Hair loss abstract
Methods for inhibiting radiation induced weight and hair loss are
provided. A Bowman Birk Inhibitor product is administered in an
effective amount, preferably orally, to inhibit cutaneous manifestations
of radiation and weight loss.
Hair loss claims
What is claimed is:
1. A method for inhibiting radiation induced weight and hair loss
in an animal receiving radiation treatment comprising administering
an effective amount of a Bowman Birk Inhibitor product wherein the
Bowman Birk Inhibitor product is produced by the steps consisting
essentially of:
(i) providing soybean solubles produced from acidic aqueous-extracted
hexane-defatted soybeans;
(ii) diluting the soybean solubles with an aqueous solution to
form a slurry;
(iii) separating the aqueous soluble portion of the soybean solubles
from the slurry to form a purified soybean soluble composition;
(iv) diluting the purified soybean soluble composition with an
aqueous solution and ultrafiltering the aqueous soluble portion
of the diluted purified soybean soluble composition at least once
to form a crude Bowman-Birk Inhibitor concentrate; and
(v) drying the crude Bowman-Birk Inhibitor concentrate to produce
a Bowman-Birk Inhibitor concentrate product; to said animal.
2. The method of claim 1 wherein said administration is oral.
3. The method of claim 1 wherein the drying method used in step
(v) is spray drying,
4. The method of claim 1 wherein the slurry of step (ii) is a 15
to 20 percent solid solution.
5. The method of claim 1 further comprising diluting the crude
Bowman-Birk Inhibitor concentrate produced in step (iv) with an
aqueous solution and separating a semi-crude Bowman Birk Inhibitor
concentrate prior to drying in step (v).
6. The method of claim 5 further comprising, prior to the drying
step, diluting the semi-crude Bowman-Birk Inhibitor concentrate
with acetone and retaining the precipitated acetone insoluble portion.
7. The method of claim 6 further comprising lyophilizing the dried
Bowman-Birk Inhibitor product.
8. A method for inhibiting radiation induced weight and hair loss
in an animal receiving radiation treatment comprising administering
an effective amount of a Bowman Birk Inhibitor product wherein the
Bowman Birk Inhibitor product is produced by the steps consisting
essentially of:
(i) providing soybean solubles from acidic aqueous-extracted hexane-defatted
soybeans;
(ii) diluting the soybean solubles with water and separating a
first aqueous soybean soluble portion;
(iii) adding acetone to the first aqueous portion to produce a
first Bowman-Birk Inhibitor precipitate concentrate;
(iv) diluting the first concentrate with water and separating a
second aqueous soybean soluble portion;
(v) adding acetone to the second aqueous portion to produce a second
Bowman-Birk Inhibitor precipitate concentrate;
(vi) drying the second Bowman-Birk Inhibitor concentrate to produce
a Bowman-Birk Inhibitor concentrate product; to said animal.
9. The method of claim 8 wherein the drying step is spray drying.
10. The method of claim 9 further comprising adding acetone to
the second Bowman Birk Inhibitor concentrate prior to the drying
step.
11. The method of claim 10 further comprising lyophilizing the
dried Bowman Birk Inhibitor concentrate product.
Hair loss description
FIELD OF THE INVENTION
This invention relates to methods of inhibiting radiation induced
weight and hair loss by administration of a Bowman-Birk Inhibitor
(BBI) product.
BACKGROUND OF THE INVENTION
Radiation induced weight and hair loss are clinical, cosmetic and
psychological problems for cancer patients. Hair loss in patients
receiving radiation therapy for cranial and extracranial lesions
may have major psycho-social consequences for the patient. Where
hair loss is due to direct radiation, it is usually irreversible.
Attempts to minimize radiation dermatitis and hair loss have been
directed at using topical radiation protectant agents (Kim, J. et
al., Seminars in Oncology 1983, 10, 86-92; Verhey, L J and Sedlacek,
R., Rad. Res. 1983, 93, 175-183); lowering skin temperature in the
radiation field (Liebner, E J et al., Am. J. Roent. Rad. Ther and
Nucl. Med. 1962, 88, 976-987); applying topical cortisone (Potera,
M E et al., Radiology 1982, 143, 775-777); and use of local anesthetics
(Ohlsen, L. et al., Acta Oncological 1987, 26, 467-476). The success
of these therapies has been minimal and none are in common clinical
usage.
Weight loss is a well recognized problem in cancer patients treated
with radiation and/or cancer chemotherapeutic agents, and has been
shown to be an independent prognostic indicator of decreased survival
rates. The cause of this weight loss is believed to be related to
both decreased caloric intake/absorption and increased energy requirements.
However, it has been found that increased caloric intake has not
improved survival for patients with a variety of advanced cancers.
Thus, the prevention of weight loss should be considered as an important
goal to decrease morbidity and mortality associated with cancer-related
therapies.
U.S. Pat. 4,793,996 (Kennedy et al.) discloses a process comprising
treating soybeans with acetone, followed by ethanol extraction and
acetone precipitation for obtaining Bowman Birk Inhibitor (BBI).
The soybeans may be defatted prior to acetone treatment. In addition,
BBI may be further purified by conventional techniques. Kennedy
et al. discovered that in the conventional process for preparing
BBI from soybeans, a factor remained which adversely affected the
ability of BBI to inhibit the malignant transformation of cells.
If the factor was removed, the resulting BBI product was capable
of inhibiting the malignant transformation of cells. It was found
to be possible to remove this factor by treating the soybeans with
acetone prior to the ethanol extraction step.
Kennedy et al., in U.S. application Ser. No. 824,719 filed Jan.
17, 1992 now U.S. Pat. No. 5,217,717 entitled "Methods of Making
Soybean Bowman-Birk Inhibitor Concentrate and Use of Same As a Human
Cancer Preventative and Therapy", which is incorporated herein
in its entirety, describe methods for producing novel BBI concentrate
products. Those BBI concentrate products are employed by the methods
of the present invention. The process described to produce those
BBI products was found to be economically superior due to the avoidance
of an aqueous alcohol extraction step and the use, in certain embodiments,
of ultrafiltration as a separation process step.
SUMMARY OF THE INVENTION
Methods for inhibiting radiation induced weight and hair loss by
administration of a Bowman Birk Inhibitor product are provided.
It has been found that hair and weight loss are minimized following
dietary supplementation with a Bowman-Birk Inhibitor product.
The methods described by the present invention may employ the use
of a BBI product produced in accordance with the following methods.
The source material for preparing the BBI products is soybean solubles.
The soybean solubles are preferably obtained from soybean flakes
or soy flour. The soybean flakes or soy flour are first subjected
to a hexane defatting step. The defatted material is subjected to
an acidic aqueous extraction step, pH from about 4 to 5, and the
insolubles are removed to produce the soybean solubles. The process
for the production of soybean solubles are well known in the art
as shown by U.S. Pat. No. 3,365,440, which is incorporated herein
in its entirety. The soybean solubles are conventionally produced
at a relatively high solids concentration, usually at a solids concentration
of at least about 50 percent by weight as recognized by the Association
of American Feed Control Officials Incorporated.
The BBI product is produced by diluting the soybean solubles with
water, preferably to about 15-25% by weight solids content, followed
by centrifugation to produce purified soybean solubles. The purified
solubles are then diluted with water, preferably to about 10-12%
by weight solids, to produce reslurried purified soybean solubles.
The reslurried solubles are then subjected to ultrafiltration to
produce a crude BBI concentrate. The crude concentrate is then diluted
with water and spray dried to produce the Bowman Birk Inhibitor
Concentrate (BBIC) product. In another process embodiment for the
production of the BBIC product, the diluted crude BBI concentrate
is subjected to another ultrafiltration step to produce a semi-crude
BBI concentrate which is then spray dried to produce the BBIC product.
In a preferred process embodiment, the semi-crude BBI concentrate
is treated with acetone to produce a BBI concentrate precipitate.
After settling and decanting the resulting purified BBI concentrate
precipitate is air dried, ground, reslurried with water, filtered
and then lyophilized or spray dried to produce the BBIC product.
The BBIC product can be produced in accordance with another process
embodiment wherein the time-consuming step(s) are eliminated by
starting with soy solubles and applying the acetone treatment to
a substrate that has a substantially higher concentration of BBI
than that in the defatted soy flour/flake of the prior art, resulting
in a more economical process for production.
It was surprising found that a Bowman-Birk Inhibitor product inhibited
radiation induced weight and hair loss.
DESCRIPTION OF THE DRAWINGS
FIGS. 1(a) and 1(b) are photographs of irradiated C57BL mice. The
mouse in FIG. 1(a) received a Bowman-Birk Inhibitor dietary supplement.
The mouse in FIG. 1(b) did not. Extensive hair loss was observed
in mice that did not receive BBI dietary supplementation; an example
of such a mouse is shown in FIG. 1(b).
DETAILED DESCRIPTION OF THE INVENTION
The present invention concerns methods of inhibiting radiation
induced weight and hair loss by administration of a Bowman-Birk
Inhibitor product. The Bowman-Birk product may be produced in accordance
with the methods described herein. The administration can be by
any acceptable and convenient mode, with oral administration preferred.
The preparation of the BBI product useful in the methods of the
present invention includes the steps of (1) providing soybean solubles
produced from acidic aqueous-extracted hexane-defatted soybeans
in the absence of an ethanol extraction step, the soybean solubles
preferably having a solids concentration of at least about 50 percent
by weight; (2) diluting the soybean solubles with an aqueous solution
to form a slurry, preferably a 15 to 20 percent by weight solid
solution; (3) separating the aqueous soluble portion of the soybean
solubles from the slurry to form a purified soybean soluble composition;
(4) diluting the purified soybean soluble composition with an aqueous
solution, preferably to about a 10-12% solid solution, and ultrafiltering
the aqueous soluble portion of the diluted purified soybean soluble
composition at least once, retaining the supernatant fluid, to form
a crude BBI concentrate; and (5) drying the crude BBI concentrate,
preferably by spray drying, and recovering the BBIC product. The
process can include an optional, additional dilution of the crude
BBI concentrate with an aqueous solution followed by an ultrafiltration
step to form a semi-crude BBI concentrate prior to the drying step.
The process can be further modified by diluting the semi-crude BBI
concentrate with acetone and retaining the precipitated acetone
insoluble portion prior to the drying step.
In accordance with one embodiment of the process to produce the
BBI product, soybean solubles are diluted with water to 18% solids
and then centrifuged to produce purified soybean solubles. The purified
solubles are diluted with water to 8% solids to produce reslurried
purified soybean solubles which are subjected to ultrafiltration
(10,000 m.w. membrane). The resulting crude BBI concentrate is diluted
with water (1:1) and then subjected to a second ultrafiltration
step (1,000 m.w. membrane) to produce a semi-crude BBI concentrate.
The semi-crude concentrate is treated with acetone (2.2:1) to produce
a BBI concentrate precipitate. After settling and decanting, the
resulting purified BBI concentrate precipitate is air dried, ground,
reslurried with water to 15% solids, filtered (Buchner funnel/Whatman
#1) and then lyophilized to produce the BBIC product.
In another embodiment of the process to produce the BBIC product,
purified soybean solubles are produced as described above and then
diluted to 10% solids. The resulting reslurried purified soybean
solubles are then treated as described in the foregoing to produce
a semi-crude BBI concentrate which is treated with acetone (1.66
to 1) to produce a BBI concentrate precipitate. The BBIC product
is produced as described above, with the exception that the filtered
precipitate is spray dried rather than lyophilized.
In still another embodiment of the process to produce the BBIC
product, soybean solubles are diluted with water to 15-20% solids
and centrifuged to produce purified soybean solubles. The purified
solubles are diluted with water to 10% solids to produce reslurried
purified soybean solubles which are subjected to ultrafiltration
(1,000 m.w. membrane). The resulting crude BBI concentrate is diluted
with water (1:1) and spray dried to produce the BBIC product.
In yet another embodiment of the process to produce the BBIC product,
soybean solubles are diluted with water to 16% solids and centrifuged
to produce purified soybean solubles. The purified solubles are
diluted with water to 10% solids. The resulting reslurried purified
solubles are then subjected to ultrafiltration (10,000 m.w. membrane),
producing a crude BBI concentrate. The crude concentrate is diluted
with water (1:1) and again subjected to ultrafiltration (1,000 m.w.
membrane) to produce a semicrude BBI concentrate which is spray
dried to produce the BBIC product.
In another embodiment of the process to produce the BBIC product,
the ultrafiltration step(s) are eliminated by starting with soy
solubles, and applying the acetone treatment to a substrate that
has a substantially higher concentration of BBI than that in defatted
soy flour/flake. In this process, insolubles are removed from acid
aqueous-extracted hexane defatted soybeans to produce soybean solubles
having a solids content of at least 50%. The soybean solubles are
diluted with water to a solids concentration of from about 15-20%
and are then centrifuged to produce purified soybean solubles. Acetone
is added to the supernatant to produce a crude BBI concentrate precipitate,
which is allowed to settle. The resulting precipitate containing
the partially purified BBI is then resuspended in water and centrifuged.
Acetone is then added to the supernatant and the resulting water
soluble, acetone insoluble precipitate allowed to settle, and then
dried to produce the BBIC product. An optional additional acetone
resuspension step can be employed before the final drying step.
The BBIC products made in accordance with the various processes
set forth herein are useful for inhibiting radiation induced weight
and hair loss. These Bowman-Birk Inhibitor products may be administered
either alone or in combination with a pharmaceutically acceptable
carrier. Oral administration, either as a dietary supplement or
a pharmaceutical composition are contemplated by the teachings of
this invention.
In studies with C57BL mice absence of hair loss, overall shiny
coats and decreased weight loss were unexpectedly found when their
diet was supplemented with a Bowman-Birk concentrate product.
Radiation induced leukemia in C57BL mice is an established animal
carcinogenesis assay system. See, for example, (Berenblum et al.,
Radiation Research 1974, 60, 501-505). Although the Bowman-Birk
Inhibitor (BBI) product did not affect the incidence of cancer in
this study, three unexpected findings were noted. At the time of
death due to leukemia (approximately 80% of both the BBI supplemented
and non-BBI supplemented mice died of leukemia in this study), the
BBI supplemented mice appeared healthier in that they had shinier
coats, had no radiation-induced hair loss (See FIG. 1), and weighed
more than the non-BBI supplemented mice. Thus, the mice on the BBI-supplemented
diet appeared to be a healthier population compared to those not
on the BBI supplemented diet.
In other studies, the effect of BBI oral administration on cutaneous
effects of radiation will be evaluated. Skin changes and hair loss
will be observed in two groups of animals; C57BL mice exposed to
whole body irradiation (WBI) and Sprague-Dawley rats following WBI.
To evaluate whether oral supplementation of BBI minimizes weight
loss due to radiation, two groups of animals will be studied: C57BL
mice exposed to WBI and Sprague-Dawley rats following WBI. Weight
loss and food intake with and without addition of BBI will be serially
monitored.
The invention is further illustrated by the following, nonlimiting
examples.
EXAMPLES
Example 1
C57BL and CD-1 mice and Sprague Dawley rats are used in the studies.
The experimental groups are as follows:
C57BL mice and Sprague Dawley rats:
WBI/gavage with 0.5% BBIC
WBI/gavage with 0.5% autoclaved BBIC
No treatment
Sham WBI/gavage with water
Gavage with 0.5% BBIC
Upon arrival in the laboratory, the animals will be randomly assorted
into treatment groups, housed 1-2 animals per cage and placed on
a standard diet, AIN-76A. American Institute of Nutrition purified
diets for rats and mice containing 20% protein. The BBIC will be
administered 5 days per week via gavage. The BBIC will be prepared
as described in the following Examples. Control animals will receive
autoclaved BBI; autoclaving is believed to eliminate all protease
inhibitor activity. The groups receiving the autoclaved BBIC preparation
will serve as isocaloric diet control groups for other animals in
the study. Animals will receive water ad libitum and will be maintained
in a controlled environmental animal facility at 25.degree. C. with
a 12 hour light-dark cycle.
The C57BL mice and Sprague-Dawley rats will be maintained for 6
months following the end of the radiation treatments. Endpoints
which are measured include visual skin and hair changes, body weight,
food intake (to assure that any observed weight loss/gain is not
due to differences in intake), progressive dermal biopsies and photographic
appearance. At the time of autopsy, animals and individual organs
will be weighed. The pancreas will be prepared for histopathological
examination. It is important to evaluate the histopathological alterations
in the pancreas because high levels of soybean protease inhibitors
in rats have previously been associated with abnormal growth in
the pancreas. However, in our studies, pancreatic histopathological
alterations have not been observed in rats, hamsters, or mice utilizing
BBI as a cancer preventative agent.
Example 2
Hair and weight loss in patients exposed to cancer therapy protocols
utilizing radiation and/or chemotherapeutic agents will also be
monitored. Many of the agents used in cancer therapy are known to
produce hair and, presumably, weight loss. Patients treated for
various malignancies with agents expected to produce hair and weight
loss will be given pills containing BBIC or a placebo (to be taken
on a daily basis). The endpoints to be monitored in the patients
will include weight and any visual skin and hair changes, with photographic
documentation. Patients will be observed over a 6-week period during
and after receiving radiation and/or chemotherapy treatments.
Example 3
139 pounds of soybean solubles from an acidic aqueous extraction
of hexane-defatted soybeans was diluted to 18% solids with 332 pounds
of water. The slurry of the diluted soy solubles was centrifuged
to remove insoluble matter, and the partially "purified"
solids were further diluted with water to a 8% solids level. These
"purified" soy solubles were then subjected to ultrafiltration
using a 1,000 MW cut-off membrane at 15 gpm and 105 psig, until
31 gallons of permeate was collected. The liquid containing the
crude BBI concentrate was again diluted with 31 gallons of water,
and the ultrafiltration step was repeated until an additional 47
gallons of permeate was collected and 45 gallons of a semi-crude
BBI concentrate remained.
At this point, 55 gallons of acetone was added to 25 gallons of
the concentrate; the BBI concentrate precipitate thus obtained was
allowed to settle for 1 hour. The liquid supernatant was then decanted,
and the precipitate containing the "purified" BBI concentrate
was placed in a Buchner Funnel under vacuum to draw off the excess
liquid. The dried precipitate was ground in a Waring blender and
reslurried to 15% solids. The reslurried suspension was then allowed
to settle and the supernatant was lyophilized. The yield was 8 pounds
of product with a Chymotrypsin inhibitor (CI) level of 135.5 mgs/g.
Example 4
87.3 pounds of soybean solubles from an acidic aqueous extraction
of hexane-defatted soybeans were diluted to 18% solids with 207.5
pounds of water. The slurry was centrifuged to remove the insoluble
sludge material; diluted to 8% solids with water; and then subjected
to ultrafiltration over a 1,000 M cutoff membrane at 15 gpm. and
100 psig. 44 pounds of permeate was collected; the crude BBI concentrate
was rediluted with 44 pounds of water, and the ultrafiltration step
was repeated. 112 pounds of permeate and 163 pounds of a semicrude
BBI concentrate were collected.
270 pounds of acetone was then added to this semicrude BBI concentrate,
and the precipitated BBI concentrate thus formed was allowed to
settle for 1 hour. The liquid was decanted and the precipitate was
placed in a Buchner funnel under vacuum to draw off the excess liquid.
It was then reslurried with water in a Waring blender, allowed to
settle, and the supernatant was spray-dried. The yield was 2.3 pounds
of product with a Chymotrypsin (CI) content of 261 mgs/g.
Example 5
90 pounds of soybean solubles from an acidic aqueous extraction
of hexane-defatted soybeans were diluted to between 15% to 20% of
solids with water. (The initial solubles contain 50-60% solids).
The slurry was centrifuged to remove 3-5% of the solids, present
as insoluble sludge. The supernatant solution was then diluted with
water to 10% solids, and subjected to ultrafiltration over a 1,000
MW cut-off membrane. One (1) pound of high-purity water was added
to this fraction containing the crude BBI concentrate for every
one (1) pound of permeate that had been removed. The ultrafiltration
was considered complete when the solids content had begun to decrease.
At that point, the BBI concentrate was spray-dried. The yield was
14 pounds of product with a CI content of 99.2 mgs/g.
Example 6
50.2 pounds of soybean solubles from an acidic aqueous extraction
of hexane-defatted soybeans was diluted to 16% of solids with 126.2
pounds of water. The slurry was centrifuged to remove 3-5% of the
solids, present as insoluble sludge. The supernatant solution was
then diluted with water to 10% solids, and subjected to ultrafiltration
over a 10,000 MW cut-off membrane. One (1) pound of high-purity
water was added to the concentrate fraction for every one (1) pound
of permeate that had been removed. When the solids content had begun
to decrease in the permeate, the permeate was also subjected to
ultrafiltration over a 1,000 MW cut-off membrane. After that, the
BBI concentrate was spray-dried. The yield was 2.6 pounds of product
with a CI content of 61.9 mgs/g.
Example 7
A slurry obtained from the whey protein stream produced during
the production of soy protein isolate was treated by ultra filtration
over a 1,000 MW cut-off membrane, as described in Example 4. A total
of 157.75 pounds of whey protein solution was used. After ultrafiltration,
the BBI concentrate fraction, containing 2.7% solids, was spray-dried.
The yield was 1.2 pounds of product, containing 187.8 mgs/g of CI.
Example 8
1000 grams of soy solubles with a solids content of 19% from an
acidic aqueous extraction of hexane-defatted soybeans were centrifuged
to remove insoluble matter. At this point, 2 liters of acetone were
added to the supernatant. The crude BBI concentrate precipitate
thus obtained was allowed to settle for 1 hour. The liquid supernatant
was then decanted. The precipitate containing the partially purified
BBI was then resuspended in 200 ml of water and centrifuged to remove
matter rendered irreversibly insoluble by acetone. 400 ml of acetone
was then added to the supernatant. The water soluble, acetone insoluble
precipitate which was formed was allowed to settle for 1 hour. The
supernatant was decanted. The major portion of water remaining in
the precipitate was removed by resuspending the precipitate in 100
ml of acetone and allowing the precipitate to settle for 30 minutes.
The supernatant was decanted. The BBI concentrate precipitate was
spread thinly on a tray and allowed to air dry to a free flowing
white powder. The yield was 5 gm of product with a chymotrypsin
inhibitor level of 200 mgs/g.
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