U.S. patent application number 10/810762 was filed with the patent office on 2005-09-29 for hmb compositions and uses thereof.
Invention is credited to Baxter, Jeffrey H., Mukerji, Pradip, Tisdale, Michael J., Voss, Anne C..
Application Number | 20050215640 10/810762 |
Document ID | / |
Family ID | 34990887 |
Filed Date | 2005-09-29 |
United States Patent
Application |
20050215640 |
Kind Code |
A1 |
Baxter, Jeffrey H. ; et
al. |
September 29, 2005 |
HMB compositions and uses thereof
Abstract
The present invention relates to methods for the prevention and
treatment of chronic inflammatory diseases, cancer, and involuntary
weight loss. In the practice of the present invention patients are
enterally administered HMB alone or alternatively in combination
with eicosapentaenoic (20:5 .omega.-3), FOS, carnitine and mixtures
thereof. HMB may be added to food products comprising a source of
amino-nitrogen enriched with large neutral amino acids such as
leucine, isoleucine, valine, tyrosine, threonine and phenylalanine
and subtantially lacking in free amino acids.
Inventors: |
Baxter, Jeffrey H.;
(Gahanna, OH) ; Mukerji, Pradip; (Gahanna, OH)
; Voss, Anne C.; (Columbus, OH) ; Tisdale, Michael
J.; (Birmingham, GB) |
Correspondence
Address: |
Ross Products Division
Abbott Laboratories
625 Cleveland Avenue
Columbus
OH
43215
US
|
Family ID: |
34990887 |
Appl. No.: |
10/810762 |
Filed: |
March 26, 2004 |
Current U.S.
Class: |
514/560 |
Current CPC
Class: |
A61P 3/02 20180101; A61K
31/19 20130101; A61P 3/04 20180101; A61P 1/16 20180101; A61P 13/12
20180101; A61P 1/00 20180101; A61K 31/19 20130101; A61P 21/00
20180101; A61P 25/18 20180101; A61P 31/18 20180101; A61P 17/02
20180101; A61K 2300/00 20130101; A61P 11/00 20180101; A61P 19/02
20180101; A61P 35/00 20180101; A61P 37/00 20180101; A61P 1/12
20180101; A61P 1/04 20180101; A61P 31/00 20180101; A61P 3/12
20180101; A61P 3/00 20180101; A61P 29/00 20180101 |
Class at
Publication: |
514/560 |
International
Class: |
A61K 031/202 |
Claims
We claim:
1. A method for the prevention or treatment of disease conditions
in patients by downregulating the expression and/or activity of
components selected from the group consisting of protein kinase C,
nuclear factor kappa-B, ubiquitin-conjugating enzymes and
components of 26S proteasome which comprises administering HMB, its
salts, metobolites or derivatives thereof.
2. The method according to claim 1 wherein the administration of
HMB, its salts, metobolites or derivatives thereof downregulates
the expression and/or activity of protein kinase C.
3. The method according to claim 1 wherein the administration of
HMB, its salts, metobolites or derivatives thereof downregulates
the expression and/or activity of nuclear factor kappa-B.
4. The method according to claim 1 wherein the administration of
HMB, its salts, metobolites or derivatives thereof downregulates
the expression and/or activity of ubiquitin-conjugating
enzymes.
5. The method according to claim 1 wherein the administration of
HMB, its salts, metobolites or derivatives thereof downregulates
the expression and/or activity of components of 26S proteasome.
6. The method according to claim 1 wherein at least one of the
components selected from the group consisting of L-carnitine, amino
nitrogen source enriched with large neutral amino acids
substantially lacking free amino acids, omega-3 fatty acids and
indigestible oligossacharide is administered in combination with
the HMB or its salts thereof.
7. The method according to claim 1 wherein the disease condition is
selected from the group consisting of cancer, cachexia,
age-associated wasting, wasting associated with long-term
hospitalisation, HIV/AIDS, arthritis, trauma, liver disease,
Crohn's disease, IBD, renal insufficiency and COPD.
8. The method according to claim 7 wherein the disease is
cachexia.
9. A composition comprising: a. HMB, its salts, metabolites or
derivatives thereof; b. carnitine; c. amino nitrogen source
enriched with large neutral amino acids; and wherein said
composition is substantially lacking in free amino acids.
10. The composition according to claim 9 wherein said HMB is
selected from the group consisting of sodium HMB, potassium HMB,
magnesium HMB, chromium HMB, calcium HMB, alkali metal HMB,
alkaline earth metal HMB and HMB lactone.
11. The composition according to claim 9 further comprising
.omega.-3 fatty acids.
12. The composition according to claim 11 wherein said .omega.-3
fatty acids are selected from the group consisting of
eicosapentaenoic acid and docosahexaenoic acid.
13. The composition according to claim 9 wherein said large neutral
amino acids comprise at least 10% of the amino nitrogen source.
14. The composition according to claim 9 wherein said free amino
acids comprise less than 0.4 gm/serving of the composition.
15. The composition according to claim 9 further comprises less
than 2 grams per serving of carnitine.
16. The composition according to claim 9 further comprising at
least 1 gram per serving of FOS.
17. The composition according to claim 9 further comprising a
nutrient selected from the group consisting of vitamins, minerals,
and trace minerals.
18. A composition comprising: a. from about 2 to 10 gm/liter
calcium HMB; b. at least 1 gram per liter of .omega.-3 fatty acids;
c. from about 1 to about 8 gm/liter carnitine; d. from about 1 to
about 25 gm/liter FOS; amino nitrogen source enriched with large
neutral amino acids, wherein said amino nitrogen source comprises
from about 10 to 60 wt/wt % large neutral amino acids; and wherein
said composition is substantially lacking in free amino acids.
19. The composition of claim 9 wherein said composition is
administered to a human or an animal.
20. The composition of claim 9 wherein said composition is selected
from the group consisting of dietary supplement, meal replacement,
nutritional bars, chews or bites and beverage.
21. A method of treating disease-associated wasting of a patient
comprising administering the composition according to claim 9 to
said patient.
Description
[0001] The present invention relates to methods for the prevention
and treatment of chronic inflammatory diseases, cancer, and
involuntary weight loss. In the practice of the present invention
patients are enterally administered HMB alone or alternatively in
combination with eicosapentaenoic (20:5 .omega.-3), FOS, carnitine
and mixtures thereof. HMB may be added to food products comprising
a source of amino-nitrogen enriched with large neutral amino acids
such as leucine, isoleucine, valine, tyrosine, threonine and
phenylalanine and subtantially lacking in free amino acids.
BACKGROUND
[0002] Undesired weight loss, particularly lean mass loss is a
relatively common occurance in critical illness, and has a
significant impact on morbidity and mortality. This is particularly
true in cancer patients, where such mass losses can become
tretment-limiting, and thus impact overall prognosis.
[0003] Cachexia is a syndrome characterized by anorexia, weight
loss, premature satiety, asthenia, loss of lean body mass, and
multiple organ dysfunction. It is a common consequence of chronic
illnesses (both malignant and non-malignant) and is associated with
a poorer prognosis in chronic obstructive pulmonary disease (COPD),
chronic heart failure (CHF), renal failure, AIDS, dementia, chronic
liver disease and cancer. It is often independent of other
indicators of disease severity. (Witte, K. K. A. and Clark, A. L.:
Nutritional abnormalities contributing to cachexia in chronic
illness, International Journal of Cardiology 85:23-31, 2002)
Pulmonary disease is often associated with cachexia, and
substantial numbers of patients suffering from COPD, particularly
emphysema, become emaciated during the course of the disease.
Weight loss is an independent risk factor for prognosis, and is
often associated with increased oxygen consumption. This has been
linked with development of inefficient muscle energy metabolism
(Kutsuzawa, T, et al.: Muscle energy metabolism and nutritional
status in patients with chronic obstructive pulmonary disease. Am.
J. Respir. Crit. Care Med. 152(2):647-652, 1995). COPD is also
associated with a general elevated systemic inflammatory response,
reflected by elevated concentrations of pro-inflammatory cytokines
and acute phase proteins in the peripheral blood (Schols, A. M., et
al: Evidence for a relation between metabolic derangements and
increased levels of inflammatory mediators in a subgroup of
patients with chronic obstructive pulmonary disease. Thorax
51:819-824, 1996; Takabatake, N., et al.: Circulating leptin in
patients with chronic obstructive pulmonary disease. Am J Respir
Crit Care Med 159:1215-1219, 1999; Dentener, M. A., et al.:
Systemic anti-inflammatory mediators in COPD: increase in soluble
interleukin I receptor II during treatment of exacerbations. Thorax
56:721-726, 2001.) Such changes are often associated with muscle
wasting syndromes.
[0004] Studies with incubated muscles and muscle extracts suggest
that the ATP-dependent ubiquitin-proteosome pathway is responsible
for most of the increased proteolysis which ultimately results in
muscle wasting. In particular, increased levels of
ubiquitin-conjugated proteins, and increases in mRNA levels for
polyubiquitin, certain proteosome subunits and the
ubiquitin-conjugating enzyme E2.sub.14K are features found in most
atrophying muscles (Schols, A. M. W. J.: Pulmonary cachexia. Intl J
Cardiology 85:101-110, 2002; Jagoe, R. T. and Goldberg, A. L.: What
do we really know about the ubiquitin-proteosome pathway in muscle
atrophy? Curr Opin Clin Nutr Metab Care 4:183-190, 2001).
[0005] The majority of patients with cancer whose disease
progresses to metastatic disease develop cachexia during their
treatment program and the cachexia contributes to their deaths. The
frequency of weight loss in cancer patients ranges from 40% for
patients with breast cancer, acute myelocytic leukemia, and sarcoma
to more than 80% in patients with carcinoma of the pancreas and
stomach. About 60% of patients with carcinomas of the lung, colon
or prostate have experienced weight loss prior to beginning
chemotherapy. Although the relationship between pretreatment
malnutrition (weight loss) and adverse outcome is established, no
consistent relationship has been demonstrated between the
development of cachexia and tumor size, disease stage, and type or
duration of the malignancy.
[0006] Cancer cachexia is not simply a local effect of the tumor.
Alterations in protein, fat, and carbohydrate metabolism occur
commonly. For example, abnormalities in carbohydrate metabolism
include increased rates of total glucose turnover, increased
hepatic gluconeogenesis, glucose intolerance and elevated glucose
levels. Increased lipolysis, increased free fatty acid and glycerol
turnover, hyperlipidemia, and reduced lipoprotein lipase activity
are frequently noted. The weight loss associated with cancer
cachexia is caused not only by a reduction in body fat stores but
also by a reduction in total body protein mass, with extensive
skeletal muscle wasting. Increased protein turnover and poorly
regulated amino acid oxidation may also be important. The presence
of host-derived factors produced in response to the cancer have
been implicated as causative agents of cachexia, e.g., tumor
necrosis factor-.alpha. (TNF) or cachectin, interleukin-1 (IL-1),
IL-6, gamma-interferon (IFN), and prostaglandins (PGs) (e.g.,
PGE.sub.2).
[0007] Weight loss is common in patients with carcinomas of the
lung and gastrointestinal tract, resulting in a massive loss of
both body fat and muscle protein, while non-muscle protein remains
unaffected. While loss of body fat is important in terms of energy
reserves, it is loss of skeletal muscle protein that results in
immobility, and eventually impairment of respiratory muscle
function, leading to death from hypostatic pneumonia. Although
cachexia is frequently accompanied by anorexia, nutritional
supplementation alone is unable to maintain stable body weight and
any weight that is gained is due to an increase in adipose tissue
and water rather than lean body mass. The same is true for appetite
stimulants, such as megestrol acetate and medroxyprogesterone
acetate, suggesting that loss of lean body mass is due to factors
other than energy insufficiency.
[0008] Skeletal muscle mass is a balance between the rate of
protein synthesis and the rate of degradation. Patients with cancer
cachexia show a depression of protein synthesis in skeletal muscle
and an increase in protein degradation, which is reflected in an
increased expression of the ubiquitin-proteasome proteolytic
pathway, the major determinant of protein degradation. Thus
skeletal muscle from cachectic cancer patients shows increased
expression of mRNA for both ubiquitin and proteasome subunits,
while proteasome proteolytic activity increased in parallel with
ubiquitin expression. The inability of anabolic stimuli to increase
lean body mass in cachectic patients suggests that protein
degradation must be attenuated before muscle mass can increase.
Eicosapentaenoic acid (EPA), downregulates the increased expression
of the ubiquitin-proteasome proteolytic pathway in the skeletal
muscle of cachectic mice, and has been shown to stabilize body
weight in cachectic patients with pancreatic cancer. When patients
consumed an energy-dense supplement containing 32 g protein and 2 g
EPA body weight increased and this was attributed solely to an
increase in lean body mass (Barber, M. D., Ross, J. A., Voss, A.
C., Tisdale, M. J., Fearon, K. C. H. The effect of an oral
nutritional supplement enriched with fish oil on weight-loss in
patients with pancreatic cancer. Br. J. Cancer, 81: 80-86,
1999).
[0009] A recent study by May et al (May, P. E., Barber, A.,
D'Olimpio, J. T., Hourihane, A. and Abumrad, N. N. Reversal of
cancer-related wasting using oral supplementation with a
combination of .beta.-hydroxy-.beta.-me- thylbutyrate, arginine and
glutamine. Am. J. Surg., 183: 471-479, 2002) showed a mixture of
HMB, arginine and glutamine to be effective in increasing body
weight in weight losing patients with advanced (stage IV) cancer.
Moreover, the increase in body weight was attributed to an increase
in fat-free mass, as observed with EPA.
[0010] The use of the polyunsaturated fatty acid eicosapentaenoic
acid is suggested for the treatment of cachexia by inhibiting
lipolytic activity of lipolytic agents in body fluids and the
activity of the enzyme guanidino-benzoatase. See Tisdale, M. J.,
and Beck, A., U.S. Pat. No. 5,457,130, issued Oct. 10, 1995; and
Tisdale, et al. Cancer Research 50: 5022-5026 (August 1990).
However, the product taught by Tisdale was in a solid dosage form,
requiring an already ill patient to swallow 12-16 capsules per day.
This method had serious drawbacks, including difficulty in
swallowing, belching, and bad odor.
[0011] HMB has been found to be useful within the context of a
variety of applications. Specifically, in U.S. Pat. No. 6,031,000
to Nissen et al. describes a composition comprising from about 0.5
g to about 30 g of HMB, wherein from about 0.5 g to about 30 g is
based on the weight of the calcium salt of HMB and from about 0.5 g
to about 50 g of free L-arginine, and from about 0.5 g to about 50
g of free L-glutamine. This patent also provides a method for the
treatment of disease-associated wasting of an animal, a method for
decreasing the serum-level of triglycerides of an animal, a method
for decreasing the serum viral load of an animal, and a method for
redistributing fat in an animal having a visceral region and a
subcutaneous region. All methods comprise administering to the
animal a composition comprising HMB and at least one free amino
acid.
[0012] U.S. Pat. No. 5,348,979 to Nissen et al. describes the use
of HMB in the nitrogen retention in human subjects. The amount of
HMB administered is effective to conserve protein as determined by
reduction in urinary nitrogen. The method can be used with patients
having a negative nitrogen balance due to disease conditions, and
also with normal elderly persons who are subject to protein loss.
The HMB may be administered orally or by intravenous infusion. An
effective amount of HMB is within the range from 0.01 to 0.20 grams
of HMB based on its calcium salt per kilogram body weight per 24
hours.
[0013] U.S. Pat. No. 5,028,440 to Nissen describes a method for
raising meat producing domestic animals to increase lean tissue
development. HMB or an edible salt thereof is administered to the
animals in an amount for a sufficient length of time to obtain a
substantial increase in lean tissue weight. The method is
particularly adapted for use with ruminants, including beef cattle
and lambs, since HMB is not subject to appreciable rumen
destruction. The method can also be practiced with other domestic
animals, including chickens, and turkeys. HMB is feed within the
range of from 0.5 to 100 mg.
[0014] U.S. Pat. No. 4,992,470 to Nissen describes the use of HMB
to be markedly more effective for activating the immune function of
T lymphocytes of mammals than .alpha.-ketoisocaproate (KIC). For
activation of the T lymphocytes, .HMB or an edible water-soluble
salt thereof is administered to the mammal by a route through which
the HMB enters the blood of the mammal. The amount administered is
sufficient for effective enhancement of the blastogenesis of their
T lymphocytes. The method is adapted for use with domestic mammals,
including particularly cattle, sheep, and swine. HMB can also be
used with humans as an immune system stimulant. HMB (Ca-HMB basis)
is orally or parenterally administered in an amount of 500 to 2,500
milligrams (mg) per human subject per 24 hours.
[0015] German patent DE 29707308 to Kunz describes the use of
branched chain amino acids in combination with HMB to promote
muscle generation in the weight training population. Kunz teaches
that a supplement of 3 gm taken daily with a protein consumption of
200 gm per day enhances the value of nutritional protein and
significantly increases the protein efficiency. Kunz also teaches
that better effects can be achieved when HMB is combined with
protein hydrolysates and/or free amino aicd mixtures rather than
with intact (pure) proteins.
[0016] U.S. Pat. No. 5,976,50 to Engel et al. describes a dietary
food supplement for weight reduction formed of a mixture of a sugar
based confectionary containing therapeutic amounts of chitosan,
kava and a fat burning nutriceutical which may include
choline/inusital, chromium picolinate, HMB, carnitine and pyruvate.
The nutriceutical ingredient mixed with the chitosan and kava
functions to burn whatever fat the body has consumed, i.e. to
metabolize better any fat that is ingested and not attracted to the
chitosan.
[0017] Commercial products designed for the weight lifting
population that contain HMB include Lean DynamX by EAS Inc. of
Golden, Colo. Lean DynamX provides a blend of ingredients that
support fat loss without the use of strong stimulants. The
ingredients include HMB, chromium picolinate, conjugated linoleic
acid, mate leaves and stems and carnitine tartrate. The powder
composition is mixed with water and taken 2-3 servings daily, with
one serving taken 30 minutes before workouts.
[0018] Additional commercial products include Mega HMB Fuel.RTM.
from Twinlab Corporation in Hauppauge, N.Y. Mega HMB Fuel.RTM.
contains 750 mg of HMB in one capsule. The suggested daily dosage
is 4 capsules to support damage to muscle cells which can occur
subsequent to intense resistance exercise.
[0019] Also of interest is U.S. Pat. No. 5,444,054 to Garleb, et
al. and a related U.S. Pat. No. 5,780,451. These documents describe
compositions and methods useful in the treatment of ulcerative
colitis. Such compositions include a protein source that can be
intact or hydrolyzed proteins of high biological value (col. 21);
an indigestible oligosaccharide such as fructooligosaccharide; and
a lipid blend containing a relatively high proportion of
eicosapentaneoic acid, which contributes to a relatively high
.omega.-3 to .omega.-6 fatty acid ratio.
[0020] Long chain fatty acid bio-pathways and physiological actions
are discussed in U.S. Pat. No. 5,223,285 to DeMichele, et al., the
entirely of which is incorporated herein by reference.
[0021] The prevention and/or treatment of cachexia remain a
frustrating problem. Both animal and human studies suggest that
nutritional support is largely ineffective in repleting lean body
mass in the cancer-bearing host. Randomized trials exploring the
usefulness of total parenteral nutrition (TPN) support as an
adjunct to cytotoxic antineoplastic therapy have demonstrated
little improvement in treatment results. See for example Brennan,
M. F., and Burt, M. E., 1981, Cancer Treatment Reports 65 (Suppl.
5): 67-68. This, along with a clear demonstration that TPN can
stimulate tumor growth in animals suggests the routine use of TPN
in cancer treatment is not justified. Kisner, D. L., 1981, Cancer
Treatment Reports 65 (Suppl. 5): 1-2.
SUMMARY OF THE INVENTION
[0022] The present invention relates to methods for the prevention
and treatment of chronic inflammatory diseases, cancer, and
involuntary weight loss. In the practice of the present invention
patients are enterally administered HMB alone or alternatively in
combination with eicosapentaenoic (20:5 .omega.-3), FOS, carnitine
and mixtures thereof.
[0023] In another embodiment, the present invention provides a
method for the treatment of the disease-associated wasting of a
patient. The method comprises administering to the patient the
above-described composition, which comprises HMB in amounts
sufficient to treat the disease-associated wasting, wherein, upon
administration of the composition to the patient, the
disease-associated wasting is treated.
[0024] In another embodiment, the present invention provides a
method for reducing tumor growth rate in a patient. The method
comprises administering to the patient the above-described
composition, which comprises HMB in amounts sufficient to reduce
tumor growth rate, wherein, upon administration of the composition
to the patient, the tumor growth rate is reduced.
[0025] In another embodiment, the present invention provides a
method for the prevention or treatment of diseases in patients by
down regulating the expression and/or activity of protein kinase C,
nuclear factor kappa-B, ubiquitin-conjugating enzymes, and
components of 26S proteasome. These methods comprise administering
to the patient HMB, its salts, metabolites or derivatives
thereof.
[0026] In yet another embodiment, HMB may be added to food products
comprising a source of amino-nitrogen enriched with large neutral
amino acids such as leucine, isoleucine, valine, tyrosine,
threonine and phenylalanine and subtantially lacking in free amino
acids.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 presents a scheme describing the potential
intracellular events in skeletal muscle involved in PIF induced
proteasome activation.
[0028] FIG. 2 presents dose-response curves for the effect of HMB
on body weight (A) and tumor volume (B) in mice bearing the MAC16
tumor. HMB (in PBS) was administered orally by gavage on a daily
regime at a concentration of 0.05 (.circle-solid.), 0.125
(.largecircle.) and 0.25 glkg (X). Control mice received PBS alone
(.diamond-solid.). The results shown are the mean.+-.SEM, where
n=20.
[0029] FIG. 3 presents the effect of HMB (0.25 glkg; .box-solid.),
EPA (0.6 glkg; X) and the combination (.largecircle.) together with
PBS controls (.circle-solid.) on body weight of mice bearing the
MAC16 tumor. Results shown are mean.+-.SEM, where n=20.
[0030] FIG. 4 presents the weight of soleus muscles (A) and rate of
protein degradation in soleus muscle (B) of mice bearing the MAC16
tumor and treated with either EPA (0.6 glkg), HMB (0.25 glkg) or
the combination for 3 days. Values shown are mean.+-.SEM, where
n=6.
[0031] FIG. 5 presents the effect of HMB and EPA on proteasome
functional activity, determined as the `chymotrypsin-like` enzyme
activity, in gastrocnemius muscle of mice bearing the MAC16 tumor
and treated for 3 days. Results are shown as mean.+-.SEM, where
n=6.
[0032] FIG. 6 presents the expression of proteasome 20S
.alpha.-subunits (A) and .alpha.-subunits (B), detected by Western
blotting, in gastrocnemius muscle of mice treated for 3 days with
PBS (Control), HMB (0.25 glkg), EPA (0.6 glkg) or the combination.
Densitometric analysis of the blots (n=6) are shown. A. control
(closed bars), HMB (open bars), EPA (hashed bars) and combination
(dotted bars).
[0033] FIG. 7 presents the expression of proteasome 19S subunits,
MSS1 (A) and p42 (B), detected by Western blotting, in
gastrocnemius muscle of mice treated for 3 days with PBS (Control),
HMB (0.25 glkg), EPA (0.6 glkg) or the combination (HMB+EPA).
Densitometric analysis of the blots (n=6) are shown.
[0034] FIG. 8 presents the expression of E2.sub.14k, detected by
Western blotting, in gastrocnemius muscle of mice treated for 3
days with PBS (Control), HMB (0.25 glkg), EPA (0.6 glkg) or the
combination (HMB+EPA). Densitometric analysis of the blots (n=6)
are shown.
[0035] FIG. 9(A) presents the effect of PIF on total protein
degradation in C.sub.2C.sub.12 myotubes in the absence (X) or
presence of either 50 .mu.M EPA (.quadrature.), or 25 .mu.M
(.largecircle.) or 50 .mu.M (.circle-solid.) HMB. Measurements were
made 24 h after the addition of PIF and are shown as mean.+-.SEM,
where n=9. 1(B) presents the chymotryptic activity of soluble
extracts of murine myotubes treated with PIF in the absence or
presence of EPA (50 .mu.M) or HMB (25 or 50 .mu.M). The symbols are
the same as in (A). The results are shown as mean.+-.SEM, where
n=9.
[0036] FIG. 10 presents the effect of EPA and HMB on PIF-induction
of 20S proteasome .alpha.-subunit (A), .beta.-subunit (B) and p42
(C). The actin loading control is shown in (D). Western blots of
soluble extracts of C.sub.2C.sub.12 myotubes 24 h after treatment
with PIF alone (lanes A-C) or with PIF in the presence of 50 .mu.M
EPA (lanes D-F), 50 .mu.M HMB (lanes G-I) or 25 .mu.M HMB (lanes
J-L) at a concentration of PIF of 4.2 nM (lanes B, E, H and K) or
10 nM (lanes C, F, I and L). Control cultures received PBS (lane
A), 50 .mu.M EPA (lane D), 50 .mu.M HMB (lane G) or 25 .mu.M HMB
(lane J). The blots shown are representative of three separate
experiments.
[0037] FIG. 11 presents the Western blot of the effect of PIF on
cytoplasmic (A) and membrane-bound (B) PKC.sub..alpha. in murine
myotubes. Cells were treated with PIF alone (lanes A-C) or with PIF
in the presence of 50 .mu.M EPA (lanes D-F), 50 .mu.M HMB (lanes
G-I) or 25 .mu.M HMB (lanes J-L) at 4.2 nM (lanes B, E, H and K) or
10 nM PIF (lanes C, F, I and L). Control cells received PBS (lane
A), 50 .mu.M EPA (lane D), 50 .mu.M HMB (lane G) or 2511M HMB (lane
J). The blots shown are representative of three separate
experiments.
[0038] FIG. 12 presents Western blots of total ERK 1/2 (p44 and
p42) (A) and active (phosphorylated) ERK 1/2 (B) in soluble
extracts of murine myotubes treated with PIF alone (lanes A-C) or
with PIF in the presence of 50 .mu.M EPA (lanes D-F), 50 .mu.M HMB
(lanes G-I) or 25 .mu.M HMB (lanes J-L) at a PIF concentration of
4.2 nM (lanes B, E, H and K) or 10 nM (lanes C, F, I and L).
Control cells received either PBS (lane A), 50 .mu.M EPA (lane D),
50 .mu.M HMB (lane G) or 25 .mu.M HMB (lane J). The blots shown are
representative of three separate experiments.
[0039] FIG. 13 persents the effect of exposure of C.sub.2C.sub.12
myotubes for 30 min on cytosolic levels of I.kappa.B.alpha. (A),
determined by Western blotting, and activation of NF-.kappa.B
binding to DNA, as determined by EMSA (B and C). The densitometric
analysis is an average of 3 replicate blots or EMSAs. (A) Myotubes
were treated with PIF alone (lanes A-E) or with PIF in the presence
of 50 .mu.M HMB at a concentration of 0 (lanes A and F), 2.1 (lanes
B and G), 4.2 (lanes C and H), 10.5 (lanes D and I) or 16.8 nM PIF
(lanes E and J). In (B) and (C) myotubes were treated with 0, 2.1,
4.2, 10.5 or 16.8 nM PIF, in the absence (dark bars) or presence
(open bars) of 25 .mu.M HMB (B) or 50 .mu.M HMB (C).
DETAILED DESCRIPTION OF THE INVENTION
[0040] The term HMB, which is also referred to as
beta-hydroxy-beta-methyl- butyric acid, or beta-hydroxy-isovaleric
acid, can be represented in its free acid form as
(CH.sub.3).sub.2(OH)CCH.sub.2 COOH. HMB is a metabolite of leucine
formed by transamination to alpha-ketoisocaproate (KIC) in muscle
followed by oxidation of the KIC in the cytosol of the liver to
give HMB.
[0041] The term "large neutral amino acids" refers to leucine,
isoleucine, valine, tyrosine, threonine and phenylalanine. Amino
acids are the building blocks of proteins. They are characterized
by the presence of a carboxyl group (COOH) and an amino group (NH2)
attached to the same carbon at the end of the compound.
[0042] The term "substantially lacking in free amino acids" refers
to compositions which contain less than 0.4 grams of total free
amino acid content in a daily dose of the composition. For example,
if the product is designed to be fed at the rate of 1 can per day,
then the one can of product contains less than a total of 0.4 grams
of free amino acids. The amino acids in question are those
naturally occuring L-isomers, consisting of one or more of the
following compounds: L-alanine, L-arginine, L-asparagine,
L-aspartic acid, L-cysteine (or L-cystine), L-glutamic acid,
L-glutamine, glycine, L-histidine, L-Isoleucine, L-leucine,
L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,
L-threonine, L-tryptophan, L-tyrosine and L-valine, or their food-
or pharmaceutically-acceptable salts, esters, salts or derivatives
(such as methyl or ethyl esters).
[0043] The term "cachexia" refers to a state of general ill health
and malnutrition. It is often associated with and induced by
malignant cancer, and is characterized by loss of appetite, loss of
body mass, especially lean body mass, and muscle wasting.
[0044] The term "fatty acids" refer to a family of carboxylic acids
having a hydrocarbon chain, generally from about 12 to 24 carbons
long. When unsaturated (having a double bond) at least one point in
the hydrocarbon chain, such fatty acids are designated by the
position of the first double bond. .omega.-3 fatty acids have a
first double bond at the third carbon from the methyl end of the
chain; and include, but are not limited to, .alpha.-linolenic acid,
stearidonic acid, eicosapentaenoic acid ("EPA"), docosapentaenoic
acid and docosahexaenoic acid ("DHA") and the like. .omega.-6 fatty
acids have a first double bond at the sixth carbon from the methyl
end of the chain; and include, but are not limited to, linoleic
acid, .gamma.-linolenic acid, arachidonic acid ("AA"), and the
like.
[0045] The term "food products" as used herein refer to delivery
vehicles that contain one or more of fats, amino nitrogen and
carbohydrates and provides some or all of the nutritional support
for a patient in the recommended daily amounts. Frequently a food
product will contain vitamins, minerals, trace minerals and the
like to provide balanced nutrition to meal repalcements, medical
foods, supplements. The food products may be in any typical form
such as beverages, powders, bars, juices, carbonated beverages,
bottled water.
[0046] The term "Reference Daily Intakes or RDI" refers to a set of
dietary references based on the Recommended Dietary Allowances for
essential vitamins and minerals. The Recommended Dietary Allowances
are a set of estimated nutrient allowances established by the
National Academy of Sciences, which are updated periodically to
reflect current scientific knowledge.
[0047] The term "patient" refers to humans, dogs, cats, and any
other non-ruminant animal.
[0048] Any reference to a numerical range in this application
should be considered as being modified by the adjective "about".
Further, any numerical range should be considered to provide
support for a claim directed to a subset of that range. For
example, a disclosure of a range of from 1 to 10 should be
considered to provide support in the specification and claims to
any subset in that range (i.e., ranges of 2-9,3-6, 4-5, 2.2-3.6,
2.1-9.9, etc.).
[0049] While not intending the invention to be limited to any
particular theory of operation, applicants describe below a
probable mechanism.
[0050] In times of extreme need (e.g., starvation and the like),
skeletal muscle is often used by the body as a reservior of amino
acids and energy. This is mediated by upregulation of the
proteolysis and downregulation of protein synthesis in muscle. The
net result of which is release of amino acids from muscle to the
general circulation for use in maintence of critical systems. When
good health and adequate nutritient availability are restored,
muscle is rebuilt. In the case of cachexia, this system is
inappropriately activated, so even in the case of nutritional
adequacy, muscle tissue proteins continue to be broken down.
[0051] One of the key proteolytic systems which are inappropriately
activated is the ubiquitin proteosome system. When normally
functioning, this system recognizes proteins which are either aged
or in some other manner either damaged or no longer needed, and
marks them for removal via conjugation with ubiquitin. Such
ubiquitinylated proteins are recognized by the proteosome, and
degraded, releasing free ubiquitin and peptides and free amino
acids in an energy-consuming process. There are a number of
signaling molecules which activate or upregulate this system,
including proteolysis-inducing factor (PIF), which is a protein
factor produced by certain cachexia-inducing tumors. Binding of PIF
to the muscle cell causes the upregulation of phospholipase A
(PLA). This in turn produces signaling factors which ultimately
activate protein kinase C, resulting in the activation of genes
(via nuclear factor kappa B, NF.kappa.B) for ubiquitin conjugation
and for certain subunits of the proteosome. The net result of all
of this signaling is the up regulation of the ubiquitin proteosome
system, and inappropriate, sustained protein degradation in the
muscle. FIG. 1 shows a detailed pathway of this activation
sequence.
[0052] Protein Kinase C
[0053] Protein kinase C is a family of calcium- and lipid-activated
serine-threonine kinases that play a key role in numerous
intracellular signaling cascades. There are at least 12 different
PKC isotypes, which are grouped into three classes based on their
primary structure and biochemical properties (CA Carter: "Protein
kinase C as a drug target: Implications for drug or diet prevention
and treatment of cancer." Current Drug Targets 1:163-183 (2000).
These are the conventional--(cPKC.alpha., .beta.I, .beta.II and
.gamma.) which require diacylglycerol, phosphatidylserine and
calcium for activation, novel (nPKC.delta., .epsilon., .eta.,
.theta. and .mu.) which require diacylglycerol and
phosphatidylserine, but are calcium independent, and the atypical
(aPKC .lambda., .tau. and .xi.) which are calcium and
diacylglycerol-independent.
[0054] PKC is synthesized as a membrane-bound proenzyme. Removal of
the pro-sequence by proteolytic cleavage, and subsequent
phosphorylation releases a competent enzyme from the membrane to
the cytosol. Subsequent interaction with the peculiar sets of
activators produces active enzyme. Thus, there are several levels
of regulation possible, including control of expression, control of
proteolytic processing, control of initial phosphorylation events
and finally, regulation of the cytosolic levels of the various
activators required for full activity.
[0055] Protein kinase C is involved in some of the signaling
pathways leading to mitogenesis and proliferation of cells,
apoptosis, platelet activation, remodelling of the actin
cytoskeleton, modulation of ion channels and secretion. In
addition, other observation that PKC is also the major receptor for
tumor-promoting phorboly esters provided a key reagent for studying
the mechanism of action of this enzyme. PKC regulates pathways
relevant to inflammation, cardiovascular, peripheral microvascular,
CNS, oncology, immune and infectious disease states, and are
considered as serious and important targets for drug development
(P. G. Goekjian and M. R. Jirousek: "Protein Kinase C in the
Treatment of Disease: Signal Transduction Pathways, Inhibitors, and
Agents in Development" Current Medicinal Chemistry 6(9): 877-903,
(1999); CA O'Brian, N E Ward, K R Gravitt and K P Gupta: "The tumor
promoter receptor protein kinase C: A novel target for
chemoprevention and therapy of human colon cancer." Growth Factors
and Tumor Promotion: Implications for Risk Assessment, pages
117-120.COPYRGT. 1995, Wiley-Liss, Inc.; F Battaini: "Protein
kinase C isoforms as therapeutic targets in nervous system disease
states." Pharmacological Research 44(5):353-361, (2001); RN Frank:
"Potential new medical therapies for diabetic retinopathy: protein
kinase C inhibitors. Am J Ophthalmol 133:693-698 (2002); M Meier
and G L King: "Protein kinase C activation and its pharmacological
inhibition in vascular disease." Vascular Medicine 5:173-185
(2000)).
[0056] NF.kappa.B
[0057] Nuclear Factor .kappa. B (NF.kappa.B) is a family of
transcription factors found in a wide variety of mammalian cells.
The mature molecule is a homo- or heterodimer, made from one or two
of the following 5 gene products (RelA (p65), p50, RelB, c-Rel and
p52)--the most common is a dimer of RelA and p50. Under
non-activated conditions, NF.kappa.B is localized in the cytosol by
association with an inhibitory protein I.kappa.B.alpha.. Upstream
signaling involves an I.kappa.B kinase, and phosphorylation of the
bound I.kappa.B.alpha. results in it's release from NF.kappa.B,
allowing the later to translocate to the nucleus, and activate
specific gene transcription. The phosphorylated I.kappa.B.alpha. is
degraded by the ubiquitin-proteosome pathway.
[0058] NF.kappa.B is widely recognized as a key regulatory molecule
associated with inflammation. Thus, it plays a key role in both
acute and chronic inflammatory diseases (A B Lentsch and P A Ward:
"Activation and regulation of NF.kappa.B during acute
inflammation." Clin Chem Lab Med 37(3):205-208 (1999)). It also
plays a role in certain aspects of other diseases, such as cancer
metastasis (V B Andela, A H Gordon, G Zotalis, R N Rosier, J J
Goater, G D Lewis, E M Schwarz, J E Puzas and R J O'Keefe:
"NF.kappa.B: A pivotal transcription factor in prostate cancer
metastasis to bone." Clinical Orthopaedics and Related Research
415S:S75-S85 (2003)). This transcription factor is involved in the
development of the diabetic syndrome (E. Ho and T M Bray:
"Antioxidants, NF.kappa.B activation and diabetogenesis."
Proceedings of the Society for Experimental Biology and Medicine
222:205-213 (1999)) and in immune development and regulation (J
Moscat, M T Diaz-Meco and P Rennert: "NF.kappa.B activation by
proptein kinase C isoforms and B-cell function." EMBO Reports
4:31-36 (2003)). Finally, NF.kappa.B is associated with control of
apoptosis and in growth and differentiation. Indeed, PIF
(proteolysis inducing factor, which is released by tumors and is
involved in cancer-induced lean mass losses) is thought to be a
regulator of enbryonic development, and triggers a signaling
cascade ultimately through NF.kappa.B (F. Delfino and W H Walker:
"Hormonal regulation of the NF.kappa.B signaling pathway."
Molecular and Cellular Endocrinology 157:1-9 (1999); T M Watchorn,
I Waddell, N Dowidar and JA Ross: "Proteolysis-inducing factor
regulates hepatic gene expression via the transcription factor
NF.kappa.B and STST3." FASEB J 15:562-564 (2001)).
[0059] It is also well known that EPA exerts it's beneficial
effects on cachexia via inhibition of the signaling resulting from
activation of PLA, in particular the release of arachidonic acid
(AA). This prevents the subsequent upregulation and activation of
the ubiquitin-proteosome pathway by removing the initial signaling
event. HMB, while not preventing the activation of PLA or the
release of AA, does prevent the upregulation of protein kinase C,
preventing all subsequent activation in the signaling pathway, also
ultimately preventing the activation of the ubiquitin-proteosome
system.
[0060] It has now been surprisingly and unexpectedly discovered
that HMB alone can reduce tumor growth rate and in combination with
sub-optimal dose levels of EPA enhance the anticachectic effect.
The combination of EPA and HMB preserve muscle mass by attenuating
protein degradation through down regulation of the increased
expression of key regulatory components of the ubiquitin-proteasome
proteolytic pathway.
[0061] The term "HMB" refers to the compound having the foregoing
chemical formula, in both its free acid and salt forms, metabolites
and derivatives thereof. While any suitable form of HMB can be used
within the context of the present invention, preferably, HMB is
selected from the group consisting of a free acid, a salt, an
ester, and a lactone; more preferably, HMB is a salt.
[0062] While any pharmaceutically suitable salt of HMB can be used
within the context of the present invention, preferably, the HMB
salt is water-soluble or becomes water-soluble in the stomach or
intestines of a patient. More preferably, the HMB salt is selected
from the group consisting of a sodium salt, a potassium salt, a
magnesium salt, a chromium salt, and a calcium salt. Most
preferably, the HMB salt is a calcium salt. However, other
non-toxic salts, such as other alkali metal or alkaline earth metal
salts, can be used.
[0063] Similarly, any pharmaceutically acceptable ester can be used
in the context of the present invention. Desirably, the HMB ester
is rapidly converted to HMB in its free acid form. Preferably, the
HMB ester is a methyl ester or ethyl ester. HMB methyl ester and
HMB ethyl ester are rapidly converted to the free acid form of
HMB.
[0064] Likewise, any pharmaceutically acceptable lactone can be
used in the context of the present invention. Desirably, the HMB
lactone is rapidly converted to HMB in its free acid form.
Preferably, the HMB lactone is an isovalaryl lactone or a similar
lactone. Such lactones are rapidly converted to the free acid form
of HMB.
[0065] Methods for producing HMB and its derivatives are well known
in the art. For example, HMB can be synthesized by oxidation of
diacetone alcohol. One suitable procedure is described by Coffman
et al., J. Am. Chem. Soc. 80: 2882-2887 (1958). As described
therein, HMB is synthesized by an alkaline sodium hypochlorite
oxidation of diacetone alcohol. The product is recovered in free
acid form, which can be converted to the desired salt. For example,
3-hydroxy-3-methylbutyric acid (HMBA) can be synthesized from
diacetone alcohol (4-hydroxy-4-methylpentan-2-one) via oxidation
using cold, aqueous hypochlorite (bleach). After acidifying the
reaction mixture using HCl, the HMBA product is recovered by
extraction using ethyl acetate, and separating and retaining the
organic layer from the extraction mixture. The ethyl acetate is
removed by evaporation and the residue dissolved in ethanol. After
addition of Ca(OH).sub.2 and cooling, crystalline CaHMB can be
recovered by filtration, the crystals washed with ethanol and then
dried. Alternatively, the calcium salt of HMB is commercially
available from TSI in Salt Lake City, Utah.
[0066] Nutritional support in the cancer patient can be categorized
as (i) supportive, in which nutrition support is instituted to
prevent nutrition deterioration in the adequately nourished patient
or to rehabilitate the depleted patient before definitive therapy;
(ii) adjunctive, in which nutrition support plays an integral role
in the therapeutic plan; and (iii) definitive, in which aggressive
nutrition support is required for the patient's existence. The
routes for providing nutrition support include an oral diet, tube
feeding and peripheral or total parenteral nutrition. The preferred
embodiment for nutritional methods and compositions of the
invention is by the oral route.
[0067] An alternate to oral feeding is tube feeding by means of
nasogastric, nasoduodenal, esophagostomy, gastrostomy, or
jejunostomy tubes.
[0068] The beneficial effects that HMB has on the lean body mass of
a patient can be achieved in a number of ways. If desired, the HMB
may be administered alone, without a carrier. The HMB may simply be
dissolved in water and consumed by the patient. Alternatively, the
HMB may be sprinkled on food, dissolved in coffee, etc. The total
daily dose for the patient will vary widely, but typically a
patient will benefit from consuming at least 2 gm/day of HMB.
Alternatively, from 20 to 40 mg/kg/day.
[0069] In a further embodiment, the HMB may be incorporated into
pills, capsules, rapidly dissolved tablets, lozenges, etc. The
active dose can vary widely, but will typically range from 250 mg
to 1 gm/dose with the patient consuming from 2 to 8 doses/day to
achieve the target of 2 gm/day minimum. Methods for preparing such
dosage forms are well known in the art. The reader's attention is
directed to the most recent edition of Remingtons Pharmaceutical
Sciences for guidance on how to prepare such dosage forms.
[0070] While the HMB may be administered as a single entity, it
will typically be incorporated into food products and consumed by
the patient during their meals or snack. If desired, the patient
may simply modify the recipe of foods they normally consume by
sprinkling on food, dissolving in coffee, etc.
[0071] In a further embodiment, the HMB will be incorporated into
beverages, bars, cookies, etc. that have been specifically designed
to enhance the palatability of the HMB and increase the selection
of alternative forms, thereby enhancing patient/consumer
acceptance.
[0072] Typically, the HMB will be incorporated into meal
replacement beverages such as Ensure.RTM., Boost.RTM.,
Glucerna.RTM., Pediasure.RTM., Pedialyte.RTM., etc. The HMB may
also be incorporated into meal replacement bars such as
PowerBars.RTM., Glucerna.RTM. bars, Choice DM.RTM. bars,
Ensure.RTM. bars, and Boost.RTM. bars, etc. Alternatively, the HMB
maybe incorporated into juices, carbonated beverages, bottled
water, etc. Additionally, the HMB may be incorporated into medical
nutritonals such as ProSure.RTM., Promote.RTM., Jevity.RTM. and
Advera.RTM. designed to support specific disease states such as
cancer, HIV/AIDS, COPD arthritis, etc. Methods for producing any of
such food products are well known to those skilled in the art. The
following discussion is intended to illustrate such food products
and their preparation.
[0073] Most meal replacement products (i.e., bars or liquids)
provide calories from fat, carbohydrates, and protein. These
products also typically contain vitamins and minerals, because they
are intended to be suitable for use as the sole source of
nutrition. While these meal replacement products may serve as the
sole source of nutrition, they typically don't. Individuals consume
these products to replace one or two meals a day, or to provide a
healthy snack. The nutritional products of this invention should be
construed to include any of these embodiments.
[0074] The amount of these nutritional ingredients can vary widely
depending upon the targeted patient population (i.e., cancer,
HIV/AIDS, arthritis, organoleptic considerations, cultural
preferences, use, etc.). As a general nonlimiting guideline
however, the meal replacement products of this invention will
contain the following relative amounts of protein, fat, and
carbohydrate (based upon the relative percentage of total
calories): a protein component, providing from 5 to 80% of the
total caloric content, a carbohydrate component providing from 10
to 70% of the total caloric content, and a lipid component
providing from 5 to 50% of the total caloric content.
[0075] The meal replacements will contain suitable carbohydrates,
lipids and proteins as is known to those skilled in the art of
making nutritional formulas. Suitable carbohydrates include, but
are not limited to, hydrolyzed, intact, naturally and/or chemically
modified starches sourced from corn, tapioca, rice or potato in
waxy or non waxy forms; and sugars such as glucose, fructose,
lactose, sucrose, maltose, high fructose corn syrup, corn syrup
solids, fructooligosaccharides, and mixtures thereof.
[0076] Suitable lipids include, but are not limited to, coconut
oil, soy oil, corn oil, olive oil, safflower oil, high oleic
safflower oil, MCT oil (medium chain triglycerides), sunflower oil,
high oleic sunflower oil, palm oil, palm olein, canola oil,
cottonseed oil, fish oil, palm kernel oil, menhaden oil, soybean
oil, lecithin, lipid sources of arachidonic acid and
docosahexaneoic acid, and mixtures thereof. Lipid sources of
arachidonic acid and docosahexaneoic acid include, but are not
limited to, marine oil, egg yolk oil, and fungal or algal oil.
[0077] Numerous commercial sources for these fats are readily
available and known to one practicing the art. For example, soy and
canola oils are available from Archer Daniels Midland of Decatur,
Ill. Corn, coconut, palm and palm kernel oils are available from
Premier Edible Oils Corporation of Portland, Organ. Fractionated
coconut oil is available from Henkel Corporation of LaGrange, Ill.
High oleic safflower and high oleic sunflower oils are available
from SVO Specialty Products of Eastlake, Ohio. Marine oil is
available from Mochida International of Tokyo, Japan. Olive oil is
available from Anglia Oils of North Humberside, United Kingdom.
Sunflower and cottonseed oils are available from Cargil of
Minneapolis, Minn. Safflower oil is available from California Oils
Corporation of Richmond, Calif.
[0078] In addition to these food grade oils, structured lipids may
be incorporated into the food product if desired. Structured lipids
are known in the art. A concise description of structured lipids
can be found in INFORM, Vol. 8, No. 10, page 1004; entitled
Structured lipids allow fat tailoring (October 1997). Also see U.S.
Pat. No. 4,871,768. Structured lipids are predominantly
triacylglycerols containing mixtures of medium and long chain fatty
acids on the same glycerol nucleus. Structured lipids and their use
in enteral formula are also described in U.S. Pat. Nos. 6,194,379
and 6,160,007.
[0079] Optionally, .omega.-3 fatty acids may comprise approximately
30% of the oil blend, preferably the .omega.-3 fatty acids largely
consist of eicosapentaenoic acid and docosahexaenoic acid. Dietary
oils used in the preparation of the nutritional composition
generally contain .omega.-3 fatty acids in the triglyceride form
and include, but are not limited to canola, medium chain
triglycerides, fish, soybean, soy lecithin, corn, safflower,
sunflower, high-oleic sunflower, high-oleic safflower, olive,
borage, black currant, evening primrose and flaxseed oil.
Optionally, the weight ratio of .omega.-6 fatty acids to .omega.-3
fatty acids in the lipid blend according to the invention is about
0.1 to 3.0. The daily delivery of .omega.-3 fatty acids should be
at least 450 mg and may vary depending on body weight, sex, age and
medical condition of the individual. As mentioned, higher levels
are desired for adult human consumption: for example, from about
0.5 to 50 gm daily, more preferably from about 2.5 to 5 gm
daily.
[0080] An unexpected advantage to combining .omega.-3 fatty acids
and HMB is the improvement in taste of the meal replacement. The
typical sources of .omega.-3 fatty acids are fish and algae oils.
Each source brings objectionable flavors to the meal replacement
product. The Inventors discovered that by adding HMB, the same or
better clinical results related to the prevention of involuntary
weight loss can be obtained even when using sub-optimal or lower
levels of .omega.-3 fatty acids in the product. Consequently, the
Inventor's have discovered that there is an inverse relationship
between the levels of .OMEGA.-3 fatty acids and HMB. For example,
if an effective does of .omega.-3 fatty acids is 3 gm delivered in
2 cans of a meal replacement, the same clinical results would be
seen in product formulated to contain 2 gm of .omega.-3 fatty acids
and 1 gm of HMB delivered in 2 cans or in product formulated to
contain 1 gm of .omega.-3 fatty acids and 2 gm of HMB delivered in
2 cans. The product formulated to contain only 1 gm of .omega.-3
fatty acids will taste much better than the product formulated with
2 or 3 gm of .omega.-3 fatty acids while achieving the same
clinical effectiveness. Further, since .omega.-3 fatty acids are
known inhibitors of M, a mediator of inflammation, a product
containing .omega.-3 fatty acids and HMB could have broader
benefits than those containing either of the ingredients alone.
[0081] Suitable protein sources include, but not limited to, milk,
whey and whey fractions, soy, rice, meat (e.g., beef), animal and
vegetable (e.g., pea, potato), egg (egg albumin), gelatin and fish.
Suitable intact protein sources include, but are not limited to,
soy based, milk based, casein protein, whey protein, rice protein,
beef collagen, pea protein, potato protein, and mixtures
thereof.
[0082] Optionally, the intact protein source is enriched in large
neutral amino acids (LNAA) comprising valine, isoleucine, leucine,
threonine, tyrosine and phenylalanine. Typically, about 40% of
casein, whey and soy protein sources are large neutral amino acids.
For example, caseinate contains about 38 wt/wt % LNAA, whey protein
concentrate contains about 39 wt/wt % LNAA and soy protein isolate
contains about 34 wt/wt % LNAA. Typically, the meal replacement is
formulated with a protein source that will deliver about 1 to 25 gm
of LNAA per day, preferably from about 1 to 20 gm of LNAA per day,
more preferably from about 4 to 20 gm of LNM per day. As an
example, a meal replacement consumed 3 times a day that contains a
protein comprising 4.8 gm LNAA will deliver 14.4 gm LNAA per
day.
[0083] The meal replacements preferably also contain vitamins and
minerals in an amount designed to supply or supplement the daily
nutritional requirements of the person receiving the formula. Those
skilled in the art recognize that nutritional formulas often
include overages of certain vitamins and minerals to ensure that
they meet targeted level over the shelf life of the product. These
same individuals also recognize that certain micro ingredients may
have potential benefits for people depending upon any underlying
illness or disease that the patient is afflicted with. For example,
cancer patients benefit from such antioxidants as beta-carotene,
vitamin E, vitamin C and selenium. The food products preferably
include, but are not limited to, the following vitamins and
minerals: calcium, phosphorus, sodium, chloride, magnesium,
manganese, iron, copper, zinc, selenium, iodine, chromium,
molybdenum, conditionally essential nutrients m-inositol, carnitine
and taurine, and Vitamins A, C, D, E, K and the B complex, and
mixtures thereof.
[0084] The conditionally essential nutrient carnitine is a
naturally occurring amino acid formed from methionine and lysine.
Its major metabolic role is associated with the transport of
long-chain fatty acids across the mitochondrial membranes, thus
stimulating the oxidation of these fuel substances for metabolic
energy. Carnitine supplementation is an important metabolic tool in
conditions such as diseases of the liver and kidney, and major
chronic illnesses or extensive injuries complicated by
malnutrition. Optionally, the meal replacements may be supplemented
with carnitine at levels sufficient to supply up to 4 gm/day of
carnitine.
[0085] The meal replacements also may contain fiber and
stabilizers. Suitable sources of fiber/and or stabilizers include,
but are not limited to, xanthan gum, guar gum, gum arabic, gum
ghatti, gum karaya, gum tracacanth, agar, furcellaran, gellan gum,
locust bean gum, pectin, low and high methoxy pectin, oat and
barley glucans, carrageenans, psyllium, gelatin, microcrystalline
cellulose, CMC (sodium carboxymethylcellulose), methylcellulose
hydroxypropyl methyl cellulose, hydroxypropyl cellulose, DATEM
(diacetyl tartaric acid esters of mono- and diglycerides), dextran,
carrageenans, FOS (fructooligosaccharides), and mixtures thereof.
Numerous commercial sources of soluble dietary fibers are
available. For example, gum arabic, hydrolyzed
carboxymethylcellulose, guar gum, pectin and the low and high
methoxy pectins are available from TIC Gums, Inc. of Belcamp, Md.
The oat and barley glucans are available from Mountain Lake
Specialty Ingredients, Inc. of Omaha, Nebr. Psyllium is available
from the Meer Corporation of North Bergen, N.J. while the
carrageenan is available from FMC Corporation of Philadelphia,
Pa.
[0086] The fiber incorporated may also be an insoluble dietary
fiber representative examples of which include oat hull fiber, pea
hull fiber, soy hull fiber, soy cotyledon fiber, sugar beet fiber,
cellulose and corn bran. Numerous sources for the insoluble dietary
fibers are also available. For example, the corn bran is available
from Quaker Oats of Chicago, Ill.; oat hull fiber from Canadian
Harvest of Cambridge, Minn.; pea hull fiber from Woodstone Foods of
Winnipeg, Canada; soy hull fiber and oat hull fiber from The Fibrad
Group of LaVale, Md.; soy cotyledon fiber from Protein Technologies
International of St. Louis, Mo.; sugar beet fiber from Delta Fiber
Foods of Minneapolis, Minn. and cellulose from the James River
Corp. of Saddle Brook, N.J.
[0087] A more detailed discussion of examples of fibers and their
incorporation into food products may be found in U.S. Pat. No.
5,085,883 issued to Garleb et al.
[0088] The quantity of fiber utilized in the formulas can vary. The
particular type of fiber that is utilized is not critical. Any
fiber suitable for human consumption and that is stable in the
matrix of a food product may be utilized.
[0089] In addition to fiber, the meal replacements may also contain
oligosaccharides such as fructooligosaccharides (FOS) or
glucooligosaccharides (GOS). Oligosaccharides are rapidly and
extensively fermented to short chain fatty acids by anaerobic
microorganisms that inhabit the large bowel. These oligosaccharides
are preferential energy sources for most Bifidobacterium species,
but are not utilized by potentially pathogenic organisms such as
Clostridium perfingens, C. difficile, or Eschericia coli.
[0090] Typically, the FOS comprises from 0 to 5 gm/serving of the
meal replacement, preferably from 1 to 5 gm/serving, more
preferably from 2 to 4 gm/serving of the meal replacement.
[0091] The meal replacements may also contain a flavor to enhance
its palatability. Artificial sweeteners may be added to complement
the flavor and mask salty taste. Useful artificial sweeteners
include saccharin, nutrasweet, sucralose, acesulfane-K (ace-K),
etc.
[0092] Meal replacements can be manufactured using techniques well
known to those skilled in the art. Various processing techniques
exist. Typically these techniques include formation of a slurry
from one or more solutions, which may contain water and one or more
of the following: carbohydrates, proteins, lipids, stabilizers,
vitamins and minerals. The HMB is typically added to the
carbohydrate slurry prior to the other minerals. The slurry is
emulsified, homogenized and cooled. Various other solutions may be
added to the slurry before processing, after processing or at both
times. The processed formula is then sterilized and may be diluted
to be dried to a powder, utilized on a ready-to-feed basis or
packaged in a concentrated liquid form. When the resulting formula
is meant to be a ready-to-feed liquid or concentrated liquid, an
appropriate amount of water would be added before
sterilization.
[0093] Solid compositions such as bars, cookies, etc. may also be
manufactured utilizing techniques known to those skilled in the
art. For example, they may be manufactured using cold extrusion
technology as is known in the art. To prepare such compositions,
typically all of the powdered components will be dry blended
together. Such constituents typically include the proteins, vitamin
premixes, certain carbohydrates, etc. The fat-soluble components
are then blended together and mixed with the powdered premix above.
Finally any liquid components are then mixed into the composition,
forming a plastic like composition or dough.
[0094] The process above is intended to give a plastic mass that
can then be shaped, without further physical or chemical changes
occurring, by the procedure known as cold forming or extrusion. In
this process, the plastic mass is forced at relatively low pressure
through a die, which confers the desired shape. The resultant
exudate is then cut off at an appropriate position to give products
of the desired weight. If desired the solid product is then coated,
to enhance palatability, and packaged for distribution. Typically
the package will provide directions for use by the end consumer
(i.e., to be consumed by a cancer patient, to help prevent lean
muscle loss, etc.).
[0095] The solid compositions of the instant invention may also be
manufactured through a baked application or heated extrusion to
produce cereals, cookies, and crackers. One knowledgeable in the
arts would be able to select one of the many manufacturing
processes available to produce the desired final product.
[0096] As noted above, the HMB may also be incorporated into
juices, non-carbonated beverages, carbonated beverages, electrolyte
solutions, flavored waters (hereinafter collectively "beverage"),
etc. The HMB will typically comprise from 0.5 to 2 gm/serving of
the beverages. Methods for producing such beverages are well known
in the art. The reader's attention is directed to U.S. Pat. Nos.
6,176,980 and 5,792,502, the contents of each which are hereby
incorporated by reference. For example, all of the ingredients,
including the HMB are dissolved in an appropriate volume of water.
Flavors, colors, vitamins, etc. are then optionally added. The
mixture is then pasteurized, packaged and stored until
shipment.
[0097] Any disease with which wasting or inflammation is associated
such as cardiovascular, peripheral microvascular, central nervous
system, oncology, immune and infectious disease states can be
treated in accordance with the present methods. Preferably, the
disease is selected from the group consisting of cancer, cachexia,
age-associated wasting, wasting associated with long-term hospital
stay, HIV/AIDS, arthritis, trauma, liver disease, Crohn's disease
or other inflammatory bowel diseases (IBD), renal insufficiency and
COPD (chronic obstructive pulmonary disease). More preferably, the
disease is cachexia.
[0098] The present invention provides, in another embodiment, a
method for the treatment of the disease-associated wasting of a
patient, such as a mammal, preferably a human. The method comprises
administering to the patient the above-described composition, which
comprises HMB in amounts sufficient to treat the disease-associated
wasting, wherein, upon administration of the composition to the
patient, the disease-associated wasting is treated.
[0099] The amount of HMB that is sufficient to treat
disease-associated wasting in a given patient can be determined in
accordance with methods well known in the art. When treating the
disease-associated wasting of a patient, desirably, the composition
comprising HMB is administered to a patient suffering from
disease-associated wasting in such an amount, in such a manner, and
over such a period of time that the patient's lean tissue mass will
increase without a concomitant decrease in the patient's fat mass.
An example, within the context of treating the cancer cachexia
associated wasting of a human, when the composition is orally
administered about twice a day for a minimum of two weeks; the dose
is sufficient to provide at least 2 gm HMB/day.
[0100] The present invention provides, in another embodiment, a
method for reducing tumor growth rate in a patient, such as a
mammal, preferably a human. The method comprises administering to
the patient the above-described composition, which comprises HMB in
amounts sufficient to reduce tumor growth rate, wherein, upon
administration of the composition to the patient, the tumor growth
rate is reduced.
[0101] The amount of HMB that is sufficient to attenuate tumor
growth in a given patient can be determined in accordance with
methods well known in the art. When treating tumor growth in a
patient, desirably, the composition comprising HMB is administered
to a patient suffering from tumor growth in such an amount, in such
a manner, and over such a period of time that the patient's tumor
growth rate will decrease. An example, within the context of
treating the tumor growth in an adult human, when the composition
is orally administered about twice a day for a minimum of two
weeks; the dose is sufficient to provide at least about 2 gm
HMB/day.
[0102] The present invention provides, in another embodiment, a
method for down regulating the expression and/or activity of
protein kinase C. Examples I-IV show that both EPA and HMB
attenuated PIF-induced activation of protein kinase C (PKC) and the
subsequent degradation of I.kappa.B.alpha. and nuclear accumulation
of nuclear factor-.kappa.B (NF-.kappa.B).
[0103] The present invention provides, in another embodiment, a
method for down regulating the expression and/or activity of
nuclear factor kappa-B. Examples I-IV show that both EPA and HMB
attenuated PIF-induced activation of protein kinase C (PKC) and the
subsequent degradation of I.kappa.B.alpha. and nuclear accumulation
of nuclear factor-.kappa.B (NF-.kappa.B).
[0104] The present invention provides, in another embodiment, a
method for down regulating the expression and/or activity of
ubiquitin-conjugating enzymes. Examples I-IV show that this was
accompanied by a reduction in the expression of E2.sub.14k
ubiquitin-conjugating enzyme. The combination of EPA and HMB was at
least as effective or more effective than either treatment alone.
These results show that both EPA and HMB preserve muscle mass by
attenuating protein degradation through down regulation of the
increased expression of key regulatory components of the
ubiquitin-proteasome proteolytic pathway.
[0105] The present invention provides, in another embodiment, a
method for down regulating the expression and/or activity of
components of 26S proteasome. Examples I-IV show that proteasome
activity, determined by the `chymotrypsin-like` enzyme activity,
was attenuated by HMB. Protein expression of the 20S a or
.beta.-subunits was reduced by at least 50%, as were the ATPase
subunits MSS1 and p42 of the 19S proteasome regulatory subunit.
EXAMPLE I
Prevention of Weight Loss and Attenuation of Protein Degradation in
Animals with Cancer Cachexia
[0106] This study evaluates the effect of HMB, in comparison with
EPA or combination, on weight loss induced by the MAC16 tumor and
the mechanisms involved. Weight loss induced by the MAC16 tumor is
primarily induced by PIF.
[0107] Pure strain male NMRI mice (average weight 25 g) were
obtained from our own inbred colony and were transplanted with
fragments of the MAC16 tumor s.c. into the flank by means of a
trochar, selecting from donor animals with established weight loss
as described in Bibby, M. C. et al. Characterization of a
transplantable adenocarcinoma of the mouse colon producing cachexia
in recipient animals. J. Natl. Cancer Inst., 78: 539-546, 1987.
Transplanted animals were fed a rat and mouse breeding diet
(Special Diet Services, Witham, United Kingdom) and water ad
libitum, and weight loss was evident 10-12 days after tumor
implantation. Animals just prior to the development of weight loss
were randomized to receive daily either EPA (in olive oil), HMB (in
PBS) or the combination as described in the figure legends
administered p.o. by gavage, while control animals received either
olive oil or PBS. EPA (98% as free acid) was purchased from Biomol
Research Laboratories Inc., PA, USA. HMB (as the calcium salt) was
obtained from Abbott Laboratories, Columbus, Ohio, USA. All groups
contained a minimum of 6 mice. Tumor volume, body weight and food
and water intake were monitored daily. Animals were terminated by
cervical dislocation when the body weight loss reached 25%, and all
studies were conducted according to the UKCCR Guidelines for the
care and use of laboratory animals. The soleus muscles were quickly
dissected out, together with intact tendons, and maintained in
isotonic ice-cold saline before determination of protein
degradation.
[0108] Freshly dissected soleus muscles were fixed via the tendons
to aluminium wire supports, under tension, at approximately resting
length to prevent muscle shortening and preincubated for 45 min in
3 ml of oxygenated (95% oxygen:5% carbon dioxide) Krebs-Henseleit
bicarbonate buffer (pH 7.4) containing 5 mM glucose and 0.5 mM
cycloheximide. Protein degradation was determined by the release of
tyrosine over a 2 h period as described in Waalkes, T. P. et al. A
fluorimetric method for the estimation of tyrosine in plasma and
tissues. J. Lab. Clin. Med., 50: 733-736, 1957.
[0109] Functional proteasome activity was determined by measuring
the `chymotrypsin-like` enzyme activity, the predominant
proteolytic activity of the .beta.-subunits of the proteasome
according to the method of Orino, E. et al. ATP-dependent
reversible association of proteasomes with multiple protein
components to form 26S complexes that degrade ubiquitinated
proteins in human HL-60 cells. FEBS Lett., 284: 206-210, 1991.
Muscles were rinsed with ice-cold PBS, minced and sonicated in 20
mM Tris. HCl, pH 7.5, 2 mM ATP, 5 mM MgCl.sub.2 and 1 mM DTT. The
sonicate was then centrifuged for 10 min at 18,000 g, at 4.degree.
C. and the supernatant was used to determine `chymotrypsin-like`
enzyme activity by the release of aminomethyl coumarin (AMC) from
the fluorogenic substrate succinyl-LLVY-AMC. Activity was measured
in the absence and presence of the specific proteasome inhibitor
lactacystin (10 .mu.M). Only lactacystin suppressible activity was
considered to be proteasome specific.
[0110] For Western blotting samples of soleus muscle cytosolic
protein (2 to 5 .mu.g), obtained from the above assay, were
resolved on 10% SDS-PAGE and transferred to 0.45 .mu.m
nitrocellulose membrane (Hybond.TM., Amersham Life Science
Products, Bucks, United Kingdom), which had been blocked with 5%
Marvel in PBS. The primary antibodies for MSS1 and p42 were used at
a dilution of 1:5000, for 20S proteasome .alpha.-subunits at 1:1500
and for .beta.-subunits at 1:1000, while the antibody for
E2.sub.14k was used at a dilution of 1:500. The secondary
antibodies were used at a dilution of 1:2000. Mouse monoclonal
antibodies to 20S proteasome subunits 1, 2, 3, 5, 6 and 7 (clone
MCP 231), 20S proteasome subunit .beta.3 (HC10), 19S regulator
ATPase subunit Rpt 1(S7, Mss1; clone MSS1-104) and 19S regulator
ATPase subunit Rpt 4 (S106, p42; clone p42-23) were purchased from
Affiniti Research Products, Exeter, United Kingdom. Rabbit
polyclonal antisera to ubiquitin-conjugating enzyme E2 (anti-UBC2
antibody) was a gift from Dr. Simon Wing, McGill University,
Montreal, Quebec, Canada. Peroxidase-conjugated goat anti-rabbit
and rabbit anti-mouse secondary antibodies were from Dako Ltd.,
Cambridge, United Kingdom. Incubation was carried out for 2 h at
room temperature, and developed by chemiluminescence (ECL;
Amersham).
[0111] A dose-response relationship of HMB on weight loss in mice
bearing the MAC16 tumor is shown in FIG. 2. Doses of HMB greater
than 0.125 g/kg caused a significant reduction in weight loss (FIG.
2A). Differences from the control group are indicated as a,
p<0.05; b, p<0.01 and c, p<0.005. Attenuation of weight
loss was not accompanied by an alteration in food and water intake.
A dose level of 0.25 g/kg was chosen for all subsequent
experiments. The effect of HMB, EPA and the combination of HMB and
EPA on weight loss in MAC16 cachectic tumour-bearing mice is shown
in FIG. 3. Differences from the control group are indicated as a,
p<0.05; b, p<0.01 or c, p<0.005. A suboptimal dose of EPA
was chosen to investigate interactions with HMB. All treatments
caused a significant increase in soleus muscle weight (FIG. 4A),
and a significant reduction in tyrosine release (FIG. 4B),
indicating a reduction in total protein degradation. Differences
from the PBS control group are indicated as a, p<0.05, b,
p<0.01 or c, p<0.005. At the doses chosen, HMB was as
effective as EPA.
[0112] Proteasome expression has been shown to be elevated in
gastrocnemius muscles of mice bearing the MAC16 tumor and this
increased gene expression has been shown to be attenuated by EPA.
The results in FIG. 5 show that functional proteasome activity, as
determined by `chymotrypsin-like` enzyme activity, was attenuated
by HMB to the same extent as EPA at the doses chosen, and that the
combination of HMB and EPA did not produce a further depression in
activity. Differences from control are indicated as c, p<0.005.
Protein expression of proteasome subunits was analysed by Western
blotting of supernatants from sonicated muscle tissues. Expression
of 20S proteasome .alpha.-subunits, the structural units of the
proteasome was attenuated by both HMB and EPA, and there was some
indication of a further decrease of band 2 for the combination
(FIG. 6A). Differences from control are shown as c, p<0.001,
while differences from HMB are shown as e, p<0.01. Expression of
the 20S proteasome .alpha.-subunits, the catalytic subunits of the
proteasome, were also attenuated by HMB and EPA, but the
combination was more effective than either agent alone (FIG. 6B).
Differences from control are shown as c, p<0.001.
[0113] Expression of MSS1, an ATPase subunit of the 19S proteasome
regulatory complex is shown in FIG. 7A. Both HMB and EPA attenuated
MSS1 expression, but the combination did not appear to produce a
further reduction. Similar results were obtained with p42, another
ATPase subunit of the 19S regulator, that promotes ATP dependent
association of the 20S proteasome with the 19S regulator to form
the 26S proteasome (FIG. 7B). Differences from control are shown as
c, p<0.001. Again both HMB and EPA appeared to be equally
effective, while the combination did appear to reduce p42
expression further. Expression of the ubiquitin-conjugating enzyme,
E2.sub.14k, was also reduced by both HMB and EPA, while the
combination caused a further reduction in expression (FIG. 8).
Differences from control are shown as b, p<0.01 and c,
p<0.001, while differences from HMB alone are shown as d,
p<0.05 and f, p<0.001. These results confirm HMB to be as
effective as EPA in attenuating loss of muscle mass, protein
degradation and down-regulation of the ubiquitin-proteasome
proteolytic pathway, and this mechanism appears to be responsible
for the preservation of muscle mass in cachectic mice bearing the
MAC16 tumor.
[0114] This study has shown that HMB is effective in attenuating
the development of cachexia or involuntary weight loss in mice
bearing the MAC16 tumor and produced a reduction in protein
degradation in skeletal muscle by down regulating the increased
expression of the ubiquitin-proteasome pathway. Thus HMB is as
effective as EPA in reducing protein expression of the 20S
proteasome .alpha. and .beta. subunits, as well as two subunits of
the 19S regulator MSS1 and p42, expression of E2.sub.14k and
proteasome proteolytic activity.
EXAMPLE II
Attenuation of Tumor Growth in Animals
[0115] The animal study described in Example I above also evaluated
the effect of HMB on tumor growth rate in MAC16 cachectic
tumor-bearing mice. The experiment was conducted as described in
Example I.
[0116] A dose-response relationship of HMB alone on tumor growth
rate in mice bearing the MAC16 tumor is shown in FIG. 2B.
Differences from the control group are indicated as a, p<0.05;
b, p<0.01 and c, p<0.005. Doses of HMB greater than 0.125
g/kg caused a significant reduction in tumor growth rate.
Attenuation of tumor growth was not accompanied by an alteration in
food and water intake.
EXAMPLE III
Attenuation of Protein Degradation in Murine Myotubes
[0117] This study examines the effect of HMB on PIF-induced protein
degradation and signalling pathways in murine myotubes to determine
the mechanism of the attenuation of the increased expression of the
ubiquitin-proteasome proteolytic pathway.
[0118] C.sub.2C.sub.12 myotubes were routinely passaged in DMEM
supplemented with 10% FCS, glutamine and 1% penicillin-streptomycin
under an atmosphere of 10% CO.sub.2 in air at 37.quadrature. C.
Myotubes were formed by allowing confluent cultures to
differentiate in DMEM containing 2% HS, with medium changes every 2
days.
[0119] PIF was purified from solid MAC16 tumors (Todorov, P. et al.
Characterization of a cancer cachectic factor. Nature, 379:
739-742, 1996.) excised from mice with a weight loss of 20 to 25%.
Tumors were homogenised in 10 mM Tris-HCl, pH 8.0, containing 0.5
mM phenylmethylsulfonyl fluoride, 0.5 mM EGTA and 1 mM
dithiothreitol at a concentration of 5 ml/g tumor. Solid ammonium
sulfate was added to 40% w/v and the supernatant, after removal of
the ammonium sulfate, was subjected to affinity chromatography
using anti-PIF monoclonal antibody coupled to a solid matrix as
described in Todorov, P. et al Induction of muscle protein
degradation and weight loss by a tumor product. Cancer Res., 56:
1256-1261, 1996. The immunogenic fractions were concentrated and
used for further studies.
[0120] Myotubes in six-well multidishes were labeled with
L-[2,6.sup.-3H] phenylalanine (0.67 mCi/mmole) for 24 h in 2 ml
DMEM containing 2% HS. They were then washed three times in PBS
followed by a 2 h incubation at 37.degree. C. in DMEM without
phenol red until no more radioactivity appeared in the supernatant.
These myotubes were then further incubated for 24 h in the presence
of PIF, with and without EPA or HMB, in fresh DMEM without phenol
red, to prevent quenching of counts, and in the presence of 2 mM
cold phenylalanine to prevent reincorporation of radioactivity. The
amount of radioactivity released into the medium was expressed as a
percentage of control cultures not exposed to PIF to determine
total protein degradation.
[0121] For measurement of arachidonic acid release, myotubes in
six-well multi dishes containing 2 ml DMEM with 2% HS were labeled
for 24 h with 10 .mu.M arachidonic acid (containing 1 .mu.Ci of
[.sup.3H] arachidonate/ml) (Smith, H. et al. Effect of a cancer
cachectic factor on protein synthesis/degradation in murine
C.sub.2C.sub.12 myoblasts: Modulation by eicosapentaenoic acid.
Cancer Res., 59: 5507-5513, 1999). Cells were then washed
extensively with PBS to remove traces of unincorporated [.sup.3H]
arachidonate and either EPA or HMB was added 2 h prior to PIF.
After a further 24 h 1 ml of medium was removed to determine the
radioactivity released.
[0122] The functional activity of the .beta. subunits of the
proteasome was determined as the `chymotrypsin-like` enzyme
activity obtained fluorimetrically according to the method of
Orino, E. et al. ATP-dependent reversible association of
proteasomes with multiple protein components to form 26S complexes
that degrade ubiquitinated proteins in human HL-60 cells. FEBS
Lett., 284: 206-210, 1991. Myotubes were exposed to PIF for 24 h
with or without EPA or HMB added 2 h prior to PIF and enzyme
activity was determined in a supernatant fraction (Whitehouse, A.
S. et al. Increased expression of the ubiquitin-proteasome pathway
in murine myotubes by proteolysis-inducing factor (PIF) is
associated with activation of the transcription factor NF-.kappa.B.
Br. J. Cancer, 89: 1116-1122, 2003) by the release of aminomethyl
coumarin (AMC) from succinyl-LLVY-AMC (0.1 mM) in the presence or
absence of the specific proteasome inhibitor lactacystin (1011M)
(Fenteany, G. et al. Lactacystin, proteasome function and cell
fate. J. Biol. Chem., 273: 8545-8548, 1998). Only lactacystin
suppressible activity was considered to be proteasome specific.
Activity was adjusted for the protein concentration of the sample,
determined using the Bradford assay (Sigma Chemical Co., Dorset,
United Kingdom) using bovine serum albumin as standard.
[0123] For Western blot analysis, cytosolic protein (2 to 5 .mu.g)
obtained for the above assay were resolved on 10% SDS-PAGE and
transferred to 0.45 .mu.m nitrocellulose membrane, which had been
blocked with 5% Marvel in PBS, at 4.degree. C. overnight. The
primary antibodies were used at a dilution of 1:100 (anti-actin and
PKC.sub..alpha.); 1:500 (anti-ERK1 and 2); 1:1000 (anti-20S
proteasome .beta.-subunit and I.kappa.B.alpha.); 1:1500 (anti-20S
proteasome .alpha.-subunit) or 1:5000 (anti-p42), while the
secondary antibodies were used at a dilution of 1:2000. Incubation
was carried out for 2 h at room temperature and development was by
ECL. Loading was quantitated by actin concentration.
[0124] DNA binding proteins were extracted from myotubes by the
method of Andrews, N. C. et al. A rapid micropreparation technique
for extraction of DNA-binding proteins from limiting numbers of
mammalian cells. Nucleic Acids Res., 19: 2499, 1991, which utilizes
hypotonic lysis followed by high salt extraction of nuclei. The
EMSA (electrophoretic mobility shift assay) binding assay was
carried out according to the manufacturer's instructions.
[0125] Since protein degradation and activation of the
ubiquitin-proteasome proteolytic pathway in mice bearing the MAC16
tumor is thought to be mediated by PIF, mechanistic studies on the
effect of HMB on protein degradation were carried out in murine
myotubes treated with PIF. PIF-induced total protein breakdown with
a typical bell-shaped dose-response curve, as previously reported
by Gomes-Marcondes, et al Development of an in-vitro model system
to investigate the mechanism of muscle protein catabolism induced
by proteolysis-inducing factor. Br. J. Cancer, 86: 1628-1633, 2002
with a maximal effect at 4 nM. The effect of EPA has been
previously shown (Smith, H. J. et al. Effect of a cancer cachectic
factor on protein synthesis/degradation in murine C.sub.2C.sub.12
myoblasts: Modulation by eicosapentaenoic acid. Cancer Res., 59:
5507-5513, 1999; Whitehouse, A. S. et al. Induction of protein
catabolism in myotubes by 15(S)-hydroxyeicosatetraenoic acid
through increased expression of the ubiquitin-proteasome pathway.
Br. J. Cancer, 89: 737-745, 2003; and Whitehouse, A. S. et al.
Increased expression of the ubiquitin-proteasome pathway in murine
myotubes by proteolysis-inducing factor (PIF) is associated with
activation of the transcription factor NF-.kappa.B. Br. J. Cancer,
89: 1116-1122, 2003) to be effective at 50 .mu.M, and the data in
FIG. 9A shows that at a concentration of 50 .mu.M both HMB and EPA
were equally effective in attenuating PIF induced protein
degradation. There was also some attenuation at 25 .mu.M HMB at
low, but not at high concentrations of PIF. Differences from
control in the absence of PIF are indicated as a, p<0.005, while
differences form control with PIF (for groups with additions of HMV
or EPA) are indicated as b, p<0.01 and c, p<0.005.
[0126] PIF-induced protein degradation has previously been shown to
be due to an increased expression of the regulatory components of
the ubiquitin-proteasome proteolytic pathway by Lorite, M. J.,
Smith, H. J., Arnold, J. A., Morris, A., Thompson, M. G. and
Tisdale, M. J. Activation of ATP-ubiquitin-dependent proteolysis in
skeletal muscle in vivo and murine myoblasts in vitro by a
proteolysis-inducing factor (PIF). Br. J. Cancer, 85: 297-302, 2001
and Gomes-Marcondes, M. C. C., Smith, H. J., Cooper, J. C. and
Tisdale, M. J. Development of an in-vitro model system to
investigate the mechanism of muscle protein catabolism induced by
proteolysis-inducing factor. Br. J. Cancer, 86: 1628-1633,
2002.
[0127] The functional activity of this pathway is measured by the
`chymotrypsin-like` enzyme activity, the predominant proteolytic
activity of the .beta.-subunits of the proteasome. PIF induced an
increase in `chymotrypsin-like` enzyme activity, which was maximal
at 4.2 nM. The effect of PIF was completely attenuated by 50 .mu.M
EPA and both 25 and 50 .mu.M HMB. (FIG. 9B, differences from
control are shown as a, p<0.001, while differences in the
presence of EPA or HMB are shown as b, p<0.001). A similar
effect was observed on expression of proteasome 20S .alpha.
subunits, .beta. subunits and p42, an ATPase subunit of the 19S
regulator that promotes ATP-dependent association of the 20S
proteasome with the 19S regulator to form the 26S proteasome (FIG.
10). In all cases expression was increased by PIF at 4.2 and 10 nM
and this was attenuated by EPA and HMB at 50 .mu.M, but not at 25
.mu.M. These results confirm that HMB attenuates protein
degradation through an effect on PIF induction of the
ubiquitin-proteasome pathway.
EXAMPLE IV
Effect on Activity of Mediator of Signaling in Inflammation and
Proteolysis
[0128] The in vitro study described in Example III above also
evaluated the effect of HMB on molecules that are key mediators in
the pathway of inflammation. This experiment was conducted as
described in Example III.
[0129] Activation of PKC has been shown to activate extracellular
signal-regulated kinase (ERK) cascade of MAPK signalling pathways
(Toker, A. Signalling through protein kinase C. Front. Biosci., 3:
1134-1147, 1998; Wolf, I and Seger, R. The mitogen-activated
protein kinase signalling cascade: from bench to bedside. IMAJ., 4:
641-647). The activated ERKs, e.g., ERK1 (or p44 MAPK) and ERK2 (or
p42 MAPK), are able to phosphorylate and consequently activate
cytosolic phospholipase A2, the rate-limiting enzyme in pathways
involving arachidonic acid release in inflammation. In addition,
PIF has been shown to induce phosphorylation of p42/44 MAPK, while
the total MAPK remained unchanged and to be involved in PIF-induced
proteasome expression (Smith, H. J. et al. Signal transduction
pathways involved in proteolysis-inducing factor induced proteasome
expression in murine myotubes. Br. J. Cancer, 89: 1783-1788, 2003).
The effect of EPA and HMB on this process is shown in FIG. 12. PIF
induced an increased phosphorylation of p42/44 that was maximal at
4.2 nM and this effect was completely attenuated by both EPA and
HMB at 50 .mu.M, but not HMB at 25 .mu.M. The ability of HMB to
attenuate ERK 1/2 phosphorylation may be important in inhibition of
PIF-induced proteasome expression by HMB.
[0130] Experiments using mutants of PKC as well as inhibitors of
this enzyme show that this forms a central mediator of
intracellular signalling by PIF. PKC is likely to be involved in
phosphorylation (and degradation) of I-.kappa.B.alpha. leading to
nuclear accumulation of NF-.kappa.B and increased gene
transcription. PIF stimulates translocation of PKC.sub..alpha. from
the cytoplasm to the plasma membrane (FIG. 11) resulting in
activation with a maximum effect at 4.2 nM PIF as with protein
degradation (FIG. 9). This process was effectively attenuated by
both EPA and HMB at 50 .mu.M; while HMB was less effective at 25
.mu.M (FIG. 11). This suggests that PIF-induced stimulation of PKC
is attenuated by HMB through inhibition of PKC.
[0131] As previously discussed PIF induces degradation of
I-.kappa.B.alpha. and stimulates nuclear accumulation of
NF-.kappa.B and this process has been shown to be attenuated by 50
.mu.M EPA (Whitehouse, A. S. et al. Increased expression of the
ubiquitin-proteasome pathway in murine myotubes by
proteolysis-inducing factor (PIF) is associated with activation of
the transcription factor NF-.kappa.B. Br. J. Cancer, 89: 1116-1122,
2003). The results in FIG. 13A show HMB at 50 .mu.M to effectively
attenuate I-.kappa.B.alpha. degradation in the presence of PIF in
murine myotubes, and prevent nuclear accumulation of NF-.kappa.B
(FIG. 13C). Differences from 0 nM PIF are shown as b, p<0.01 and
c, p<0.001. Only partial inhibition of binding of NF-.kappa.B to
DNA was observed when HMB was used at a concentration of 25 .mu.M
(FIG. 13B). Differences from 0 nM PIF b=p<0.01 and c=p<0.001.
Differences between 50 uM HMB and PIF treated against PIF alone at
the same concentration e=p<0.01 and f=p<0.001. These results
suggest that the overall effect of HMB is comparable to that of EPA
in preventing movement of NF-.kappa.B into the nucleus with
concomitant activation of gene expression.
[0132] Thus HMB appears to be an effective agent in the treatment
of cytokine induced inflammation and muscle wasting in cancer
cachexia. HMB appears to exert its effect by inhibition of PKC
activity, and resultant stabilization of the cytoplasmic
I.kappa.B/NF-.kappa.B complex. Since these molecules are key
mediators in the pathway of inflammation, HMB appears to be an
anti-inflammatory compound.
EXAMPLE V
Composition of a Nutritional Product to Prevent Involuntary Weight
Loss
[0133] The specific list of materials for manufacturing the
nutritional product of this Example is presented in Table 1. Of
course, various changes in specific ingredients and quantities may
be made without departing from the scope of the invention.
1TABLE 1 LIST OF MATERIALS AMOUNT INGREDIENT (KG) WATER 316
ULTRATRACE/TRACE MINERAL PREMIX 0.06 ZINC SULFATE 0.033 MANGANESE
SULFATE 0.0082 SODIUM MOLYBDATE 0.00023 CHROMIUM CHLORIDE 0.00029
SODIUM SELENITE 0.000098 POTASSIUM CHLORIDE 0.072 SODIUM CITRATE
2.89 POTASSIUM IODIDE 0.00009 POTASSIUM CITRATE 1.5 CORN SYRUP 7.68
MALTODEXTRIN 53.6 MAGNESIUM PHOSPHATE DIBASIC 0.26 CALCIUM
PHOSPHATE TRIBASIC 0.99 MAGNESIUM CHLORIDE 1.2 SUCROSE 11.9
FRUCTOOLIGOSACCHARIDE 5.9 MEDIUM CHAIN TRIGLYCERIDE 2.6 CANOLA OIL
1.5 SOY OIL 0.87 57% VITAMIN A PALMITATE 0.007 VITAMIN DEK PREMIX
0.04 VITAMIN D 0.0000088 D-ALPHA-TOCOPHEROL ACETATE 0.036
PHYLLOQUINONE 0.00006 CARRAGEENAN 0.03 SOY LECITHIN 0.6 SODIUM
CASEINATE 15.5 CALCIUM CASEINATE 4.2 CALCIUM HMB MONOHYDRATE 2.6
MILK PROTEIN ISOLATE 14 REFINED DEODORIZED SARDINE OIL 6.9 ASCORBIC
ACID 0.12 45% POTASSIUM HYDROXIDE 0.13 TAURINE 0.12 WATER SOLUBLE
VITAMIN PREMIX 0.11 NIACINAMIDE 0.017 CALCIUM PANTOTHENATE 0.01
THIAMINE CHLORIDE HYDROCHLORIDE 0.003 PYRIDOXINE HYDROCHLORIDE
0.003 RIBOFLAVIN 0.002 FOLIC ACID 0.0004 BIOTIN 0.00034
CYANOCOBALAMIN 0.000038 ASCORBYL PALMITATE 0.03 CHOLINE CHLORIDE
0.25 L-CARNITINE 0.0681 N&A MARSHMALLOW VANILLA 1.6 N&A
DULCE DE LECHE 0.27
[0134] The liquid nutritional product of the present invention was
manufactured by preparing three slurries which are blended
together, combined with refined deodorized sardine oil, heat
treated, standardized, packaged and sterilized. The process for
manufacturing 454 kg (1,000 pounds) of the liquid nutritional
product, using the List of Materials from Table 7, is described in
detail below.
[0135] A carbohydrate/mineral slurry is prepared by first heating
about 62.6 kg of water to a temperature in the range of about
71.degree. C. to 77.degree. C. with agitation. The HMB is added to
the water and dissolved by agitaiting the resultant solution for at
least five minutes. The required amount of potassium citrate and
ultratrace/trace mineral premix is added to the water and dissolved
by agitating the resultant solution for at least 10 minutes. The
following minerals are then added, in the order listed, with high
agitation: magnesium chloride, potassium chloride, sodium citrate,
potassium iodide, magnesium phosphate and tricalcium phosphate. The
slurry is allowd to mix under moderate agitation until completely
dissolved or dispersed. The corn syrup, sucrose and maltodextrin
are then added to the slurry with agitation. Add the required
amount of FOS and allow to mix. The completed carbohydrate/mineral
slurry is held with high agitation at a temperature in the range of
about 60-66.degree. C. for not longer than 8 hours until it is
blended with the other slurries.
[0136] An oil slurry is prepared by combining and heating the
medium chain triglycerides (fractionated coconut oil), canola oil
and soy oil to a temperature in the range of about 32-43.degree. C.
with agitation. The vitamin DEK premis is added and allowed to mix
until completely dispersed. The required amounts of following
ingredients are added: sly lecithin, vitamin A, ascorbyl plamitate,
and vitamin E. The carrageen is added and allowed to mix until
completely dispersed. The completed oil slurry is held under
moderate agitation at a temperature in the range of about
32-43.degree. C. for not longer than 8 hours until it is blended
with the other slurries.
[0137] A protein slurry is prepared by first heating about 196.78
kg of water to a temperature in the range of about 60-63.degree. C.
with agitation. The calcium caseinate and sodium caseinate and milk
protein isolate are blended into the slurry using a mixing
apparatus. The completed protein slurry is held under agitation at
a temperature in the range of about 54-60.degree. C. for not longer
than 2 hours before being blended with the other slurries.
[0138] The oil and the protein slurry are blended together with
agitation and the resultant blended slurry is maintained at a
temperature in the range of about 54-66.degree. C. After waiting
for at least five minutes the carbohydrate/mineral slurry is added
to the blended slurry from the preceding step with agitation and
the resultant blended slurry is maintained at a temperature in the
range of about 54-66.degree. C. The refined deodorized sardine oil
is then added to the slurry with agitation. (In a most preferred
method of manufacture the sardine oil would be slowly metered into
the product as the blend passes through a conduit at a constant
rate.) Preferably after at least 5 minutes the pH of the blended
slurry is determined. If the pH of the blended slurry is below
6.55, it is adjusted with dilute potassium hydroxide to a pH of 6.5
to 6.8.
[0139] After waiting a period of not less than one minute nor
greater than two hours the blended slurry is subjected to
deaeration, Ultra-High-Temperature (UHT) treatment, and
homogenization, as described as follows: use a positive pump for
supplying the blended slurry for this procedure; heat the blended
slurry to a temperature in the range of about 66-71.degree. C.;
deaerate the blended slurry to 25.4-38.1 cm of Hg; emulsify the
blended slurry at 61-75 Atmospheres; heat the blended slurry to a
temperature in the range of about 120-122.degree. C. by passing it
through a plate/coil heat exchanger with a hold time of
approximately 10 seconds; UHT heat the blended slurry to a
temperature in the range of about 144-147.degree. C. with a hold
time of approximately 5 seconds; reduce the temperature of the
blended slurry to be in the range of about 120-122.degree. C. by
passing it through a flash cooler; reduce the temperature of the
blended slurry to be in the range of about 71-82.degree. C. by
passing it through a plate/coil heat exchanger; homogenize the
blended slurry at about 265 to 266 Atmospheres; pass the blended
slurry through a hold tube for at least 16 seconds at a temperature
in the range of about 74-85.degree. C.; and cool the blended slurry
to a temperature in the range of about 1-70.degree. C. by passing
it through a large heat exchanger.
[0140] Store the blended slurry at a temperature in the range of
about 1-7.degree. C., preferably with agitation.
[0141] Preferably at this time appropriate analytical testing for
quality control is conducted. Based on the test results an
appropriate amount of dilution water (10-38.degree. C.) is added to
the blended slurry with agitation.
[0142] A vitamin solution and flavor solution are prepared
separately and then added to the blended slurry.
[0143] The vitamin solution is prepared by heating about 3.94 kg of
water to a temperature in the range of about 43-66.degree. C. with
agitation, and thereafter adding the following ingredients, in the
order listed: Ascorbic Acid, 45% Potassium Hydroxide, Taurine,
Water Soluble Vitamin Premix, Choline Chloride, and L-Carnitine.
The vitamin solution is then added to the blended slurry with
agitation.
[0144] The flavor solution is prepared by adding the marshmallow
and dulce de leche flavor to about 7.94 kg of water with agitation.
A nutritional product according to the present invention has been
manufactured using an artificial marshmallow flavor distributed by
Firmenich Inc., Princeton, N.J., U.S.A. and a natural &
artificial dulce de leche flavor distributed by Firmenich Inc. The
flavor solution is then added to the blended slurry with
agitation.
[0145] If necessary, diluted potassium hydroxide is added to the
blended slurry such that the product will have a pH in the range of
6.4 to 7.0 after sterilization. The completed product is then
placed in suitable containers and subjected to sterilization. Of
course, if desired aseptic processing could be employed.
EXAMPLE VI
Composition of a Nutritional Product to Control Glycemic
Response
[0146] Table 2 presents a bill of materials for manufacturing 1,000
kg of a liquid nutritional product, which provides nutrients to a
person but limits resulting insulin response. A detailed
description of its manufacture follows.
2TABLE 2 Bill of Materials for a Liquid Nutritional Ingredient
Quantity per 1,000 kg Water QS Maltodextrin 56 kg Acidc casein
41.093 kg Fructose 28 kg High oleic safflower oil 27.2 kg Maltitol
syrup 16 kg Maltitol 12.632 kg Fibersol .RTM. 2(E) 8.421 kg
Caseinate 6.043 kg Fructooligosaccharide 4.607 kg Soy
polysaccharide 4.3 kg Canola oil 3.2 kg Tricalcium phosphate 2.8 kg
Magnesium chloride 2.4 kg Lecithin 1.6 kg Sodium citrate 1.18 kg
Potassium citrate 1.146 kg Sodium hydroxide 1.134 kg Magnesium
phosphate 1.028 kg Calcium HMB monohydrate 5.7 kg m-inositol 914.5
gm Vitamin C 584 gm Potassium chloride 530 gm Choline chloride
472.1 gm 45% Potassium hydroxide 402.5 gm UTM/TM premix 369.3 gm
Potassium phosphate 333 gm Carnitine 230.5 gm Gellan gum 125 gm
Ttaurine 100.1 gm Vitamin E 99 gm Lutein Esters (5%) 92 gm WSV
premix 75.4 gm Vitamin DEK premix 65.34 gm 30% Beta carotene 8.9 gm
Vitamin A 8.04 gm Pyridoxine hydrochloride 3.7 gm Chromium chloride
1.22 gm Folic acid 0.64 gm Potassium iodide 0.20 gm Cyanocobalamin
0.013 gm WSV premix(per g premix): 375 mg/g niacinamide, 242 mg/g
calcium pantothenate, 8.4 gm/g folic acid, 62 mg/g thiamine
chloride hydrochloride, 48.4 gm/g riboflavin, 59.6 mg/g pyridoxine
hydrochloride, 165 mcg/g cyanocobalamin and 7305 mcg/g biotin
Vitamin DEK premix(per g premix): 8130 IU/g vitamin D.sub.3, 838
IU/g vitamin E, 1.42 mg/g vitamin K.sub.1 UTM/TM premix(per g
premix): 45.6 mg/g zinc, 54 mg/g iron, 15.7 manganese, 6.39 mg/g
copper, 222 mcg/g selenium, 301 mcg/g chromium and 480 mcg/g
molybdenium
[0147] The diabetic liquid nutritional products of the present
invention are manufactured by preparing four slurries that are
blended together, heat treated, standardized, packaged and
sterilized.
[0148] A carbohydrate/mineral slurry is prepared by first heating
about 82 kg of water to a temperature of from about 65.degree. C.
to about 71.degree. C. with agitation. With agitation, the required
mount of calcium HMB is added and agitated for 5 minutes. The
required amount of sodium citrate and gellen gum distributed by the
Kelco, Division of Merck and Company Incorporated, San Diego,
Calif., U.S.A. is added and agitated for 5 minutes. The required
amount of the ultra trace mineral/trace mineral (UTM/TM) premix
(distributed by Fortitech, Schnectady, N.Y.) is added. The slurry
is greenish yellow in color. Agitation is maintained until the
minerals are completely dispersed. With agitation, the required
amounts of the following minerals are then added: potassium
citrate, potassium chloride, chromium chloride, magnesium chloride
and potassium iodide. Next, the first maltodextrin distributed by
Grain Processing Corporation, Muscataine, Iowa, U.S.A. and fructose
are added to slurry under high agitation, and are allowed to
dissolve. With agitation, the required amounts of maltitol powder
distributed by Roquette America, Inc., Keokuk, Iowa, maltitol syrup
distributed by AlGroup Lonza, Fair Lawn, N.J.,
fructooligosaccharides distributed by Golden Technologies Company,
Golden, Colo., U.S.A. and a second maltodextrin distributed by
Matsutani Chemical Industry Co., Hyogo, Japan under the product
name Fibersol.RTM. 2(E) are added and agitated well until
completely dissolved. The required amount of tricalcium phosphate
and magnesium phosphate are added to the slurry under agitation.
The completed carbohydrate/mineral slurry is held with agitation at
a temperature from about 65.degree. C. to about 71.degree. C. for
not longer than twelve hours until it is blended with the other
slurries.
[0149] A fiber in oil slurry is prepared by combining and heating
the required amounts of high oleic safflower oil and canola oil to
a temperature from about 40.5.degree. C. to about 49.degree. C.
with agitation. With agitation, the required amounts of lutein
esters from Cognis of LaGrange, Ill. is added. Agitate for a
minimum of 15 minutes. With agitation, the required amounts of the
following ingredients are added to the heated oil: lecithin
(distributed by Central Soya Company, Fort Wayne, Ind.), Vitamin D,
E, K premix (distributed by Vitamins Inc., Chicago, Ill.), vitamin
A, vitamin E and beta-carotene. The required amounts of soy
polysaccharide distributed by Protein Technology International, St.
Louis, Mo. is slowly dispersed into the heated oil. The completed
oil/fiber slurry is held under moderate agitation at a temperature
from about 55.degree. C. to about 65.degree. C. for a period of no
longer than twelve hours until it is blended with the other
slurries.
[0150] A first protein in water slurry is prepared by heating 293
kg of water to 60.degree. C. to 65.degree. C. With agitation, the
required amount of 20% potassium citrate solution is added and held
for one minute. The required amount of acid casein is added under
high agitation followed immediately by the required amount of 20%
sodium hydroxide. The agitation is maintained at high until the
casein is dissolved. The slurry is held from about 60.degree. C. to
65.degree. C. with moderate agitation.
[0151] A second protein in water slurry is prepared by first
heating about 77 kg of water to a temperature of about 40.degree.
C. with agitation. The caseinate is added and the slurry is
agitated well until the caseinate is completely dispersed. With
continued agitation, the slurry is slowly warmed to 60.degree. C.
to 65.degree. C. The slurry is held for no longer than twelve hours
until it is blended with the other slurries.
[0152] The batch is assembled by blending 344 kg of protein slurry
one with 84 kg of protein slurry two. With agitation, the 37 kg of
the oil/fiber slurry is added. After waiting for at least one
minute, 216 kg of the carbohydrate/mineral slurry is added to the
blended slurry from the preceding step with agitation and the
resultant blended slurry is maintained at a temperature from about
55.degree. C. to about 60.degree. C. The pH of the blended batch is
adjusted to a pH of 6.45 to 6.75 with 1N potassium hydroxide.
[0153] After waiting for a period of not less than one minute nor
greater than two hours, the blend slurry is subjected to
deaeration, ultra-high-temperature treatment, and homogenization.
The blended slurry is heated to a temperature from about 71.degree.
C. to about 82.degree. C. and deaerated under vacuum. The heated
slurry is then emulsified through a single stage homogenizer at 900
to 1100 psig. After emulsification, the slurry is heated from about
99.degree. C. to about 110.degree. C. and then heated to a
temperature of about 146.degree. C. for about 5 seconds. The slurry
is passed through a flash cooler to reduce the temperature to from
about 99.degree. C. to about 110.degree. C. and then through a
plate cooler to reduce the temperature to from about 7.degree. C.
to about 76.degree. C. The slurry is then homogenized at 3900 to
4100/400 to 600 psig. The slurry is held at about 74.degree. C. to
about 80.degree. C. for 16 seconds and then cooled to 1.degree. C.
to about 7.degree. C. At this point, samples are taken for
microbiological and analytical testing. The mixture is held under
agitation.
[0154] A water soluble vitamin (WSV) solution is prepared
separately and added to the processed blended slurry.
[0155] The vitamin solution is prepared by adding the following
ingredients to 9.4 kg of water with agitation: WSV premix
(distributed by J.B. Laboratories, Holland, Mich.), vitamin C,
choline chloride, L-carnitine, taurine, inositiol, folic acid,
pyridoxine hydrochloride and cyanocobalamin. The required amount of
45% potassium hydroxide slurry is added to bring the pH to between
7 and 10.
[0156] Based on the analytical results of the quality control
tests, an appropriate amount of water is added to the batch with
agitation to achieve desired total solids. Additionally, 8.8 kg of
vitamin solution is added to the diluted batch under agitation. The
product pH may be adjusted to achieve optimal product stability.
The completed product is then placed in suitable containers and
subjected to terminal sterilization.
EXAMPLE VII
Composition of a Pediatric Nutritional Product
[0157] Table 3 presents a bill of materials for manufacturing 771
kg of a pediatric enteral nutritional of the instant invention. A
detailed description of its manufacture follows.
3TABLE 3 Bill of materials for vanilla pediatric nutritional
Ingredient Quantity per 771 kg Stock PIF Slurry High oleic
safflower oil 40.7 kg Soy oil 24.4 kg MCT oil 16.3 kg Lecithin
840.2 g Monoglycerides 840.2 g Carrageenan 508.9 g Caseinate 32.8
kg Stock OSV blend DEK premix 83.3 g Vitamin A 7.1 g Lutein esters
(5%) 92 g Stock PIW slurry Water 530 kg Caseinate 11.3 kg Whey
protein 11.9 kg Stock MIN slurry Water 18 kg Cellulose gum 1696 g
Calcium HMB monohydrate 4.4 kg Magnesium chloride 2.7 kg Potassium
chloride 1.0 kg Potassium citrate 2.7 kg Potassium iodide 0.25 g
Dipotassium phosphate 1.45 kg Final blend PIW slurry 251 kg PIF
slurry 53 kg MIN slurry 12.6 kg Sodium chloride 127.4 g Sucrose
77.6 kg Tricalcium phosphate 2.5 kg Water 167 kg Stock WSV solution
Water 31.7 kg Potassium citrate 3.74 g UTM/TM premix 172.2 g WSV
premix 134.1 g m-inositol 176.7 g Ttaurine 145.5 g L-carnitine
34.92 g Choline chloride 638.7 g Stock ascorbic acid solution Water
18.6 kg Ascorbic acid 550.0 g 45% KOH 341 g Stock vanilla solution
Water 38.5 kg Vanilla flavor 4.3 kg DEK premix: (per gm premix)
12,100 IU vitamin D.sub.3, 523 IU vitamin E, 0.962 mg vitamin
K.sub.1 UTM/TM premix: (per gm premix) 132 mg zinc, 147 mg iron,
10.8 mg manganese, 12.5 mg copper, 0.328 mg selenium, 0.284 mg
molybdenum WSV premix: (per gm premix) 375 mg niacinamide, 242 mg
d-calcium pantothenate, 8.4 mg folic acid, 62 mg thiamine chloride
hydrochloride, 48.4 mg riboflavin, 59.6 mg pyridoxine
hydrochloride, 165.5 mcg cyanocobalamin, 7305 mcg biotin
[0158] The stock oil soluble vitamin blend (OSV blend) is prepared
by weighing out the specified amount of DEK premix into a screw
cap, light protected container large enough to hold 54 g of oil
soluble vitamins. Using a plastic pipette, the required amount of
vitamin A is added to the DEK aliquot. The container is flushed
with nitrogen prior to applying the lid.
[0159] The stock protein in fat slurry (PIF) was prepared by adding
the required amounts of high oleic safflower oil, soy oil and MCT
oil to the blend tank. The mixture is heated to 40.5.degree. C. to
49.degree. C. with agitation. With agitation, the required amounts
of lutein esters from American River Nutrition of Hadley, Mass. is
added. Agitate for a minimum of 15 minutes. The emulsifiers,
lecithin (distributed by Central Soya of Decatur, Ind.) and
monoglycerides (distributed by Quest of Owings Mills, Md.), are
added and mixed well to dissolve. All of the OSV blend is then
added. The containers are rinsed out 4 to 5 times with the oil
blend to assure complete transfer of the vitamins. The carrageenan
(distributed by FMC of Rockland, Me.) and the caseinate are added.
The slurry is mixed well to disperse the protein. The PIF slurry is
held up to six hours at 60-65.degree. C. under moderate agitation
until used.
[0160] The stock protein in water slurry (PIW) is prepared by
adding the required amount of water to a blend tank. The water is
held under moderate agitation and brought up to 76-82.degree. C.
The required amount of caseinate is added to the water under high
agitation and mixed on high until the protein is fully dispersed.
The protein slurry is allowed to cool to 54-60.degree. C. before
proceeding. Once cooled the required amount of whey protein is
added and mixed well until fully dispersed/dissolved. The PIW
slurry is held up to two hours at 54-60.degree. C. until used.
[0161] The stock mineral solution (MIN) is prepared by adding the
required amount of water to a blend tank and heated to
60-68.degree. C. The cellulose gum blend (distributed by FMC of
Newark, Del.) is added to the water and held under moderate
agitation for a minimum of five minutes before proceeding. The
calcium HMB is added and agitated for a minimum of five minutes
before proceeding. The mineral salts magnesium chloride, potassium
chloride, potassium citrate, potassium iodide and dipotassium
phosphate are added one at a time with mixing between each addition
to ensure the minerals dissolved. The completed MIN solution is
held at 54-65.degree. C. under low to moderate agitation until
used.
[0162] The final blend is prepared by adding the specified amount
of PIW slurry to a blend tank and heated under agitation to
54-60.degree. C. The specified amount of PIF slurry is added to the
tank and mixed well. The specified amount of MIN solution is added
to the blend and mixed well. The specified amount of sodium
chloride is added to the blend and mixed well. The specified amount
of sucrose is added to the blend and mixed well to dissolve. The
tricalcium phosphate is added to the blend and mixed well to
disperse. The specified amount of additional water is added to the
blend and mixed well. The completed final blend is held under
continuous agitation at 54-60.degree. C. If necessary, the pH is
adjusted to 6.45-6.8 with 1N KOH.
[0163] After waiting for a period of not less than one minute nor
greater than two hours, the blend slurry is subjected to
deaeration, ultra-high-temperature treatment, and homogenization.
The blended slurry is heated to a temperature from about 68.degree.
C. to about 74.degree. C. and deaerated under vacuum. The heated
slurry is then emulsified at 900 to 1100 psig. After
emulsification, the slurry is heated from about 120.degree. C. to
about 122.degree. C. and then heated to a temperature of about
149.degree. C. to about 150.degree. C. The slurry is passed through
a flash cooler to reduce the temperature to from about 120.degree.
C. to about 122.degree. C. and then through a plate cooler to
reduce the temperature to from about 74.degree. C. to about
79.degree. C. The slurry is then homogenized at 3900 to 4100/400 to
600 psig. The slurry is held at about 74.degree. C. to about
85.degree. C. for 16 seconds and then cooled to 1.degree. C. to
about 6.degree. C. At this point, samples are taken for
microbiological and analytical testing. The mixture is held under
agitation.
[0164] Standardization proceeds as follows. The stock vitamin
solution (WSV) is prepared by heating the specified amount of water
to 48-60.degree. C. in a blend tank. Potassium citrate, UTM/TM
premix (distributed by Fortitech of Schenectady, N.Y.), WSV premix,
m-inositol, taurine, L-carnitine and choline chloride are each
added to the solution in the order listed and allowed to mix well
to dissolve or disperse each ingredient. 14.2 kg of the vitamin
solution is added to the processed mix tank.
[0165] The stock vanilla solution is prepared by adding the
specified amount of water to a blend tank. The specified amount of
vanilla (distributed by Givaudan Roure of Cincinnati, Ohio) is
added to the water and mixed well. 18.5 kg of vanilla solution is
added to the processed mix tank and mixed well.
[0166] The stock ascorbic acid solution is prepared by adding the
required amount of water to a blend tank. The specified amount of
ascorbic acid is added and mixed well to dissolve. The specified
amount of 45% KOH is added and mixed well. 8.4 kg of ascorbic acid
solution is added to the mix tank and mixed well.
[0167] The final mix is diluted to the final total solids by adding
92.5 kg of water and mixed well. Product is filed into suitable
containers prior to terminal (retort) sterilization.
EXAMPLE VIII
Composition of a Complete Nutritional Supplement
[0168] Table 4 presents a bill of materials for manufacturing 1,000
kg of a typical vanilla flavored meal replacement liquid. A
detailed description of its manufacture follows.
4TABLE 4 Bill of Materials for Vanilla Liquid Nutritional
Ingredient Quantity per 1,000 kg Water QS Corn Syrup 33 kg
Maltodextrin 28 kg Sucrose 19.4 kg Caseinate 8.7 kg Calcium HMB
monohydrate 5.7 kg High Oleic Safflower Oil 4.1 kg Canola Oil 4.1
kg Soy Protein 3.7 kg Whey Protein 3.2 kg Caseinate 2.9 kg Corn Oil
2.0 kg Tricalcium Phosphate 1.4 kg Potassium Citrate 1.3 kg
Magnesium Phosphate 952 gm Lecithin 658 gm Magnesium chloride 558
gm Vanilla Flavor 544 gm Sodium Chloride 272 gm Carrageenan 227 gm
Choline chloride 218 gm UTM/TM Premix 165 gm Potassium Chloride 146
gm Ascorbic Acid 145 gm Sodium Citrate 119 gm Potassium Hydroxide
104 gm Lutein (5%) 46 gm WSV Premix 33 gm Vit DEK Premix 29 gm
Vitamin A 3.7 gm Potassium Iodide 86 mcg WSV premix(per g premix):
375 mg/g niacinamide, 242 mg/g calcium pantothenate, 8.4 gm/g folic
acid, 62 mg/g thiamine chloride hydrochloride, 48.4 gm/g
riboflavin, 59.6 mg/g pyridoxine hydrochloride, 165 mcg/g
cyanocobalamin and 7305 mcg/g biotin Vitamin DEK premix(per g
premix): 8130 IU/g vitamin D.sub.3, 838 IU/g vitamin E, 1.42 mg/g
vitamin K.sub.1 UTM/TM premix(per g premix): 45.6 mg/g zinc, 54
mg/g iron, 15.7 manganese, 6.39 mg/g copper, 222 mcg/g selenium,
301 mcg/g chromium and 480 mcg/g molybdenium
[0169] The liquid meal replacement products of the present
invention are manufactured by preparing three slurries that are
blended together, heat treated, standardized, packaged and
sterilized.
[0170] A carbohydrate/mineral slurry is prepared by first heating
the required amount of water to a temperature of from about
65.degree. C. to about 71.degree. C. with agitation. The required
amount of calcium HMB is added and agitated for a minimum of 5
minutes. With agitation, the required amount of potassium citrate
and ultra trace mineral/trace mineral (UTM/TM) premix (distributed
by Fortitech, Schnectady, N.Y.) is added. The slurry is greenish
yellow in color. Agitation is maintained until the minerals are
completely dispersed. With agitation, the required amounts of the
following minerals are then added: magnesium chloride, potassium
chloride, sodium chloride, sodium citrate, potassium iodide,
magnesium phosphate and tricalcium phosphate. Next, the
maltodextrin distributed by Grain Processing Corporation,
Muscataine, Iowa, U.S.A., sucrose and corn syrup are added to
slurry under high agitation, and are allowed to dissolve. The
completed carbohydrate/mineral slurry is held with agitation at a
temperature from about 65.degree. C. to about 71.degree. C. for not
longer than eight hours until it is blended with the other
slurries.
[0171] A protein in fat slurry (PIF) is prepared by combining and
heating the required amounts of high oleic safflower oil and canola
oil to a temperature from about 40.5.degree. C. to about 49.degree.
C. with agitation. With agitation, the required amounts of free
lutein from Kemin Foods of Des Moines, Iowa is added. Agitate for a
minimum of 15 minutes. Add the following ingredients are added to
the heated oil: lecithin (distributed by Central Soya Company, Fort
Wayne, Ind.), vitamin A, and Vitamin D, E, K premix (distributed by
Vitamins Inc., Chicago, Ill.). The required amount of carrageenan
is dry blended with the required amount of whey protein and add to
the agitating lipid mixture and allowed to agitate for a minimum of
10 minutes. The required amount of soy protein is added to the
blend slowly to assure proper mixing. The completed oil/protein
slurry is held under moderate agitation at a temperature from about
40.degree. C. to about 43.degree. C. for a period of no longer than
two hours until it is blended with the other slurries.
[0172] A protein in water slurry is prepared by first heating about
required amount of water to a temperature of about 40.degree. C.
with agitation. The caseinate is added and the slurry is agitated
well until the caseinate is completely dispersed. With continued
agitation, the slurry is slowly warmed to 60.degree. C. to
65.degree. C. The slurry is held for no longer than twelve hours
until it is blended with the other slurries.
[0173] The batch is assembled by blending required amount of
protein slurry with required amount of the carbohydrate/mineral
slurry and allowed to agitate for 10 minutes. With agitation, the
required amount of the oil/protein slurry is added and agitate for
at least 10 minutes. The pH of the blended batch is adjusted to a
pH of 6.66 to 6.75 with 1N potassium hydroxide.
[0174] After waiting for a period of not less than one minute nor
greater than two hours, the blend slurry is subjected to
deaeration, ultra-high-temperature treatment, and homogenization.
The blended slurry is heated to a temperature from about 71.degree.
C. to about 82.degree. C. and deareated under vacuum. The heated
slurry is then emulsified through a single stage homogenizer at 900
to 1100 psig. After emulsification, the slurry is heated from about
99.degree. C. to about 110.degree. C. and then heated to a
temperature of about 146.degree. C. for about 5 seconds. The slurry
is passed through a flash cooler to reduce the temperature to from
about 99.degree. C. to about 110.degree. C. and then through a
plate cooler to reduce the temperature to from about 71.degree. C.
to about 76.degree. C. The slurry is then homogenized at 3900 to
4100/400 to 600 psig. The slurry is held at about 74.degree. C. to
about 80.degree. C. for 16 seconds and then cooled to 1.degree. C.
to about 7.degree. C. At this point, samples are taken for
microbiological and analytical testing. The mixture is held under
agitation.
[0175] A water soluble vitamin (WSV) solution is prepared
separately and added to the processed blended slurry.
[0176] The vitamin solution is prepared by adding the following
ingredients to 9.4 kg of water with agitation: WSV premix
(distributed by J.B. Laboratories, Holland, Mich.), vitamin C,
choline chloride, L-carnitine, taurine, inositiol, folic acid,
pyridoxine hydrochloride and cyanocobalamin. The required amount of
45% potassium hydroxide slurry is added to bring the pH to between
7 and 10.
[0177] Based on the analytical results of the quality control
tests, an appropriate amount of water is added to the batch with
agitation to achieve the desired total solids. Additionally, 8.8 kg
of vitamin solution is added to the diluted batch under
agitation.
[0178] The product pH may be adjusted to achieve optimal product
stability. The completed product is then placed in suitable
containers and subjected to terminal sterilization.
EXAMPLE IX
Composition of a Beverage
[0179] To produce a 1000 kg batch of ready-to-drink beverage,
987.31 kg of water is placed in a vessel fitted with an agitator.
At ambient temperature, the requried amount of potassium benzoate
is added and allowed to completely dissolve. The reuqired amount of
calcium HMB is added and allowed to completely dissolve. The
following ingredients are then added in the order listed. Each
ingredient is completely dissolved before the next ingredient is
added.
5TABLE 5 Ready-to-drink beverage Potassium benzoate 0.30 kg Calcium
HMB monohydrate 5.7 kg Potassium Citrate 0.15 kg Citric Acid 2.89
kg Lactic Acid 1.41 kg Aspartame 0.55 kg Calcium Glycerophosphate
6.06 kg Coloring Agents 0.0019 kg Natural and artificial flavors
1.00 kg Ascorbic acid 0.33 kg
[0180] The ascorbic acid was added just before filling into 12-oz.
aluminum cans. The beverages may be carbonated prior to filling
into aluminum cans. The solution is deaerated and then transferred
to a "carbo-cooler" where it is cooled and carbonated to
approximately 2.5 volumes of carbon dioxide.
EXAMPLE X
Composition of an Electrolyte Replacement Product
[0181] The following example explains how to manufacture a
ready-to-drink rehydration solution. The ORS had the composition
outlined in Table 6.
6TABLE 6 Ready-to-drink Rehydration Solution Ingredient Quantity
per 454 kg Water 437 kg Dextrose, Monohydrate 10 kg Fructose 2.4 kg
Citric Acid 1.2 kg Sodium Chloride 0.937 kg Potassium Citrate 1 kg
Sodium Citrate 492.0 g Calcium HMB monohydrate 5.7 kg Fruit Flavor
226.8 g Zinc Gluconate 80.62 g Sucralose 179.2 g Acesulfame
Potassium 38.1 g Yellow #6 7.2 g
[0182] Weigh out the required amount of filtered water and add to
blend tank. Heat the water to 43-54.degree. C., with moderate
agitation. While maintaining moderate agitation, the calcium HMB is
added and allowed to mix for a minimum of 5 minutes. With continued
moderate agitation add the required amount of dextrose. Agitate
until dissolved. Add the required amount of fructose. Agitate until
dissolved. Add the required amount of the following ingredients, in
the order listed, to the dextrose/fructose blend and agitate until
dissolved: zinc gluconate, sodium citrate, sodium chloride,
potassium citrate, and citric acid. Add the required amount of
sucralose (distributred by McNeil Speciality Products Company of
New Brunswick, N.J.) and acesulfame potassium (distributed as
Sunsette by Hoechst Food Ingredients of Somerset, N.J.) and agitate
until dissolved. Add the yellow #6 and the fruit punch flavor to
the batch until dissolved. Cool the blend to 1.1-7.2.degree. C. and
hold with low agitation. Fill the required number of one liter
plastic bottles, apply the foil heat seal to the bottle opening,
and retort to food grade sterility standards.
[0183] Alternatively, the cooled blend is encapsulated within a
sealable freezable packaging material and sealed such as by heat
sealing. A single dose of rehydration solution is packaged in a
hermetically sealed freezable pouch. Various types of packaging
materials which can be used to practice the invention, such as that
used in traditional freezer pops, would be readily apparent to the
skilled artisan. The wrapping material is preferably a type which
will allow markings, such as product identification, ingredients,
etc., to be placed on the exterior surface thereof. The rehydration
formulation is shipped and stored, preferably in multiple units
thereof, in this condition. It is contemplated that multiple units
or freezer pops will be packaged together for purposes of
commercialization.
[0184] Prior to administration, a package of liquid rehydration
solution is frozen. Following freezing, the package is opened and
the contents thereof eaten. Since the frozen rehydration
formulation will normally be administered at ambient temperatures,
the amount of rehydration liquid contained in each package is
preferably an amount which can be consumed in its entirely while
still in the frozen state. Preferably 20-35 ounces, more preferably
2.0 to 2.5 ounces per package. In a particularly preferred
embodiment, 2.1 ounces of sterile rehydration solution is
encapsulated within an rectangular, e.g., 1".times.8," freezable
wrapper material. Clear plastic wrapper material is preferred.
* * * * *