Note: Descriptions are shown in the official language in which they were submitted.
1 309662
This invention relates to non-steroidal anti-inflammatory
drug (hereinafter referred to as NSAID) compositions containing
protectants against gastrointestinal injury induced by such
NSAIDs and to processes for administering such composition. More
particularly it concerns compositions and processes of the
aforesaid type that employ beta adrenergic agonists as the
protectants. The compositions of this invention are useful in
treating conditions and symptoms that are classically treated by
the administration of NSAIDs e.g. headache pain, pain and
inflammation associated with arthritis and other systemic
diseases, elevated body temperatures etc. This invention also
relates to a process for inhibiting gastric acid secretion and
the treatment of peptic ulcers.
Acetylsalicylic acid (ASA) such as Aspirin (trademark) and
other NSAIDs have long been the most popular drugs for the
management of pain, inflammation and fever. However, one of the
drawbacks in their use is the gastrointestinal injury and/or
bleeding that sometimes accompanies their administration to
individuals. This may become a particularly severe problem where
large and sustained doses of NSAIDs must be given to control the
symptoms, as for exampla, in the case of the management of
arthritis.
It has now been found that NSAID induced gastrointestinal
injury and particularly gastrointestinal mucosal injury can be
significantly reduced when beta adrenergic agonists are admin-
istered concurrently with a NSAID. The beta adrenergic agonists
form a fairly well defined class of pharmaceutically effective
compounds that are characterized by the fact that they act by
stimulating beta adrenergic receptor sites. These receptor sites
are of two types referred to as the beta 1 and beta 2
,.1 ~
1 309662
~ite~. Beta adrenergic agoni6ts may act on one or the other or
on both type6 of ~ites. Any o~ these are e~fective in
practicing the pre~ent invention.
It has also been found that gastric acid secretion can be
inhibited, and peptic ulcers can be treated by the
administration of a beta adrenergic agonist. Typical agonists
of this type which can be used in accordance with the invention,
include isoproterenol, metaproterenol, terbutaline, albuterol,
fenoterol, bitolterol, isoetharine, colterol, or ritodrine or
their pharmaceutically acceptable 6alts.
A number o~ beta adrenergic agonists are known in the prior
art which are useful for the purpose~ o~ this invention. Of
special interest are isoproterenol which i6 a mixed bata 1 and
beta 2 agonist and terbutaline which i8 a beta 2 agonist. By
way of illustrating other beta adrenergic agonists that may be
employed herein, the following are given metaproterenol,
albuterol, ritodrine. All of these ~ay be employed as such or
as pharmaceutisally acceptable salts.
The NSAIDs al50 ~orm a well known class of druys that are
anti-inflammatory analgesics. These have the common properky of
inhibitiny the fo~mation of prostaglandins which have a
protective affect on the gastrointestinal mucosa. See Goodman
and Gilman "The Pharmacological Basi6 for Therapeutics" 7th
Edition, p. 678. It is because of this inhibiting e~ect that
the oral administrat$on o~ drugs o~ this class tend to result in
gastrointestinal injury and/or bleeding, the problem that the
present invention seeks to reduce or eliminate.
A number of NSAIDs are known in the prior art to which the
present invention has application. The most aommonly known
group are the salicylates o~ which ASA is the prime
example. Another group of NSAIDs that have utility in con-
nection with the instant invention are the proprionic acid
1 ~(3~66~
derivative~. Included in thi~ group, ~or example, arQ ibuprofen
and naproxe~. A further group of NS~IDs employable herein are
the fenama~e~ and compound~ closely related to them struc~
turally. ~hesa may be illustrated by such compound~ a
mePenamlc acid, meclofenamake sodium, diclo~enac and its sodium
salt. Also belonging to the class o~ NSAIDs with which the
present invention iB concerned are the indolz derivatives (e.g.
indomethacin); pyrrolealkanoic acid derivatives (e.g. tolmetin);
pyrazalone derivatives (e.g. phenylbutazone); oxicams (e.g.
piroxicam); etc.
The quantitative relationship o~ the NSAID to beta
adrenergic agonist contained in the present products may be
expressed on the basis o~ th~ average daily dose of tha product
i.e. milligrams/per Kg of body weight/per day. In this case the
average do~e for the products will generally be from about lO
mg/Kg/day to about lO0 mg/~g/day o~ NSAID and from about 0.0003
mg/Kg/day to about 500 mg/Xg/day of one or a combination of beta
adrenergic agonist~ with the preferred range being ~rom about 15
mg/~g/day to about 75 mg/Xg/day of NS~ID and from about
0.01 mg/Xg/day to about lO mg/KgJday o~ ~aid bata adrenergic
agonists.
The unit dosage forms ~or the present product~ will be
formulated for convenient oral admlnistration. Each such unit
will generally contain from about 200 mg to about 600 mg o~
NSAID and fro~ about 0.7 mg to.about 70 mg of ons or a
combination o~ adrenergic agonists. For therap~utic use th~
beta adrenergic agonist will normally be administered a~ a
pharmaceutical composition comprislng as the essential active
ingredient at least one of such agonist in its basia form or in
the form o~ a non-toxic pharmaceutically acceptable acid
addition salt, in association with a pharmaceutically acceptable
carrier. The pharmaceutical composition can be administered
orally in~ranasally, parenterally, or by rectal suppository. A
wide variety o~ pharmaceutical forms may be employed. Thus, if
a ~olid carrier is used, the preparation may b~ a tablet, placed
1 3 i(~ ~ !6 ~ ,~
. .
in a hard gelatin tabsule in powder or yranular form, or in a
form o~ a trochs, caplet or capsule. I~ a liquid carrier i~
employed, the preparation may be in a form of a ~yrup, emulsion,
soft gelat~n capsule, ~terile solution for in~ection, or an
aqueous or non-aqueoua liquid suspension. The pharmaceutical
compositions are prepared by conventional techniques appropriate
to the desired preparation.
The dos2ge of the composition~ ~f this invention w~ll depend not
only on such factors as the weight of the patient, but al30 in the
degrea of gastric acid lnhibition desired and the potency of the
particular compound being utilized. The dec~sion as to the
particular dosage to be employed i~ within the discretion and the
routine knowledga of the physician. In thQ Heidenhain Pouch ~og test
described below, cimet~dina has an oral ED50 ~ approximately 3.3
moles/kg. Th~ usual human adult oral dose o~ cimetidine i9 3 00 mg,
given four times a day~ The usual human adult starting oral dosag~
o~ the compound~ of this invention are readily dete~mined ~rom their
oral ED50 in thi~ same test. Thu3, i~ the oral ~D50 i~ 0-33
moles/kg. the usual starting oral dosage would be approximately 30
mg. given four tlmes a day, ~tc. Si~llar calculat~ons may b~ made
for intravenous dosages. These starting dosages (and the number of
ti~e~ administered per day) may, o~ course, be varied by titration of
ths dosage to tha particular circum~tances of the specific patient.
With th2 preferred co~pound~ o~ thi~ lnventlon, each oral dosage unit
will contain the active ingredient in an amount o~ from about 0.5 mg.
to about 300 mg. and mostly pre~erably from about 190 mg. to about
100 mg. The active ingredient will preferably be administQred in
egual dose~ from ona to ~our time~ a day.
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1 ~.
1 309662
Depending upon the do~age ~o~ employed the product~ o~ 'chi~
in~ention may also conta~n other ad~uvants that may be useful in
formulating the particular dosage form or in its administration.
Thus, ~or example, when a~m~nistered a~ a tablet the products o~ this
invention may al~o contain lubricant~ exclpiants, binding agen~s,
di~lntegrating agents, flavoring agents, etc. In addition these
products may also contaln other pharmaceutically active ingredient
such ag deconge~tants~ analgesic ad~uvant~, antihis~amines,
expectorants, antitussives, diuretics, other analgesics, other
anti-lnflammatory agent~, other antipyretic~, other anti- rheumatics,
antioxidants, vasodilators, ~moot~ muscle relax- ant~, skeletal
muscle relaxants, bronchodilators, vitamin~, traca minerals, amino
acids, biological peptide~ etc.
The product~ o~ thi~ ~nYsngion may takQ a variety o~ form~,
A~ indicated above th~y ~ay ag~u~ the ~orm o~ tablat~ How-
ever, tha NSAID and the bata adrenergic agonists may ba in
powdered or granular ~orm containad in edible capsule~ such as
qalatin cap ule~. The present products may al~o taks th2 form
o~ suspension~ or ~olutions o~ the above ingredients in a
sultable liquid medium or o~ powders packaged in 8U~ table pap~r
envelope~. .
~ he mechanism by which the beta adrenergic agonists serve aæ
protectants again~t gastrointestinal injury when admlnistered
as the solQ pharmaceuticaly active ingredient, or coadmin-
istered with the NSAID is not clearly understood. one factor
seems rairly certain and that 1~ that it is not necassarily
~imply related to the inhibition of gastric acid ~ecretion.
This is made evident by the observation that, for example, with
1 3Q~6~
certa~n level~ of isoproterenol, gastric in~ury protection has
been noted notwithstanding the ~act that the pH o f the s omach
content was essentially the same as the pH of the control
stomach. Similarly, as reported by M.H. Steven et al ln the
Abstract of Paper~; I'Gastroenterology" Vol. 88, No. 5 Part 2 at
page 1600, although isoproterenol had a role in the regulation
of gastric secretion stimulated by gastrin and acetylcholine,
histamine-stimulated acid secretion was resistant to inhibition
by isoproterenol. In passing, it mlght be noted that Steven et
al were not measuring the effect of the coadministratlon of
ASA with isoproterenol.
Determination of Gastric Antisecretory
Activity in the Gastric ~istula Rat.
Male Long Evans rats weighing about 240-260 grams at the
time of cannula implementation are used. The design and
implementation of the ~tainless steel cannula into the anterior
wall of the fore-stomach are carried out essentially as
described by Pare et al. tLaboratory Animal Science, 27, 244
(1977). The ~iatula components ar~ designed and the operative
procedure is carried out exactly as described in the above
reference. Poæt operatively the animals are indiv~dually housed
in solid bottom cages with sawdust and are allowed food and
water ad libitum throughout the entire recovery period. Animals
are not used for test purposes for at least 15 days a~ter the
operative procedure.
The animals are fasted but allowed water ad llbitum for 20
hour~ before the testing procedure ~ 9 to begin. Immediately
prior to collection, the cannula i8 opened and the stomach
washed gently with 30-40 ml of warm ~aline or dlstilled water to
remove any residual contents. The catheter is then ecrewed into
the cannula in place o~ the plugging screw and the rat i6 placed
in a clear plastic rectangular cage measuring 40 cm long, 15 cm
wide and 13 cm high. The bottom o~ the cage has a slit
..
-
1 30966~
approximately 1.5 cm wide and 25 cm long running down the center
to accommodate the catheter which hangs through it. In this way
the rat i8 not restricted and can move freely about the cage
during collection periods. The remainder o~ the assay i
carried out as described by Ridley et al. [Research Co~m. Chem.
Path. Pharm., 17, 365 (1977)].
Gastric secretions collected durinq the first hour a~ter
washing the stomach are discarded as they may be contaminated.
For oral evaluation, the catheter is then removed from the
cannula and replaced with the plugging screw. Water (2 ml/kg)
i6 administered orally via gastric intubation and the animal is
returned to the cage ~or 45 minutes. After thls time the
plugging screw is removed and replaced with a catheter to which
a small plastic vial has been attached to collect the gastric
secretions. A two hour sample iOE collected (this represents the
control secrPtion), the catheter removed and replaced with the
plugging ~crew. The test drug is now administered orally in a
volume of 2 ml/kg via gastric intubation. Forty five minutes
later the plugging ~crew is again removed, replaced with the
catheter attached to a small plastic vial and another 2 hour
Eample is compared to those of the control sample in order to
determine the effect~ of the test drug.
~ hen te~t compounds are to be evaluated parenterally, the
animal i8 in~ected lp or 6C with the test compound vehicle in a
volume of 2 ml/kg immediately after discarding the initial 60
minute collection. A two hour sample is collected ~control
~ecretion) and the animals are in~ected either ip or 6C with
1 309662
the test compound in a volume o~ 2 ml/kg. An additional two hour
sampl2 i~ collected and its secret~ons are compared to those of th~
control period to datermine drug ePfect~.
The ~amples are centrifuged and pla ed in ~a gra~uated centrifuge
tube for volume determination. Titratabl~ acLdity is measured by
titrating a one ml sample to pR 7.0 with 0.2N NaO~, using an
autoburet and an electrometric pH meter (radiometer). Titratable
ac~d output i~ calculated ln microequivalent3 by multiplying the
volume in milliliters by the acid concentration in mllllequivalents
per liter.
Results are expressed a~ percent inhibition relative to control
reading. Dose re~ponse curves are constructed and ED50 values are
calculated by regression analyse~. At least three rat~ are used at
each dosage level and a minimum of three dosage levels are utilized
for determination of a do~a responaive curve.
Two o~ the ~tandard an~mal model~ for determin~ng gastric
antisecretory activity o~ antagon~st are the Gastric Fistula ~at and
the ~Pidenhain Pouch Dog. Comparative data between metaproterPnol, a
known beta adrenergic agon$3t, and cimetidine, a known H2 receptor
antagoni~t and e~ectivQ inhibitor of ga~tric secretlon, i8 ~hown in
table3 I and II below:
-- 8 --
1 3096~2
Table I
Gastric antisecretory actiY~ty ln the Gastric Fistula Rat
ED50 values ~mg/kg) for oral treatment
Metaproterenol Cimetidine Potence Ratio
~Cimetidine = 1
23.6 (15 - 42) * 12 (9.0 - lB) * 0.58
*(95% confidence limit)
Determination of Gastric Antisecretory Activity
in the Heidenhain Pouch Dog
Prior to surgery, hematology and blood chemistry profiles are
obtained and an assessment made a~ to the general health of selected
female dog~. Dogs are vaccinated with Tissue Vax 5 (DHLP-Pitman-Moore)
and housed in general animal guarters for an observation period of four
weeks, 80 incipient diseases may become apparent. Dogs are fa~tPd with
water ad libitium 24 hours prior to surgery.
Anesthesia i~ induced with ~odium pentothal (Abcott) 25-30 mg/kg
iY. Subsequent anesthesia is maintained with methoxyflurane
(Pitman-Moore~. A midline linea alba inci~ion fro~ xiphoid to umbilcu~
provides good exposure and ease o~ ~losureO The stomach is pulled up
into the operative field, the greater curvature ~tretched out a~
multiple points and clamp~ placed along the 6elQrted line of in~i~ion.
The pouch i8 made from the corpu~ of the stomach 80 that true parietal
cell ~uice is obtained. About 30% o~ the COrpU8 volume i8 resected.
The cannula is made of light-weight, biologically inert material such
as nylon or Delrin with dimensions and attachments a~ter DeVito and
Harkins [J. Appl. Phy~iol., 14, 138 (1959)]. Post operatively, dogs
1 309662
are medicated with antibiotics and an analgesic. They ar~ allowed 2-3
months for recovery. Experiments are carried out in the following way:
Dogs are fasted overnight (18 houræ3 with water ad libitum prior to
each experiment. The dogs are placed in a sllng and a saphenous vein
cannulated for drug administration. Histamine as the base (100
g/kg/hr) and chlorpheniram~ne maleate ~0.25 mg/kg/hr) are infused
continuou~ly (in a volume of 6 ml/hr~ with a Harvard infusion pump.
A Ninety m~nute infusion is allowed for the dogs to reach a steady
state of acid output. At thi3 time the drug or normal saline (control)
is administered ¢oncomitantly with the secretagogue in a volume of 0.5
ml/kg over a 30 second period. When oral studies are to be carried
out, the drug is administered via gastric gavage in a volume of 5
ml/kg. Infusion of the secretagogue is continued and 15 minute samples
of the gastric ~uice are taken for 4.5 hours. Each sample is measured
to the nearest 0.5 ml and titratable acidity i~ det~rmined by titrating
a 1 ml sample to p~ 7.0 with 002N MaOH, using an autoburet and an
electrometric pH meter (or radiometer~. Titratable acid output i8
calculated in microe~uivalents by multiplying the volume in milliliters
by the acid concentration in a m~llequ~valents per liter.
Results are expressed a~ percent inhibition relative to control
reading~. Dose response curves are constructed and ED~ values are
calculated by re~ression analy~e~ From 3 to 5 dogs are used at each
dose level and a minimum of three dosage levels are utilized for
determination of a dose response curve.
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.~i~ . - - ...............
-`` 1 30~6~
Table II
Gastric Antisecretory Activity
in the Heidenhain Pouch Dog
ED50 values (mg/kg for oral treatment)
Metaproterenol Cimetidine PotQncy Ratio
(Cimetidine - 1
0.05 0.71 14.2
The potency ratios ln the rat and canlne antisecretoxy model
describe~ above are 0.58 and 14.2, respectively.
Cytoprotective ackivity was evaluated in five te3t models.
Metaproterenol was comparPd to cimetidine as a protectant. The term
"cytoprotection" describes the phen~menon whereby some agents protect
the gastric mucosa against injury induced by a variety o~ injurious
stimuli. These assays are prlmarily used as secondary models to
evaluate agents which have confirmed anti-gastric secretory activity
and/or activity in primary (antisecretory) model In the present
case, the beta adrenergic agonist metaproterenol was compared to the
cytoprotective activity o~ cimetldineO The injuriou~ sti~uli in our
present test were provided by ethyl alcohol, ASA and stress,
hydrochloric acid, and indomethacin. The respective assays were
carried out as described below:
Eth~l alcohol-
The method employs 3.0 ml/kg ethyl alcohol (100%) a~ thenecrotizing agent and provides additional data besides ga~tric lesions.
`~ ~ 3 0 ~ !2
Adult male Long-Evans rats weighing 275 300 grams (~lue Spruce
Farms, Alton, New York) are used. Animals are housed individually in
stainless steel cages with wire-mesh bottom~. The housing battery is
arranged into six groups of five each, individually numb2red. Food and
water are removed 24 and 1 1/2 houxs, respectively, prior to exposure
to ETOH. Animals receive the test compounds ~ither p.o., 6 . C , or ip.
(3 12 ml/kg) 60 minutes before administration o~ 100% ethanol
(3.0 ml/kg/ by gavage). For some purposes, different water removal and
drug pretreatment times may be used. Sixty minutes after ethanol
administration the animals are sacrificed by administration of T 61
(Hoechst), 0.2 ml i.p.
An abdominal incision is made, and after clamping the esophagus
~ust above the esophageal sphincter with forceps, the stomach is
carefully lifted ~rom the abdominal cavity. Two cuts are made,
approximately 1/4 3~ below the pyloric valve and 1~4ll above the
esophageal ~phincter, and the stomach remoYed rapidly without a~y loss
o~ gastric contents. The size of the stomach including contents i8
noted as small ~ medium or large. Stomachs are cut open along the
greater curvature and the content3 expressed into graduated centrifuge
tubes. Gastric ~uice samples are centrifuged and total ~olume, and
contentR of solid and mucus are estimated to the nearest 0.1 ml Na+
and K~ content o~ the gastric ~uice as well as pH are determined.
Under these conditions, ethanol produces prominent ~acroscopic
lesions in the fundic stomach, but gross macroscopic changes are only
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! `~- ` .. . ~ . .. .
1 ~9~b2
var~ rarely observed in the forestomach. An occasional control animal
shows some degrae of redness or hyperemia and/or s~ell petechiae in the
forestomach. Lesions characteristic of ~undic damage are not usually
observPdO For these reasons, the forestomach (along with nongastric
tissue) is removed and not examined. In addition, absence of the
forestomach facilitat~s the photocopy and gross examlnation o~ the
remaining stomach.
The remalning fundic-pyloric stomach remnant is rinsed in water and
placed flat in a standard position. The tissue is photographed with a
Polaroid camera with a close-up lens. Scoring of lesions is done from
this permanent record. Each photograph includes a reference scale in
mm. Individual ulcers are measured by total lesion area (mm2) and
these scores are added together to determine total ulcer area for a
stomach. Small well defined but quite visible ~pot (petechiae) are
often present; a conglomerate area estimate i8 made for these and added
to the total. Some variability in ulcer scoring between persons may
occur because even optimal quality photographs will show minor shadows,
shades, overexposed ~pots and irregularly shaped ulcers.
Standard photographs are about 2 X actual siza. An estimate Qf
real ulcer area (mm23 is obtained after dividing the area score by
~our. For each treatment group, the ~ean area i8 calculated. From
this, the percent inhibition of le~ion formation, I ls calculated as:
I = Lesion Score (controls~ - Lesion Score~.(treated3 X 100
Lesion Score (Controls)
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- I 1 309662
The mean ulcer score ~or a control group will be used to calculate
"I" but normally ~ndividual lesion ~coreR ar~ used in drug treatment
groups. In some cases, the observed treatment ~core iæ greater than
the mean control valve, in which case that ~core i~ taken as the mean
control valve (exacerbation o~ lesion formation is not considered). In
rare instances, the da~a from an animal will be discarded if, e.g.,
~eces are ~ound in the stomach~
In dose-response studies, the ED50 (dose at which 50%
inhibition of lesion formation occur6) is determined by probit
analysis.
ASA + Stress
. . .
Male Lvng Evans rats (approximately 275-300 g~s) are housed 6 per
cage and acclimated at least three days while maintained on an ad
libitum schedule of food and water~ Prior to starting an experiment,
rats are deprived o~ food for twenty-four hours and water for one
hour. Thlrty mlnute~ be~ore 6ubjecting animals to cold restraint
stress, each rat i~ dosed by gastric intubation (6 ml/kg) with
ASA (80 mg/Xg) homogenized with methy-cellulos~ (0.1%) in water.
TeYt agents are admini~tered 60 or 30 min prior to the ASA
mixture either orally or parenterally. Each control o~ drug treated
group normally con~ists of 5 rata. The multi-stress method entails
placing the dosed rat in a ventilated plastic tub2 closed at one end
-- lg --
1 30966~
with a rubber ~topper. The open end ~8 then closed with a 6econd
stopper and $he tube is place in an environmental cha~ber maintained
at 4-5C for two hour~. The tube contains ~everal 3~8" holes for
breathing purposes and iB about ten inches long and two inches wide.
At the end of ~he two-hour cold-restraint period, each animal is
sacrificed with an injection of T-61 euthanasia solution (Hoechst).
An abdominal incision is mada and the stomach removed by cutting
just above the esophageal ephincter and just below the pyloric valve.
The ~tomach is opened along the greater curvature and the stomach
contents di6carded. Under the conditions described, ASA plus
cold reskraint stress produce6 spotty and narrow dark bands of linear
macroscopic lesions which are usually found in the corpus ~undic
stomach. There~ore, forestomach and other accessory ti sue which
show no macroscopic leslon damage are trimmed. The remaining fundic
portion is rlnsed wlth water and placed flat in a standard poæition
for photographing. The stomach along with a millimeter reference
scale i5 photographed at 2X actual size with a Polaroid camera.
Scoring of leslon~ i8 done from thi~ permanent r~cord.
The lesion ~core i8 determlned by measuring and adding together
the total millimeter lengths of all visible erosions. Small spots
(petechia) are scored as 1.0 mm each and added to the aggregate. An
estimate of real lesion length i~ obtained by dividing the aggregate
score by two. For each treatment gxoup, a mean lesion score is
determined. From this, the percent inhibltion of lesion ~ormation,
I, is calculated as:
- 15 -
.~
1 30~6~
I = Le~ion Scor~ ~controls~ - Les~on Score (t_eated) X 100
~esion Score Control~
The mean lesion ~core for a control group will be used to
calculat~ "I" but indivldual lesion score~ will usually ba used in
drug treatment groups. Observed treatment ~cores tha~ exceed th~
mean control value are taken a~ the mean control value without
considering ~xacerbation o~ le~ion ~ormatlon.
In doso response studie~, thQ ED50 (dose at which 50%
inhibition o~ lesion formation occurs) i~ determlned by probit
analy~is.
HCl:
The present method to produce ga~tr~c lesions in rats employs 3.0
ml/kg 0.75N HCl as the necrotlzing agent and provide additional data
besides gastric le~ions.
Adult male Long~Evan~ rats weighing 275-3~0 gram3 ~Blue Spruce
Farms, AltQn~ Ne~ York) are u~ed. An~mals are housad individually in
6tainless ~teel cages with wir~ mesh boktom~. Th~ hou~ing battery is
arranged into 5iX group o~ ~iv~ each, indlvidually numbered. Food
and water are removQd 24 and 1 1~2 hours, respectiv21y, prior to
HCl. Animal~ receive th~ test compounds either p.o., ~.c., or i.p.
(3-12 ml/kg) 60 minute~ befor~ administration of 0.75N HCl (3.0
ml/kg, by gavage). For some purposes, di~fer~nt water removal and
- 16 -
:
: ''' `
1 30`~6`~
-
.
drug pretreatment times may ba used. Sixty ~inutes after ~Cl th~
animal~ are sacrificed by admini~tration o~ T-61 (Hoechst), 0.2 ml,
i .p.
An abdominal incision i~ made, and after clamplng the esophagu~
~ust above the esophageal sphincter with tweezer~, the stomach is
carefully lifted ~rom the abdomina~ cavity. Tw~ cut8 are made,
approximately 1/4 " below the pyloric valve and 1/4 ~i above the
esophageal sphincter, and the stomach rapidly removed without any
1088 0~ gastric contents. The size o~ the stomach including content~
is noted as small, medium or large. Stomachs are cut open along the
greater curvature and the content~ expressed into graduated
centrifuge tube~. Gastric ~uic~ samples are centrl~uged and total
volume, and contents o~ olid~ and mucus are e~timated to the neare~t
0.1 ml. Na~ and K~ contents o~ the gastric jUiCQ as well as pH
are determined.
Under these condition~, 0.75N HCl produce~ prominent macroscopic
lesions in the fundic sto~ach, but gros~ macroscopic changes are only
very rarely observed in the ~orestomach. An occas~onal control
animal ~how~ 80mQ degra~ o~ radnes~ or hyperemia and/or ~mall
petechiae ln the iorestomach, but never lesion~ charactsristlc o~
fundlc damage. Fox these reasons, the fore~tomach (along with
nongastric ti~sue) i~ removed and not examinad. In addition, absenc2
o~ the fore~to~ach ~acilitates the. photography and examination of the
remaining stomach. W~t wsight of th~ whola stomach i9 determined
be~ore removal of the ~ore~tomach.
- 17 -
096`62
~ h~ rema~ning fundic-pyl~ric ~tomach remnant 1~ rinsed in water
and placed ~lat ln a ~tandard po8ition~ ~h~ tissue i8 photographed
with a Polarold camera with a c~o~e-up lens. Scoring of lesions i~
done from this permanent record. Each photograph includ8~ a
referencs scale in m~. Individual ulcer~ are measured by total
lesion length in mm and these scores ar~ added tog~ther to determine
total ulcer length for a stomachc Small well d~ined but quite
visiblc ~pot~ (petechiae) are often present: a conglomerate length
estima~e i~ made for these and added to the total. Some variability
ulcer scoring between persons may occur because even optimal qual~ty
photographs will show minor shadows, shades, overexposed spots and
irregularly shaped ulcers.
Standard photograph~ are about 2 x actual size. An estimate of
real ulcer length (mm~ i~ obtained after div~ding th2 length score by
two. For each treatment group, the mean length 1~ calculated. From
this, the percen~ inhibition of lesion formation, I, i~ calculated
as:
I = Lesion score (controls) - Lesion Score (treated~ X 100
Lesion Score (controls~
The ~ean ulcer score for a control group will be used to
calculate 'II" but normally lndividual lQsion scores ara u6ed in drug
treatment group~. In ~ome casas, th~ obs~rved treat~ent ~cor~ 13
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greater than the mean control value (~xacerbation o~ lesion ~ormation
1~ not considared). In rare lnstance~, tha data ~rom an animal will
be discarded i~, s.g., fece~ are ~ound in the ~tomach.
In dose-response studie~, thQ ED50 (dose at which 50~
inhibition o~ lesion ~ormation occurs) iQ determined by probit
analysis.
IndomethaCin
Male Long Evan~ rat~ (approx~mately 275-300 ~s~ ar~ housed 6 per
cage and acclli~ate~ a~ lea~t thre~ dayg ln ~ c~trolled environment
whilo maintained on an ad libitum schedule o~ food and water. Prior
to the exper~ment, rats are deprived o~ food Por twenty-four hours
and water ~or 1 1/2 hour. On thQ day of t~e exper~menk, rat-~ are
randomly placed individually into a Rtainles~ steel cage with a wire
mesh botto~. Control or drug treated groups consist o~ 5 rats earh.
Grouped rats receive a test drug or vehicle admlnistered orally 30 or
60 minutes prior to ths lesionlng agent. Gastric le~io~s are induced
by administratlon o~ indo~ethacln ~olution (30 mg~kg3 by gavage ~3. a~
ml/kg). The indom~thacln i8 prepared ~y di~olving 100 mg powdar ln
1.0 ml of lN Na~C03 and heating un~11 d~Q301ved. Water i8 added to
giv~ a ~inal lndo~ethacin concentration o~ 10 mg/ml in O.lN
NaHC03. Four hsurs a~ter indo~thacln ths rat~ are ~acri~ic~d by
in~erting T-61 euthanasia ~olution (HoPchst)O ~o determ~nQ duration
or mechanlsm o~ act~on, different ti~e~ or routes o~ compound
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1 ~ 6 6 ~
administration are used,
The ~tomach~ are removed, opened along khe curvatur~, r~nsed with
water and ~pread out ~lat ~n a standard position for examination and
photographing.
Under the conditions described, indomethacin normally produces
prominent macroscopic lesion~ in the ~undic glandular secr~ting
portion of ths ~tomach.
Nonlesioned forestomach and other accessory tiS9UQB are trimmed
of~ to improve standard placement. The remaining ~undic tis~u~ along
with a millimeter reference scale i~ photograph~d at 2X actual size
with a Polaroid camera with a close-up lens. Scoring o~ le~ion~ i~
donQ ~rom thls permanent record.
For ea h animal the severity o~ gastric le~on~ i3 defined a~ the
8um 0~ the maximu~ continuou~ lengths of tha lesions (in mm). Barely
visible spot~ tpetechia) are ~cored as 1.O mm each an~ added to the
total length. Real lesion length ~mm) i8 obtained aftar dividing th~
total length score by two.
The mean lesion score i5 calculated ~or each treatment group.
From this, the percent inhibition o~ lesion ~ormation, I, i~
calculated:
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,~
3 ~ 6 6 ~
I = Lesion score (cont,r,ols) - Lesion score (treated~ X 100
Lesion Score (controls)
The mean lesion ~core for a control group will be usPd to
calculate "I" but individual lesion ~cores will usually be used in
drug treatment groups. Observed treatment scores that exceed the
mean control value are taken as the mean control value without
considering exacarbation of lesion formation~
In dose response studies, the ED50 (dose at which 50%
inhibition of lesion formation occurs) is determined by probit
analysis.
The results of the gastroprotective studies in the rat models
with respect to ED50 values (mg/kg) for the various irritants, by
oral treatment, are ~ummarized in Table III.
Table III
Irritant Metap,r,oterenol Cimetidine Potency Ratio
tCi~etidine = 1)
ETOH 0.19 (0.03-0.47)* 223 (191-256) 1174
ASA ~ Stress 11.3 (7.29-16.2) 4.5 (2.0-7.1) 0.4
HCl 8.9 (4.4-17.8) 225 (176-284) 25.3
Indomethacin 2.0 (1.0-3~7) 5.5 (3.9-7.3) 2.8
* (95~ confidenae limit)
In three out of four tests, metaproterenol was more effective than
cimetidine (potency ratio range 2.75-1174~.
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` 1 309662
In vlew o~ the above test results, using metaproterenol which is a
typical beta adrenergic agoni~t used commonly for testing such
materials, beta adrenerglc agonist~ are indicated as good gastro-
protectant and antisecretory agents useful in the treatment of
gastro-intestinal disorders such a~ gastroesopha~eal reflux disease
(GERD), dyspepsia, undue gastrlc acid secretion, gastritis and peptic
ulcer.
The following Exa~ples are given to further illustrate the present
invention. It is to be understood, however, that this invention is
not limited thereto.
EXAMPLES
1 2 3 _ 4
ASA 325 mg 325 mg 325 mg325 mg
metaproterenol3.33 mg - - _
i60pro~Qrenol - 10 mg - -
albuterol - - 2.67 ~g
terbutaline - - - 1.67 mg
~ o test the effectiveness of the beta adrener~ic
agonists in protecting the 6tomach against NSAID induced ~ucosal
in~ury each ~eta adrenerglc agonist is admini~tered to dogs
si~ultaneously and orally with, for example, aspirin in
capsules. A ~tandard dose of 975 mg of ASA is admin-
istered with varying doses of the beta adrenergic agonist in a
capsule and the stomach lining of the dogs were examined
endoscopically and rated for the degree of ln~ury.
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..,~
1 309662
All test Pormulations are prepared on the day of the tests.
The capsules are placed in the back of the dog's throatO A
catheter with attached funnel is positioned in the doy's stomach
and 50ml of deionized water is administered.
Healthy ad~llt beagle dogs of either sex are ~elected for
testing. Dogs are housed individually ln stainless steel cages
with grid floore to allow excreta to pass through. Room
temperature in the holding rooms and test laboratories is
maintained between 65-F and 85F and relative humidity of
between 30% and 80%, Room light~ remain on from 6:00 AM to 4:00
PM.
Each dog 18 tralned to stand in a ~tanchion with ~ling
support and to accept a bit tied in its mouth. A gastroscope is
then passed through the bit into the dog' stomach. This
training requires ken days to two weeks in most dogs.
To determine whethar a dog i8 suitable for test purposes,
its stomach i8 examlned for a norDal mucosa, and its yastric
responsivaness to ASA is evaluated (as under Test
Procedure). An acceptable gastric irritation ~core in the
antrum must be 5 or greater, 2 hours after dosage.
Food i6 withheld from test dogs ~or 24 hrs. before the test
and during the te~t and water is allowed ad lib. The dogs are
moved into a holding area away from the kennel. Fasted dogs of
either ~ex are examined gastroscop~cally to ensure that their
~tomachs have normal healthy mucosal linings. The dogs are
dosed orally with test formulations, which are flushed into
their stomachs with 50 ml. of deionized water. They are then
re-examined two hour~ later for ga~tric petechiae and signs o~
bleeding according to the following scale:
0 = uniform, pale to dark pink mucosa
1 = darker pink or blotchy mucosa
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1 30~`662
2 ~ peteahia6 and/or light red 6treaks
3 - ~w small le~ion~
4 = many or connected ~mall lasions ~striati~ns)
5 = ~ew large lesion~
6 = ~any large lesion~
7 = masslve hemorrhagic dama~e
Severity of bleeding for each treatment and at each time is
calculated as the mean gastric irritation 6core.
In addition to the endoscopic observatlon of the gastric
mucosa of each dog a qualitative deæcript$on of gastric ~luid is
recorded and a pH measurement i8 made of the ga~tr~c fluid. All
of thess are don~ 2 hours after administration o~ the test
product.
A ~ase line t~ established by measuring the various para-
meters after the administration of 975 mg of ASA by itself.
The resting stomach ha~ an irritation score of O and a pH of 5
to 5.5. ASA alone produced injury which scored at
approximately 5.5 a~ter 2 hours and the gastric pH at this time
i8 about 3.1. After 4 hour~ these values were 3.7 for the
irritation factor and the pH was 4.8. Thi~ indicate~ that a
certain a~ount of healing takQs place between the 2nd and 4th
hour after administration.
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. ~ ,,
, 1 3nq6~
The re~ults o~ the various tests are summarized in Table IV below.
able I
Test Composition 2 Hr. Score
In~ury _ pK
ASA (975 ~) 5.6 3.1
ASA ~975 ~g) + isoproterenol (7.5 mg) 3.8 3.5
ASA (975 mg) + isoproterenol (15 mg) 2.7 3.8
~SA (975 mg) + isoproterenol (30 mg) 1.3 5.0
ASA (975 mg) + terbutaline (1.25 mg) 4.0 2.9
ASA (975 mg) + terbutaline ~5.0 mg) 1.4 4.0
ASA (975 mg) + terbutaline ~10.0 mg) 1.2 4.~
ASA (975 mg) + albuterol (8 mg) 1.0 5.4
ASA (975 mg~ + metaproterenol (20 mg) 0.75 5.7
An examinat$on of these data shows that the beta adrenergic
agonists provide significant protection against ASA induced
~ucosal in~ury particularly at thQ 2 hour leval after admin-
istration. This protection, moreovsr, does not appear to be
particularly x~lated to the p~ of the gastric contents.
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