U.S. patent application number 11/472059 was filed with the patent office on 2006-10-26 for biodegradable glucosamine-muramyl peptides for apoptosis modulation.
Invention is credited to Todor Vassilev Dimitrov, Vladimir I. Slesarev.
Application Number | 20060241025 11/472059 |
Document ID | / |
Family ID | 33130662 |
Filed Date | 2006-10-26 |
United States Patent
Application |
20060241025 |
Kind Code |
A1 |
Slesarev; Vladimir I. ; et
al. |
October 26, 2006 |
Biodegradable glucosamine-muramyl peptides for apoptosis
modulation
Abstract
The present invention includes apoptosis modulating
glucosamine-muramyl-peptides, obtained by specific endopeptidase
digestion of gram positive bacteria, methods of preparation of
thereof and medical food compositions for management and treatment
of conditions caused by TNF alpha cytotoxicity.
Inventors: |
Slesarev; Vladimir I.;
(Boyds, MD) ; Dimitrov; Todor Vassilev; (Chestnut
Hill, MA) |
Correspondence
Address: |
HOGAN & HARTSON LLP
ONE TABOR CENTER, SUITE 1500
1200 SEVENTEENTH ST
DENVER
CO
80202
US
|
Family ID: |
33130662 |
Appl. No.: |
11/472059 |
Filed: |
June 21, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10409846 |
Apr 9, 2003 |
|
|
|
11472059 |
Jun 21, 2006 |
|
|
|
Current U.S.
Class: |
514/18.9 ;
514/19.3; 514/20.9; 530/322 |
Current CPC
Class: |
C07K 9/005 20130101;
C12P 21/06 20130101; A61K 38/00 20130101 |
Class at
Publication: |
514/008 ;
530/322 |
International
Class: |
A61K 38/14 20060101
A61K038/14; C07K 9/00 20060101 C07K009/00 |
Claims
1. A Biodegradable glucosamine-muramyl-peptides of the general
formula ##STR9## where R1 is lysine or ornithine residue, R2 is
amino acid residue selected from the group of D-alanine, L-alanine,
D-aspartic acid, L-glycine, L-serine, D-serine, and L-threonine,
and R3 is amino acid residue selected from the L-glycine,
D-asparagine, D-aspartic acid, and D-glutamine.
2. The composition of claim 1, wherein the biodegradable compound
is ##STR10##
3. The composition of claim 1, wherein the biodegradable compound
is ##STR11##
4. The composition of claim 1, wherein the biodegradable compound
is ##STR12##
5. The composition of claim 1, wherein the biodegradable compound
is ##STR13##
6. The composition of claim 1, wherein the biodegradable compound
is ##STR14##
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application claiming
priority of U.S. patent application Ser. No. 10/409,846 filed 9
Apr. 2003, which is incorporated herein in its entirety by this
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to apoptosis modulating
glucosamine-muramyl-peptides, obtained by specific endopeptidase
digestion of gram positive bacteria, methods of preparation of
thereof and medical food compositions for management and treatment
of conditions caused by TNF alpha cytotoxicity.
BACKGROUND OF THE INVENTION
[0003] Apoptosis, or programmed cell death, is a naturally
occurring process that plays a strong role in ensuring the
development and maintenance of multicellular organisms by
eliminating unwanted cells. However, if this process goes over
stimulated, cell loss and degenerative disorders such as rheumatoid
arthritis, chronic heart, liver and renal failure, adult
respiratory distress syndrome, cachexia caused by cancer, stroke,
heart attack and heart failure can result. (Sharma, R., et al, Int.
J. Cardiol., 2002, 85:161-171, Argiles, J. M. et al., Int. J.
Biochem. Cell Biol., 2003, 35:405-409, Castellanos, M, et al.,
Stroke, 33:982-987, Cusack, M. R., et al., Amer. Coll. Cardiol.,
2002, 39:1917-23, Agnoletti, L., et al, Circulation, 1999,
100:1983-1991).
[0004] Mediators, which can trigger apoptosis include TNF alpha,
Fas and transforming growth factor beta, neurotransmitters, growth
factor withdrawal, loss of extracellular matrix attachment and
extreme fluctuations in intracellular calcium levels (Afford and
Randhawa, Mol. Pathol., 2000, 53:55-63). Among them TNF alpha
killing pathways are considered as a most common route of apoptosis
signaling. This cytokine is also involved in cancer related
fatigue, leucopenia, anemia, and thrombocytopenia (Kurzrock, R.,
Cancer, 2001, 92: 1684-8). TNF alpha is also implicated in the
development of the neurodegenerative disorders such as Alzheimer's
and Parkinson's disease, ALS, rhinitis pigmentosa and multiple
sclerosis (McGuire S O et al., Exp. Neurol., 2001, 169:219-230).
Its pathogenetic role is crucial in the development of the
extensive brain, lung, and heart damage (Barone F. C., et al,
Stroke, 1997, 28:1233-1244; Armstrong, L. et al., Thorax, 1997,
52:442-446). Plasma concentrations of TNF alpha are persistently
elevated among patients after myocardial infarction (Ridker, P., et
al, Circulation, 2000, 101:2149-2153)
[0005] With the identification of the systemic TNF alpha response
as a major component in the pathogenesis of the septic shock
syndrome (Jaecshke H., et al J. Immunol., 1998, 160:3480-3486),
much of the recent work has focused on modulating of this response.
High-flow haemofiltration was offered to clean both endotoxin and
cytokines (Sharma, V. K., et al., Expert Opin. Investig. Drugs,
2003, 12:139-152). Hypotensive and proinflammotory effects of TNF
alpha were inhibited by soluble receptors/receptor antagonists and
anti-inflammatory cytokines such as interleukine10. The current
experimental therapies involve transforming growth factor-beta,
granulocyte colony-stimulating factor, interferon phi and anti TNF
alpha antibody (Stamm, C., et al, Circulation, 101, Suppl. 1,
350-351). Moreover, less known cytokines such as macrophage
migration inhibitory factor and high mobility group I protein will
be clinically tested (Zanotti, S., et al., Expert Opin. Investig.
Drugs, 2002, 11:1061-75).
[0006] Another preliminary study has demonstrated that recombinant
GM-CSF upregulates HLA-DR expression on monocytes, thus reversing
the immunoparalysis in patients with severe sepsis. Moreover, there
was a concurrent increase of whole blood TNF response. (Nierhaus,
A., et al., Intensive Care Med, 2003, 21).
[0007] The same situation was noticed in rats with burn shock.
Nonsurvival group had lower levels of serum G-CSF and higher
content of TNF alpha compared with survival. Supplement of GM-CSF
could significantly improve animal survival with burn wound
infection following severe burn shock (Yan R., et al. Zhonghua Zhen
Xing Shao Shang Wai Ke Za Zi, 1997, 13:368-372). Serum TNF alpha
was also elevated after soft tissue trauma and hemorrhagic shock,
which leads to the acute respiratory distress syndrome (ARDS)
{Jarrar D, et al. Am. J. Physiol. Lung Cell Mol. Physiol., 2002,
283:799-805}.
[0008] Ischemia/reperfusion (I/R) induces a cytokines response and
production of reactive oxygen species, which affects the organs
remote to the sites of I/R. Yet, hepatic TNF alpha was implicated
in playing major role in the liver damage during renal surgery
(Serteser, M., et. al., J. Surg. Res. 2002, 107:234-40).
[0009] Intravenous injection of whole lactobacillus reduced
tachyarrhythmia significantly and improved recovery of the
ischemized rat heart (Oxman, T., et al, Am. J. Physiol. Heart.
Circ. Physiol., 278, H1717-H1724).
[0010] However, a broad variety of biodegradable glucosaminemuramyl
peptides were not isolated and tested for cytoprotective
effects.
SUMMARY OF INVENTION
[0011] The present invention is based on the discovery that
hydrolysis of the peptide bonds and peptide cross link of the gram
positive bacteria leads to release of the novel glucosamine muramyl
peptides, with strong potency towards inhibition of TNF alpha
cytotoxicity. Consequently, in one aspect the invention provides
new biodegradable glucosaminemuramyl tri, tetra-, penta-, hexa-,
and octapeptides, which possess apoptosis modulating properties and
are useful for treating all conditions with elevated serum lactate
dehydrogenase activity. Examples of such conditions are ischemic
reperfusion injury, atherosclerosis, heart attack, cerebral
infarction, and chronic heart failure. Applicants also demonstrated
that enhanced cytoprotective properties of these muramyl peptides
are caused by the presence of two and more D-amino acids covalently
bound L-amino acids.
[0012] Another aspect of the present invention is to provide a
method for isolation of high purity biodegradable
glucosaminemuramyl peptides, which comprises the bacterial wall
isolation with subsequent lysozyme and endopeptidase hydrolysis and
purification with preparative high pressure liquid chromatography
(HPLC).
[0013] Yet, another aspect of this invention is a preparation of
the medical food for dietary management of all conditions caused by
elevation of TNF alpha and LDH. Novel medical food consisting of
the hydrolyzed bacterial wall or whole bacteria can be used for
reduction of the systemic cytokine toxicity, which leads to
elevation of LDH and cachexia. Such medical food may be used to
reduce LDH associated malignancy and damage caused by radiation
therapy and chemotherapy. Specifically, the present food may be
recommended for those patients who suffer from common
postchemotherapy toxicity such as leukocytopenia, thrombocytopenia,
and high bilirubin with elevated liver enzymes. Further, the
present invention provides nutrition for reducing cancer fatigue
and muscle dystrophy.
[0014] In a related aspect, the present invention provides a food
useful for treating patients suffering from hepatotoxicity caused
by chemicals, anesthetics, drugs, and alcohol. Glucopeptide
fortified food and drink may be especially beneficial for people
with concurrent liver cirrhosis, thus preventing severe fatigue and
brain damage caused by ammonia. Furthermore, presented invention
provides the food for metabolic detoxifications of the cancerogenic
chemicals and mutagens.
[0015] Still another aspect of the present invention includes
dietary methods of inhibiting dermal apoptosis, thereby reducing
clinical symptoms of psoriasis.
[0016] While another aspect of this invention is to provide a
method of lung protection in the patients with pulmonary diseases
such as adult respiratory distress syndrome, fibrosis, and
cardiogenic lung edema.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] For further details, reference is made to the discussion
which follows, in light of the accompanying drawings, wherein:
[0018] FIG. 1 illustrates the cytoprotective potency of the novel
GMHP in comparison with GMDP.
[0019] FIG. 2. demonstrates inhibition of TNF alpha cytotoxicity by
D-amino acids in combination with the same concentration of
N-acetyl-glucosamine.
DETAILED DISCRIPTION OF THE INVENTION
[0020] The present invention relates to the novel
glucosaminemuramyl three-, tetra-, and pentapeptides, obtained by
specific endopeptidase digestion, medical food and drinks,
containing, as effective component, glucopeptide extracted from
Gram positive bacteria. This invention also provides medical food
for specific inhibition of TNF alpha cytotoxicity, which may be
administered orally to humans in single dose as small as 1 mg/kg.
The dosage of 2-10 mg/kg may be preferable. For the safety reasons,
glucopeptide complexes from lactic acid bacteria such as
Lactobacillus or Bifidum may be preferable.
[0021] Glucosaminemuramyl tetrapeptide is a basic unit of the
glucoproteins-peptidoglycans. In the cell wall they are bound to
teicholic acid and polysaccharides by a phosphate diester band.
Peptidoglycan strands are cross-linked to each other to form a
large sheet that surrounds the cell. Cross links are formed between
two tetrapeptides on adjacent glycan strands. The .epsilon.-NH2
group of diaminopimelic acid in the third position on 1 glycan
bonds with COOH group of D-alanine on an adjacent glycan in
Lactobacillus Plantarum. Majority bacteria of the species of
lactobacillus have L-lysine or L-ornithine instead of
diaminopimelic acid. The peptide bond forms a cross-link between
adjacent glycan strands.
[0022] It is important to point out that 60-90% of the total basic
units are bound to the adjacent glycan strands in the gram positive
bacteria. Obviously, there is a need to hydrolyze this cross link
in order to release novel biologically active glucosaminemuramyl
peptides. Recently, bromelain and lysine endopeptidases have been
offered for digestion of Brevibacterium flavum, Corynebacterium
herculis, and Corynobacterium glutamicum (Shionoya et al, 2003,
U.S. Pat. No. 6,506,388). However, neither bromalain, nor
lysine-endopeptidase can cleave the direct cross link between
diaminopimelic acid and D-alanine in the peptidoglycan chain of
these bacteria. As a result, the suggested enzymes are useless for
preparation of glucosamine muramyl tri- tetra-, and pentapeptides
from these bacteria. Recently, only glucosaminemuramyl-L-alanine,
D-glutamyl-diaminopimelic acid (tripeptide) and, tetrapeptide from
L. Plantarum have been isolated (Takase et al, U.S. Pat. No.
4,545,932, 1985). Tripeptide (glucosaminemuramyl-L-alanine,
D-glutamyl-diaminopimelic acid) was prepared by applying
diminopimelic acid-D-alanine peptidase. Immunostimulating
properties of this tetrapeptide were well characterized.
[0023] However, absolute majority of yogurt bacteria of genus
lactobacillus and bifidum have a different structure of their basic
peptidoglycan unit. Glucosaminemuramyl tetrapeptide contains
L-lysine, or L-ornithine in the third position instead of
diaminopimelic acid. Moreover, the peptide subunits of the
peptidoglycan are cross-linked weather by single D-isoasparaginyl
residues, or complex residues of L- or D-amino acids, selected from
the group of serine, alanine, threonine, glycine, and glutamine.
Therefore, there is a real practical need to release from large
peptidoglycan chain a significant amount of biologically active
glucosamine di-, tri-, tetra-, penta-, and hexapeptides.
[0024] The presence of two, or three D-amino acids could enhance
the cytoprotective properties described for
glucosamine-muramyl-dipeptide. For this purpose, inventors have
proposed to hydrolyze cross link peptide bonds. Cleavage of this
bond can also be done by hydrolysis of carboxyl bond of the
aspartic acid or carboxyl bonds of L-lysine in the third position.
A broad variety of specific endopeptidase which cleaves the
carboxyl bond of glutamic acid, glycine, serine, threonine, lysine,
aspartic acid, alanine-alanine can be used. They are of plant,
bacterial, or animal origin.
[0025] In the present invention, the bacteria are necessarily
specific bacteria, because they produce a different cross link
residues, which require a specific endopeptidase to hydrolyze a
cross bond. However, in view of the safety and the utilization of
waste material, lactate producing bacteria are preferable one, for
example. Lactobacillus acidophilus, bulgaricus, fermentum or
Bifidobacterium infantis.
[0026] Culture of bacteria belonging to genera of Lactobacillus,
Bifidobacterium, and Streptococcus thermophilus can be made by a
known method in an appropriate medium. Centrifuged cells, freeze
dried cells, heat killed cells, and spray-dried cells can be used
for the purpose of production of novel glucosamine muramyl
peptides. They can be prepared whether by digestion of isolated
bacterial wall preparations, or by digestion of the whole bacteria
with following purification by gel filtration on sephadex. The
washed bacterial cells should be treated with pronase or papain to
dissolve the surface proteins then they are boiled for 10 min in
ion detergent. 5% sodium dodecyl sulphate can be recommended as a
detergent for separation of bacterial wall from protoplast. The
protoplast is discarded as supernatant after centrifuging at 10000
rpm for 20 min. Elimination of the protoplast effectively purify
the active decomposed material of the present invention as in the
examples.
[0027] The peptide cross link bond of these bacterial wall a can be
hydrolyze by trypsin and glycine-endopeptidase, which are also food
additives. Temperature 27-30.degree. C., pH 6.0 during 4-6 hours
are considered the optimal conditions. Glycan moiety can be
decomposed by lysozyme. The lysozyme hydrolysis is considered
optimal under temperature of 55.degree. C., pH=6.0 during 48
hours.
[0028] Both enzymes can be eliminated by ultrafiltration with 3000
D cutoff. Highly concentrated solution of the novel
glucosaminemuramyl peptides can be obtained after reverse osmosis.
Novel biodegradable glucosaminemuramyl peptide can be identified by
analytical reverse phase HPLC with following isolation and
purification by preparative HPLC.
[0029] The centrifuged cells for whole bacteria digestion are
purified by pronase or papain treatment. Then biomass is diluted in
the distilled water in the ratio 1:10 for lysozyme hydrolysis. The
protoplast (as a pellet fraction) is discarded after centrifuging
at 4000 rpm for 1 hour. The supernatant is concentrated by factor 4
after ultrafiltration with 3000 D cutoff and nanofiltration with
reverse osmosis.
[0030] Routine methods for purifications of glycopeptides complexes
can be employed. More specifically, hydrolysis obtained by
aforementioned methods, is applied to anion-exchange column to
remove lysozyme and high-molecular nuclear acids. Further, protease
and nuclease can be used for degradation of the remaining proteins
and nuclear acids, respectively. Hydrophobic chromatography may be
used to remove enzymes by passing them through a column with resin.
Glucosaminemuramyl peptide composition may be fractioned by gel
chromatography.
[0031] A broad variety of specific endopeptidases can be applied to
hydrolyze a cross interpeptide bond. For the safety reason trypsin
(E.C.3.4.21.4) and glycine-endopeptidase (E.C.3.4.22.25) are
preferable. For example, glycine-endopeptidase or peptidase B was
isolated from Papaya Carica. Unlike papain, it cleaves the bond on
carboxyl terminal of glycine, a residue, which crosses links the
basic peptidoglycan units of Bifidobacterium Infantis, B. Breve, B.
Asteroides, B. Parvulorum, B. Globosum, and Streptococus Viridance.
Such specifically targeted cleavage of cross link bond results in
the releasing of the novel biodegradable peptides:
N-acetyl-glucosamine-N-acetyl-muramyl-L-Ala-D-isoGlu-L-Lys-D-Al- a,
N-acetyl-glucosamine-N-acetyl-muramyl-L-Ala-D-Glu-L-Lys-D-Ala,
##STR1##
[0032] Trypsin and lysyl endopeptidase ((E.C.3.4.24.50) cleave the
bond on the carboxyl end of lysine. Such specific cleavage leads to
release to a novel tripeptide
N-Acetyl-N-glucosamine-N-Acetyl-muramyl-L-Ala-isoGlu-L-Lys and
hexapeptide: ##STR2## after proteolysis of the petidoglycan of
Lactobacillus Bulgaricus, L. helveticus, L. jugurti, L. lactis, L.
Acidophilus, L. salivarius, L. delbruckii, L. leichmannii, L.
jensenii, L. casei, L. rhamnosus, L. tolerans, L. fusiformis, L.
pseudoplantarum, L. coryneformis, L. torquens, L. curvatis, L.
xylosus, L. zeae, L. brevis, L. buchneri, L. fructovoranse, L.
malefermentans, L. pastorianus, L. parvus, L. frigidus, L.
hilgardii, Bifudobacterium eriksonii, B. coryneforme, and B.
indicum.
[0033] Short term digestion of peptidoglycan of the same bacteria
with flavastacin (E.C.3.4.24.76) cleaves peptide bond on N terminal
of aspartic acid. It causes a release of another novel
biodegradable pentapeptide: ##STR3##
[0034] The flavostatin digestion of the B. Bifidum results in the
release of another novel hexapeptide: ##STR4##
[0035] Flavastacin hydrolysis of peptidoglycan of L. fermentum
leads to release of novel pentapeptide: ##STR5##
[0036] Yet, another endopeptidase saccharalysine (E.C.3.4.24.37)
can be used for cleavage of cross peptide Ala-Ala link.
Saccharalysine hydrolysis of Lactobacillus coprophilus,
Bifidobacterium globosum, Streptoccocus thermophilus, Leuconostoc
paramesenteroides, and amelibiosus leads to release the novel
##STR6##
[0037] The saccharilysine hydrolysis of cross peptide link in the
peptidoglycan chain of Lactobacillus minor, Bifidobacterium
adolescentis, Leuconostoc lactis, L. lactophilum, L. cremoris and
L. mesenteroides leads to the novel ##STR7##
[0038] The cross link cleavage of peptidoglycan of Bifidobacterium
lactentis, B. longum, B. suis by the same endopeptidase can lead to
another novel biodegradable compound is ##STR8##
[0039] Yet another aspect of this invention is to prepare
biodegradable GMDP, which is commercially available as semi
synthetic or synthetic drug. Enzymatic hydrolysis of every gram
positive or negative bacteria can be accomplished with
peptydyl-lysine metallopeptidase or V8 endopeptidase
(E.C.3.4.21.82) derived from S. aureous. Peptydyl-lysine
metallopeptidase (E.C.3.4.24.20) cleaves the N terminal of lysine.
V8 endopeptidase hydrolysis carboxyl bond of glutamic acid, thus
releases natural GMDP.
[0040] The antiapoptotic properties for the semi synthetic analog
GMDP were demonstrated previously (Slesarev V., Ellithorpe R., and
Dimitrov T., Med Oncol., 1998,). However, biodegradable glucosamine
tri-, tetra-. penta-, hexa-, and octapeptide never been isolated
and tested for the inhibition of TNF alpha cytotoxicity. It also
worthy to point out, they have more than one D-amino acid, which
are capable of enhancing cytoprotective properties. The inventors
have demonstrated that the presence of at least two D-amino acids
increases cytoprotective potency in comparison with GMDP.
[0041] Daily peptidoglycan dosage in the range of from 50 mg to
2000 mg may be found to be acceptable for dietary management of TNF
alpha cytotoxicity with optimal range of 200-1500 mg per day. Daily
isolated disaccharide tetra-, penta-, and hexapetide dosage in the
range of from 10 mg to 100 mg would acceptable with optimal range
10-50 mg.
[0042] When the glucosaminemuramyl peptides of the present
invention are applied as a main medical agent, it can orally be
used in powder, tablets, dispersion, capsules, confectionery,
drinks or the like. When they are used as a pharmaceutical or
cosmetic agent, it can be administered orally, rectally, or
vaginally in sprays, tablets, suppositories, or capsules.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Specific production embodiments are presented hereinafter.
Example 1
Isolation of
N-acetyl-glucosamine-N-acetyl-muramyl-L-Ala-D-isoGlu-L-Lys-D-Asp-D-Ala
from Lactobacillus bulgaricus
1. Biomass Preparation
[0043] Fermentation of Lactobacillus bulgaricus was done in 300 L
fermenter with working volume of 220 L. Liquid nutrient medium was
composed of 10 g/L of yeast extract, 20 g/L glucose, 20 g/L
pepton+beef extract, 2 g/L K.sub.2HPO4, 5 g/L CH.sub.3COONa, 0.2
g/L MgSO.sub.4, 0.05 g/L MnSO4, and 0.1 g/L Tween 80.
[0044] Fermenting was made anaerobic conditions at temperature
40+-3.degree. C. Fermenting period was 22 hours. Biomass was
separated from the suspension on centrifuge Sorvall 3B at 4000
r/min. Moisture biomass yield was 1.6 kg, dried one -440 g. Wet
biomass was rinsed twice by 1 L of distilled water, then was
suspended in 1 L of CH3COONa for 2-4 hours, and again was rinsed by
water using centrifuge Servile-5B at 8000 r/min. Biomass was
exposed for 1-2 hours in 1 L of CH.sub.3COONa at 60-80.degree. C.
and rinsed by distilled water. Then, 50-70% ethanol was used to
rinse the biomass until supernatant liquid becomes colorless. 96%
ethanol was used to stabilize the biomass. This processing was
needed to preserve the biomass at room temperature for following
hydrolysis. Moisture biomass should be stored at -60.degree. C.
2. Hydrolysis.
[0045] 1.6 kg moist biomass was rinsed by distilled water and 0.5M
CH.sub.3COOH, then resespended in 2.5 L H.sub.2O and kept at
90.degree. C. for 20-30 min. After that, it was diluted in 10 L
H.sub.2O+70 g NaHCO.sub.3 (to achieve .sub.PH=6.0) and added 8 g of
lyzosyme (Canadian Inovatech, Inc., Vancouver, Canada). Hydrolysis
was done for 11.5 H in the shaking incubator at 54.degree. C. Then,
3 g of trypsin was added for 6 hours.
[0046] 110 ml CH.sub.3COOH was added to achieve pH=4.0 and was
centrifuged on Beckman J-6 g at 4000 rpm for 30 min.
3. First Ultrafiltration.
[0047] Cartridge with the membrane capable of retaining compounds
with molecular weight less than 3000 D and with S=0.09M.sup.2. at
speed 2.5 L/h (Millipore Corp, USA) were used. 1.8 L solution with
retained nuclear acids, phospholipids, and lysozyme was wasted. 10
L was passed through column with micro pore cationite in H-form in
order to eliminate residual lysozyme and pigments.
4. Second Gel Chromatography
[0048] Sephadex G-25 and G-50 was used for separation of this novel
glucosaminemuramyl pentapeptide. A distinct pick of neutral
glucopeptide fraction was identified. This fraction of 1000 D
molecular weight was collected and freeze dried. Amino acid
analysis revealed L-Ala-, D-isoGly, D-Asp, and L-Lys in the ratio
2:1:1. The ratio of L-Ala and D-Ala was 1:1
Example 2
Inhibition of TNF Alpha Cytotoxicity by GMHP
[0049] A549 cells (human lung cancer) were seeded in six-well
plates, and after 24 h (70% confluence) treated with 25 ug/ml
cycloheximide (CHX) and either 100 U/ml human TNF (Beoringer) or an
agonist monoclonal antibody to FAS (Panerva) in concentration 200
ng/ml. GMDP, and GMHP were added just prior to the cytokine in the
concentrations discussed in the figure legends. Twenty hours after
the treatment 20 ul of the cultured supernatant was removed and
tested for the LDH activity in 96 well plates in triplicate.
Samples were assayed on an EL340 Microplate reader (Biotec
Instruments, Inc) at 490-nm wavelength. FIG. 1 demonstrates the
effect of GMDP and GMHP on LDH release. One can see enhanced
potency of GMHP in comparison with GMDP. The level of the LDH
activity was comparable to LDH background release by control intact
cells.
Example 3
Effect of D-amino Acid on the Inhibition of TNF Alpha
Cytotoxicity
[0050] The purpose of this experiment was to show the synergistic
effect of NAG and D-glutamine on LDH release. Technically the
experiment was similar to example 1. Both ingredients were added in
concentration 1 ug/ml. One can see, that L-glutamine does not
protect cells from TNF alpha cytotoxicity (FIG. 2). Almost 50%
inhibition of LDH activity was noticed for NAG+D-glutamine
composition. This example explains why GMHP possesses more potency
compared to GMDP. The glucosamine muramyl hexapeptide has 3 D-amino
acids while GMDP has only one D-isoglutamine.
* * * * *