U.S. patent number 3,852,413 [Application Number 05/268,641] was granted by the patent office on 1974-12-03 for labelled sulfated amylopectins and method of determining abnormal gastrointestinal mucosa.
This patent grant is currently assigned to G. D. Searle & Co.. Invention is credited to Peter S. Cammarata.
United States Patent |
3,852,413 |
Cammarata |
December 3, 1974 |
LABELLED SULFATED AMYLOPECTINS AND METHOD OF DETERMINING ABNORMAL
GASTROINTESTINAL MUCOSA
Abstract
Described herein are dye and radionuclide labelled anionic
polysaccharide compositions, useful as diagnostic aids in the
qualitative and quantitative detection in mammals of abnormal
mucosa, particularly of the gastrointestinal variety, a process for
their preparation and a method for their use. Preferred embodiments
are the compositions of: sodium amylosulfate combined with
technetium-99m and sodium amylosulfate combined with methylene
blue.
Inventors: |
Cammarata; Peter S. (Skokie,
IL) |
Assignee: |
G. D. Searle & Co.
(Chicago, IL)
|
Family
ID: |
27183532 |
Appl.
No.: |
05/268,641 |
Filed: |
July 3, 1972 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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52707 |
Jul 6, 1970 |
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Foreign Application Priority Data
Current U.S.
Class: |
424/1.69;
250/303; 536/2; 530/322; 530/370; 424/1.73; 424/9.1 |
Current CPC
Class: |
A61K
51/06 (20130101); A61K 49/04 (20130101); A61K
49/006 (20130101); A61K 2123/00 (20130101) |
Current International
Class: |
A61K
51/02 (20060101); A61K 49/00 (20060101); A61K
49/04 (20060101); A61K 51/06 (20060101); A61k
027/04 () |
Field of
Search: |
;424/1,7,9,180
;250/16T,71.5S ;260/209.5 ;23/23B |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Nuclear Science Abstracts, Vol. 24, No. 1, Jan. 15, 1970, page 87,
item 767..
|
Primary Examiner: Padgett; Benjamin R.
Attorney, Agent or Firm: Schubert; Elliot N.
Parent Case Text
This application for Letters Patent is a continuation-in-part of
Applicant's prior copending application, Ser. No. 52,707 filed July
6, 1970, now abandoned.
Claims
What is claimed is:
1. A composition comprising an alkali metal salt of sulfated potato
starch amylopectin, which is characterized by a molecular weight of
about 1-30 .times. 10.sup.7 and a sulfate content of about 1-1.8
sulfate groups per glucose unit, chemically combined with a
diagnostically effective, physiologically suitable labelling
agent.
2. The composition according to claim 1 wherein the alkali metal
salt is the sodium salt possessing about 1.6 sulfate groups per
glucose unit and characterized also by a molecular weight of about
6.3 .times. 10.sup.7.
3. A composition according to claim 1 wherein the alkali metal salt
is the sodium salt possessing about 1.8 sulfate groups per glucose
unit and characterized also by a molecular weight of about 12.5
.times. 10.sup.7.
4. The composition according to claim 1 wherein the diagnostically
effective, physiologically suitable labelling agent is a thiazin
dye.
5. The composition according to claim 1 wherein the diagnostically
effective, physiologically suitable labelling agent is methylene
blue.
6. A composition according to claim 1 comprising an alkali metal
salt of sulfated potato starch amylopectin, which is characterized
by a molecular weight of 1-30 .times. 10.sup.7 and a sulfate
content of 1-1.8 sulfate groups per glucose unit chemically
combined with 0.01 to 5 percent by weight of methylene blue.
7. A composition according to claim 1 wherein the diagnostically
effective, physiologically suitable labelling agent is a
radionuclide.
8. A composition according to claim 1 wherein the diagnostically
effective, physiologically suitable labelling agent is
iodine-125.
9. The composition according to claim 1 wherein the diagnostically
effective, physiologically suitable labelling agent is
iodine-131.
10. The composition according to claim 1 wherein the diagnostically
effective, physiologically suitable labelling agent is
technetium-99m.
11. A composition comprising the sodium salt of sulfated potato
starch amylopectin, which is characterized by a molecular weight of
about 6.3 .times. 10.sup.7 and a sulfate content of about 1.6
sulfate groups per glucose unit, chemically combined with
technetium-99m.
12. A method of detecting abnormal gastrointestinal mucosa which
comprises administering to a mammal a diagnostic dose of a
composition comprising a sulfated polysaccharide, which
differentially binds to the gastrointestinal mucosa, and which is
characterized by a molecular weight of at least one million and a
sulfate content of substantially 1-2 sulfate groups per
monosaccharide unit, chemically combined with a diagnostically
effective, physiologically suitable labelling agent, followed by
examination of the mucosa in order to determine those portions
containing bound labelled sulfated polysaccharide.
13. The method according to claim 12 wherein the polysaccharide is
an alkali metal salt of sulfated potato starch amylopectin, said
salt possessing about 1-1.8 sulfate groups per glucose unit and
characterized also by a molecular weight of about 1-30 .times.
10.sup.7.
14. The method according to claim 12 wherein the diagnostically
effective, physiologically suitable labelling agent is methylene
blue, which is visualized upon endoscopic examination of the mucosa
in order to determine those portions containing bound labelled
sulfated polysaccharide.
15. The method according to claim 12 wherein the diagnostically
effective, physiologically suitable labelling agent is
technetium-99m and the examination is conducted with a radiation
imaging device in order to determine those portions containing
bound labelled sulfated polysaccharide.
16. The method of detecting abnormal gastrointestinal mucosa in
mammals which comprises administering to the mammal a diagnostic
dose of a composition comprising the sodium salt of a sulfated
potato starch amylopectin possessing 1.8 sulfate groups per glucose
unit and characterized also by a molecular weight of 6.3 .times.
10.sup.7, chemically combined with the diagnostically effective,
physiologically suitable labelling agent, technetium-99m, followed
by examination with a radiation imaging device in order to
determine those portions containing bound labelled sulfated
polysaccharide.
Description
The detection and accurate measurement of abnormal mucosa (i.e.
mucous membrane), in mammals, especially humans, can be an
invaluable aid to the medical practitioner in the diagnosis of a
great number of disorders associated with the presence of such
abnormal mucosa. Illustrative of such disorders are gastric and
duodenal ulcers, carcinoma, benign lesions comprehending those with
diffuse mucosal changes frequently interpreted as gastritis,
oesophagitis, pre-ulcer, ulcerated colitis and similar pathological
conditions. In the past, the most common method employed for the
diagnosis of ulcers and stomach carcinoma was barium radiology
including the familiar barium meal followed by X-ray examination.
Unfortunately, the barium method suffers from several drawbacks
including the well known dangers of X-ray examination, and
inaccuracy and inconvenience in usage. For example, the barium
method is not susceptible of visualizing ulcers unless gross
changes have occured in the mucosa and the ulcerated area is
susceptible of planar visualization. Consequently, it is difficult
to detect gastric ulcers because it is frequently impossible to
assess the dimensional aspects of the depth and angles of the
abnormal area upon barium radiological examination. Conversely, in
certain subjects it has been found that barium radiology has shown
abnormally deep rugal folds, particularly on the greater curvature
of the body of the stomach, supposedly indicative of gastric
ulcers, whereas the presence of ulceration could be confidently
excluded from the areas suspected upon appropriate fibroscopic
examination, particularly utilizing as a diagnostic aid the
labelled compositions of this invention. In the case of X-ray
examination of the type employed in the barium meal method, i.e.
where the radiological source of energy is outside the subject, in
contradistinction to the source being within the subject as
hereafter described, there is the ever present danger of excess
radiation which can be harmful to both the radiological technician
and the subject, being particularly acute in the case of pregnant
patients.
The object of this invention is to provide a safe, simplified,
highly accurate, and convenient method for the detection in mammals
of abnormal mucosa, particularly of the gastrointestinal variety,
as well as a labelled composition suitable for use in this method.
According to this method, a labelled polysaccharide composition,
which binds strongly and differentially to mucosa, is administered
to the mammal and timely subsequent examination of the mammal by a
suitable visualizing technique provides definitive detection and
quantification of abnormal mucosa present in the tested area.
Polysaccharides suitable for the formulation of the compositions of
this invention are anionic polysaccharides having a high charge
density, conveniently of the order of 0.97 .times. 10.sup.-.sup.6
to 0.50 .times. 10.sup.-.sup.6 Z.sub.OH /(A.degree.).sup.3
(negative charge per Angstrom.sup.3) which characteristically bind
differentially to mucosa and include sulfated and phosphorylated
polysaccharides having a molecular weight of at least 1,000,000.
Among such sulfated polysaccharides are sulfated starch, sulfated
mequite gum, sulfated psyllium seed, sulfated amylopectin, sulfated
cellulose, sulfated amylose and sulfated hyaluronic acid. Among the
sulfated polysaccharides having specifically desirable application
in this invention are those containing one to two sulfate groups
per monosaccharide unit, for example per glucose unit in the case
of starch, starch fractions, and particularly corn and potato
amylopectin. It has been observed that polysaccharides lacking a
high charge density lack the ability of the polysaccharide
component of the instant compositions to differentially and
strongly bind to mucosa, particularly without causing deleterious
side effects to the subject, for example degraded carrageenin.
Particularly preferred sulfated polysaccharides for use in this
invention are the alkali metal, and especially sodium, salts of
sulfated potato starch amylopectin, said alkali metal salts
possessing about 1-1.8 sulfate groups per glucose unit and
characterized also by a molecular weight of about 1-30 .times.
10.sup.7. (The expression "molecular weight" as used in this
application refers to weight average molecular weight.) A very
preferred polysaccharide is the sodium salt of sulfated potato
starch amylopectin possessing substantially 1.6 sulfate groups per
glucose unit and characterized by a molecular weight of about 6.3
.times. 10.sup.7, which material is known by the non-proprietary
name "sodium amylosulfate". The preparation of the aforementioned
alkali metal salts of sulfated potato starch amylopectin and in
particular sodium amylosulfate, are described in U.S. Pat. No.
3,271,388, issued Sept. 6, 1966. The latter sulfated
polysaccharides are preferred components of the labelled
compositions of this invention because of their superior binding
properties which promote not only their uniform binding with the
labelling agent, but also their differential adhesion to mucosa,
which most importantly enables the resultant labelled compositions
of this invention to be characterized by this same advantageous
binding property permitting optimal physiological and diagnostic
results. Physiologically, for example, the labelled composition of
this invention is not only safe, in view of its low toxicity and
negligible absorption from the gastrointestinal tract, but
simultaneously provides therapeutic treatment for the abnormal area
as well as amazingly accurate visualization of the abnormality.
The choice of an appropriate labelling agent for use in the instant
composition is dependent upon numerous factors which affect the
diagnostic effectiveness and physiological suitability of the
labelling agent and the ultimate composition. Moreover, each of
these critical factors must be coordinated with the selection of
polysaccharide and visualizing instrument which is to be employed.
Consequently, considering the magnitude and complexity of choices
available, the safe, simplified, highly accurate and convenient
labelled composition provided by this invention is most unexpected
against the background of known materials which have been employed
for such purposes previously. Throughout this application the
expression "diagnostically effective" as applied to the labelling
agent of the instant composition shall refer to that combination of
properties, discussed with particularity hereinafter with respect
to both the dye and the radionuclide labelling agents, which enable
the agent, as well as the composition in which it is incorporated,
to permit definitive detection and quantification of mucosal
abnormalities by facilitating a satisfactory visualization of the
abnormality by the imaging instrument employed. The expression
"physiologically suitable" as applied to the labelling agent of the
instant composition refers to those properties discussed with
particularity hereinafter with respect to both the dye and
radionuclide labelling agents, which enable the agent, as well as
the composition in which it is incorporated to produce a
diagnostically effective response while assuring patient and
technician safety. It has been found that in view of their
respective properties, certain radionuclides and certain dyes can
meet the stringent requirements of "diagnostically effective" and
"physiologically suitable" and consequently constitute the
appropriate labelling agents of the instant compositions.
Specifically, in the selection of an appropriate dye for use in
this invention from the standpoint of diagnostic effectiveness, one
must ascertain that they exhibit strong light absorption and a
capacity to provide the desired contrast. Suitably such properties
are present in materials having at least 1/10 the light absorption
as does methylene blue. As a measure of its absorption, it is noted
that methylene blue has a molecular extinction coefficient of
80,000 at 658 millimicrons in water. In assessing physiological
suitability, appropriate dyes are those which have been found to
substantially lack toxicity in mammals at diagnostic doses, i.e.
doses sufficient to permit visualization of the abnormality upon
endoscopic examination, and which chemically combine with the
selected polysaccharide to form a uniformly stained composition.
Such dyes include basic aniline dyes, including azins (e.g.,
phenosafranine, methylene violet, diethylphenosafranine, etc.),
pyronins (e.g., acridine red 3B, pyronin B, etc.), oxazins (e.g.,
brilliant cresyl blue, cresyl fast violet, nile blue [sulfate]),
basic azo dyes (e.g., Janus green, indazole blue, Bismark brown,
etc.), triphenyl- and diphenylnaphthylmethanes (e.g., malachite
green, pararosaniline chloride, pararonsaniline acetate, methyl
violet, ethyl green, Victoria blue, etc.), phthalocyanines (e.g.,
alcian blue, luxol fast blue, etc.), and preferably the thiazins
(e.g., thionin, azure A, B and C, methylene green, toluidine blue
and especially methylene blue). Other polysaccharide staining dyes
can also be used including fluorochrome stains such as the
fluoresceins, fat stains such as sudan black B, the tetrazoles
(e.g., blue tetrazolium), and food dyes such as F. D. and C. No. 1
(food and drug color No. 1). As indicated, the thiazins are the
preferred dyes for the dye labelling component of the instant
diagnostic compositions, with methylene blue being most preferred,
in view of their suitable extinction coefficient, which is both
strong as well as providing the desired contrast, and their
substantial lack of toxicity. A very preferred embodiment of this
invention comprises the labelled composition containing preferably
no greater than 2 percent, but conveniently within the range of
about 0.01 to 5.0 percent, by weight of methylene blue and
preferably no less than 90 percent by weight of sodium
amylosulfate. Said very preferred embodiment is especially
advantageous as a diagnostic reagent in comparison with methylene
blue used alone because of the former's superior binding qualities.
That is, the intensity of color in the stained area is greater and
persists longer than methylene blue when employed as a stain apart
from the instant composition.
The choice of an appropriate radionuclide for use as an instant
labelling agent is likewise dependent upon its ability to be
diagnostically effective and physiologically suitable. With respect
to a radionuclide, the term "physiologically suitable" comprehends
the properties of: (1) short half life, (2) essentially pure gamma
emission and no substantial emission of beta-particles, and (3)
chemical compatibility with the selected polysaccharide such that
negligible absorption of the resultant composition occurs from the
gastrointestinal tract. The importance of a short half life, that
is one no greater than 60 days and more preferably of considerably
low magnitude such as 2-24 hours, stems from the fact that the
radiation dose to the patient should be an intense burst of high
energy in order to maximize effectiveness and safety, since high
energy radiation is better tolerated by mammals than low energy
radiation. The use of an essentially pure gamma emittor provides an
additional safety factor because it has been observed that
radiation damage is frequently the result of the absorption of beta
emissions, rather than gamma emissions. Generally gamma energy of
0.03-2 Mev is desirable for an optimal and safe result. Among those
characteristics maximizing the diagnostic effectiveness of a
radionuclide labelling agent is its chemical ability to bind
irreversibly to the polysaccharide carrier. Such property is
essential to an effective diagnostic aid since less than
essentially quantitative and irreversible combination would
provide: (1) an aggregate of label in the composition which would
not permit accurate visualization of the abnormality, or (2)
loosely bound or unbound radionuclide or nuclide labelled
composition which would result in the loss of the material
throughout the body of the mammal being studied and, perhaps,
necessitate the administration of an unusually high dosage of of
labelled composition in order to achieve any useful visual result.
For obvious reasons of safety an unusually high radiation dosage is
to be avoided. A significant factor which must be considered in the
selection of a diagnostically effective radionuclide for use in the
instant labelled composition is its ability to be visualized on
existing instrumentation. Many types of radiation imaging equipment
provide varying tolerances of isotope energy, most commonly in the
range of 20-2000 Kev, though this wide range is not necessarily
available on any one instrument currently marketed. Among possible
radionuclides for use in this invention are iodine-131, iodine-125,
iodine-123, indium-111, indium-113m, iodine-132, indium-114m,
ytterbium-169, gallium-67, dysprosium-157, mercury-203,
mercury-197, gold-198, bismuth-204, chromium-51 and most preferred,
in view of its short half life, ideal lack of beta emission and
ability to be imaged on both rectilinear scanners and scintillation
cameras, is technetium-99m. Additional radionuclides, which because
of unusually long half life, failure to be adequately imaged on
present detectors, etc. are less desirable, though nevertheless of
potential usefulness as advances in radiation imaging
instrumentation may provide better energy resolution enabling use
of lower dosages of radionuclides, include selenium-75, gallium-68,
cobalt-57, samarium-153, and lutetium-177.
The dye labelled compositions can be manufactured by mixing the dye
with the selected polysaccharide in a suitable medium followed, if
desired, by dialysis of the resulting product to enhance its
purity. Alternatively other conventional methods of staining
carbohydrate materials may be employed.
The radionuclide labelled compositions of this invention can be
prepared by methods analagous to those used for the labelling of
proteins, such as albumin, and fats such as triolein, as well as
other radiolabelled carbohydrate materials known in the art. As an
illustration of such a procedure the preferred polysaccharide
material of this invention, sodium amylosulfate, was combined with
.sup.125 I-sodium iodide in a suitable solvent such as acetone, in
the optional presence of an oxidizing agent, such as
(N-chloro-p-toluenesulfonamido) sodium. After stirring and cooling,
the resultant composition is optionally dialyzed against water to
provide greater purity of the .sup.125 I-sodium anylosulfate
composition product thereof. The technetium-99m labelled
compositions of this invention are most conveniently prepared by
utilization of a reducing agent such as sodium borohydride,
ascorbic acid, a source of stannous ion, illustratively stannous
chloride, or most preferably a source of ferrous ion, such as
ferric chloride and ascorbic acid or ferrous chloride. The most
preferred procedure entails the addition of a few drops of
hydrochloric acid to a saline solution of .sup.99m Tc-sodium
pertechnetate, followed by the addition of a reducing agent such as
ferric chloride coupled with ascorbic acid. The mixture is
thereafter permitted to stand for several minutes, after which time
upon adjustment of pH, the desired sulfated polysaccharide, such as
sodium amylosulfate, is added to the reaction mixture, and the pH
suitably adjusted to provide the desired technetium-99m labelled
sodium amylosulfate. Any unbound technetium-99m may be removed by
dialysis or by passing a solution of the product through an ion
exchange or gel filtration column.
As indicated above, the selection of radionuclide labelling agent
for incorporation in the composition of this invention is dependent
upon the visualizing instrument which is to be used for the
detection of abnormal mucosa. Described hereinafter are several
types of radiation imaging devices currently in use, each of which
can be used with a least one or more of the instant radionuclide
labelled compositions. Specifically, the technetium-99m labelled
compositions of this invention are detectable on all of the
instruments discussed hereinafter and consequently these
radionuclide labelled polysaccharides are particularly preferred
embodiments of this invention. Two basic types of radiation imaging
devices are rectilinear instruments or scanners, whose detecting
heads move back and forth over the scanning area; and scintillation
cameras, which are characterized by their ability to produce a
unitary image of the object under study at any given moment.
With respect to the rectilinear scanner, the concentration of
radionuclide in the target organ is outlined by a corresponding
pattern of dots. Various black and white printout systems are often
employed. However, systems in which the color of the printout
changes with the count rate may be of particular value when
scanning areas of gradual change in concentration. A typical
rectilinear scanner is Nuclear-Chicago Corporation's PHO/DOT
Isotope Scanner, which automatically produces a display of the
location and concentration of isotopelabelled compositions within
selected organs or areas of the body. Data is recorded on X-ray
film by a photorecording system (photoscans) and is also printed on
paper by a dot recording system (dotscans). The PHO/DOT has the
ability to visualize isotopes within the energy range of 20-2000
Kev.
The scintillation camera, commonly known as the Anger camera, is
described in its basic embodiment in U.S. Pat. No. 3,011,057. Such
camera devices have the feature of viewing at least a substantial
portion of the object under study at any one time, thus
significantly reducing examination time in comparison to
rectilinear scanners. A typical scintillation camera,
Nuclear-Chicago Corporation's PHO/GAMMA Scintillation Camera,
includes an image or detector head containing a sodium iodide
thallium activated scintillation crystal and a matrix of
photomultiplier tubes; interchangeable collimators (multiaperture
or pinhole); and image data computation, display and control
modules. The distribution of radionuclides is often imaged on
Polaroid film (scintiphotos). The PHO/GAMMA instrument has the
ability to visualize isotopes within the energy range of 50-680
Kev.
Very recent improvements of the scintillation camera are those
having tomographic imaging capabilities. These involve various
additions to the basic Anger camera system such as those described
in Canadian Pat. Nos. 872386 and 872387, in German
Offenlegungsschrift No. 2011164 and in an article by G.
Muehllehner, "A Tomographic Scintillation Camera", Physics in
Medicine and Biology, Vol. 16., No. 1, pp. 87-96 (1971).
Nuclear-Chicago Corporation's PHO/GAMMA Tomocamera.sup.tm Accessory
System comprises the conventional PHO/GAMMA Scintillation Camera, a
rotating slanted hole collimator assembly, a floating-top
radiographic table and an associated operating control module, and
has the ability to image data from four distinct parallel
enatomical planes in a single display.
The instant compositions can be combined with pharmaceutically
acceptable carriers suitable for the administration of such a
diagnostic aid. Obviously, the chemical properties of the
particular labelling agent chosen for use in the composition will,
in practice, limit the type of carriers with which it can be
combined. For example, the instant technetium-99m composition,
because of the short half life of technetium-99m, can most
conveniently be used as a liquid of relatively few ingredients,
since compounding time for complicated formulations would reduce
the usefulness and practicality of the end product. The term
"pharmaceutically acceptable carrier" as used herein in reference
to the compositions of this invention means a solid or a liquid
composed of a single substance or a number of substances which may
be solids, liquids or a combination of solids and liquids each of
which is less toxic than an equal weight of the active ingredient
present in the composition when measured in the same mammalian host
using the same method and conditions of administration. The
concentration of the active ingredient in the composition is not
critical, but for economy of preparation, when a dye is the
labelling component, the concentration should be at least 0.5
percent by weight and preferably 1-80 percent. An illustration of a
suitable radionuclide composition is that containing 0.5 mg to 10
mg. of sodium amylosulfate per kilogram weight of the mammalian
patient species (1.7mg./kg. to 5mg./kg. being the more preferred
range) and 0.1 to 10 mCi "milliCuries" of a radionuclide such as
technetium-99m (more preferably in the amount of 2 milliCuries).
These compositions can be administered either orally or
parenterally. For oral administration, tablets, lozenges, capsules,
dragees, pills and powders are suitable, while aqueous and
non-aqueous solutions or suspensions are appropriate for both oral
(e.g. in the case of radionuclide labelling agents via a stomach
tube) and parenteral (e.g. rectal) administration. Acceptable
pharmaceutical carriers are exemplified by chewable tablets, sugars
such as lactose or sucrose, starches such as corn starch or potato
starch, cellulose derivatives such as sodium carboxymethyl
cellulose, ethyl cellulose, methyl cellulose or cellulose acetate
phthalate, talc, calcium phosphates such as dicalcium phosphate or
tricalcium phosphate, sodium sulfate, calcium sulfate, polyvinyl
alcohol, acacia, stearic acid, alkaline earth metal stearates such
as magnesium stearate, vegetable oils such as peanut oil,
cottonseed oil, sesame oil, olive oil, corn oil, and oil of
theobroma, water, agar, alginic acid, benzyl alcohol, isotonic
saline and phosphate buffer solution as well as other non-toxic
compatible substances used in pharmaceutical formulations. Some
typical formulations as well as the method of preparation thereof,
are presented below.
__________________________________________________________________________
Formulation I
__________________________________________________________________________
Liquid Composition Containing 2% Methylene Blue: Sodium
Amylosulfate
__________________________________________________________________________
Amount Ingredient (Conc. 250 mg/5 ml.) (Conc. 500 mg/5 ml.)
__________________________________________________________________________
Composition of methylene 250 mg. 500 mg. blue with sodium amylo-
sulfate (in a ratio of preferably 2:100, res- pectively) Distilled
Water 5 ml. 5 ml.
__________________________________________________________________________
To the composition of methylene blue with sodium amylosulfate was
added slowly, with stirring, 100 ml. of distilled water. To this
slurry was added, with stirring, sufficient distilled water to
bring the mixture to the volume of 1 liter and stirring was
continued until solution was complete. The solution was then poured
into amber bottles of the desired quantity, for example, 5
milliliter portions, sampled and assayed.
______________________________________ Formulation II
______________________________________ Chewable Tablet Composition
Containing 2% Methylene Blue: Sodium Amylosulfate
______________________________________ Amount Ingredient Per Tablet
Per Batch of 1000 ______________________________________
Composition of methylene 500 mg. 500 g. blue with sodium amylo-
sulfate (in a ratio of pre- ferably 2:100, respectively) Dextrose
USP, anhydrous 1.66 mg. 1660 g. Polyvinylpyrrolidone 67.4 mg. 67.4
g. Hydrogenated cottonseed oil 22.6 mg. 22.6 g. Flavor (e.g.
Florasynth en- 1.76 mg. 1.76 g. trapped black cherry flavor)
______________________________________
500 Grams of the composition of sodium amylosulfate with 2 percent
methylene blue (powder form) was blended with 1660 grams of
dextrose, and 67.4 grams polyvinylpyrrolidone in a large bowl for 5
minutes. That mixture was thereafter comminuted at high speed,
remixed, granulated with 300 ml. of ethanol, and dried for 4 hours
at 60.degree.C. After oscillation through a No. 12 screen, followed
by the addition of 22.6 grams of hydrogenated cottonseed oil and
1.76 grams of Florasynth entrapped black cherry flavor, the mixture
was blended for 5 minutes and a sample was taken for assaying.
Thereafter it was compressed into tablets of the appropriate
size.
The method of this invention for the detecting in mammals of
abnormal mucosa, particularly of the gastrointestinal variety,
comprises the administration of a diagnostic dose of the instant
novel diagnostic composition. The term "diagnostic dose" is defined
as the amount of active ingredient that will adequately adhere to
the mucosa and effect labelling thereof sufficient to permit
detection upon examination, endoscopically in the case of dye
labelling agents and by radiation imaging devices in the instance
of radionuclide labelling agents. This dose will vary with the
particular labelling agent chosen, the type and location of the
particular disorder being diagnosed, the route of administration,
the particular polysaccharide used, and the subject's physical
characteristics, e.g. body weight and physiological state such as
hypersecretory. A typical diagnostic dosage for a subject of about
22 to about 136 kg. body weight for the composition of sodium
amylosulfate with up to 2 percent methylene blue is 100 to 500
milligrams, administered about 1/4 hour prior to endoscopic
examination for gastric ulcer. For a subject of identical weight
and disorder, 2 milliCuries of techetium-99m combined with 250 mg.
of sodium amylosulfate, administered 1-2 hours prior to examination
with a radiation imaging device, would be a typical diagnostic
dosage.
Application of the instant method to the diagnosis of abnormal
gastric mucosa, particularly ulcers, has been found to be
especially effective when the preferred dye labelled composition of
this invention, i.e. sodium amylosulfate with no greater than 2
percent by weight methylene blue, is administered to the patient 15
minutes prior to endoscopic examination employing a gastroscope
(i.e. a hollow tubular instrument designed to pass into the stomach
by way of the mouth and esophagus and fitted with optical and
lighting equipment that permits visual inspection of the stomach)
to which a camera suitable for color photography has been affixed.
As will be apparent to those skilled in endoscopic identification,
the light source for the gastroscope may be visible or ultraviolet
lighting dependent upon the particular dye employed in the
composition. In a very preferred embodiment of this invention,
methylene blue, which may be viewed by visible light, serves as the
dye.
In one clinical illustration of this very preferred method and
diagnostic aid of this invention, a human subject, previously known
to possess an ulcer as diagnosed by barium radiology, was
administered 5 ml. of an aqueous solution containing 250 mg. of the
composition comprising 2 percent methylene blue with sodium
amylosulfate. Fifteen minutes later, gastroscopic examination of
the patient accompanied by color photography of the area in
question revealed the ulcer. The visualization of the ulcer, both
to the naked eye and in the color photographs, was marked by
sharpness of color, namely bright blue, at the edges of the ulcer
crater, while the crater was differentially deprived of such color.
Moreover, the shape and character of the ulcer as well as an
indication of depth was apparent upon inspection. Thus, in contract
and preference to X-ray pictures produced via the barium meal
method, wherein the crater itself is often poorly defined or
distorted, the shape and character of the ulcer or other
abnormality in the mucosa is easily detected and quantified. Hence
the instant diagnostic composition and method are particularly
useful in detecting aberations of gastric mucosa since the
characteristics of the stomach (its asymmetry, flabbiness and
variability in shape and motility) make it difficult to examine by
radiological techniques employing a source of radioenergy ouside
the subject, such as the barium meal method. Most effective and
desirable results are obtained when the radioenergy source is
placed within the subject, as with the instant compositions wherein
the labelling agent is a radionuclide, most preferably
technetium-99m, as illustrated in Examples 3 to 11 described
hereinafter.
The following examples are given for the purpose of illustrating
the preparation of the instant diagnostic composition according to
the present invention. It will be understood that the invention is
not to be construed as limited in spirit or in scope by the details
contained therein, as many modifications in materials and methods
will be apparent from this disclosure to those skilled in the art.
In the following examples, temperatures are given in degrees
Centigrade (.degree.C.)
EXAMPLE 1
A 1 percent aqueous solution of sodium amylosulfate (i.e., the
sodium salt of sulfated potato starch amylopectin, possessing
substantially 1.6 sulfate groups per glucose unit, characterized by
molecular weight of about 6.3 .times. 10.sup.7 and having been
prepared according to the method described in Example 4 of U.S.
Pat. No. 3,271,388) was placed inside a dialysis bag, and the bag
was then placed inside a container having a 0.5 percent solution of
methylene blue (U.S.P.) in water. The ratio of methylene blue
solid, in the outer compartment, to sodium amylosulfate solid, in
the inner compartment, was 2/100. The two solutions were allowed to
equilibrate for 24 hours, after which time practically all the
methylene blue had diffused into the dialysis bag containing the
sodium amylosulfate solution. At the end of this time, the dialysis
bag was removed from the exhausted methylene blue outer solution
and placed in a large container of hydrochloric acid at pH 2, where
it was dialyzed for 24 hours. No detectable methylene blue was
removed by 24 hour dialysis against the acid solution, proving the
irreversible binding of methylene blue to sodium amylosulfate under
acid conditions analogous to that of gastric juice. At the end of
the acid dialysis period, the contents of the dialysis bag were
neutralized to a pH of 7.4 with sodium hydroxide and
lyophilized.
Alternatively, upon completion of the 24 hour equilibration
described above, the dialysis bag containing the sodium
amylosulfate-methylene blue solution was removed from the exhausted
methylene blue outer solution and was adjusted to a pH of 7.8 with
sodium hydroxide and the solution was thereafter lyophilized for 72
hours. The lyophilized material was first extracted with ethanol
and then with redistilled acetone, followed by filtration. The
resultant filtercake was dried in vacuo for 48 hours to provide a
composition, identical with that prepared in the preceding
paragraph containing 2 percent methylene blue with about 93 percent
sodium amylosulfate, which composition is characterized by
ultraviolet absorption maxima at about 246, 285 and 575
millimicrons. It will be observed that the methylene blue component
individually displays ultraviolet absorption maxima at about 609
and 667 millimicrons. Thus by comparison with the maxima described
above for the prepared composition, this metachromatic behavior is
evidence that the composition consists of a unique composition of
matter distinguishable from its component, methylene blue.
EXAMPLE 2
A 0.1 percent solution of methylene blue in water was pumped
slowly, over a 48 hour period, at a rate of 0.1 ml./min., into a
rapidly stirred 1.0 percent solution of the alkali metal salt of
sulfated potato starch amylopectin possessing 1-1.8 sulfate groups
per glucose unit and being characterized also by a molecular weight
of 1-30 .times. 10.sup.7 in water. After the solutions were
combined, the dyed salt solution was dialyzed against acid, then
against water, and finally lyophilized and purified as described in
the above example to provide a composition containing 2 percent
methylene blue and an alkali metal salt of sulfated potato starch
amylopectin possessing 1-1.8 sulfate groups per glucose unit and
being characterized also by a molecular weight of 1-30 .times.
10.sup.7.
EXAMPLE 3
To a suspension of 503 mg. of sodium amylosulfate in 10 ml. of
acetone was added a solution of 1 mCi of carrier-free .sup.125
I-sodium iodide in 10 ml. of acetone. To that mixture was then
added 10 .mu.1. of an aqueous solution of Chloramine-T, i.e.
(N-chloro-p-toluenesulfonamido)sodium [concentration = 2 mg./ml.],
and the resultant mixture was stirred at 60.degree.C. for about 2
hours, then allowed to cool to room temperature over a period of 1
hour. The mixture was diluted with approximately 50 ml. of water
and then dialyzed against three 5.5 liter portions of water during
a period of 4 days, using cellulose dialyzer tubing. The contents
of the tubing were then lyophilized, affording approximately 0.45
g. of .sup.125 I-sodium amylosulfate composition having a specific
activity of 18.9 .mu.Ci/g.
A solution containing the .sup.125 I-sodium amylosulfate product
described above in 50 ml. of water was administered
intragastrically to a 7.8 kg. female dog (Beagle). After waiting 5
minutes, the stomach region was visualized by means of a
Nuclear-Chicago Corporation PHO/DOT Isotope Scanner. The dot and
photo scans demonstrated the binding effect of .sup.125 I-sodium
amylosulfate to the muscosa observed.
EXAMPLE 4
To a suspension of 503 mg. of sodium amylosulfate to 10 ml. of
acetone was added a solution of 1 mCi of carrier-free .sup.125
I-sodium iodide in 10 ml. of acetone. That mixture was stirred at
60.degree.C. for about 2 hours, then allowed to cool to room
temperature over a period of 1 hour. The resultant mixture was
transferred to dialysis tubing with approximately 2 ml. of acetone
and 60 ml. of water and then dialyzed against 5.5 liters of water
for about 2 hours. The contents of the tubing were lyophilized.
There were obtained approximately 409 mg. of .sup.125 I-sodium
amylosulfate composition having a specific activity of 18.0
.mu.Ci/g.
EXAMPLE 5
501.5 Mg. of sodium amylosulfate, 5.1 ml. of water containing 1 mCi
of carrier-free .sup.131 I-sodium iodide, and an aqueous solution
of 4.4 .times. 10.sup.-.sup.11 millimoles of Cloramine-T were
combined and the required amount of water needed to bring the total
amount of water to 50 ml. was added. The resulting mixture,
containing .sup.131 I-sodium amylosulfate, was administered
intragastrically to a 7.8 kg. dog (Beagle) and the stomach region
was visualized by means of a Nuclear-Chicago Corporation PHO/GAMMA
Scintillation Camera. Scintiphotos were taken over a period of 21/2
hours. Binding to the gastrointestional mucosa was observed
throughout the length of the visualization period.
EXAMPLE 6
A solution of 3.69 mCi of carrier-free .sup.99m Tc-sodium
pertechnetate in 2 ml. of saline (obtained by elution from a
.sup.99 Mo-.sup. 99m Tc generator) was acidified with 3 drops of 2
N aqueous hydrochloric acid solution. To that solution were added
10 mg. of ferric chloride hexahydrate, followed by 8 mg. of
ascorbic acid. The acidity of the resulting solution was adjusted
to approximately pH 5 by addition of 14 drops of 1N aqueous sodium
hydroxide solution. 500 Mg. of sodium amylosulfate were then added
and the mixture was stirred for about 5 minutes. At the end of that
time, the mixture was passed through a small Sephadex
chromatographic column in order to remove free technetium. The
first 25 ml. of eluate were discarded and the next 50 ml. were
collected. The latter eluate contained .sup.99m Tc-sodium
amylosulfate with an activity, corrected to zero time, of 0.0426
mCi/ml. (total radioactivity = 2.1 mCi). 43 Ml. of that solution
(radioactivity = 1.8 mCi) were administered to a 7.8 kg. dog
(Beagle) for gastrointestinal visualization using a Nuclear-Chicago
Corporation PHO/GAMMA Scintillation Camera. Scintiphotos were taken
beginning at about 4 minutes after administration and continuing
for 31/2 hours. Binding to the gastrointestinal mucosa was observed
throughout the length of the visualization period.
EXAMPLE 7
A solution of 3.4 mCi of carrier-free .sup.99m Tc-sodium
pertechnetate in 2 ml. of saline (obtained by elution from a
.sup.99 Mo-.sup. 99m Tc generator) was stirred with 500 mg. of
sodium amylosulfate in 50 ml. of water for 5 minutes. The resultant
mixture was poured into dialysis tubing and dialyzed against three
5.5 liter portions of water during a period of about 51/2 hours.
There was thus obtained 25 ml. of a .sup.99m Tc-sodium amylosulfate
composition having a total radioactivity, corrected to zero time,
of about 0.12 mCi.
EXAMPLE 8
A solution of 3.4 mCi of carrier-free .sup.99m Tc-sodium
pertechnetate in 2 ml. of saline (obtained by elution from a
.sup.99 Mo-.sup. 99m Tc generator) was stirred while adding 3 drops
of 2 N aqueous hydrochloric acid solution. There were then added,
with stirring, 10 mg. of ferric chloride, followed by 8.5 mg. of
ascorbic acid. The acidity of the resulting solution was adjusted
to pH 4.5-5.5 with 1 N aqueous sodium hydroxide solution. 503 Mg.
of sodium amylosulfate were added and stirring was continued for
about 30 minutes. The resultant solution was transferred to
dialysis tubing and dialyzed against three 4 liter portions of
water during a period of about 51/2 hours. There were obtained 28
ml. of a .sup.99m Tc-sodium amylosulfate composition having a total
radioactivity, corrected to zero time, of 1.24 mCi.
EXAMPLE 9
A solution of 3.86 mCi of carrier-free .sup.99m Tc-sodium
pertechnetate in 2 ml. of saline (obtained by elution from a
.sup.99 Mo-.sup.99m Tc generator) was acidified with 3 drops of 2 N
aqueous hydrochloric acid solution. To that solution were added
17.5 mg. of ferric chloride, followed by 16 mg. of ascorbic acid.
The acidity of the resulting solution was adjusted to pH 4.5-5.5 by
the addition of approximately 14 drops of 1 N aqueous sodium
hydroxide solution. 500 Mg. of sodium amylosulfate were then added
and the mixture was stirred for about 1 hour. At the end of that
time, the mixture was passed through a small Sephadex column. The
first 20-30 ml. eluted from the column were discarded. The next 50
ml. which were eluted were collected. There were thus obtained 50
ml. of a .sup.99m Tc-sodium amylosulfate composition having a total
radioactivity, corrected to zero time, of 3.36 mCi.
EXAMPLE 10
A solution of 3.86 mCi of carrier-free .sup.99m Tc-sodium
pertechnetate in 2 ml. of saline (obtained by elution from a
.sup.99 Mo-.sup. 99m Tc generator) was acidified with 3 drops of 2
N aqueous hydrochloric acid solution. To that solution were added
17.5 mg. of ferric chloride, followed by 16 mg. of ascorbic acid.
The acidity of the resulting solution was adjusted to pH 4.5-5.5 by
the addition of approximately 14 drops of 1 N aqueous sodium
hydroxide solution. 500 Mg. of the sodium salt of sulfated potato
starch amylopectin possessing 1.8 sulfate groups per glucose unit
and being characterized also by a molecular weight of 12.5 .times.
10.sup.7 were then added and the mixture was stirred for about 1
hour. At the end of that time, the mixture was passed through a
small Sephadex column. The first 25 ml. eluted from the column were
discarded. The next 50 ml. which were eluted were collected. There
were thus obtained 50 ml. of a composition containing the sodium
salt of sulfated potato starch amylopectin possessing 1.8 sulfate
groups per glucose unit and being characterized by a molecular
weight of 12.5 .times. 10.sup.7, labelled with technetium-99m.
EXAMPLE 11
A solution of 3.69 mCi of carrier-free .sup.99m Tc-sodium
pertechnetate in 2 ml. of saline (obtained by elution from a
.sup.99 Mo-.sup.99m Tc generator) was acidified with 3 drops of 2 N
aqueous hydrochloric acid solution. To that solution were added 10
mg. of ferric chloride, followed by 8 mg. of ascorbic acid. The
acidity of the resulting solution was adjusted to approximately pH
5 by the addition of 14 drops of 1 N aqueous sodium hydroxide
solution. 500 Mg. of the alkali metal salt of sulfated potato
starch amylopectin possessing 1-1.8 sulfate groups per glucose unit
and being characterized also by a molecular weight of 1-30 .times.
10.sup.7 were then added and the mixture was stirred for about 5
minutes. At the end of that time, the mixture was passed through a
small Sephadex column. The first 25 ml. of eluate were discarded
and the next 50 ml. were collected. The latter eluate contained the
aforementioned alkali metal salt of sulfated potato starch
amylopectin, labelled with technetium-99m.
* * * * *