A pharmaceutical composition and process for administering non-steroidal anti-inflammatory drugs which are protected against injury to gastrointestinal tract by beta adrenergic agonists.

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. 
Aspirin 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 example, 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 administered 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 
sites. Beta adrenergic agonists may act on one or the other or on both 
types of sites. Any of these are effective in practicing the present 
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 salts. 
A number of beta adrenergic agonists are known in the prior art which are 
useful for the purposes of this invention. Of special interest are 
isoproterenol which is a mixed beta 1 and beta 2 agonist and terbutaline 
which is 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 may be employed as such 
or as pharmaceutically acceptable salts. 
The NSAIDs also form a well known class of drugs that are anti-inflammatory 
analgesics. These have the common property of inhibiting the formation of 
prostaglandins which have a protective affect on the gastrointestinal 
mucosa. See Goodman and Gilman "The Pharmacological Basis for 
Therapeutics" 7th Edition, p. 678. It is because of this inhibiting effect 
that the oral administration of drugs of 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 commonly known group are the 
salicylates of which aspirin is the prime example. Another group of NSAIDs 
that have utility in connection with the instant invention are the 
proprionic acid derivatives. Included in this group, for example, are 
ibuprofen and naproxen. A further group of NSAIDs employable herein are 
the fenamates and compounds closely related to them structurally. These 
may be illustrated by such compounds as mefenamic acid, meclofenamate 
sodium, diclofenac and its sodium salt. Also belonging to the class of 
NSAIDs with which the present invention is concerned are the indole 
derivatives (e.g. indomethacin); pyrrolealkanoic acid derivatives (e.g. 
tolmetin); pyrazalone derivatives (e.g. phenylbutazone); oxicams (e.g. 
piroxicam); etc. 
The quantitative relationship of the NSAID to beta adrenergic agonist 
contained in the present products may be expressed on the basis of the 
average daily dose of the product i.e. milligrams/per Kg of body 
weight/per day. In this case the average dose for the products will 
generally be from about 10 mg/Kg/day to about 100 mg/Kg/day of NSAID and 
from about 0.0003 mg/Kg/day to about 500 mg/Kg/day of one or a combination 
of beta adrenergic agonists with the preferred range being from about 15 
mg/Kg/day to about 75 mg/Kg/day of NSAID and from about 0.01 mg/Kg/day to 
about 10 mg/Kg/day of said beta adrenergic agonists. 
The unit dosage forms for the present products will be formulated for 
convenient oral administration. Each such unit will generally contain from 
about 200 mg to about 600 mg of NSAID and from about 0.7 mg to about 70 mg 
of one or a combination of adrenergic agonists. For therapeutic use the 
beta adrenergic agonist will normally be administered as a pharmaceutical 
composition comprising as the essential active ingredient at least one of 
such agonist in its basic form or in the form of a non-toxic 
pharmaceutically acceptable acid addition salt, in association with a 
pharmaceutically acceptable carrier. The pharmaceutical composition can be 
administered orally intranasally, parenterally, or by rectal suppository. 
A wide variety of pharmaceutical forms may be employed. Thus, if a solid 
carrier is used, the preparation may be a tablet, placed in a hard gelatin 
tabsule in powder or granular form, or in a form of a troche, caplet or 
capsule. If a liquid carrier is employed, the preparation may be in a form 
of a syrup, emulsion, soft gelatin capsule, sterile solution for 
injection, or an aqueous or non-aqueous liquid suspension. The 
pharmaceutical compositions are prepared by conventional techniques 
appropriate to the desired preparation. 
The dosage of the compositions of this invention will depend not only on 
such factors as the weight of the patient, but also in the degree of 
gastric acid inhibition desired and the potency of the particular compound 
being utilized. The decision as to the particular dosage to be employed is 
within the discretion and the routine knowledge of the physician. In the 
Heidenhain Pouch Dog test described below, cimetidine has an oral 
ED.sub.50 of approximately 3.3 moles/kg. The usual human adult oral dose 
of cimetidine is 300 mg, given four times a day. The usual human adult 
starting oral dosages of the compounds of this invention are readily 
determined from their oral ED.sub.50 in this same test. Thus, if the oral 
ED.sub.50 is 0.33 moles/kg. the usual starting oral dosage would be 
approximately 30 mg. given four times a day, etc. Similar calculations may 
be made for intravenous dosages. These starting dosages (and the number of 
times administered per day) may, of course, be varied by titration of the 
dosage to the particular circumstances of the specific patient. With the 
preferred compounds of this invention, each oral dosage unit will contain 
the active ingredient in an amount of from about 0.5 mg to about 300 mg. 
and mostly preferably from about 1.0 mg. to about 100 mg. The active 
ingredient will preferably be administered in equal doses from one to four 
times a day. 
Depending upon the dosage form employed the products of this invention may 
also contain other adjuvants that may be useful in formulating the 
particular dosage form or in its administration. Thus, for example, when 
administered as a tablet the products of this invention may also contain 
lubricants, excipients, binding agents, disintegrating agents, flavoring 
agents, etc. In addition these products may also contain other 
pharmaceutically active ingredients such as decongestants, analgesic 
adjuvants, antihistamines, expectorants, antitussives, diuretics, other 
analgesics, other anti-inflammatory agents, other antipyretics, other 
anti- rheumatics, antioxidants, vasodilators, smooth muscle relax- ants, 
skeletal muscle relaxants, bronchodilators, vitamins, trace minerals, 
amino acids, biological peptides etc. 
The products of this invention may take a variety of forms. As indicated 
above they may assume the form of tablets. However, the NSAID and the beta 
adrenergic agonists may be in powdered or granular form contained in 
edible capsules such as gelatin capsules. The present products may also 
take the form of suspensions or solutions of the above ingredients in a 
suitable liquid medium or of powders packaged in suitable paper envelopes. 
The mechanism by which the beta adrenergic agonists serve as protectants 
against gastrointestinal injury when administered as the sole 
pharmaceuticaly active ingredient, or coadministered with the NSAID is not 
clearly understood. One factor seems fairly certain and that is that it is 
not necessarily simply related to the inhibition of gastric acid 
secretion. This is made evident by the observation that, for example, with 
certain levels of isoproterenol, gastric injury protection has been noted 
notwithstanding the fact that the pH of the stomach content was 
essentially the same as the pH of the control stomach. Similarly, as 
reported by M. H. Steven et al in the Abstract of Papers; 
"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 might be noted 
that Steven et al were not measuring the effect of the coadministration of 
aspirin with isoproterenol. 
Determination of Gastric Antisecretory Activity in the Gastric Fistula Rat. 
Male Long Evans rats weighing about 240-260 grams at the time of cannula 
implementation are used. The design and implementation of the stainless 
steel cannula into the anterior wall of the fore-stomach are carried out 
essentially as described by Pare et al. (Laboratory Animal Science, 27, 
244 (1977). The fistula components are designed and the operative 
procedure is carried out exactly as described in the above reference. Post 
operatively the animals are individually 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 after the operative procedure. 
The animals are fasted but allowed water ad libitum for 20 hours before the 
testing procedure is to begin. Immediately prior to collection, the 
cannula is opened and the stomach washed gently with 30-40 ml of warm 
saline or distilled water to remove any residual contents. The catheter is 
then screwed into the cannula in place of the plugging screw and the rat 
is placed in a clear plastic rectangular cage measuring 40 cm long, 15 cm 
wide and 13 cm high. The bottom of the cage has a slit 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 is not restricted and canmore 
freely about the cage during collection periods. The remainder of the 
assay is carried out as described by Ridley et al. [Research Comm. Chem. 
Path. Pharm., 17, 365 (1977)]. 
Gastric secretions collected during the first hour after 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) is administered orally via gastric 
intubation and the animal is returned to the cage for 45 minutes. After 
this 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 is collected (this represents the control 
secretion), the catheter removed and replaced with the plugging screw. The 
test drug is now administered orally in a volume of 2 ml/kg via gastric 
intubation. Forty-five minutes later the plugging screw is again removed, 
replaced with the catheter attached to a small plastic vial and another 2 
hour sample is compared to those of the control sample in order to 
determine the effects of the test drug. 
When test compounds are to be evaluated parenterally, the animal is 
injected lp or sc 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 secretion) and the animals are injected 
either ip or sc with the test compound in a volume of 2 ml/kg. An 
additional two hour sample is collected and its secretions are compared to 
those of the control period to determine drug effects. 
The samples are centrifuged and placed in a graduated centrifuge tube for 
volume determination. Titratable acidity is measured by titrating a one ml 
sample to pH 7.0 with 0.2N NaOH, using an autoburet and an electrometric 
pH meter (radiometer). Titratable acid output is calculated in 
microequivalents by multiplying the volume in milliliters by the acid 
concentration in milliequivalents per liter. 
Results are expressed as percent inhibition relative to control reading. 
Dose response curves are constructed and ED.sub.50 values are calculated 
by regression analyses At least three rats are used at each dosage level 
and a minimum of three dosage levels are utilized for determination of a 
dose responsive curve. 
Two of the standard animal models for determining gastric antisecretory 
activity of antagonists are the Gastric Fistula Rat and the Heidenhain 
Pouch Dog. Comparative data between metaproterenol, a known beta 
adrenergic agonist, and cimetidine, a known H.sub.2 receptor antagonist 
and effective inhibitor of gastric secretion, is shown in tables I and II 
below: 
TABLE I 
______________________________________ 
Gastric antisecretory activity in the Gastric Fistula Rat 
ED.sub.50 values (mg/kg) for oral treatment 
Potence Ratio 
Metaproterenol 
Cimetidine (Cimetidine = 1) 
______________________________________ 
23.6 (15-42) 12 (9.0-18)* 
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 as to the general health of selected female dogs. Dogs 
are vaccinated with Tissue Vax 5 (DHLP-Pitman-Moore) and housed in general 
animal quarters for an observation period of four weeks, so incipient 
diseases may become apparent. Dogs are fasted with water ad libitium 24 
hours prior to surgery. 
Anesthesia is induced with sodium pentothal (Abcott) 25-30 mg/kg iv. 
Subsequent anesthesia is maintained with methoxyflurane (Pitman-Moore). A 
midline linea alba incision from xiphoid to umbilcus provides good 
exposure and ease of closure. The stomach is pulled up into the operative 
field, the greater curvature stretched out at multiple points and clamps 
placed along the selected line of incision. The pouch is made from the 
corpus of the stomach so that true parietal cell juice is obtained. About 
30% of the corpus volume is resected. The cannula is made of light-weight, 
biologically-inert material such as nylon or Delrin with dimensions and 
attachments after DeVito and Harkins [J. Appl. Physiol., 14, 138 (1959)]. 
Post operatively, dogs are medicated with antibiotics and an analgesic. 
They are allowed 2-3 months for recovery. Experiments are carried out in 
the following way Dogs are fasted overnight (18 hours) with water ad 
libitum prior to each experiment. The dogs are placed in a sling and a 
saphenous vein cannulated for drug administration. Histamine as the base 
(100 g/kg/hr) and chlorpheniramine maleate (0.25 mg/kg/hr) are infused 
continuously (in a volume of 6 ml/hr) with a Harvard infusion pump. 
A Ninety minute infusion is allowed for the dogs to reach a steady state of 
acid output. At this time the drug or normal saline (control) is 
administered concomitantly 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 juice are 
taken for 4.5 hours. Each sample is measured to the nearest 0.5 ml and 
titratable acidity is determined by titrating a 1 ml sample to pH 7.0 with 
0.2N NaOH, using an autoburet and an electrometric pH meter (or 
radiometer). Titratable acid output is calculated in microequivalents by 
multiplying the volume in milliliters by the acid concentration in a 
millequivalents per liter. 
Results are expressed as percent inhibition relative to control readings. 
Dose response curves are constructed and ED.sub.50 values are calculated 
by regression analyses. 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. 
TABLE II 
______________________________________ 
Gastric Antisecretory Activity 
in the Heidenhain Pouch Dog 
ED.sub.50 values (mg/kg for oral treatment) 
______________________________________ 
Potency Ratio 
Metaproterenol 
Cimetidine 
(Cimetidine = 1) 
______________________________________ 
0.05 0.71 14.2 
______________________________________ 
The potency ratios in the rat and canine antisecretory models described 
above are 0.58 and 14.2, respectively. 
Cytoprotective activity was evaluated in five test models. Metaproterenol 
was compared to cimetidine as a protectant. The term "cytoprotection" 
describes the phenomenon whereby some agents protect the gastric mucosa 
against injury induced by a variety of injurious stimuli. These assays are 
primarily used as secondary models to evaluate agents which have confirmed 
anti-gastric secretory activity and/or activity in primary (antisecretory) 
models. In the present case, the beta adrenergic agonist metaproterenol 
was compared to the cytoprotective activity of cimetidine. The injurious 
stimuli in our present test were provided by ethyl alcohol, aspirin and 
stress, hydrochloric acid, and indomethacin. The respective assays were 
carried out as described below: 
Ethyl alcohol 
The method employs 3.0 ml/kg ethyl alcohol (100%) as the necrotizing agent 
and provides additional data besides gastric lesions. 
Adult male Long-Evans rats weighing 275-300 grams (Blue Spruce Farms, 
Alton, N.Y.) are used. Animals are housed individually in stainless steel 
cages with wire-mesh bottoms. The housing battery is arranged into six 
groups of five each, individually numbered. Food and water are removed 24 
and 11/2 hours, respectively, prior to exposure to ETOH. Animals receive 
the test compounds either p.o., s.c., or i.p. (3-12 ml/kg) 60 minutes 
before administration of 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 just above 
the esophageal sphincter with forceps, the stomach is carefully lifted 
from the abdominal cavity. Two cuts are made, approximately 1/4" below the 
pyloric valve and 1/4" above the esophageal sphincter, and the stomach 
removed rapidly without any loss of gastric contents. The size of the 
stomach including contents is noted as small, medium or large. Stomachs 
are cut open along the greater curvature and the contents expressed into 
graduated centrifug tubes. Gastric juice samples are centrifuged and total 
volume, and contents of solids and mucus are estimated to the nearest 0.1 
ml Na.sup.+ and K.sup.+ content of the gastric juice as well as pH are 
determined. 
Under these conditions, ethanol produces prominent macroscopic lesions in 
the fundic stomach, but gross macroscopic changes are only very rarely 
observed in the forestomach. An occasional control animal shows some 
degree of redness or hyperemia and/or smell petechiae in the forestomach. 
Lesions characteristic of fundic damage are not usually observed. For 
these reasons, the forestomach (along with nongastric tissue) is removed 
and not examined. In addition, absence of the forestomach facilitates the 
photocopy and gross examination of the remaining stomach. 
The remaining 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 (mm.sup.2) and these 
scores are added together to determine total ulcer area for a stomach. 
Small well defined but quite visible spots (petechiae) are often present; 
a conglomerate area estimate is 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 
spots and irregularly shaped ulcers. 
Standard photographs are about 2 X actual size. An estimate of real ulcer 
area (mm.sup.2) is obtained after dividing the area score by four. For 
each treatment group, the mean area is calculated. From this, the percent 
inhibition of lesion formation, I is calculated as: 
##EQU1## 
The mean ulcer score for a control group will be used to calculate "I" but 
normally individual lesion scores are used in drug treatment groups. In 
some cases, the observed treatment score is greater than the mean control 
valve, in which case that score is taken as the mean control valve 
(exacerbation of lesion formation is not considered). rare instances, the 
data from an animal will be discarded if, e.g., feces are found in the 
stomach. 
In dose-response studies, the ED.sub.50 (dose at which 50% inhibition of 
lesion formation occurs) is determined by probit analysis. 
Aspirin+Stress 
Male Long Evans rats (approximately 275-300 gms) 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 of 
food for twenty-four hours and water for one hour. Thirty minutes before 
subjecting animals to cold restraint stress, each rat is dosed by gastric 
intubation (6 ml/kg) with aspirin (80 mg/kg) homogenized with 
methy-cellulose (0.1%) in water. Test agents are administered 60 or 30 min 
prior to the aspirin mixture either orally or parenterally. Each control 
of drug treated group normally consists of 5 rats. The multi-stress method 
entails placing the dosed rat in a ventilated plastic tube closed at one 
end with a rubber stopper. The open end is then closed with a second 
stopper and the tube is place in an environmental chamber maintained at 
4.degree.-5.degree. C. for two hours. The tube contains several 3/8 holes 
for breathing purposes and is about ten inches long and two inches wide. 
At the end of the two-hour cold-restraint period, each animal is 
sacrificed with an injection of T-61 euthanasia solution (Hoechst). 
An abdominal incision is made and the stomach removed by cutting just above 
the esophageal sphincter and just below the pyloric valve. The stomach is 
opened along the greater curvature and the stomach contents discarded. 
Under the conditions described, aspirin plus cold restraint stress 
produces spotty and narrow dark bands of linear macroscopic lesions which 
are usually found in the corpus fundic stomach. Therefore, forestomach and 
other accessory tissue which show no macroscopic lesion damage are 
trimmed. The remaining fundic portion is rinsed with water and placed flat 
in a standard position for photographing. The stomach along with a 
millimeter reference scale is photographed at 2X actual size with a 
Polaroid camera. Scoring of lesions is done from this permanent record. 
The lesion score is determined 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 is obtained by dividing the aggregate score by two. For each 
treatment group, a mean lesion score is determined. From this, the percent 
inhibition of lesion formation, I, is calculated as: 
##EQU2## 
The mean lesion score for a control group will be used to calculate "I" but 
individual lesion scores 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 exacerbation of lesion 
formation. 
In dose response studies, the ED.sub.50 (dose at which 50% inhibition of 
lesion formation occurs) is determined by probit analysis. 
HCl: 
The present method to produce gastric lesions in rats employs 3.0 ml/kg 
0.75N HCl as the necrotizing agent and provides additional data besides 
gastric lesions. 
Adult male Long-Evans rats weighing 275-300 grams (Blue Spruce Farms, 
Alton, N.Y.) are used. Animals are housed individually in stainless steel 
cages with wire-mesh bottoms. The housing battery is arranged into six 
groups of five each, individually numbered. Food and water are removed 24 
and 11/2 hours, respectively, prior to HCl. Animals receive the test 
compounds either p.o., s.c., or i.p. (3-12 ml/kg) 60 minutes before 
administration of 0.75N HCl (3.0 ml/kg, by gavage). For some purposes, 
different water removal and drug pretreatment times may be used. Sixty 
minutes after HCl 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 just above 
the esophageal sphincter with tweezers, the stomach is carefully lifted 
from the abdominal cavity. Two cuts are made, approximately 1/4 below the 
pyloric valve and 1/4 above the esophageal sphincter, and the stomach 
rapidly removed without any loss of gastric contents. The size of the 
stomach including contents is noted as small, medium or large. Stomachs 
are cut open along the greater curvature and the contents expressed into 
graduated centrifuge tubes. Gastric juice samples are centrifuged and 
total volume, and contents of solids and mucus are estimated to the 
nearest 0.1 ml. Na.sup.+ and K.sup.+ contents of the gastric juice as 
well as pH are determined. 
Under these conditions, 0.75N HCl produces prominent macroscopic lesions in 
the fundic stomach, but gross macroscopic changes are only very rarely 
observed in the forestomach. An occasional control animal shows some 
degree of redness or hyperemia and/or small petechiae in the forestomach, 
but never lesions characteristic of fundic damage. For these reasons, the 
forestomach (along with nongastric tissue) is removed and not examined. In 
addition, absence of the forestomach facilitates the photography and 
examination of the remaining stomach. Wet weight of the whole stomach is 
determined before removal of the forestomach. 
The remaining 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 length in mm and these 
scores are added together to determine total ulcer length for a stomach. 
Small well defined but quite visible spots (petechiae) are often present; 
a conglomerate length estimate is made for these and added to the total. 
Some variability ulcer scoring between persons may occur because even 
optimal quality photographs will show minor shadows, shades, overexposed 
spots and irregularly shaped ulcers. 
Standard photographs are about 2 X actual size. An estimate of real ulcer 
length (mm) is obtained after dividing the length score by two. For each 
treatment group, the mean length is calculated. From this, the percent 
inhibition of lesion formation, I, is calculated as: 
##EQU3## 
The mean ulcer score for a control group will be used to calculate "I" but 
normally individual lesion scores are used in drug treatment groups. In 
some cases, the observed treatment score is greater than the mean control 
value (exacerbation of lesion formation is not considered). In rare 
instances, the data from an animal will be discarded if, e.g., feces are 
found in the stomach. 
In dose-response studies, the ED.sub.50 (dose at which 50% inhibition of 
lesion formation occurs) is determined by probit analysis. 
Indomethacin 
Male Long Evans rats (approximately 275-300 gms) are housed 6 per cage and 
accilimated at least three days in a controlled environment while 
maintained on an ad libitum schedule of food and water. Prior to the 
experiment, rats are deprived of food for twenty-four hours and water for 
11/2 hour. On the day of the experiment, rats are randomly placed 
individually into a stainless steel cage with a wire mesh bottom. Control 
or drug treated groups consist of 5 rats each. Grouped rats receive a test 
drug or vehicle administered orally 30 or 60 minutes prior to the 
lesioning agent. Gastric lesions are induced by administration of 
indomethacin solution (30 mg/kg) by gavage (3.0 ml/kg). The indomethacin 
is prepared by dissolving 100 mg powder in 1.0 ml of lN NaHCO.sub.3 and 
heating until dissolved. Water is added to give a final indomethacin 
concentration of 10 mg/ml in 0.1N NaHCO.sub.3. Four hours after 
indomethacin the rats are sacrificed by injecting T-61 euthanasia solution 
(Hoechst). To determine duration or mechanism of action, different times 
or routes of compound administration are used. 
The stomachs are removed, opened along the curvature, rinsed with water and 
spread out flat in a standard position for examination and photographing. 
Under the conditions described, indomethacin normally produces prominent 
macroscopic lesions in the fundic glandular secreting portion of the 
stomach. 
Nonlesioned forestomach and other accessory tissues are trimmed off to 
improve standard placement. The remaining fundic tissue along with a 
millimeter reference scale is photographed at 2X actual size with a 
Polaroid camera with a close-up lens. Scoring of lesions is done from this 
permanent record. 
For each animal the severity of gastric lesions is defined as the sum of 
the maximum continuous lengths of the lesions (in mm). Barely visible 
spots (petechia) are scored as 1.0 mm each and added to the total length. 
Real lesion length (mm) is obtained after dividing the total length score 
by two. 
The mean lesion score is calculated for each treatment group. From this, 
the percent inhibition of lesion formation, I, is calculated: 
##EQU4## 
The mean lesion score for a control group will be used to calculate "I" but 
individual lesion scores 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 ED.sub.50 (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 ED.sub.50 values (mg/kg) for the various irritants, by oral treatment, 
are summarized in Table III. 
TABLE III 
______________________________________ 
Potency 
Ratio 
(Cimeti- 
Irritant Metaproterenol 
Cimetidine dine = 1 
______________________________________ 
ETOH 0.19 (0.03-0.47)* 
223 (191-256) 
1174 
Aspirin + 
11.3 (7.29-16.2) 
4.5 (2.0-7.1) 
0.4 
Stress 
HC1 8.9 (4.4-17.8) 225 (176-284) 
25.3 
Indome- 2.0 (1.0-3.7) 5.5 (3.9-7.3) 
2.8 
thacin 
______________________________________ 
*(95% confidence limit) 
In three out of four tests, metaproterenol was more effective than 
cimetidine (potency ratio range 2.75-1174). 
In view of the above test results, using metaproterenol which is a typical 
beta adrenergic agonist used commonly for testing such materials, beta 
adrenergic agonists are indicated as good gastroprotectant and 
antisecretory agents useful in the treatment of gastro-intestinal 
disorders such as gastroesophageal reflux disease (GERD), dyspepsia, undue 
gastric acid secretion, gastritis and peptic ulcer.

The following Examples 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 
______________________________________ 
aspirin 325 mg 325 mg 325 mg 325 mg 
metaproterenol 
3.33 mg -- -- -- 
isoproterenol 
-- 10 mg -- -- 
albuterol -- -- 2.67 mg -- 
terbutaline 
-- -- -- 1.67 mg 
______________________________________ 
To test the effectiveness of the beta adrenergic agonists in protecting the 
stomach against NSAID induced mucosal injury each beta adrenergic agonist 
is administered to dogs simultaneously and orally with, for example, 
aspirin in capsules. A standard dose of 975 mg of aspirin is administered 
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 injury. 
All test formulations are prepared on the day of the tests. The capsules 
are placed in the back of the dog's throat. A catheter with attached 
funnel is positioned in the dog's stomach and 50ml of deionized water is 
administered. 
Healthy adult beagle dogs of either sex are selected for testing. Dogs are 
housed individually in stainless steel cages with grid floors to allow 
excreta to pass through. Room temperature in the holding rooms and test 
laboratories is maintained between 65.degree. F. and 85.degree. F. and 
relative humidity of between 30% and 80%. Room lights remain on from 6:00 
AM to 4:00 PM. 
Each dog is trained to stand in a stanchion with sling support and to 
accept a bit tied in its mouth. A gastroscope is then passed through the 
bit into the dog's stomach. This training requires ten days to two weeks 
in most dogs. 
To determine whether a dog is suitable for test purposes, its stomach is 
examined for a normal mucosa, and its gastric responsiveness to aspirin is 
evaluated (as under Test Procedure). An acceptable gastric irritation 
score in the antrum must be 5 or greater, 2 hours after dosage. 
Food is withheld from test dogs for 24 hrs. before the test and during the 
test and water is allowed ad lib. The dogs are moved into a holding area 
away from the kennel. Fasted dogs of either sex are examined 
gastroscopically to ensure that their stomachs 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 hours later for gastric petechiae and signs of bleeding 
according to the following scale: 
0=uniform, pale to dark pink mucosa 
1=darker pink or blotchy mucosa 
2=petechias and/or light red streaks 
3=few small lesions 
4=many or connected small lesions (striations) 
5=few large lesions 
6=many large lesions 
7=massive hemorrhagic damage 
Severity of bleeding for each treatment and at each time is calculated as 
the mean gastric irritation score. 
In addition to the endoscopic observation of the gastric mucosa of each dog 
a qualitative description of gastric fluid is recorded and a pH 
measurement is made of the gastric fluid. All of these are done 2 hours 
after administration of the test product. 
A base line is established by measuring the various parameters after the 
administration of 975 mg of aspirin by itself. The resting stomach has an 
irritation score of 0 and a pH of 5 to 5.5. Aspirin alone produced injury 
which scored at approximately 5.5 after 2 hours and the gastric pH at this 
time is about 3.1. After 4 hours these values were 3.7 for the irritation 
factor and the pH was 4.8. This indicates that a certain amount of healing 
takes place between the 2nd and 4th hour after administration. 
The results of the various tests are summarized in Table IV below. 
TABLE IV 
______________________________________ 
2 Hr. Score 
Test Composition Injury pH 
______________________________________ 
aspirin (975 mg) 5.6 3.1 
aspirin (975 mg) + isoproterenol (7.5 mg) 
3.8 3.5 
aspirin (975 mg) + isoproterenol (15 mg) 
2.7 3.8 
aspirin (975 mg) + isoproterenol (30 mg) 
1.3 5.0 
aspirin (975 mg) + terbutaline (1.25 mg) 
4.0 2.9 
aspirin (975 mg) + terbutaline (5.0 mg) 
1.4 4.0 
aspirin (975 mg) + terbutaline (10.0 mg) 
1.2 4.6 
aspirin (975 mg) + albuterol (8 mg) 
1.0 5.4 
aspirin (975 mg) + metaproterenol (20 mg) 
0.75 5.7 
______________________________________ 
An examination of these data shows that the beta adrenergic agonists 
provide significant protection against aspirin induced mucosal injury 
particularly at the 2 hour level after administration. This protection, 
moreover, does not appear to be particularly related to the pH of the 
gastric contents.