antagonists of the PZX~ Purine Receptor (Ciut Neurotransmitter
Receptor)
Compounds that bind the P2x, subtype of purinoceptor found in gut tissue may
also be
used in the methods described herein. The P2x~ purinoceptor is a
neurotransmitter receptor
present in smooth muscle of the gut and is involved in the control of gut
motility (see,
Examples, Fig. 2). Adenosine triphosphate (ATP) is a naturally occurnng ligand
of the P2h,
receptor. In the duodenum and ileum of the small intestine, stimulation of
purinergic motor
neurons releases ATP. ATP initially acts as an agonist to the PZx,
purinoceptor in that the
first ATP molecule to bind to a PZx~ purinoceptor on a smooth muscle cell
signals an
inhibition of the smooth muscle, which then relaxes As noted above, such
relaxation is a
component of gut motility that can be detected and measured using the method
of Krantis et
al. (1996). However, a molecule of ATP appears to remain bound to the PZX,
pur7noceptor
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and thereby desensitizes the muscle to additional relaxation by ATP because
other ATP
molecules cannot bind the receptor to signal additional relaxation events
(Smits et al., Br. ,l
Pharmacol., 303: 695-703 ( 1996)). Accordingly, the Pz:m receptor mediated
pathway for gut
motor activity is blocked, resulting in an observable attenuation of all gut
motor activity,
which cannot be influenced by additional ATP (development of tachyphylaxis).
ATP is,
therefore, a "desensitizing" agonist, which prevents any further relaxations,
which are critical
in both the fed pattern and fasting pattern of gut motility. ATP is capable of
binding to all
types of purinergic receptors. The synthetic ATP analog, ex,p-methylene ATP,
is also a
desensitizing agonist, but is specific for P2x species of purinoceptors.
Furthermore, since the
PZx, subtype of purinoceptor is the PZx species of receptor involved in the
relaxation
component of gut motility, studies using a,a-methylene ATP provide data that
accurately
reveal the specific neurophysiological features ofrelaxatian in gut motility
(see, Examples).
In contrast to a desensitizing agonist of the Pzx, receptor, such as ATP or
a,(3-
methylene ATP, a "non-desensitizing" agonist ofthe Plx, receptor has receptor
binding
1 S properties that are necessary to provide continual stimulation of
relaxations for gut motor
activity. In particular, a non-desensitizing agonist ofthe PZx, receptor is a
compound that
binds but does not block the receptor. Each molecule of a non-desensitizing
agonist is able to
bind the PZx, receptor, evoke a relaxation, and then dissociate to be replaced
by another of its
kind, which in turn signals another relaxation, and so on. 'thus, non-
desensitizing agonists of
the PZx, receptor are able to stimulate relaxation events as long as molecules
of the non-
desensiting agonist are available for binding to the Pzx, receptor. Non-
desensitizing agonists
of the P2x, receptor stimulate the fed pattern of gut motility. 'Chus, non-
desensitizing agonists
of the P2xi receptor are chemical alternatives to using DQN, other
trichothecenes, or
trichothecene analogs in the methods oftreating obesity described herein
A desensitizing agonist (see, above) or an antagonist of the P2x, receptor
blocks the
receptor and attenuates gut motility Such compounds may be used to prevent or
inhibit the
fed pattern of gut motility and, thereby, inhibit satiety. Inhibiting satiety
will promote longer
eating or feeding time because the feeling of fullness is not evoked.
According to the
invention, an antagonist, such as 2',3'-O-(2,4,6-trinitrophenyl) adenosine
.'i'-lriphosphate
("TNP-ATP"), or a desensitizing agonist, such as a,~3-methylene ATP, that acts
at P2X1
receptors in gut tissue is useful in methods of prolonging eating time and
increasing weight
gain. Such a goal is particularly useful in the meat and poultry industry
where increasing
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weight of livestock or decreasing the time required to bring an animal to a
marketable weight
is commercially desirable and advantageous.
A number of structural analogs of ATP and some of their pharmacological
characteristics and receptor binding properties are known (see review by
Harden et al., in
Annu. Rev. Pharmacol. Toxicol., 35: 541-579 (1995)). Such compounds may serve
as
candidate compounds that can be further screened for the ability to bind the
P2xi subtype
purinoceptor and affect gut motility. Alternatively, the chemical structures
of such known
ATP analogs may be further modified to make other ATP analogs that can then be
screened
for the ability to act as non-desensitizing agonists, desensitizing agonists,
or antagonists of
the P2x, receptor that can be used in the various methods described herein.
The ATP analog TNP-ATP is a PZx purinoceptor antagonist that has been used in
vitro as a PZx subtype selective antagonist (whole tissue IC'.5o in 11M range)
to determine the
role of Pzxl and P2~, homomeric and Pzx2i3 heteromeric purinoceptors (see,
Lewis et al.,13r.
J. I'harmacol., I24: 1463-1466 (1998); Virginio et al., Mol. 1'harmacol., 53
969-973
(1998)). P2~ receptors arc reported to be expressed only on sensory neurones
(Evans et al.,
Semin. NeuroSCi., 8: 217-223 (1996)). As shown herein, the ~l'NP-ATP
antagonist is useful to
show the direct involvement of the P2xi subtype purinoceptor in regulating the
fed pattern of
gut motility in vivo. Since TNP-ATP is able to prevent or inhibit the fed
.pattern of gut
motility induced by DON, other trichothecene compounds, and derivatives
thereof, this PZxi
antagonist may, itself, be used in methods of the invention for prolonging
onset of satiety and
for increasing food uptake. Such methods are particularly useful for preparing
livestock and
poultry for market. Furthermore, TNP-ATP may he used as a parent molecule for
producing
derivative compounds having enhanced or improved properties affecting gut
motility.
Another class of compounds that may serve as a source of agonists or
antagonists of the
Pzx, receptor useful in this invention are anthroquinone-sulfonic acid
derivatives originally
described by Bohme et al. (Chromatogr., Gy: 209-213 (1972)). Such derivatives
may be
viewed as ATP analogs and include a triazinyl moiety, which has been shown to
antagonize
certain ATP-mediated actions in the guinea pig (see, Kerr and Krantis, Prac.
Austr. Physiol.
Soc., 10: 156P (1979)). It is expected that those anthroquinone-sulfonic acid
derivatives that
are capable of binding the Pzxr receptor and regulating (that is, either
stirrmlating or
inhibiting) patterns ofgut motility may be useful in various methods described
herein.
Another approach for developing non-desensitizing agonists of the Pzx,
receptor that
are useful in treating obesity is to develop compounds liom sulfonyl ureas,
for example, by
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replacing the triphosphate moiety of the parent compound (adenosine 5'-
tetrahydrogen
triphosphate) with unique, innovative acidic functionalities that are known to
mimic the
charge distribution in diphosphate or triphosphate, but that have never before
been combined
with the adenosine molecule. Importantly, the adenosine-S02-NH-CO moiety is
available for
combinatorial chemistry on a polymer base (Chiron Technologies). Compounds
that bind the
P2xi receptor and stimulate the fed pattern of gut motility are useful in
methods of treating
obesity according to the invention.
Screening Methods
Various methods may be employed to identify those trichothecenes,
trichothecene
analogs, non-desensitizing P2xi receptor agonists, PZxi receptor antagonists,
and other gut
motility-regulating compounds that are useful in the methods and compositions
of the
mvent~on.
Specific antibodies for detecting DON and 15-acetyl DON have been made and
used
in ELISAs (enzyme linked immunadsorbent assays) (see, Sinha et al., J. Agric.
Food Chem.,
43: 1740-1744 (i995)). Thus, antibodies to DON and other trichothecenes may be
employed
in various immunological procedures, such as EI_,ISA, to rapidly screen for
derivatives or
related trichothecenes that rnay also be employed in the methods described
herein.
Structure-function relationships for a large number of 12,13-
epoxytricothecenes have
been determined using in vitro cell cultures of Vero (Green Monkey kidney)
cells, murine
erythroleukemia (MEL) cells, and rat spleen lymphocytes. Fer example, such
cell culture
systems were used to test the ability of various 12, l3-epoxytricothecenes to
inhibit
peptidyltransferase activity and, hence, protein synthesis (see, for example,
Erlich and Daigle,
Biochim. Biophy.~. Acta, 923: 206-213 (1987); Ratter et al., J. Toxicol. Errv.
Health, 48:1-34
(1996)). In particular, tricothecenes bind to the 60S subunit ofthe eukaryotic
ribosome and,
thereby, interfere with peptidyltransferase. The degree of structural
substitution on the
trichothecene sesquiterpene affects the binding characteristics to the
peptidyltransferase and
hence the degree of inhibition of this enzyme (Erlich et al., 1987; Ratter et
al., 1996).
Cell cultures as described above can be employed to test or screen compounds
of
unknown activity as possible candidate compounds useful in the compositions
and methods
of treatment described herein. Such cell-based testing and screening methods
are particularly
useful to test and characterize various trichothecene or derivative compounds
of unknown
activity, such as newly synthesized or discovered compounds having structural
features of
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known trichothecenes, such as DON or other nivalenol-related trichothecenes.
Other
compounds that are not structurally related to known tr~ichothecenes may also
be screened
using such cell cultures.
In cell-based screening methods, each test compound may be compared to one or
more standard preparations of a known trichothecene, such as DON, which is
typically
prepared in stock solutions (10 pglml) in dimethyi sulfoxide. The
concentration of dimethyl
sulfoxide is adjusted so that it is always 1% (v/v) or less during incubations
with the cells.
Tricothecenes are generally stable for up to one year at room temperature
(27°C).
Preferably, candidate compounds may also be screened and characterized using
the
method of Krantis et al (1996), which uses miniaturized foil strain gauges and
a
computerized data analysis system to precisely and simultaneously record
relaxations and
contractions of smooth muscle in the gut This is a means of screening
compounds directly
for their effects on gut motility. As noted above, the method of Krantis et
al. (1996) is able to
provide an actual recording of the effect of a compound on fed and fasting
patterns of gut
1 S motility in vivo, ex vivo, or irr vitro (see, Examples 1 and 2). Such
methods of screening or
identifying a new compound for use in the compositions and methods of the
invention may
also comprise comparing the effect that a candidate compound has on gut motor
activity with
the effect that a known trichothecene, such as DON, has on gut motor activity.
Binding Assays and Screens for P2Xr Aazonists or Antagonists
Candidate compounds (also referred to as "lead" or "test" compounds) can also
be
tested or screened for the ability to bind or block the PZx~ subtype of purine
receptor, which is
the purinergic receptor expressed on smooth muscle and particularly involved
in controlling
the relaxation component of gut motility in the small intestine Much is now
known about
the structure of Pz-purinoceptors (see, for example, Virginio et al., Mol.
I'harmacol., 53:969-
973 (1998); Humphrey et al., Naurryrr Schmiedeberg's Arch. Pharrnacol.,
3~2v585-596
(1995); Bo et al., Br. J. Pharmacol., 112:1151-I 159 (1994); and reviews in
Burnstock, G.,
C'iba Found. Symp., 198: 1-28 (1996) (P2 receptor classification) and in
Surprenant, A., Ciba
H'ourrd Symp., 198: 208-219 ( 1996) (functional properties of native and
cloned Pzx
receptors)). The ability of a compound to bind the PZx~ purinoceptor may also
be compared
with that of a known receptor ligand, such as ATP or an ATP analog such as a,
~i-methylene
ATP or TNP-ATP.
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Successful radiolabeling of cell surface receptors for extracellular stimuli
depends on
the availability of ligands of high affinity, stability, and protein-binding
specificity. Until
recently, there were no selective antagonists for specific subtypes of Pi
purinoceptors, and the
several compounds that had been shown to competitively inhibit Pz
purinoceptors (for
example, suramin, reactive blue 2) did so with only micromolar at~inity and
lacked
specificity in that they interacted with many other proteins. A consistent
problem has been
that binding assays have been carried out under conditions, for example, with
membranes,
which are very different from the conditions in which biological responses to
the receptor can
be measured. Accordingly, direct correlations between binding constants and
receptor
activity constants have been difficult to make. Agonists ofFz-purinoceptors
also present
problems, since their binding affinities are only slightly higher than their
affinities for other
ATP binding proteins, and they are subject to hydrolysis by nucleotide
hydrolyzing enzymes.
[3H]-labeled a,[i-methylene ATP has been used as a radioligand for Pzx-
purinoceptors
in preparations of urinary bladder and vas deferens smooth muscle. Generally,
agonist
binding aWnities follow those observed in intact tissues. 1~or example, the
differences in
apparent binding affinity of a,(3-methylene ATP aad 2-methyl-S-ATP for
competition at the
vas deferens binding site were only about 30-fold, and many nucleotides that
are supposedly
not Pzx agonists also fully inhibit radioligand binding. The density of
binding sites labeled
with [~H] a.,[3-methylene ATP far exceeded that observed with all other
neurotransmitter
2.0 receptors.
Enteric smooth muscle expressing Pzx, receptors can be dissociated, and the
isolated
smooth muscle cells maintained in primary culture. These cultures can be used
in a binding
assay for lead or candidate compounds that are able to act as agonists or
antagonists of the
PzX, receptor. Alternatively, embryonic kidney 293 cells which express the P2m
receptors
may be used (Virginio et al, Mol. 1'hurmacol~ 53:969-9"73 (1998)}. This
receptor subtype is
also expressed in platelets and megakaryoblastic cell lines (Vial et al.,
77rromb. Haemost., 78:
1500-1504 (1997)), as well as in PILfiO cells (6uell et al, Blouci 87: 2659-
2664 (1996)).
Accordingly, any cell, including recombinantly modified cells, that expresses
the Pzx~
receptors in culture may be useful in screens for agonists or antagonists of
the Pzxl receptor.
'The Pzxi purine receptor has been purified (Valero et al., Natr~re, 371: 516-
519
(1994)) and cloned (Sun et al., J. Biol. Chem., 273; 11544-11547 (1998)). 'fhe
binding
characteristics for recombinant PzX, receptors have been described (Michel et
al., Br. J.
Pharmacol., I18 1806-1812 (1996)) A purified Pzxi receptor may be attached by
any of a
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variety of linking agents to a solid substrate, such as the surface of a well
in a microtiter plate,
a resin particle, or the surface of an assay chip. Such arrangements allow
very small
quantities of compounds to be tested for the ability to bind to the receptor-.
Furthermore, the
robotic technology that is available for screening samples in microtiter
plates and assay chips
permits hundreds or thousands of compounds to be accurately and continuously
screened in
hours with minimal supervision by the skilled practitioner.
Lead or candidate compounds identified as having an activity in one of the
above
screening methods can be further evaluated using an in vitro assay, ex vivo
gut organ assay,
and/or an in vivo assay for gut motility, for example, by the method of
Krantis et al. (1996)
(see, Example 1). In an in vitro gut organ bath assay, portions of a gut
organ, for example,
segments of the duodenum, jejunum, and ileum of the small intestine, are
excised from an
animal and placed in a physiological maintenance medium, such as Krebs
solution at
physiological body temperature Individual gut segments are usually mounted to
record
circular muscle activity, preferably at two attachment paints. A compound may
be injected,
mixed, or applied to the extricated gut organ segments, and the efhect on the
organ's motility
measured. 1n an ex vivo gut organ assay, the gut organs of an anesthetized
animal are
exposed, but maintained intact and at physiological conditions. A test or lead
compound may
then be conveniently applied (topically) directly on the organ, and the effect
on the organ's
motility monitored. In an in vivo assay, a compound may be injected into or
ingested by an
animal, and the et~'ect on gut motility measured directly.
Sources of compounds to be tested or screened for use in the compositions and
methods described herein include, without limitation, small molecule
collections,
combinatorial libraries, growth media or cell extrac,-ts from fungal,
bacterial, and various
eukaryotic cell cultures or fermentations, and biological fluids, tissues, and
serum samples
from humans and other animals.
Methods of Treatment, Pharmaceutical Compositions. Modes of Administration
The invention provides pharmaceutical compositions, which are used in methods
of
treating obesity. Other compositions of this invention are formulated for
administering to
animals to promote weight gain, which is especially useful in raising
commercial livestock
and poultry for market Humans and other vertebrate animals have the same basic
~,~rt
neurophysiology with respect to controlling gut motility. Accordingly, animals
that can be
treated using the methods described herein include, without limitation, humans
and other
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primates, swine, cattle, sheep, birds (poultry and other birds), horses, cats,
dogs, and rodents,
including hamsters, guinea pigs, rats, and mice. Both pharmaceutical
compositions and
compositions for administration to other animals described herein contain an
effective
amount of a compound to achieve the desired effect on gut motility without
significant or
undesirable side effects.
According to the invention, obesity is treated by administering to a human or
other
animal an effective amount of DON or other trichothecene, trichothecene
derivative,
trichothecene analog, or non-desensitizing agonist of the Pzxi purinoceptor to
stimulate or
activate the fed pattern of gut motility and, thereby, signal satiety. In
contrast, a P2xt
purinoceptor antagonist, such as TNP-ATP, or a desensitizing agonist, such as
oc,(3-methylene
ATP, may be administered to a human or other animal to prevent or inhibit the
fed pattern of
gut motility and, thereby, prolong eating time and promote weight gain.
Such compositions may be in any of a variety of forms particularly suited for
the
intended mode of administration, including solid, semi-solid or liquid dosage
forms, for
example, tablets, lozenges, pills, capsules, powders, suppositories, liquids,
powders, aqueous
or oily suspensions, syrups, elixirs, and aqueous solutions Preferably, the
pharmaceutical
composition is in a unit dosage form suitable for single administration of a
precise dosage,
which may be a fraction or multiple of a dose which is calculated to produce
the desired
affect on gut motility. The compositions will include, as noted above, an
effective amount of
the selected compound in combination with a pharmaceutically acceptable
carrier and/or
buffer, and, in addition, may include other medicinal agents or pharmaceutical
agents,
carriers, diluents, fillers and formulation adjuvants, or combinations
thereof, which are non-
toxic, inert, and pharmaceutically acceptable. In liquid mixtures or
preparations, a
pharmaceutically acceptable buffer, such as a phosphate buffered saline may be
used. By
"pharmaceutically acceptable" is meant a material that is not biologically,
chemically, or in
any other way, incompatible with body chemistry and metabolism and also does
not
adversely affect any other component that may be present in the pharmaceutical
composition.
For solid compositions, conventional nontoxic solid carriers include, for
example,
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharin,
talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.
Pharmaceutically
acceptable liquid compositions can, for example, be prepared by dissolving or
dispersing an
active compound that regulates gut motility as described herein and optimal
pharmaceutical
adjuvants in an excipient, such as, water, saline, aqueous dextrose, glycerol,
ethanol, and the
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like, to thereby form a solution or suspension. If desired, the pharmaceutical
composition to
be administered may also contain minor amounts of nontoxic auxiliary
substances such as
wetting or emulsifying agents, pH buffering agents and the like, for example,
sodium acetate,
triethanolamine oleate
Standard methods of preparing dosage forms are known, or will be apparent, to
those
skilled in this art (see, for example, Remington's Pharmaceutical Sciences
(Martin, E.W. (ed.)
latest edition Mack Publishing Co., Easton, PA). 1n the case of DON and other
trichothecenes, a dose is prepared that does not result in emesis (vomiting).
Such sub-emetic
doses are readily determinable as has been demonstrated in animal studies
(see, Examples 1
and 2).
The primary active ingredient of a composition of this invention is a compound
which
is a trichathecene, trichothecene analog, an agonist of the Pzx, receptor, or
an antagonist of
the P2X, receptor that affects (modulates) gut motility. 'frichothecenes such
as DON are
clearly capable of exerting their activity on gut motility when ingested.
Accordingly, a
preferred composition of this invention is formulated for oral administration.
Such
compounds may also be administered parenterally, for example, by intravenous,
intramuscular, or intraperitoneal injection.
For oral administration, which is preferred, compositions of the invention may
be
formulated as fine powders or granules containing of the compound that affects
gut motility
and may also contain diluting, dispersing, and/or surface active agents.
C;ornpositions for oral
administration may also be presented in water or in a syrup as a solution or
suspension, in
pills, tablets, capsules or sachets in the dry state, or in a nonaqueous
solution or suspension
wherein suspending agents may be included. Binders and lubricants may also be
used in
compositions for oral administration. Where desirable or necessary, flavoring,
preserving,
suspending, thickening, or emulsifying agents may be included. Tablets and
granules are
preferred oral administration forms, and these may be ccxated.
Parenteral administration, if used, is generally a method of injection.
lnjectable
preparations can be prepared in conventional forms, either liquid solutions or
suspensions,
solid forms suitable for solution or suspension in liquid prior to injection,
or as emulsions.
For most purposes, a compound useful in regulating gut motility may be
injected
intravenously in a pharmaceutically acceptable buffer. However, it is within
the scope of this
invention that such a compound may alternatively be prepared as a bolus, which
may contain
a mordant for gradual release from an injection site. One approach for
parenteral
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administration involves use of a slow release or sustained release system,
such that a constant
level of dosage is maintained (see, for example, LJ S. Patent No. 3,710,795).
The exact, effective amount of a compound useful in regulating gut motility in
the
compositions and methods described herein will vary from subject to subject,
depending on
the age, weight and general condition of the subject, the degree of obesity
being treated, the
particular compound used, its mode of administration, and the like. Thus, it
is not possible to
specify an exact amount for an ideal dose applicable to all individuals.
However, it is
expected that generally a trichothecene such as DON will be used or tested in
a range of 0.01
- 100 mglkg body weight. Furthermore, the useful dosage selected for a
particular individual
will be a sub-emetic dose, that is, a dose that does not, evoke vomiting in
that individual. For
commercial pharmaceutical compositions, it is understood that a
pharmaceutically efTective
and suitable amount of trichothecene, trichothecene derivative, trichothecene
analog, PZx~
receptor agonist, or P2xl receptor antagonist will be determined, in the case
of human use, by
the healthcare professional in studies acceptable to the standards of the
United States Food
and Drug Administration (or comparable agency). For use in animals, an
appropriate
composition will be determined and formulated according to the standards and
practices for
commercial livestock feed or veterinary medicine.
Additional embodiments and features of the invention will be apparent from the
following non-limiting examples.
Examples
Example 1
The following example shows that DON acts at sites outside the gut and
interferes
with specific intrinsic neural pathways of the stomach and small intestine,
giving rise to
altered patterns of motor activity. These findings show that DON induces loss
of appetite (as
illustrated by feed refusal in animals) and support a method of inducing such
loss of appetite.
Normal gastrointestinal motility is dependent on the intrinsic (enteric)
neural
networks of the gut wall, with modulatory inputs from the periphery and
central nervous
system (CNS). The intrinsic circuitry coordinates reflex activity, such as the
peristaltic
3'D reflex, or complex patterned motor activity, such as interdigestive
migrating myoelectric
complexes (MMCs) that occur within the fasting pattern and segmentation
driving fed
pattern.
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The effects of DON on the pathways contralling interdigestive spantaneous
motor
activity of the rat stomach and intestine were examined. Even though rats do
not posses an
emetic reflex, they do exhibit the sickness and discomfort associated with
vomiting
(Andrews, Br. J. Anaesth., 69: 2s-19s (1992), Rapley, W.A. and Hirst, M.,
Abstract in lcrb.
Anim. Sci., 38: 504 (1988)). The effects of low-threshold levels ofDON on the
interdigestive
motor pattern in vivo were examined. Additional information was obtained from
in vitro gut
bath experiments to obtain adjunct pharmacological information.
Experiments were performed on male Sprague-Dawley rats (Charles River)
weighing
250-350 grams. All experimental protocols were carried out according tc~ the
guidelines of
the Animal Care Committee at the University of Ottawa.
Individual rats, fasted for 24 hours with free access to water, were
anesthetized using
a Halothane (4%) in oxygen mixture. Rats were maintained under Halothane (2%)
anesthesia
on a heated scavenging table to preserve body temperature at 37°C. The
right carotid artery
was exposed and cannulated using PE50 tubing to monitor blood pressure via a
pressure
1 S transducer (P23TD, Gould Statham, OH) connected to an IBM PC data
acquisition system.
The jugular vein was cannulated (PE 50 tubing) to allow for intravenous (i.v.)
injections.
However, due to the shart half life of various drugs, and to evade hepatic
first-pass
metabolism, an intra-arterial route of administration was often necessary, In
animals where
drugs were to be introduced by close intra-arterial (i.a.) injections, a
cannula (PE 10 tubing)
was inserted from the femoral artery and fed in a retrograde directian to
position the tip at the
level of the superior mesenteric artery.
A median laparotomy exposed the gastrointestinal segments of interest. Foil
strain
gauges (Showa type NI I, Durham Instruments, Pickering, ON) were sequentially
attached
using Vet Bond glue to the gastric antrum (2~ cm proximal to the pyloric
sphincter); to the
anti-mesenteric border of the duodenum (1-2 cm distal to the gastroduodenal
junction); and
lateral to the anti-mesenteric border of the ileum (just proximal to the ilex-
cecal junction).
All foil strain gauges were oriented parallel to the longitudinal muscle layer
since this affords
the most sensitive setting for recording circumferential motor activity. Wire
leads from the
foil strain gauges were exteriorized and attached to an IBM PC data
acquisition system via a
3-channel interface box. The detailed method for recording and analyzing rn
vivo motility
using foil strain gauges coupled to a computer based data acquisition system,
has been
described by Krantis et al. (1996). A schematic diagram of this method for
recording gut
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motility is shown in Figure 1. After completion ofthe surgery, the rats were
turned over to
the prone position, and Halothane was maintained at 1 % for the remainder of
the experiment
Lx vivo organ preparations were made using the same surgical procedures
described
above, except rats were left in the supine position to maintain the foil gauge
attachment sites
exposed. This allowed for local administration of drugs directly onto the
serosal surface of
the gut. Regular application of warmed saline kept the exposed gut segments
moist.
Gut organs for use in in vitro gut organ baths were prepared according to
McKay and
Krantis (Can. J. Physiol. Pharmacol., 69: 199-204 (1991)). Kats were
euthanized, and 4-5
cm segments of the proximal duodenum, jejunum, and ileum were quickly removed,
carefully
cleared of contents, any mesenteric attachments dissected away, and then
placed in an organ
bath containing Krebs solution of the following composition (mM): Na' (
151.0), K+ (4.6),
Mg2+ (0.6), Ca2+ (2.8), CI- (i 34 9), HCO~' (24.9), HzP04~ ( 1.3), S04Z-
(O.ci), and glucose (7.7).
This solution was maintained at 37° C and continuously gassed with 95%
02 : 5% COz to
give a pH 7.4.
Individual gut segments were then mounted horizontally to record circular
muscle
activity at two attachment points on the mesenteric border 25 mm apart, each
opposite a frog
heart clip tethering the segment to the bottom of the organ bath, and each
connected to Grass
isometric force transducers by thin polyester string. Mechanical activity
detected by the
transducers was monitored directly by a MacLab Macintosh data acquisition
system (Apple
Z.O Corp., Toronto, Ontario).
Each attachment point was placed under a resting tension of 1 gram and the
segment
allowed to equilibrate for 60 minutes before drug treatments. Organ bath
preparations were
washed with renewals of the bathing Krebs solution every 15 minutes and
between drug
challenges. Subsequent drug challenges were tested only after an equilibration
period of at
2.5 least 5 minutes or until the basal tone recovered tc~ 90% of the resting
tension
The motor activity recorded in the in vivo and ex vrvo experiments was
acquired,
digitized, and stored by an IBM data acquisition system that calculated, in
addition to other
variables, the amplitude and frequency of motor responses (Krantis et al.,
1996). Qualified
responses were marked depending on their capacity to satisfy a set of~six
numerical
30 parameters (separately for contraction and relaxation), based on user-
defined threshold values
that must be satisfied within limited time periods. 'these parameters were
continuously
monitored over sequential two minute periods and adjusted as deemed necessary
to
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efl3caciously mark motor responses with 95-100% accuracy. The data were then
output and
organized into tabular form for statistical analysis.
A one-way ANOVA with a Tukey multiple analysis test was used to compare mean
values, using Statgraphics Plus 5.0 software (Statistical Graphics Corp.) A
probability of
less than 0.05 (p<0.05) was regarded to be significant. Al) values are
expressed as mean ~
S.E.M of experiments.
All the drugs utilized in the in vivo and ex vivo experiments, including DON,
were
dissolved in physiological saline (0.9%). The infused concentrations
(delivered at a rate of
0.5 ml/min) were: a,p-methylene adenosine triphosphate (oc,~i-methylene ATP,
300 mg/kg),
N-w-nitro-L-arginine methyl ester (L-NAME, 10 rnglkg;l, BRL 43694
(granisetron, 80
mg/kg), pentolinium (5 x 10-s M) and hexamethonium (18 mg/kg, s c.). All drugs
were
purchased from Sigma Chemical Company, Toronto, ON; except for a,~i-methylene
ATP,
which was obtained from RBI, Natick, MA; and DON was provided by Dr. Dave
Miller,
Agriculture Canada, Ottawa, ON, where it was biosynthetically produced and
purified
according to the methods ofMiller et al. (Can. J. Mrcrnhiol., l9: 1171-1178
(1983); Miller
and Blackwell, Can. ,I. Bot., 64: 1-5 (1986); Miller and Arnison ((:an. .l.
~'lant Path., 8v 147-
150 (1986)). The label "mg/kg" (or mg~kg'1) refers to milligrams per kilogram
of body
weight ofthe individual subjectlanimal.
The concentrations of drugs applied in the organ bath preparations (in vitro)
or
topically in the ex vivo experiments were: carbachol (0.5 rrrM), papaverine
(10 mM), ATP
(0.5 mM), DMPP (50 mM), 3-APS (0.5 mM) (Sigma Chemical Company, Toronto, ON)
and
DON (20 ~. Drug volumes were never more than 1 °ro of the bath
volume.
Under control conditions, the stomach and small intestine in anesthetized rats
displayed typical spontaneous motor activity. In the gastric antrum, this
consisted of
oscillatory contractile and relaxant motor responses. In the proximal
duodenum, spontaneous
motor activity was patterned into periods of intense "grouped" activity (1-.5
minutes
duration), comprised primarily of high amplitude, high frequency relaxations
and
contractions. T"he periods of "grouped" molar activity were separated by
"intergroup"
activity (3-10 minutes duration), comprised primarily of low amplitude, low
frequency
relaxations and contractions. In contrast to the "intergroup" activity,
"grouped" activity was
propagatory at a rate of 3.4 ~ 0.6 cm~min 1 in the aboral direction. Control
spontaneous
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motor activity of the distal ileum consisted primarily of randomly occurring
contractions
and/or relaxations of relatively high amplitude and low frequency.
DON, administered systemically in a 1 or 2 mg~kg ~ bolus, did not affect
control
motor activity. However, at 10 mg~kg', DON disrupted spontaneous motor
patterns, as
described below. Treatment with 20 mg~kg' of DON did not yield significantly
greater
effects.
Gastric Antrum
Within 2 minutes following intravenous injection, DON (10 mg~k~ i, i.v., n---
7)
inhibited antral motor activity (Figure 4), attenuating (p<:0.05 ) both
spontaneous contractions
(to 20 ~ 6 % of control) and relaxations (to 27 ~ 11 % of" control). This
effect was transient,
as motor activity recovered to 90% of the control level within 18 -~- 3
minutes. A proximate
reapplication of DON was typically without effect
Proximal Duodenum
Within 2 minutes of systemic DON (10 mg~kg ~, n = 12) injection, spontaneous
duodenal motor activity transformed from the control pattern of alternating
"grouped" and
"intergroup" activity to a period (46 -~- 15 minutes) of sustained "grouped"-
like activity (Fig.
5). This hyperactivity was not significantly different in amplitude or
frequency from the
control "grouped" motor activity. Within 60 minutes following the
administration of DON,
the control pattern of alternating "grouped" and "intergroup" activity
recovered. Subsequent
injection of DON (n=6) did not significantly alter the motor pattern,
indicative of the
development of tachyphylaxis. This tachyphylaxis to DON was relatively short-
lived, such
that retesting DON (n = 5) 30 minutes later, again elicited hyperactivity
(p<0.05); however
the duration of this activity (24 ~ 14 min) was considerably reduced compared
to the duration
of the initial DON induced hyperactivity.
Distal Ileum
Systemic injection ofDON (10 mg~kg', n - 9), within 2 minutes, evoked
hyperactivity of ileum. The frequency and amplitude of contractile and
relaxant motor
responses were significantly (p<0.05) increased. Thus, the pattern of gut
motility in the
ileum in the presence of DON resembled the characteristic fed pattern of gut
motility that
occurs when food has been ingested and needs to be propelled through the gut.
This effect
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lasted 63 ~ 22 minutes. Thereafter, motor activity gradually recovered to
control levels. In
parallel to the duodenum, tachyphylaxis to readministration of DON (n = 6)
also developed in
the ileum. This tachyphylaxis persisted for up to 90 minutes, after which
time, hyperactivity
could again be induced by readministration of DON (n = 6).
Effects ofLocall~Administered DON
~x vivo preparations exhibited patterned motor activity similar to that of the
in viva
preparations. Direct (topical) application of 20 mM DON (a concentration
considerably
greater than the in vivo dose) to the serosa of the gastric antrum (n = 3),
proximal duodenum
(n 3), or distal ileum (n = 3) did not evoke any c<>mparable motor responses
to those seen in
vivo. The vitality of the gut regions examined was verified by observing
predictable
responses to pharmacological stimuli that are known to act directly on smooth
muscle, such
as, papaverine (10 mM), which relaxes smooth muscle, and carbachol (0 5 mM),
which
induces cholinergic muscarinic receptor mediated contractions. Topically
applied DON did
not interfere with the action of these drugs.
Isolated gut bath preparations (n -- 5) of duodenum, jeaunum, and ileum
reacted with
either a contraction or a relaxation to an application of carbachol (0.5 mM)
or papaverine (10
mM), respectively. In addition, the gut segments exhibited relaxant responses
to the putative
non-andrenergic, non-cholinergic (NANC) inhibitory transmitter ATP (0.5 mM)
and to neural
stimulation, using the GABAA receptor agonist 3-.APS (0.5 mM) or the nicotinic
receptor
agonist DMPP (50 mM). However, in these same preparations, DON (20 mM) was
ineffective. Furthermore, DON did not interfere with the responsiveness of
these gut
segments to the pharmacologic agents tested.
Pharmacolog~ofDON Induced Hyperactivity
L-NAME' In anesthetized rats exhibiting spontaneous motor activity, the nitric
oxide
(NO) synthase inhibitor, L-NAME, attenuates NU-mediated '°intergroup"
relaxations and
enhances "grouped" activity of the duodenum (unpublished observations).
Therefore, the
effect of L-NAME on the action of DON was examined. Systemically administered
L-
NAME (I0 mg~kg', n = 5), did not alleviate (p>0 05) DON induced hyperactivity
of
frequency and amplitude of relaxations in the duodenum (see. Figure 6A,
frequency, and
Figure 6B, amplitude). In the ileum, L-NAME always enhanced both the frequency
and
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amplitude of relaxations of spontaneous motor activity to the same level as
with the presence
of DON alone (see, Figure 6C, frequency, and Figure 6D, amplitude).
Purinoceptor tachyphylaxis: Spontaneous duodenal "grouped" relaxations are
specifically mediated through P2x-receptor related purinergic transmission
(unpublished
observations). This was confirmed here following desensitization of the f2x-
purinoceptors
with prolonged exposure to the specific agonist, a,G3-methylene ATP. Initial
injection ofcx,(3-
methylene ATP (300 mg~kg ~, i.a, n = 3) induced a prominent relaxation.
Following recovery
to baseline tone, re-challenge with a,(3-methylene ATP was without effect,
indicative ofthe
development of tachyphylaxis. Under these conditions, spontaneous duodenal
"grouped"
relaxations and deal relaxations were specifically blocked. 1n addition, DON
induced
hyperactivity was also abolished during a,p-tnethylene ATP induced
tachyphylaxis in bath
the duodenum (n = 8) and ileum (n = 4).
Nicotinic receptors: Cholinergic nicotinic mechanisms are fundamentally
involved in
the control of intestinal motility (see, Furness and Costa, Neurnsci., 5: 1-
;'0 (1980); Gershon,
Ann. Rev. Neurosci., 4: 227-272 (1981)). In vivo, treatment with the
ganglionic nicotinic
antagonists, pentolinium (50 mM topical application, n -- 2) or hexamethonium
( 18 mg/kg,
s.c., n = 2, not shown), significantly reduced the frequency and amplitude of
the DON
induced hyperactivity in the duodenum and ileum.
Granisetron: In the duodenum, systemically administered 5-HT3 receptor
antagonist
granisetron (80 mg~kg', n = 8) attenuated (p<0.05) the frequency and amplitude
of
spontaneous contractions and relaxations of the "grouped" activity (compare
broadly spaced
diagonal bars (granisetron alone) with open bars oh control grouped activity
in Figures 7A -
7D). This effect of granisetron persisted for up to 30 minutes However, since
the duodenum
did not exhibit desensitization to the actions of this drug, granisetron was
repeatedly
readministered to maintain blockade of the "grouped" activity. Under these
conditions, DON
(l0 mg~kg ~, n=5) consistently induced (p<0.05) hyperactivity (compare
narrowly spaced
diagonal bars (DON alone) with filled bars (granisetron + DON) in Figures 7A-
7D).
Similarly in the ileum, granisetron (80 mg~kg', i a.) attenuated (pv0.05) gut
motility,
both spontaneous contractile and relaxant motor rE;sponses to 40 ~ 18% (n -=
4) and 27 ~ 10%
3~ (n = 3), respectively, of control levels; however, it did not antagonize
the DUN induced
hyperactivity (not shown).
These experiments characterized motor patterns at the level of the stomach and
small
intestine in anesthetized rats exposed to the mycotoxin DON. Systemic
injection of DON
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disrupted the gastric antrum oscillatory motor activity, replacing it with a
quiescent pattern;
and in the duodenum, DON induced a hyperactivity in place of the spontaneous
cyclic pattern
of propagatory and non-propagatory motor activity. DON also caused
hyperactivity of the
existing motor pattern in the ileum. T'he patterned activity induced by D(:)N
was reminiscent
of a typical "fed pattern" motor activity Low levels of DON were used, i.e.,
levels that did
not induce emesis or emetic behavior. The action of DON was maximal with 10
mg/kg of
DON; this dose is comparable to other studies using rodents, where up to 40
mg/kg (i.v.) of
DON was used to induce alterations in feeding (Rapely et al., Lab. Anim. Sci.,
38: 5041
{1988)).
A progressive tolerance to DON was evident in the rats examined. DON-induced
hyperactivity in the small intestine lasted up to 60 minutes, then full
restoration of control
motor patterns followed. Subsequently, responsiveness to routinely applied
pharmacological
stimuli was maintained; except for DON, which was inei~'ective after proximate
successive
applications, characteristic of the development of tachyphylaxis to DON.
However, this
tachyphylaxis was not sustained, possibly due to the high rate of DON
detoxification in rats
(Prelusky et al., Fund Appl. Tbxicol., 10: 276-286 {1988)).
Many drugs, in particular emetic agents, alter intestinal motility at the
level ofthe gut
where they activate vagal afferents projecting to autonomic ganglia and/or the
vomiting
center of the central nervous system (CNS), which in turn reflexively
stimulate the gut
(Castex et a1_, Bruin Res., 6R8: 149-160 ( 1995), Cubeddu et al., Sem. Oncnl.,
19: 2-13
(1992)). However, results from the ex vivo and in vitro experiments presented
herein indicate
that while the isolated gut segments were sensitive to a variety of
pharmacological stimuli,
directly administered DON was without effect. Thus, DON must exert its effects
indirectly,
from sites outside the gut. This finding aligns with certain reports in the
literature of a
2.5 delayed time-to-onset of DON induced effects following intragastric versus
intravenous
injection (30 min versus 15 min, respectively) (Coppock et al., Am. J. Vet..
Kes., 46: 169-174
(1985), Foresyth et al., Appl. Fnviron. Microbiol, ..?4: 547-552 (1977),
Prelusky et al.,
Natural. Toxins, l: 296-302 (1993)).
Under normal circumstances, feeding interrupts the fasting cyclic motor
pattern at all
levels of the gut, replacing it with continuous, irregular low level activity
(segmentation, fed-
pattern). As mentioned above, segmentation is characterized by narrow annular
contractions
interposed between relaxations in the small intestine, and reduced motility in
the gastric
antrum. The fed pattern functions to mix intestinal contents and delay
anterograde propulsion
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to enhance substrate absorption (L,undgren et al., Uig. Dis. Sci., 34: 264-283
(1989)). Fed
pattern motility is activated by peripheral autonomic ganglia via primarily
vagal inputs and is
controlled, to a lesser extent, by the CNS (Chung et al., Cart. .l: Physiol.
f'harmacol., 70:
1148-1153 (1992), Tanaka et al., J. Surg. Re.s., 53 588-595 (1996), Yoshida et
al , J.
Pharmacol Isxp. Therap., 256: 272-278 (1991)). Over-activation of autonomic
nerves
accelerates the onset and increases the duration ofthe fed pattern,
concurrently increasing the
frequency and amplitude of propagatory motor activity (Hall et al., Am. J.
Physio., 250:
6501-6510 (1986), Johnson et al., Am. J. Surg:, i67: 80-88 (1994)). This motor
activity is
similar to the DON induced hyperactivity in the small intestine, observed
above. In addition,
DON induced inhibition of antral motor activity and delay of gastric emptying
(Fioramonti et
al., .l. Pharmacol. Exp. 7Tterap., 2b6: 1255-1260 ( 1993)), is also
characteristic of the fed
pattern (Hall et al., 1986). Taken together, these results indicate that DON,
from extrinsic
sites, stimulates pathways mediating the fed pattern, either via peripheral
autonomic ganglia
or vagal efferents.
Fed pattern motility can be partially activated by the suppression of
inhibitory nervous
influences (L.undgren et al., 1989). Hence, DON, acting outside the gut, could
stimulate
hyperactivity by eliminating the tonic suppression of the enteric neural
circuits controlling
gut motility. NO (nitric oxide) is proposed to be a tonically released
inhibitory mediator that
modulates gastrointestinal motility (Daniel et al., Am. .I. Physia., 26h: G:31-
G39 (1994),
Gustafsson et al., J. Aut. Nerv. Sys., 44: 179-187 ( 1993), liryhorenko et
al., J. Pharmacol.
Exp. Therap., 271: 918-926 (1994)). In the in viva experiments, treatment with
the NO
synthesis inhibitor L-NAME (10 mg/kg, i.v.) mimicked to some degree the
effects of DON,
by potentiating specific motor activity of the duodenum and ileum. However, L-
NAME
treatment did not affect DON actions in the gut.
These experiments provide valuable insights into the pathways mediating DON
effects in the gut While the NO synthase inhibitor, L.,-NAME, selectively
blocked
spontaneous "intergroup" duodenal relaxations, and potentiated grouped motor
activity, it did
not affect DON induced hyperactivity. By contrast, a,p-methylene ATP induced
tachyphylaxis, which selectively attenuated "grouped" relaxations, also
prevented DON
induced hyperactivity in the duodenum. There is strong evidence suggesting
that ATP and
NO are NANC inhibitory neurotransmitters in the rat duodenum (Katsuragi et
al., .I.
t'harmacol. Exp. 7fterup., 259: 513-518 (1991), Manzini et al., Ear. J.
Pharmacol., I23: 229-
236 (1986), Postorino et al., J. Aulon. Pharmacol., 1.5: 65-71 (1995),
Windschief et al., tir.
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J. Pharmacol., IIS: 1509-1517 (1995)). 'This targeting of purinergic
relaxations by DON
was also apparent in the ileum, where DON induced hyperactivity was blocked by
a, ~i-
methylene ATP tachyphylaxis. Until now, the identity ofthe transmitter
mediating
spontaneous ileal relaxations in vivo has not been examined in the rat.
However, there is a
large body of in vitro functional evidence for both ATP and NO to mediate NANC
relaxations in the rat ileum (Belai et al., Cell. Tiss. Re.s., 278: 197-200
(1994), Fargeas et al.,
Gasiroenterol., 102: 157-162 (1992), Mahmod and Huddart, Comp. Biochem.
f'hysiol.,
106C: 79-85 (1993), Smits et al., Br. .l. Pharmacol_, 118v 695-703 (i996)).
Nicotinic receptor blockade abolished both spontaneous and DON induced motor
activity in the small intestine, which explains results from a several
previous studies.
Nicotinic ganglionic transmissions are known to mediate cholinergic
stimulation of both
excitatory and inhibitory intramural neurons that modulate and process enteric
neural signals
(Bornstein et al., Clin. Exp. Pharmacol. I'hysiol., 21 v 441-452 (1994),
Gershon, Ann. Rev.
Neurnsei., 4' 227-272 (1981)). In fact, cholinergic neurons mediate all
motility patterns of
the gastrointestinal tract, including the peristaltic retlex and MMCs.
Therefore, treatment
with nicotinic antagonists would effectively block most if not all enteric
neural circuits.
5-HT3 sites on vagal afferents are not likely to be involved in DON actions
related to
gut motor activity, since DON was ineffective when applied directly onto the
exposed gut of
a whole animal. 5-HT3 receptors are also localized to myenteric neurons that
are involved in
the enteric circuits regulating the interdigestive motor pattern (Hoyer,
Neurop.sychopharmacol., 3: 371-383 (1990), Yoshida et al., 1991), and these
neurons may
occur within the enteric pathways} targeted by DON. Treatment with
granisetron, a potent
and specific 5-HT3 receptor antagonist, at a sufficiently high dose that
abolished spontaneous
"grouped" activity in the duodenum and motor activity of the ileum, did not
affect DON-
induced hyperactivity. Therefore, DON and 5-HT' operate through dif~'erent
pathways that
target common enteric elements in the small intestine. However, both the DON
activated
pathway and the 5-HT3 receptor-dependent pathway converge on the same
population of
inhibitory purinergic motor neurons.
These findings provide an explanation for DON-induced feed refusal. The
effects of
"low" threshold levels of DON are manifest in the disruption of gut motility;
antral motor
activity is diminished, and motor activity of the small intestine is
intensified. Together, these
motor patterns exemplify the "fed state", a state normally associated with
satiation. Under
these circumstances, a human or other animal ceases to eat. This DON-induced
fed pattern is
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transient, and the interdigestive cyclic pattern soon recovers, presumably
because DON
plasma levels fall below threshold.
A consistent feature of this study, was the targeting by DON of enteric P~x-
purinoceptor mediated inhibitory enteric motor innervation. This innervation
also involves
nicotinic receptors, but, as described above, the ubiquitous distribution and
involvement of
nicotinic sites within enteric neural circuits precludes the use of nicotinic
antagonists to
counteract the actions of DON. More promising however, is the specificity of
DON in
activating P2x-purinoceptor related activity. P2x-purinergic sites represent a
highly restricted
component of enteric pathways, and hence targeting these sites may represent a
simple
approach for counteracting DON effects in the gut.
Example 2
The effects of DON on spontaneous motor activity ofthe gastrointestinal tract
in
swine in vrvo: involvement of enteric P2x-purinoceptors.
This example demonstrates that the trichothecenc; DON affects gut motility by
acting
at a site in the peripheral nervous system and that the affect of DON may be
counteracted by
the P2xr purinoceptor desensitizing agonist, a,(3-methylene ATP, which binds
with high
affinity to the PZa, purinoceptor on gut tissue. The intense binding by this
purinergic ATP
analog not only desensitizes the PZxr purinoceptor regulation of gut motility
from DON, but
also can shut down the pathway of regulation of gut motility ef~'ectively as
an P2.~,
purrnoceptor antagonist.
Male Yorkshire pigs (10-1 S kg live weight) weaned for one week were fasted
for 12
hours overnight, with free access to water. On the morning of the surgery,
animals were
sedated using Ketamine (8 mg/kg) via an intramuscular injection. Ketamme is a
dissociative
anaesthetic, which causes an increase in blood pressure and skeletal tone, and
the trachea will
be stiff. A cataleptic sedation is produced with a lack of awareness of the
surroundings.
However, salivary secretions are increased and hence airway obstruction is a
hazard; yet
atropine cannot be used. Anesthesia was induced using a Halothane-oxygen
mixture by
application of a face mask. Topical pharyngeal anaesthesia was provided using
1-2 daces of
lidocaine aerosol (10 mg per dose, Xylocain, Sigma). Animals were then
intubated, and a
surgical plane of anesthesia was achieved using Halothane (3-4%) in oxygen
(200 ml/min)
via a closed non- rebreathing circuit. A catheter was inserted into a
superficial ear vein for
electrolyte replacement (0.9% saline) and intra venous drug injections. The
femoral artery
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was also cannulated for intra-arterial drug injections. PE 205 tubing was fed
in a retrograde
direction such to place the tip of the cannula at the level of the superior
mesenteric artery.
Blood pressure was also monitored through this arterial catheter by way of a
pressure
transducer (P23TD, Gould Statham, OH, USA) connected to an anline IBM data
acquisition
system. The animals were then subjected to a laparotomy, and foil strain
gauges (Showy type
N 11, Durham lnstruments, Pickering, ON) were affixed, using Vet Bond glue as
described in
Example 1, onto the serosa of the gastrointestinal tract. One strain gauge was
placed on the
gastric antrurn (5-10 cm distal to the pylorus); a second gauge was placed on
the anti-
mesenteric border of the proximal duodenum (2-10 cm from the pylorus), and a
final gauge
was attached onto the serosa of the distal ileum (2-10 cm distal to the
cecum). All three foil
strain gauges were oriented parallel to the axis of the longitudinal muscle.
Leads from the
strain gauges were exteriorized and attached to the IBM data acquisition
system, via an
interface box. Following completion of the surgery, the pigs were turned over
to their side,
and a light plane of anesthesia was maintained for the remainder of the
experiment by 1-2%
1 S Halothane.
Motor activity was continuously recorded from all foil strain gauges
simultaneously
using data acquisition software (AD1000 analog to digital conversion card,
Real Time
Devices Inc., Dr. Frank Johnson, Institute of Medical. Engineering, University
of Ottawa) and
an IBM compatible computer. Qualified motor responses were selected based on
their
capacity to satisfy two sets (for contractions and relaxations) of six
numerical values. These
values defined threshold duration and magnitude parameters that efllicaciously
marked motor
activity based on the user's visual inspection of the recordings. The user is
able to
continuously monitor these parameters over sequential two minute periods and
adjust the
values as deemed necessary to efficaciously mark motor responses within 95-100
2.5 accuracy. The data acquisition software output the frequency, amplitude,
area, time to peak
and duration of both contractile and relaxant motor respanses.
A one-way ANOVA with a Tukey multiple analysis was used for comparison
between mean values, using Statgraphics Plus 5.0 program. A probability of
less than 0.05
(p<0.05) was regarded to be significant. All values are expressed as mean ~
S.E.M. of
experiments.
All the drugs utilized in the in vivo and ex vivo experiments, including DON,
were
dissolved in physiological saline (0.9%). The infused concentrations
(delivered at a rate of
0.5 ml/min) were: a,~-methylene ATP (300 ~g/kg), L-NAME (10 mglkg),
granisetron (BO
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~g/kg), pentolinium (5 x 10-5 M) and hexamethonium ( L 8 mg/kg, s.c.). All
drugs were
purchased from Sigma Chemical Company, Toronto, ON; except for a,,/3-methylene
ATP and
methylthio ATP, which were obtained from RBI, Natick, MA; and DON was provided
by Dr.
Dave Miller, Agriculture Canada, Ottawa, ON, where it was biosynthetically
produced and
'_> purified according to the methods ofMiller and Arnison, 1986.
Spontaneous Motor Activity of the Crastrointestinal Tract
Stomach: Motor activity in the gastric antrum generally consisted of
oscillatory
contractions and relaxations. These were either present fbr the entire
duration of the control
recording or they occurred randomly. A motor pattern analogous to MMta was not
evident
in the stomach recordings. A summary of the spontaneous motor activity is
shown in Table
1.
Duodenum: Spontaneous motor activity consisted of an irregular pattern of
contractile and/or relaxant motor activity (Table l ). Occasionally, activity
reminiscent of
1'.s MMCs, consisting of phase III propagatory type activity ("grouped"
activity) and quiescent
periods, were evident. The duration of the "grouped" activity was
approximately S minutes,
however the cycle length could not be accurately determined, since the
"grouped" activity did
not arise more than 2 or 3 times during the control period, which only lasted
up to 2 hours in
our experiments. MMCs in fasted pigs are known to have a cycle length of 70-
115 minutes.
In our pigs, the "grouped" activity consisted of relatively high amplitude,
high frequency
relaxations and contractions: frequency of contractions: 11 9 ~ 0.5
events/min; amplitude of
contractions: 0.08 ~ 0.01 g; frequency of relaxations: 12.9 ~ 0.8 events/min;
amplitude of
relaxations: 0.07 ~ 0.01 g.
Ileum: The ileum usually exhibited random contractile and/or relaxant motor
activity
('Table 1). MMC-like activity was rarely observed In one third (n = 5) of the
pigs, motor
activity of the ileum was in a quiescent state; however in these experiments,
the ileum proved
to be responsive to DON treatment.
TABLE 1. Characteristics of spontaneous interdis~estive motor activities in
anesthetized pies
Parameters Stomach Duodenum Ileum
Amp. contraction0.16f0.04 0.07U.OI 0.050.01
Amp. relaxation0.190.03 0.071).01 0.050.01
Freq. contraction4.810.5 6.7-l).6 3.2+_0.5
Freq. relaxation5.50.3 6.81).5 3.60.5
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Values represent the mean~SEM of data obtained from 12 pigs.
Amp: Amplitude (grams tension); Freq: Frequency (eventslmin.); Dur:
Duration (seconds)-
DON was administered at 0.1 mg/kg (n = 3 ), 0.7 mg/kg (n = 2) and 1.0 mg/kg (n
=
10), via either an intravenous (i.v.) or infra-arterial (i.a.) route Within 5
minutes following
injection, DON (n = 6) decreased (p<0.05) the frequency and amplitude of the
spontaneous
contractile and relaxant motor responses. The duration of this DON induced
inhibition lasted
from 10 minutes up to an indefinite period of time. By contrast, in three
pigs, DON increased
(p<0.05) the frequency and amplitude of the spontaneous motor activity by 182
~ 40 % and
206 ~ 38 % respectively; this effect lasted up to 30 minutes before recovering
to the control
pattern. This differential action of DON was not obviously correlated to
either the dose
injected or the route of administration. Furthermore, neither the dose
injected nor the route of
administration induced any changes to the mean arterial blood pressure, which
continued a
1 i steady level for the duration of the control and DON treatment periods.
The effects of DON were more consistent in the duodenum (n -- 21 ), where it
always
potentiated (p<0.05) the spontaneous motor activity. A systemically
administered dose of
DON greater than or equal to 1 mg/kg consistently induced significant
potentiation; it also
represents the dose which increased the frequency, as well as the amplitude,
of duodenal
motor activity. The frequency of the DON induced hyperactivity typically
remained elevated
for the entire duration of the experiment, while the amplitude of the rnotor~
responses
progessively recovered to control levels.
Dose effects of DON on frequency and amplitude parameters of contractile and
relaxant motor activity were examined for dosages of 0.1 mg/kg, n = 3; 1.0
mg/kg, n = 12; 10
2S mg/kg, n = 3. A clear dose-response effect was evident only for the
amplitude of the motor
activity In addition, enhanced motor activity due to DO:N at 10 rng/kg was not
significantly
different from the effects of DON at 1 mglkg.
Systemically administered DON induced a dose-dependent increase in the ileal
motor
activity. DON was given at doses of 0.1 mg/kg (n = 3); 0. .' mgJkg (n = 3); l
.0 mg/kg (n =
12); and 10 mg/kg (n = 4). In the ileum, the dose-response effect was evident
in both
frequency and amplitude parameters of the motor activity. However, maximal
effects of
DON occurred at a dose equal to or greater than 1 mg/kg, where both the
frequency, as well
as the amplitude, of the contractile and relaxant spontaneous motor activity
were significantly
(p<0.05) increased. Thirty to sixty minutes following the DON enhanced
activity, the
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frequency and amplitude of the present motor activity started to decrease,
however, two hours
after the initial injection of DON, the motor activity was still significantly
higher than
control .
:p Effects of a.~ -methylene ATP against DON Induced Hyperactivity
a.,p-methylene ATP was always administered during the DON induced
hyperactivity.
This afforded an internal control for DON action. At the dose of 300 pg/kg,
a,~3-methylene
ATP, given intra-arterially, induced only a transient (less than 1 minute)
increase in the mean
arterial blood pressure.
Administration ofa,(~-methylene ATP (30t) pg/kg, i.a.) always induced an
initial
relaxant response in the stomach, however, it did not counteract the effects
of DON on gastric
motor activity.
Upon injection, a,/3-methylene ATP (175 ~gkg, i.a.) usually induced a small
phasic
relaxation of the duodenum. However, DON induced hyperactivity did not appear
to be
l:> affected. A higher dose of a,~-methylene ATP (300 wg/kg, i.a.) more
consistently induced
an initial phasic relaxation (0.5 ~ 0. I g, n = 10), with subsequent transient
(3-10 minutes)
reduction in the DON induced hyperactivity. a,(3-methylene ATP significantly
decreased the
amplitude, but not the frequene.y, ofthe DON induced relaxations and
contractions (see, Fig.
8). When a,~-methylene ATP (300 pg/kg, n = 3) was readministered 10-20 minutes
following the initial injection, the duodenum again relaxed. However, there
appeared to be
no further effect on the DON induced hyperactivity.
Analogous results were observed in the ileum, where a,~3-methylene A'TP (300
p,g/kg,
i.a.) induced a large phasic relaxation (1.2 ~ 0.2 g, n - 6~ upon injection,
and reduced DON
induced hyperactivity. 'The efficacy of a,(3-methylene ATP in reducing the
amplitude, as
2:5 well as the frequency, of DON induced relaxations and contractions in the
ileum is presented
in Figure 9. In three trials, a,~3-methylene ATP (3t)0 ~gikg) was
readministered within 10
minutes of the initial dose, to test for the development of tachyphylaxis.
'Che amplitude of the
relaxation to a,/3-methylene ATP was reduced by 68 ~ 18°'o compared to
the initial
administration of this agent.
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Example 3
Non-adrenergic, non-cholinergic (NANC) cantrol of interdigestive motor
activity in
the rat small intestine, in vivo.
This example provides a study of the neural pathways controlling
intcrdigestive motor
activity in different regions of the rat small intestine in viva The data in
this example, along
with the data in Examples 1 and 2, above, indicate that modulation of gut
motility is being
controlled by neural circuitry as shown in the schematic representations
ofFigures 2 and 10.
The migrating motor complex (MMC) is associated with interdigestive propulsion
of
1~0 intestinal contents, and like peristalsis, involves sequential activation
of excitatory and
inhibitory pathways. The neural circuitry underlying peristalsis comprises
excitatory
(primarily cholinergic) and inhibitory non-adrenergic, non-cholinergic (NANC)
motor
neurons innervating the gastrointestinal smooth muscle, as well as excitatory
and inhibitory
interneurones (Costa and Brookes, Am. Ciastroeriterol., 89: S129-5137 (1994)).
However,
little was previously known about the intramural neurons controlling
interdigestive motility,
mainly since MMCs are not easily assessed in vitr~a. Moreover, analysis of
MMCs in vivo
has for the most part focussed on the contractile activity only. In vivo
motility studies such as
those described herein revealed that propagatory intestinal motor activity,
characteristic of
MMCs, consist of contractions as well as relaxations
We determined the extent of cholinergic and 5-HT involvement, as well as the
role of
ATP, VIP and NO, in spontaneous motor activity of the duodenum and ileum.
Male Sprague-Dawley rats (250-350 g) were fasted for 24 hrs with free access
to
water. For surgery, anesthesia was induced with 2% Halothane in 500 ml/min
oxygen, and
body temperature was maintained constant at 37°C using a
thermostatically controlled heated
table and a thermal blanket. The right carotid artery was exposed and
cannulated to monitor
blood pressure via a pressure transducer (P23ID, Uould Statham, OH). The right
jugular vein
was cannulated for intravenous drug injections. An intra-arterial (i.a.) route
of drug
administration was often favored, due to the short half life of many drugs and
to evade
hepatic first-pass metabolism. For this, a cannula was inserted from the right
femoral artery
and fed in a retrograde direction to position the tip at the level of the
superior mesenteric
artery.
Animals were prepared for assessment of motility in viva as described above.
Foil
strain gauges were sequentially attached using Vet Bond glue onto the anti-
mesenteric border
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of the duodenum, 1-2 cm distal to the gastroduodenal junction, and lateral to
the anti-
mesenteric border of the ileum, just proximal to the ileocecal junction. In 6
rats, 2 or 3 foil
strain gauges were attached 2 cms apart to the proximal duodenum. From these
experiments,
we extrapolated the propagation velocity ofthe "grouped" activity. All
intestinal placed foil
strain gauges were oriented parallel to the longitudinal muscle layer since
this affords the
most sensitive setting for recording circumferential motor activity. Rats were
allowed to
recuperate from the surgery for one hour, then control motor activity was
recorded for
another hour before the administration of any drugs. All surgical and
experimental protocols
were carried out according to the Canadian Council on Animal Care guidelines
administered
1 ~~ by the Animal Care Committee at the University of Ottawa.
Data acquisition and statistical analysis was carried out as described above.
All the drugs were dissolved in 0.5 ml of physiological saline (0.9%). The
doses
(delivered within 1 minute) were: a,, (3-methylene ATP (300 pg/kg), methyl-S
ATP (360
pg/kg), N-w-vitro-L-arginine methyl ester (L-NAME, 10 mg/kg), vasoactive
intestinal
peptide (VIP, 4-10 Ilglkg), BRL 43694 (granisetron, 80 mglkg), atropine (4-6
mg/kg) and
hexamethonium ( 18 mg/kg s.c.), all purchased frorn Sigma, except for oc, ~3-
methylene ATP
and methyl-S ATP, which were obtained from RBI; granisetron which was a gift
from Dr.
R.K. Harding.
Region specific patterns of spontaneous motor activity were easily
characterized in
the rat small intestine. In the duodenum (n --= 8), this consisted of
reoccurring cycles of
propagating "grouped" and non-propogating "intergroup°' motor
activities, with a cycle length
of 5.4 ~ 0.4 min. "Grouped" activity was typified by an intense period
(approximately 2-4
min) of contractile and/or relaxant motor activity that propagated caudally at
a rate of 3.4 t
0.6 em~miri', in a manner reminiscent of MMCs. 'The "intergroup" activity
consisted of
randomly occurring, low amplitude, low frequency relaxations and/or
contractions.
Spontaneous motor activity of the ileum consisted of either relaxations (50%
of all
animals tested) or contractions only (30% of all animals tested); in the
remaining
experiments, contractile and relaxant motor activity occurred together. The
predominance of
one type of motor response (contraction vs. relaxation) is thought to be
indicative of the
intrinsic tone of the smooth muscle; where tissue with high tone shows mainly
relaxant
activity, whilst tissue with low tone more readily shows contractions.
Generally, the
spontaneous ileal relaxations and contractions occurred at a relatively low
frequency, and
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were of relatively high amplitude. In 10% of the experiments, the ileum
displayed periodic
bursts of high frequency motor responses comparable to phase III MMC activity.
The substituted derivatives of ATP, a, ~3-methylene ATP and methyl-S ATP, have
differential at3inities for PZx- and PZY-purinoceptors, respectively
(Burnstock and Kennedy,
Gen. Pharmacol., 16: 433-440 (1985)). Tissues develop tachyphylaxis following
prolonged
exposure to these agents, and thus in this manner it was possible to
discriminate between Pzx
and PzY receptor-mediated responses. Upon injection, a,(3-methylene A'IP (300
pg/kg, i.a )
evoked a phasic relaxation in the duodenum {1.0 t 0. t g, n = S);
subsequently, it selectively
attenuated (p<0.05) the frequency and amplitude of "grouped" relaxations by 73
~ 7% and 48
~ 5%, respectively. The effects of a,(3-methylene ATP on spontaneous duodenal
contractions were variable and could not be analyzed.
In the ileum, a,~i-methylene ATP (300 pglkg, i.a., n = 8) evoked an initial
phasic
relaxation. Proximate rechallenge with a,(3-methylene ATP did not elicit
another response,
indicative of the development oftachyphylaxis. During this period, spontaneous
ileal
I 5 relaxations were (p<0.05, n = 8) attenuated for up to 30 minutes.
Spontaneous contractions
were not affected by a,~i-methylene ATP treatment. Methyl-S ATP (360 ug/kg,
i.a., n = 4)
also attenuated (p<0.05) ileal relaxations, however, methyl-S ATP did not
evoke an initial
phasic relaxation.
L-NAME ( 10 mg/kg, i.v., n = 8), selectively attenuated the frequency and
amplitude
of spontaneous "intergroup" relaxations ofthe duodenum 44 ~_ 8% and 66 ~ 1%
respectively.
In the ileum, L-NAME potentiated both the contractile (n = 6j and relaxant (n
= 8) motor
activity. This effect often persisted for the entire duration of the
experiment The relaxations
potentiated by L-NAME were attenuated {p<0.05, n = 4-6) by either a,(3-
methylene ATP (by
59 t 12%) or methyl-S-ATP treatment (by 70 ~ 3°,%).
Spontaneous contractions and relaxations of "grouped" and "intergroup" motor
activity, as well as motor activity of the ileum, were all attenuated (p<0.05,
n = 4) for up to
20 minutes by the nicotinic receptor antagonist hexamethonium. L-NAME-enhanced
activity
was also attenuated (p< 0.05, n ---- 6) by hexamethonium. Atropine (4-6 mg/kg,
i.a., n = 4),
attenuated the spontaneous ileal contractions by 87 ~ 3% and 89 t 7%
respectively.
Duodenal contractions were similarly affected.
VII' (4-10 pg/kg, i.a.) evoked a phasic relaxation (n = 8) in the duodenum.
Subsequently, VIP transiently inhibited (p<0.05) duodenal "intergroup" motor
activity, and
CA 02374358 2002-O1-04
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potentiated (p<0.05) the "grouped" activity. In the ileum, VIP consistently
evoked only a
slow contraction which recovered to control level within 6 minutes.
Concomitant with this
contraction, spontaneous (n = 4) and L-NAME-enhanced (n = 6) relaxations were
attenuated
(p<0.05) for up to 8 minutes. The frequency and amplitude of the spontaneous
relaxations
were reduced to 33 t 8% and 21 t 5°ro of control, respectively. The
frequency and amplitude
of the L-NAME induced relaxations were reduced to 32112°~a and 14 ~ 3%
of control,
respectively.
Within 5 minutes, granisetron (80 lrg~kg', i.v. or i.a.) attenuated (p<0.05)
spontaneous
duodenal "grouped" relaxations (n = 9) and contractions (n = 4), but did not
affect the
"intergroup" motor activity. The "grouped" motor activity was reduced for up
to 40 minutes;
subsequently, the control pattern of interdigestive motility progressively
recovered.
Granisetron treatment also attenuated (p<0.05, n - 4) the spontaneous ileal
contractions and
relaxations. The interdigestive motor pattern of the ileum progressively
recovered to control
levels within approximately 6U minutes. The amplitude of the L-NAME enhanced
ileal
motor activity was also attenuated (p <0 05, n -- 6) by 76 t 8%, in the
presence of granisetron.
Grouped relaxations were sensitive to a,~3-methylene ATP treatment, while
"intergroup" relaxations were inhibited in the presence of the NO-synthase
inhibitor
L-NAME. By contrast, our results show that NO is not the mediator of
spontaneous deal
relaxations. Others have shown that in isolated rat ileal preparations,
application of ATP
evokes relaxations and AT'P-desensitization reduces these relaxations (Smits
et al., Rr. J.
1'harmacnl., I iR: 695-703 ( 1996)). In this study, systemic injection of the
PZx-purinoceptor
agonist a,/3-methylene ATP, induced an initial relaxation ofthe ileum. A
proximate re-
administration ofa,(3-methylene ATP did not induce a response, indicative of
the
development of tachyphytaxis. Concomitant with the induced tachyphylaxis,
spontaneous
ileal relaxations were inhibited. Hence, ATP, via PZx-sites, is the
transmitter mediating
spontaneous NANC relaxations in the rat ileum.
ATP exhibits multiple enteric neural functions, since in addition to mediating
P2x-
purinoceptor dependent relaxations in the duodenum and ileum, ATP via P2~,~-
purinoceptors
can stimulate NO-mediated non-propagating "intergroup~" relaxations in the
duodenum
(Glasgow et al., Am. J. Physinl, 27h (Ga.slrointest. Liver Physiol., 38): G$89-
6896 (1998)).
In the present example, the PzY-purinoceptor agonist, methyl-S-A'rP, inhibited
spontaneous
ileal relaxations. However, in contrast to a,~3-metitylene ATP, methyl-S-ATP
did not evoke
an deal relaxation upon injection. This indicates that P2.~-purinoceptors are
not present on the
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smooth muscle, or else are not active within the inhibitory motor
innervation(s) of the ileum.
T'he data support the view that in the rat ileum, PZY~-purinoceptors are
involved in the
activation of pathways mediating tonic inhibition of the purinergic NANC motor
neurons
targeting PZx purinoceptors. PZV-purinoceptors may be present on nitrergic
interneurones
subserving tonic inhibition, or on other interneurones within this
prejunctional input. The
nitrergic and purinergic interneurones may also represent the same population,
since ATP and
NO synthase are co-localized in myenteric neurons in the rat ileum (Belai and
Aurnstock,
Cell Tiss. Res., 278: 197-200 (1994)).
VIP is a NANC inhibitory transmitter in numerous gut regions (Bojo et al.,
fur. J.
Pharmacol., 236: 443-448 (1993); Mule et al., J. Az~torr. Pluzrmacol., 12: 81-
88 (i992). In these
experiments, VIP evoked a transient relaxation in the rat duodenum. 'the
subsequent
development oftachyphylaxis to VIP inhibited the contractile and relaxant
"intergroup" activity
and enhanced the "grouped" motor activity. The data indicate that the initial
VIP-evoked
relaxations are dependent on NO and sensitive to VIP desensitization. Thus,
VII'ergic
interneurones must be targeting direct motor innervations (the nitrergic and
cholinergic motor
neurons) ofthe "intergroup" activity, as well as the nitrergic prejunctional
modulatory inputs of
the "grouped" activity.
In these experiments, treatment with VIP inhibited the spontaneous relaxations
of the
ileum. Since VIP did not evoke a relaxation upon injection, it is unlikely
that VIPergic
neurons mediate direct inhibitory input to the ileal smooth muscle (Smits et
al., Br. J.
I'harmacol., I18: 695-703 ( 1996)). Irt vivo experiments in the canine ileum,
suggest that VIP
plays a major role in the tonic inhibition of circular muscle motor activity
via an inhibitory
neural action (Fox-Threlkeld et al., Peptides, l2: 1039-1045 ( 1991 )). The
data support the
view that VIP targets the NO-dependent prejunctional modulation of the
purinergic inhibitory
motor innervations in both the duodenum and ileum. These purinergic motor
pathways
specifically generate the propagatory motor activity of the small intestine.
Furthermore, VIP
specifically inhibits phase III activity of MMCs, and VII' antagonists
initiate phase III
activity (Hellstrorn and Ljung, Neurogastroenterol. Motil., 8: 299-306 (1996).
1'he
conclusion is that VIP simultaneously stimulates excitatory motor inputs and
inhibitory
nitrergic prejunctional inputs of the purinergic motor neurons in the rat
ileum.
All components of the interdigestive motor complex are dependent upon vago-
sympathetic integrity (Chung et al., Am. J. Physio. , 267: 6800-6809 ( 1994);
Galligan et al.,
J. Pharmacol. Exp. Therap., 238: 1 I 14-1125 (1986)). The cholinergic
interneurones
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controlling gastrointestinal motility, act via nicotinic synapses, whereas ACh
(acetyl choline)
actions on smooth muscle are via muscarinic receptors. Atropine inhibited al)
spontaneous
contractions of the small intestine. The results from this study indicate that
all of the
spontaneous interdigestive motor activity of the duodenum and ileum is
sonically driven and
is sensitive to nicotinic receptor blockade.
In vitro studies show neuronally derived 5-HT stimulates purinergic NANC
relaxations (Briefer et al., Naunyn-Schmiedebergs Arch. Pharamacol., 3.~1 v
126-135 (1995);
Briefer et al., J. Pharmacol. Exp. Tlrerap., 279: 641-648 (1995)), and
cholinergic contractions
(Briefer et al., Eur. J. P'harmacnl., 308: 173-180 (1996)). The in viva
results in this study
show that 5-HT3 receptors are involved within the motor pathways mediating the
spontaneous
cholinergic contractions and purinergic relaxations in the duodenum and ileum.
An arrangement of cholinergic, nitrergic, (:~ABAergic, purinergic and
V'IPergic neural
elements within the proposed tonic and modulatory pathways controlling
spontaneous motor
activity in the rat duodenum and ileum is presented schematically in the
simplified wiring
diagram of Figure 10, which also shows key locations for P2~ and PZY receptors
in the
pathway. Stereotypic motility patterns are elicited when driver circuits
activate excitatory
and inhibitory motor pathways, as determined by "enteric neural programs".
However, a
continuous drive from inhibitory interneurones maintains myogenic activity
quiescent. This
coordinated inhibition and disinhibition is mediated by the inhibitory
nitrergic inputs, and it is
precisely the control of these prejunctional neuronal pathways, along with the
topically active
motor pathways, which generates cyclical (interdigestive) motility patterns
upon an existing
baseline of motor activity-. The GABAergic/nitrergic combination pathway
circuit is not
present in the ileum.
Example 4
Effects of DON and DON-based derivatives on spontaneous gastrointestinal motor
activity.
This example demonstrates the ability ofDON-based derivatives to induce the
fed
pattern of gut motility in a manner similar to DON.
DON derivatives were selected for a comparative study with DON in rats using
methods described in the previous examples for testing DUN and recording its
effect on gut
motility. One of the representative DON derivatives was 3-acetyl DON
(C,7H2207). Other
new DON-based derivatives were also selected for study: isopropylidine DON
(i.e., 3-
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hydroxy-7,15-isopropylidine-12,13-epoxy-9-tricothecin-8-one, having the
formula C,gHZaO~,
designated EN139491); isopropylidine-3-acetyl-DON (i.e_., 3-acetyoxy-7,15-
isopropylidine-
12,13-epoxy-9-tricothecin-8-one, having the formula C2oHz~07, designated
EN139492); DUN
carbonate (i.e., 3-hydroxy-12,13-epoxy-9-tricothecin-8-one-7,15 carbonate,
having the
formula C,6Hix07> designated EN 139494); 3-acetyl-DON carbonate (i.e., 3-
acetoxy-12,13-
epoxy-9-tricothecin-8-one-7,15 carbonate, having the formula C,gHzoUs,
designated
ENI39495); 3-acetyl-DON benzylidene acetal (i.e., 3-acetoxy-7,15-benzylidene-
12,13-
epoxy-9-tricothecin-8-one, having the formula Cz4H2~OR, designated EN 139496);
and DON-
benzylidene acetal (i.e., 3-hydroxy-7,15-benzylidene-12,13-epoxy-9-tricothecin-
8-one,
having the formula CZZH1a07, designated EN 139497). T'he chemical structures
of DON and
these representative DON derivatives are shown irr Figures 3A and 3B.
~nthesis of Novel DON Derivatives
Isopropylidine DON (EN 13949I):
To a solution of SO mg (0 168 mmol) of deoxynivalenol (DON) and 70 mg of 2,2-
dimethoxypropane in 2.0 ml of anyhdrous acetone at 0°<". was added
approximately 1 mg of
p-toluenesulfonic acid. The reaction mixture was stirred and allowed to warm
to room
temperature. The progress of the reaction was monitored by thin layer
chromatogaphy
(TLC), and the reaction was judged to be complete after 5 hours. The solvent
was
evaporated, and the crude product was partitioned between 2 rnl of water and S
ml of ethyl
acetate. The organic layer was washed with 2 ml c~f saturated NaHC03, followed
by 2 ml of
saturated brine, and then dried with anhydrous magnesium sulfate. The solvent
was
evaporated, and the residue was chromatographed on silica gel using a 6.4
mixture of ethyl
acetate-hexane mixture. This yielded 38 mg (67%) of a white solid.
~H NMR analysis (CDCl3, 200 MHz): 8: 6.81 (m, 1H), 4.82 (m, IH), 4.45 (s, 1H),
4.40 (m,1H), 3.85 (bs, 2H), 3.61 (d, J = 8.0 Hz, IH), 2.91 (d, .1 -= 8.0 Hz,
1H), 1.91-2.10 (m,
2H), 1.99 (s, 3H), 1.49 (s, 3H), I .26 (s, 3H), 0.99 ( ,3H) 'fhe peak for the
OH group could
not be located in this spectrum.
Mass spectroscopy data confirmed the structure of isopropyl idine DON (EN
139491 )
as shown in Figure 3A.
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Isopropylidine-3-acetyl-DON (EN139492):
This compound was prepared in 62% yield starting with 70 mg of 3-acetyl-DON
and
87 mg of 2,2-dimethoxypropane. The product was obtained as a white solid after
silica gel
chromatography using 1:5 ethyl acetate-hexane mixture as eluent.
S IH NMR analysis (acetone-d6, 200 MHz): b 6.6'7 (d, J = 8.0 Hz, IH), 5.09 (m,
1H),
4.81 (m, 2H), 3.85 (d, J = 8.0 Hz, 1H), 3.51 (bs, 2H), 3.12 {m, 2H), 2.61 (dd,
J = 8.0 Hz, 16.0
Hz, 1H), 2.01 (s, 3H), 1.99 (dd, J - 8.0 Hz, 1H, 16 0 Hz), 1.82 (s, 3H), 1.'?5
{s, 6H), 1.15 (s,
3H).
Mass spectroscopy data confirmed the strucrture of isopropylidine-3-acetyl-DON
(EN139492) as shown in Figure 3A.
DON-7,15-carbonate (EN 139494) v
Triphosgene (5 mg, 0.016 mmol) in 1 ml of CHzCl2 was added, dropwise to a
solution
of 10 mg (0.033 mmol) of DON and 0.015 ml of pyridine in 1 ml of anhydrous
dichloromethane at -78 c'C. The reaction mixture was warmed to room
temperature and
stirred for another 6 hours. 'Che solvent and remaining volatile reagents were
evaporated, and
residue was purified by silica gel column chromatography using ethyl acetate
as eluent. The
yield of DON-7,15-carbonate, a white solid, was 10 mg (99 %).
1H NMR analysis (acetone-d6, 200 MHz): b 6.71 (d, .I =- 8.0 Hz, IH), 5 49 (s,
1H),
4.81 (d, J = 8.0 Hz, 1 H), 4. 51 (m, 3H), 4.31 (d, J = 16.0 H-r_, 1 H), 3 51
(d, J = 4.0 Hz, I H),
3.21 (m, 2H), I .90 2.21 (m, 2H), I .86 (s, 3H), I .01 (s, 31-I).
Mass spectroscopy data confirmed the structure of DON-7;15-carbonate
(EN139494)
as shown in Figure 3B.
3-acetyl-DON-7,15-carbonate (EN-139495):
This compound was prepared in 99% yield starting with 20 mg of 3-acetyl-DON,
0.023 ml of pyridine, and 10 mg oftriphosgene. 'fhe product was obtained as a
white solid
after silica gel chromatography using 7:3 ethyl acetate-hexane mixture as
eluent.
'H NMR analysis (CDCl3, 200 MHz): &: 6 61 (d.. J = 8 0 Hz, IH), 5.36 {m, 1H),
5.29
3 0 (s, 1 H), 4.49 (d, J = 8.0 Hz, l I-i), 4.41 (d, J = 16.0 Hz, 1 H), 4. I 9
(d, J = 16.0 Hz, 1H), 3 . 95 (d,
J = 4.0 Hz, 1H), 3.20 (m, 2H), 2.39(s, 1H), 2.12 (s, 31-1], 1.92 (m, 1H), 1.92
(s, 3H), 1.12 (s,
3H).
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Mass spectroscopy data confirmed the structure of 3-acetyl-DON-7,15-carbonate
(EN139495) as shown in Figure 3B.
7,15-benzylidene-3-acetyl DON acetal (EN-139496):
This compound was prepared in 95 % yield starting with 20 mg of 3-acetyl-DON
and
13 mg of benzaldehyde dimethyl acetal. 'hhe product was obtained as a white
solid after
silica gel chromatography using 4:6 ethyl acetate-hexane; mixture as eluent
'H-NMR analysis (CDCl3, 200 MHz): 8: ~ 45 (nt, 5H}, 6.81(d, J = 8.0 Nz, 1 H),
5.39
(s, 1 H), 5.10 (m, 1 H), 4.90 ( s, 1 H), 4.3 5 (d, J = 8, 0 Hz, LH), ~'1. 31
(d, J = 16.0 Hz, 1 H), 3 . 81
(d, J - 16.0 Hz), 3.81(d, J -- 16 Hz> 1H), 3.21 (m, 2H), 2,20-2 45 (m, 2H),
2.01(s, 3I-~, 1.91(s,
3H), 1.31(s, 3H).
Mass spectroscopy data confirmed the structure of isopropylidine-3-acetyl-DON
(EN139496) as shown in Figure 3B.
7,15-benzylidene-DON acetal (EN 139497}:
To a solution of 25 mg (0.084 mmol) DON and 20 mg (0.126 mmol) benzaldehyde
dimethyl acetal (20 mg, 0.126 mmol) in 2 ml of anhydrous acetonitrile .was
added
approximately 1 mg of p-toluenesulfonic acid. T'he reaction mixture was
stirred at room
temperature for 2 hours, and the solvent was evapurated 1'he crude residue was
taken in
ethyl acetate (5 ml) and was washed with saturated sodium bicarbonate solution
(2 ml),
followed by water (2 ml). The organic layer was separated, dried over
anhydrous MgSO.~,
and concentrated to give a crude product, which was purified by column
chromatography
(7:3, ethyl acetate-hexane) to afford 27 mg, (87 %) of the title compound as a
white solid.
'H NMR (Acetone-d6, 200 MHz) analysis fi: 7.45 (m, SI-1~, 6.75 (d, J °
8.0 Hz, 1H),
2 5 5.3 5 (s, 1 H), 4.95 (s, 1 H), 4. 51 (d, J = 8.0 Hz, l I-I), 4.49 (m, 1
H), 4.25 (d> J = l 6. 0 Hz, 1H),
3.85 (d,1= 16.0 Hz, 1H), 3.45 (d, J -= 5.0 Hz, 1H), 3.11 (m, 2H), 2.'10 (m,
2H), 1_85 (s, 3H),
1.25 (s, 3H) The peak for the OH group could not be located in this spectrum.
Mass spectroscopy data confirmed the structure of isopropylidine-3-acetyl-DON
(EN 139497) as shown in Figure 3B.
DON administered systemically (10 mg/kg, i.v.)> induced a typical fed pattern
of gut
motor activity in the rat gastroduodenum (see, Figure 11 ). DON abruptly
attenuated the
gastric antral (site S1) motor activity and induced a sustained hyperactivity
in the duodenum
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(proximal duodenal site D1). Within 60 minutes, the control motor pattern
recovered.
Occasionally, tricothecene-induced hyperactivity was characterized by an
initial high
frequency motor activity where the amplitude although larger than
"intergroup'" responses
was smaller than the MMC motor activity. This is shown in Figure 1 l, where
this initial
period lasted 3-10 minutes; thereafter the amplitude increased to MMC levels.
3-Acetyl-DON ( 10 mg/kg of body weight (bw), i.v.) induced a typical fed
pattern
motor activity in the rat gastroduodenum (n = 4) Figure 12 shows the time to
onset of action
( 1 min) and duration (40 t- 4 min) of effects were similar to those for DON.
Figure 13 shows
the effects of intravenously administered 3-acetyl DON on spontaneous motor
activity in the
rat gastric antrum (S 1 ) and proximal duodenum (D2). Within 60 minutes, the
control motor
pattern recovered.
Within 30 seconds of intravenous inj ection of EN 13949 l ( 10 mg/kg bw),
there
developed a long lasting (40 ~ 1.75 min, n -- 6) hyperactivity in the duodrnum
and a
simultaneous and parallel attenuation of motor a~~tivity in the gastric
antrum, typical of the
effect seen with DON. Figure 14 shows typical in vivo recordings of the motor
activity in the
rat duodenum (D 1 ) and gastric antrum (S I ) illustrating the action of the
compound
EN139491 on the fasting pattern of gut motor activity. The top panel of the
recording shows
minutes of normal fasting pattern motor activity without any drug treatment.
During this
period, the duodenum displayed a typical pattern of tow frequency spontaneous
motor
20 activity together with propagating motor activity (MMC). The gastric antrum
displayed a
typically rhythmic motor activity. The second panel of the recording shows
activity at the
time of injection with EN139491. Within 30 seconds of injection, a long
lasting (40-60 min)
hyperactivity in the duodenum and a simultaneous and parallel attenuation of
motor activity
in the gastric antrum developed. This EN139491 induced motor activity was
typical of fed
pattern motor activity. Recovery of fasting pattern motor activity is shown in
the bottom
panel of the recording in Figure 14. Figure 15 shows the recording of the
effects that
EN139491 had on duodenal motor activity recorded at D2 (i.e., 1.5 cm distal to
the D1 strain
gauge). The recording of the induction and duration of fed pattern al D2 by
EN139491 as
shown in Figure l5 was similar to the results recorded at duodenal site D1 as
shown in Figure
14.
A closer analysis of the features of the individual relaxation and contraction
components of the fed pattern of gut motor activity induced by EN139491 was
also made. In
this analysis, the amplitude and the frequency of the relaxation or
contraction component of
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MMCs and ofthe fed pattern induced by EN139491 at duadenal site D1 were
expressed as a
percent of the respective frequency and amplitude of the relaxation or
contraction component
observed in the normal "intergroup" activity of the fasting pattern, which
serves as an internal
control for each animal. The results of the analysis indicated that both
amplitude (Figure 16)
and frequency (Figure 17) of the relaxation component of the fed pattern
induced by
EN139491 were comparable to those seen in the spontaneous "grouped" MMC
activity of the
gut. Likewise, both amplitude (Figure 18) and frequency (Figure 19) of the
contraction
component of the fed pattern induced by EN139491 were comparable to those seen
in the
spontaneous "grouped" MMC activity afthe gut. These results indicated that the
fed pattern
induced by EN139491 had the same features as the fed pattern induced by DON.
As in the case EN139491, within 30 seconds of intravenous injection ofthe DON
derivative EN 139492 ( 10 mg/kg bw}, a long lasting (48. 5 _t 2 min, n = 6;1
hyperactivity was
induced in duodenum sites D 1 and D2 and a simultaneous and parallel
attenuation of motor
activity in gastric antrum site S 1 occurred. An example of these effects on
in vivo gut motor
activity is presented in Figure 20. Figure 20 shows a typical W vivo recording
of the motor
activity at the rat duodenum D1 and D2 sites and the gastric antrum S1 site
illustrating the
action of EN 139492 on the fasting pattern of gut motor activity. The top
panel of the
recording shows more than 40 minutes ofnormal tasting pattern motor activity
in the absence
of DON or a DON derivative. During this period, the duodenum displayed a
typical pattern
of low frequency spontaneous motor "intergroup" activity together with
propagating
"grouped" motor activity (1.e., "MMC"). The gastric antrum displayed a
typically rhythmic
motor activity. Within 30 seconds of injection, a long lasting hyperactivity
in the duodenum
was initiated and a simultaneous and parallel attenuation of motor activity in
the gastric
antrum developed.
A closer analysis of the fed pattern of gut motor activity induced by ENl
39492,
revealed that the frequency and amplitude of relaxation and contraction
components were at
least comparable to the frequency and amplitude of relaxation and contraction
components of
the MMC activity ofthe gut (data not shown). Thus, as with EN139491, the DON
derivative
EN139492 is able to induce a fed pattern of gut motor ac.-tivity that is
comparable to the fed
pattern induced by DON, the structural parent of EN 139491 and EN139492
By the same methods employed to study EN139491 and EN139492 above, the DON
derivative compounds referred to as DON carbonate (EN 139494) and 3-acetyl DON
carbonate (EN 139495) (see, Figure 3B} were also tested and shown to be able
to induce the
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fed pattern of gut motor activity at levels that are at lease; comparable to
those induced by the
structural parent DON. Intravenous injection of the tricothecene based
derivative ENI39495
(10 mg~kg-r) induced a typical in vivo fed pattern motor activity in the gut
recorded from the
proximal duodenum (D~) and gastric antrum (Si) of halothane-anaesthetized male
Sprague
Dawley rats, n - 4. The effect of EN139495 was evident within 40 seconds of
injection, and
the duration of action was 40-60 minutes. At S,, contraction amplitude and
frequency were
reduced to 59 ~ 8.7% and 64.25 t 12.0% SEM of control motor activity,
respectively. The
gastric antral relaxation amplitude and frequency were also reduced to 28.4 t
3.4% and 48.0
~ 10.5% SEM, respectively. In the intestine (Dr), there was a profound
hyperactivity
contraction amplitude and frequency were increased to 1 19.0 ~ 12.0% and 1598
8 ~ 421.9%
SEM of control motor activity, respectively. Relaxation amplitude and
frequency were also
increased to 331.0 ~ 39.8% and 724.4 ~ 180.75% SEM, respectively.
In approximately 20% of experiments, the 3-acetyl-DON and EN 139491 induced
hyperactivity did not show an immediate large amplitude activity. An example
of this is
shown in Figure I 5. Although there was an initial high frequency motor
activity, the large
amplitude motor activity was delayed, replaced by responses larger than
"intergroup"
responses but smaller than the MMC motor activity. When this "delay" in
amplitude
occurred, it usually lasted 3-10 minutes. Thereafter, the amplitude of the
induced
hyperactivity increased to MMC levels as shown in Figures 16-19. The initial
differential
action of these DON derivative compounds did not affeca the duration of the
induced fed
pattern of gut motor activity.
A proximate reapplication of DON or its derivatives within 90 minutes of the
first
injection was typically without effect. After 120 minutes, DON and its
derivatives were
again effective and could induce the fed pattern of gut motor activity.
The foregoing results demonstrate the effectiveness of DON and a familiar
derivative
of DON, 3-acetylated DON ( which is reported to have the lowest toxicity of
all the
tr-icothecenes), to induce a fed pattern of gut motor activity was tested. In
addition, two new
DON-based derivatives (EN139491 and EN139492) were synthesized and shown to be
capable of inducing a fed pattern of gut motor activity in a manner at least
comparable to
DON. All derivatives (tested intravenously as a single 1 ml bolus at a dose of
10 mg~kg'r)
displayed a similar profile of action to that of a single bolus i.v. injection
of DON: within 1
minute of intravenous injection in anesthetized Sprague Dawley rats, the
spontaneous fasting
motor pattern of the gastroduodenum changed to a typical fed pattern motor
activity. In the
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gastric antrum, oscillatory motor activity was replaced with a quiescent
pattern; and in the
duodenum, DON induced a sustained hyperactivity in place ofthe cyclic
"grouped" MMC
pattern. This effect lasted 40-60 minutes, and the spontaneous motor activity
then recovered
to a fasting pattern of motor activity. Neither DON nor its derivatives caused
any discernible
e$'ects on blood pressure, heart rate, or respiratory rate.
Example 5
Effects of the selective Pzx~-zx3 purinoceptor antagonist 2',3'-O-(2,4,6-
trinitrophenyl)
adenosine triphosphate (7T1P-ATP).
This example demonstrates the direct involvement of the Pzx, purinoceptor
present on
the smooth muscle of gut tissue in regulating gut motor activity and the
ability of the P2x,-zx3
purinoceptor antagonist TNP-ATP to block the Pzxt purinoceptor and, thereby,
to inhibit the
fed pattern of gut motor activity induced by DON or DON-based derivatives.
In previous examples, the pharmacology of the intrinsic motor inhibitory
innervation
of the rat and porcine gastroduodenum was characterized using specific nitric
oxide (NO)
synthesis inhibitors and inhibitors of purinoceptor-mediated responses, such
as the general Pz
receptor antagonist, suramin, and the general PZX agonise, a,~i-methylene ATP,
and the PzY-
agonise, methyl-thiol-ATP. Proximate re-challenge for each of these
purinoceptor agonists
was marked by a profound tissue taehyphylaxis, which was exploited to block
the respective
purinoceptor. The results showed relaxations within the patterned spontaneous
motor activity
of the rat proximal duodenum were differentially dependent upon either NO or
ATP:
duodenal "grouped" MMC relaxations were sensitive to a,~3-rnethylene ATP
treatment, whilst
"intergroup" relaxations were inhibited by treatment with the NO synthesis
inhibitor
L-NAME. In addition, the data showed that NO was not the mediator of
spontaneous ileal
relaxations. These ileal relaxations were dependent upon ATP acting via P2~-
purinoceptors
similar to the MMC related relaxations.
The previous examples also showed that interference of either motor component
effectively prevented UON-induced fed pattern of motor activity. In this
example, we further
investigate the hypothesis that the relaxations occuring within the DON-
induced fed pattern
motor activity in the intestine are mediated by the PZxi purinoceptor subtype.
This study
employed a new purinoceptor antagonist, TNP-ATP (Lewis et al., Rr. J.
1'harmacol., 124:
1463-1466 (1998)) which has been used in vitro as a subtype selective
antagonist (whole
tissue ICso in wM range) to determine the role of Pzxl and .P2~3 homorneric
and Pzx2is
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heteromeric purinoceptors (Virginio et al., Mol. Pharmacol., .53: 969-973
(1998)). PZ~
receptors are reported to be expressed only on sensory neurones (Evans and
Suprenant,
femin, Neurosci., 8: 217-223 (1996)). 'Chis study represents the first use of
the PZx,-selective
antagonist TNP-ATP in vivo. The aim was to determine the role ofPzx~ receptors
in control
of patterned gastrointestinal motor behaviour and test directly the hypothesis
that the Pzxi
purinoceptor subtype mediates DON-induced hyperactivity in the gut.
Four doses of TNP-ATP were tested in the irr vivo rat model as described
above.
Sprague Dawley rats were continuously monitored for blood pressure,
respiratory rate, pallor,
and general well being. 'fNP-ATP had no discernible effects on these
parameters throughout
an experiment, which often lasted up to 6hrs.
TNP-ATP had no effect on spontaneous gastric motor activity (data not shown).
By
contrast, TNP-ATP injected intravenously as a single bolus significantly and
specifically
affected spontaneous duodena! relaxations. At 2.S mg/kg, the actions of 'I'NP-
ATP were
barely observable. By contrast, 4.5 and 5 mglkg appeared to be supramaximal
doses and the
effectiveness inconsistent. Occasionally, there were also some non-specittc
actions on gut
motor activity at these higher doses.
TNP-ATP adminstered at 3.5 mg/kg was found to be reproducibly effective and
specific in its actions. This was chosen for subsequent evaluation in the
model. 'typical in
vivo recordings showing the effects of intravenously injected TNP-ATP {3.5
mg~kg'1) on
spontaneous motor activity in the rat duodenum (at duodenal site Dl) is shown
in Figure 21.
TNP-ATP did not evoke any response upon injection. 1-lowever, within 1 minute
of injection,
MMC related relaxations were reduced. "Intergroup" motor activity was not
significantly
affected. 'There was recovery of M1V1C related relaxant motor activity to
within 90% of
control level within 30 minutes of TNP-ATP injection.
In vivo recordings showing the etTects of intravenously injected TNP-ATP (3.5
mg~kg'') on DON-induced fed pattern motor activity in the rat stomach and
duodenum are
presented in Figures 22 (recording duodenal site D 1) and 23 (recording
duodenal site D2 and
gastric antral site S1). Consistent with all other experiments, TNP-ATP did
not evoke any
response upon injection. However, within 1 minute of injection, the effects of
DON (10
mg~kg'~, i.v:) were significantly reduced. This inhibitory action of TNP-ATP
consisted of an
initial profound effect (up to 80% inhibition) lasting approximately 5
minutes, followed by a
longer period of less profound (up to 40% inhibition), but significant
antagonism of DON
actions.
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The ability of TNP-ATP to counter DON actions is shown graphically in Figures
24-
27. The bar graphs of Figures 24-27 show the effects of intravenous treatment
with TNP-
ATP on DON-induced relaxations and contractions recorded t) om the proximal
duodenum
(D1). The effects ofTNP-ATP on the amplitude (Figure 24) and frequency (Figure
25) of
DON-induced relaxations were compared to the amplitude and frequency of the
relaxation
component of "grouped" MMC and the control "intergroup" motor activity, which
was set as
100%. Likewise, the ef~'ects of TNP-ATP on the amplitude (Figure 26) and
frequency
(Figure 27) ofDON-induced contractions were compared to tree amplitude and
frequency of
the contraction component of "grouped" MMC and the control "intergroup" motor
activity.
A consistent feature of the TNP-ATP effect was the specific attenuation of the
amplitude, but
not the frequency, of DON-induced duodenal hyperactivity (compare, open bars
with
checkered bars in Figures 24-27). The effect of TNP-ATP on the frequency of
the relaxation
component of DON-induced hyperactivity in the duodenum was evident within 2U
seconds of
administration of TNP-ATP, and maximal attenuation of the iiequency of the
relaxation
component was attained within 2 minutes. The DON-evoked hyperactivity resumed
within
35 minutes of TNP-ATP administration and recovered to 90% of the pre-'TNP-ATP
administration level.
The effectiveness ofthe selective PZx, purinoceptor antagonist TNP-ATP was
evaluated. An intravenous injection of a single bolus of this antagonist
rapidly and
specifically attenuated MN(C related relaxations; indicative of the
involvement of PZx~
purinoceptors in gut motor activity. Intravenous injection with a single bolus
injection of
TNP-A.TP reduced (transiently) the DON-induced fed pattern in a dose-dependent
manner.
These results confirm the trend of the data employing a general P2x receptor
antagonist. PZx-
purinergic sites represent a highly restricted component of enteric pathways
The results
2.5 clearly show that the P2xi receptor subtype mediates intrinsic purinergic
inhibitory
innervation of the duodenum and that blocking these receptors may represent a
simple
approach for counteracting DON effects in the gut Furthermore, the data
support the notion
that these receptor sites are potential targets for developing agents to
modify feeding
behavior.
All publications cited in the text are incorporated herein by reference.
