Note: Descriptions are shown in the official language in which they were submitted.
W094/~ 2 1 6 4 ~ 3 7 PCT~S94/06653
T~ERAP~ulIC COMPOSITIONS AND MET%ODS OF ~SB
Field of the Inventlon
The invention relates to admixture compositions of
short chain organic fatty acids which have b ological effects.
For example, they are useful in the treatment of numerous
pathologies that affect mAmmAls. The invention also relates to
methods of treatment of such pathologies, wherein appropriate
therapeutically effective amounts of the compositions are
A~m; n; stered.
Back~round of the Invent~on
A wide variety of carboxylic acid cont~;ning
compositions are biologically active, and several short chain
organic acids which are naturally occurring acids have previously
been implicated in the treatment of various pathological
conditions. For example, Nordman, U.S . Patent No. 3,291,6~9,
discloses the treatment of hepatic ~mmo~; A intoxication with a
mixture of L-arginine and malic acid. U.S. Patent No. 3,718,664
discloses the use of thioctic acid derivatives in the treatment
of acidosis.
Sloa~, U.S. Patent No. 4,381,307, di~closes soft
tertiary amine ester derivatives that have biological effects,
while U.S. Patent No. 4,760,078 discloses a 1,2, dithiol-3-thione
derivative.that has an ;mmllnomodulating effect.
Rubenstein, U.S. Patent No. 4,971,760, discloses the
sterilization of blood, blood constituents, and transplant
tissues with a disinfectant that includes lactic acid and sodium
chlorite. Naphtholic acid derivatives were discovered to be
useful in enhancing oxygen availability to m~mmAlian tissue by
Suh et al., U. S. Patent No. 5,015,663.
WOg4/2~ 2 1 6 4 8 3 7 PCT~S94/06653
Hoffler et al., U.S. Patent No. 5,043,357, disclose a
virucidal agent that is predomln~ntly comprised of ethanol and/or
alcohol but which includes a minor amount of a short chain
organic acid. Further, U.S. Patent No. 5,093,140 discloses an
aqueous bactericide that contains organic acids. This solution
is used in the scalding or washing stages of meat dressing.
~ross et al., U. S. Patent No. 5,100,652 disclose an oral
disinfectant that contains organic acids. See also Comroe et
al., The Lung, 1955 Yearbook of Medical Publications Inc., Chptr.
4; John West, Respiratory Physiology, The Williams & Wilkins Co.,
Chptr. 6; Hypoxia, Metabolic Acidosis and Circulation,
ARTCF...Oxford; Arnold et al, "Excessive Intracellular Acidosis
of Skeletal Muscle on Exercise in a Patient with a Post-Viral
Exhaustion/Fatigue Syndrome" The Lancet: 6/23/84; Schweckendiek,
W., ~Heilung von Psoraiasis vulgaris", Med. SschS, 13:103-4,
1959; G.E. Abraham et al., "Rationale for the Use of Magnesium
and Malic Acid", Journal of Nutritional Medicine, 3:49-49 (1992);
Kuroda, Z. and Akano, M., "Antitumor and Anti-intoxication
Activities of Fumaric Acid in Cultured Cells n, G~nn, 727:77-782
(19~1); Chemucal Abstracts 93945Y, 116, Mar 1992 "Antifungal
Activity of Fumarates in Mice Infected with C.Albicansn; Bauer et
al., "Cllnishe Wochenschrift 1991 69(2), pp. 722-4; for further
details of several pathological processes which are susceptible
to treatment according to the present invention.
It has now been discovered that ~m~tures of specific
short chain organic acids have broad therapeutic effects.
Summary of the Inventlon
According to the present invention, there i~ provided
a composition which comprises an ~m; ~ture of sorbic acid,
aconitic acid, malic acid, fumaric acid, crotonic acid, and
optionally aconitic acid.
Further contemplated by the present invention are
methods for treating various pathological conditions such as
viral infections including, but not limited to, cytomegalovirus
and hepatitis infections; acidosi~; tumors; and bacterial and
W094/~ 2 1 6 4 ~ 3 7 PCT~S94/06653
fungal infections, by administering various therapeutically
effective amounts of the admixture described above.
Detailed Descri~tion of the Tnventlon
Organic acids are a large group of compounds, many of
which can be derived from proteins, carbohydrates, and fats.
Although many occur naturally, many can be synthetically produced
without great difficulty.
Sorbic acid is a crystalline diolefinic acid having the
formula C6H802- Typically, it is used as mold and yeast
inhibitor; a fungistatic agent for foods, especially cheeses; or
to improve the characteristics of drying oils, the gloss of alkyd
type coatings, or the milling properties of cold rubber.
Aconitic acid is a white crystalline acid having the
formula C6~06. Typically, it is used in manufacturing itaconic
acid or as a plasticizer for buna rubber and plastics.
Malic acid is a crystalline dicarboxylic acid having
the formula C4H40~ and is typically found in three isomeric
crystalline forms.
Fumaric acid is a crystalline unsaturated dicarboxylic
acid having the formula C4~0~. Fumaric acid is typically used as
a substitute for tartaric acid in beverages and baking powders,
as a replacement or partial replacement of citric acid in soft
drinks, and as an antioxidant.
Crotonic acid is an unsaturated aliphatic acid having
the formula C4~02. It exists in the crystalline form, and is
typically used in the manufacture of copolymers with vinylacetate
that are used in lacquers and paper sizing, and in the
manufacture of softening agents for synthetic rubber.
It has now been diccovered that these acids, in
combination, haye significant therapeutic value. The ~m;~ture~
of these acids are prepared by conventional means known to those
skilled in the skilled art. The order of addition of the acids
to the admixture does not affect the final composition.
Although a wide range of proportions of the individual
components of the admixture is effective, in a typical
21 64~37
W094/2U~ PCT~S94/06653
composition, each of the four required individual acids will
independently comprise from about 1 to about 50 parts by weight
and, optionally, aconitic acid will comprise from 0 to about 50
parts by weight, based upon 100 parts by weight of the five acids
combined. Preferably, sorbic acid comprises from about 5 to
about 40 parts by weight, aconitic acid comprises from 0 to about
50 parts by weight, malic acid comprises from about 1 to about 30
parts by weight, fumaric acid comprises from about 1 to about 30
parts by weight, and crotonic acid comprises from about 1 to
about 30 parts by weight, based upon 100 parts by weight of the
five acids combined. Most preferably, sorbic acid comprises from
about 20 to about 30 parts by weight, aconitic acid comprises
from about 35 to about 45 parts by weight, malic acid comprises
from about 10 to about 20 parts by weight, fumaric acid comprises
from about 10 to about 20 parts by weight, and crotonic acid
comprises from about 1 to about 20 parts by weight, based upon
100 parts by weight of the five acids combined. Special mention
is made of an ~m; xture wherein sorbic acid comprises about 24
parts by weight, aconitic acid comprises about 40 parts by
weight, malic acid comprises about 16 parts by weight, fumaric
acid comprises about 16 parts by weight, and crotonic acid
comprises about 4 parts by weight, based upon 100 parts by weight
of the five acids combined.
Suitable ph~r~ceutically active carriers may be
combined with the admixture. Such carriers include those known
to those skilled in the art and can be added at any point in the
mixing process. If a ph~rm~ceutically active carrier is used,
the carrier will comp~ise from about 1 to about 99 parts by
weight and the acid admixture will comprise from about 99 to
about 1 part by weight, based upon 100 parts by weight of acid
admixture and carrier combined.
The acid admixture with or without the carrier can be
formulated into dosage unit forms including, but not limited to,
capsules and tablets. Method~ of preparation of dosage unit
forms would be known to those skilled in the art. Agents which
W094/~ 2 1 6 4 ~ 3 7 PCT~S94/06653
facilitate the manufacture and/or use of such dosage unit forms
may be added such as plasticizers, lubricants, excipients,
diluents and the like.
Methods of treatment of various pathologies
contemplated by the present invention involve the A~m;n-stration
of therapeutically effective amounts of the admixture to m~mm~ls
in need of such treatment. ~m;n;~tration i8 by means known to
those skilled in the art. Preferably, A~m;n;stration is systemic
and most preferably, it is oral.
Pathologies which are effectively treated with the
organic acid admixture described herein include but are not
limited to acidosis; viral infections including, but not limited
to, cytomaglovirus and hepatitis infections; tumors; and
bacterial and fungal infections, including but not limited to S.
aureus, E. coli, and C. albicans.
All of these pathologies are well known to those
skilled in the art, and their diagnosis would be evident to one
skilled in the art. For example, acidosis is reflected by
abnormally acidic blood pH and/or urine pH. Normal blood pH
ranges from about 7.3 to about 7.38, and normal urine pH ranges
from about 6 to about 8. Viral, bacterial, and fungal infections
can be confirmed by appropriate assay methods. Other diseases
such as chronic fatigue syndrome are only diagnosed through
clinical observations. Various opportunistic infections which
are characteristic of AIDS including, but not limited to,
cytomegalovirus, hepatitis, and the like are effectively treated
as described herein.
The amount of the admixture necessary to treat acidosis
is an anti-acidosis effective amount. Similarly, the amounts of
the admixture necessary to treat a viral infection, and
particularly hepatitis and cy~3megalovirus infections are an
anti-viral effective amount, and particularly anti-hepatitis and
an anti-cytomegalovirus effective amounts. The amount required
to treat tumors, and bacterial and ~ungal infections are anti-
21 64837
W094/28~ PCT~S94/06653
tumor, anti-bacterial, and anti-fungal effective amount~,
respectively.
The actual amounts of the admixture to be administered
will depend independently upon the age, weight, sex, sensitivity,
medical condition, including but not limited to stage of a
particular infection or disease, or the like, of an individual.
However, the amount will be a safe non-toxic amount.
These amounts can be determ;ne~ by experimentation
well-known in the art such as by establishing a matrix of dosages
and frequencies and assigning a group of experimental subjects to
each point in the matrix. Typically, that amount will range from
about 1000 to about 2000 milligrams per day for a 150 pound man
and preferably about 1500 milligrams per day for a 150 pound man.
Appropriate dosages for different body weights can be calculated
from this.
A daily dosage identifies the average amount of the
mixture ~m;n;stered to an individual. Although the daily dosage
may actually be ~m;n;~tered daily, it need not be ~m;n;stered
daily. The daily dosage i8 merely an average dosage that an
individual receives when the mixture is ~m; n; stered over a
period of time. The daily dosage can be ~m;n;stered in divided
portions so that the total amount ~m; n; stered is the daily
dosage.
Description of the Preferred ~hodlments
The following examples illustrate the invention without
limitation. All amounts are given as parts by weight (pbw).
Example 1
An ~m;~ture of 24 parts by weight of sorbic acid, 40
parts by weight of aconitic acid, 16 parts by weight of malic
acid, 16 parts by weight of fumaric acid, and 4 parts by weight
of crotonic acid was applied, in various dilutions, to cut filter
paper, and the filter paper was overlayed on plates streaked with
bacteria. Results are illustrated in Table 1.
W094/2~3 2 1 6 4 8 3 7 PCT~S94/06653
Table 1 - Bacterial Assays
Admixt Aspergi Staph. Pseudom Escheri Candida E~
ure llus aureus onas chia albican
5 Diluti aeruqin coli s
on osa
Full - 1.6 cm - 1.1 cm 1.6 cm 2.2
Streng
th
10 1:10 - - - - - 4.6
1:100 ~ - 5.2
The (-) negative means that no inhibition was observed.
Example 2
A patient had a temperature of 101F and symptoms of
acute urinary and prostate infection. The patient was treated
with 1500 mg/day of an A~mi~ture of 24 pbw of sorbic acid, 40 pbw
of aconitic acid, 16 pbw of malic acid, 16 parts by weight of
fumaric acid, and 4 pbw of crotonic acid based upon 100 pbw of
total ~mï ~ture for ten days. Symptoms and temperature subsided
by the 4th or 5th day. Antimicrobial and alkalizing activity and
therapeutic effects of the admixture are illustrated in Table 2.
Cultures which were positive for streptococci were negative by
the third week.
W094/~3 2 ~ 6 4 8 3 7 PCTNS94/06653
Table 2 - Urinary Infection
Initial Treatment
~aboratory Test Day 1 Day Day 32
5 URINALYSIS
pH 5.5 8 8
Protein 1+ 0 0
WBC 20-30 2-4 2-4
Bacteria many 0 few
Urine Culture Strept. + + 0
Blood PSA (normal 0-4) 21.9 5.1 3.22
WBC 10.4 6.1
Example 3
Six HIV positive individuals with average urine pH at
breakfast, lunch, and ~; nner of 6.0, as measured by pHydrion
tape, were administered 1500 mg/day of an admixture of 24 pbw of
sorbic acid, 40 pbw of aconitic acid, 16 pbw of malic acid, 16
pbw of fumaric acid, and 4 pbw of crotonic acid.
After one month, average urine pH was raised to 6.3.
Previously, it was fixed at 6Ø Results are illustrated in
Table 3 below.
W094l~3 2 1 6 4 8 3 7 PCT~S94/06653
Table 3 - Acidosis
Urine pH Urine pH Urine pH
Patient Breakfast Lunch Dinner
"A" 6.0 6.2 6.2
6.0 6.2 6.0
6.4 6.2 6.2
"B" 6.2 6.4 6.2
6.0 6.0 6.4
6.2 6.4 6.2
"C" 6.0 6.0 6.2
6.0 6.2 6.0
6.0 6.0 6.2
"D" 6.0 6.0 6.2
6.0 6.2 6.2
6.0 6.0 6.2
"E" 6.0 6.4 6.4
6.2 6.2 6.2
6.0 6.0 6.2
nFn 6.0 6.4 6.4
6.2 6.2 6.2
6.0 6.2 6.2
Example 4
A patient with urine culture po~itive for
cytomegalovirus infection was treated with 1500 mg/day of an
admixture of 24 pbw of sorbic acid, 40 pbw of aconitic acid, 16
pbw of malic acid, 16 pbw of fumaric acid, and 4 pbw of crotonic
acid, based upon 100 pbw of total A~m;Yture for six weeks. After
six weeks, a cytomegalovirus urine culture was negative.
Example 5
A patient having cancer of the prostate with a PSA of
17 was treated with Zolodex~. PSA was lowered to 2.4. Seven
months after Zolodex treatment wa~ discontinued, PSA rose to 13.
The patient then receive~ 1500 mg/day of an A~m;~ture
of 24 pbw of sorbic acid, 40 pbw of aconitic acid, 16 pbw of
malic acid, 16 pbw of fumaric acid, and 4 pbw of crotonic acid
based upon 100 pbw of total admixture, and PSA was lowered to
21 64837
W094l~ PCT~S94/06653
10
1.65. Nightly urination decreased from 5-6 times/night to two
times/night and the patient's general energy increased 80~.
Example 6
A patient was diagnosed with a pancreatic mass. The
patient also had nausea, pain, an inability to eat, weight loss,
and weakness.
The patient, who weighed about less than eighty pounds,
was given 50 mg/day of an admixture of 24 pbw of sorbic acid, 40
pbw of aconitic acid, 16 pbw of malic acid, 16 pbw of fumaric
acid, and 4 pbw of crotonic acid based upon 100 pbw of total
admixture for two months. The patient' 9 nausea abated, her
appetite improved, she slept more restfully, and she did not
urinate as frequently at night. The epigastric mass was no
longer palpable.
Example 7
A patient underwent a resection of a recurrent bladder
tumor, at which time, the pathological specimen revealed Grade
II/III moderately differentiated papillary transitional cell
carcinoma with invasion of the muscularis of the urinary bladder
and of the prostate. The patient was placed on a protocol of
1500 mg/day of an admixture of 24 pbw of sorbic acid, 40 pbw of
aconitic acid, 16 pbw of malic acid, 16 pbw of fumaric acid, and
4 pbw of crotonic acid based upon 100 pbw of total admixture. A
repeat cystoscopy two months later showed only several small
papillary le8ions in the urinary bladder and a small lesion in
the prostatic urethra. Rectal eY~min~tion was unremarkable. CAT
SCAN confirmed the improvement observed by cystoscopy.
Example 8
A patient was diagnosed with long st~n~;ng Hepatitis C.
The patient was treated with 1500 mg/day of an ~m; Yture of 24
pbw of sorbic acid, 40 pbw of aconitic acid, 16 pbw of malic
acid, 16 pbw of fumaric acid, and 4 pbw of crotonic acid based
upon 100 pbw of total admixture. After one month, pain in the
patient's side subsided. The pat~ent was no longer nauseated and
W094/~ 2 1 6 4 8 3 7 PCT~S94/06653
11
generally felt better. His SGOT laboratory value went down from
68 to 37.
Example 9
An admixture of 24 pbw of sorbic acid, 40 pbw of
aconitic acid, 16 pbw of malic acid, 16 pbw of fumaric acid, and
pbw of crotonic acid based upon 100 pbw of total admixture was
t ated for virucidal effectiveness against Cytomegalovirus. The
vlral inoculum was diluted 1:10 with the admixture. The test
material/virus mixture was sampled at 3 and 6 minutes. The
treated viral mixture was neutralized and titered. An initial
viral titer was determ;n~d.
The challenge organism was Cytomegalovirus, (CMV) and
the host cell line was MRC-5, ATCC CCL-171. Reagents and media
were EMEM with Earle's salts ~upplemented with heat inactivated
fetal bovine serum (FBS), glut~m;n~, and penicillin streptomycin
(MEM complete); sterile phosphate buffered saline; and newborn
calf serum (NBCS).
The virus stock culture was titered and adjusted to
contain approximately 107 CCID50/ml (50% cell-culture infectious
doses per milliliter) and stored in liquid nitrogen until use.
The virus stock was thawed and was diluted 10-fold in MEM through
107. One ml aliquots of appropriate dilutions were plated onto
5 confluent MRC-5 monolayers. The cells were incubated at 37 +
1C, in 5~ CO2, for 2 hours to allow the virus to adsorb to the
cells. After adsorption, the medium was withdrawn from the
monolayers, the cells were washed once with PBS, and refed with
MEM. The monolayers were incubated for 14 days at 37 + 1C, in
5~ CO2, and subsequently were observed for virus-specific
cytopathogenic effects (CPE).
The test solution was tested by combining the virus
with the admixture at a 1:10 dilution. This quspension was
agitated continuously at a constant temperature of 25C and
samples were withdrawn at 3 and 6 minutes to determine a survivor
curve and to calculate a D-value.
W094/~ 2 1 6 4 8 3 7 PCT~S94/06653
12
The sampled suspensionC were neutralized with a 1:1
dilution in new-born calf serum and were diluted ten-fold in MEM.
One ml aliquots of the neutralized mixture were plated onto MRC-5
monolayers (four wells per dilution) and were incubated at 37 +
1C in a 5~ CO2 in air atmosphere for 2 hours. Following the
incubation period, the fluids were removed. The monolayers were
washed with PBS, refed MEM and were incubated at 37 + 1C in a 5
CO2 in air atmosphere for thirteen days.
Control virus stock was titered at the time of the test
as described, to confirm that the titer was in an acceptable
range. The maintenance of the virus titer was evaluated using
PBS instead of the test solution. Samples were taken less than
15 seconds after the initiation of the test (zero time control)
and after 6 minuteq (final time control). The average CCID50/ml
for each titer was determined by the Reed and Muench method.
American ~ournal of Hygiene, 1938, 27:493. The D-value was
calculated by the Stumbo method. Stumbo, C.R.,
Thermobacteriology in food processing, New York Ac~Pm;c Press
(1973), pp. 235-247
No CPE was observed at 3 minutes. A D-value calculated
using this time resulted in a D-value of 45 seconds. This is an
estimate since the first sampling was done at 3 minutes (no data
were collected at less than 3 minutes).
When a survivor curve was determ;ned in this test, the
admixture provided data used to calculate a D-value of 45 seconds
(estimated). A conclusion can be drawn that the admixture was
antiviral against cytomegalovirus.
Results are illustrated in Tables 4, 5, and 6 below.
W094/~U~3 2 1 6 4 ~ 3 7 PCT~S94/06653
Table 4
Test and Control Titers Expressed as CPE
Day 13 Results
PBS TEST SOLUTION
DILUTION VIRUS STOCK Zero 6 min 3 min 6 min
10-l NA ++++ ++++ ---- ----
1o-2 ++++ ++++ ++++
10-3 ++++ ++++ ++++ _ _ _ _ _ _ _ _
0 10 4 ++++ + _ + _ + _ + _ _ _ _ _ _ _ _ _
1 o-5 ++++ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
-------- -------- --___ _ __
1 o-7 - - - _ N/A N/A N/A N/A
CCID50/ml 3.2 x 105 1 X 104 1 X 104
+ = CPE observed
- = No CPE observed
CT = Cytotoxicity observed
20 0 = No Cytotoxicity observed
Table 5
Control and Test Titers at Time Intervals
After Exposure to Scam Disinfectant
SAMPLE CCID501ml
Sample 3.2 x 105
PBS at Zero time 1 x 104
PBS at 6 minutes 1 x 104
3 minutes No CPE observed
6 minutes No CPE observed
W094/~3 2 1 6 4 8 3 7 PCT~S94/06653
14
Table 6
Determ-n~tion of the D-Value
D = Tx - To = 3 - 0 = 3 = 0.75 min = 45 seconds
Log Ao - Log Ax Log 104 - 0 4
Tx = end of time interval
To = beginning of time interval
AO = CCID50/ml at To
AX = CCID50/ml at Tx
Example 10
A 36 year old male with well controlled HIV infection
had progressively deteriorating kidney function from no
demonstrable cause. He averaged 5x nocturia and ran a
consistently 3 plus albllm;nllria with urinary specific gravity
fixed at 1.005 (normal to 1.030).
After approximately 4 months of 1500 mg/day of-
admixture of 24 pbw of sorbic acid, 40 pbw of aconitic acid, 16pbw of malic acid, 16 pbw of fumaric acid, and 4 pbw of crotonic
acid based upon 100 pbw of total admixture, his kidney function
normalized. His three plus alb~lm~n-lria gradually became
negative; he became able to sleep through nights, and the
specific gravity of his urine by concentration test increased to
1.0020. His urinary pH, which had been fixed at 5-6 resumed a
normal pattern of morning aciduria, increasing to neutral or
alkaline during the day ~ccording to his diet and activity.
Accompanying this was a total regression of his long-
existing bilateral massively swollen parotid glands; the non-
specific diagnosis derived from multiple biopsies had been cystic
parotitis. The presumptive retro-diagnosis for parotitis and the
kidney insufficiency was severe cytomegalovirus (CMV) infection,
suggested by high titers of CMV antibodies.
The above mentioned patents, test methods, and
publications are hereby incorporated by reference in their
entirety.
W094/~U~3 2 1 6 4 8 3 7 PCT~S94/06653
Many variations of the present invention will sugges~
themselves to those skilled in the art in light of the above
detailed description. All such obvious variations are within the
full intended scope of the appended claims.
.
