Method using sturgeon notochord for alleviating the symptoms of arthritis

This invention provides a composition comprising notochord and extracts thereof in therapeutic amounts. The invention more specifically relates to a method of treating arthritis in mammals, more particularly rheumatoid arthritis in humans through the enteral administration of notochord, notochord extracts or mixtures thereof. In a preferred embodiment, collagen obtained from sturgeon is enterally administered to a human at from 1.0 .mu.g to 1.05 gms per day.

The present invention pertains to a method for treating the symptoms of 
arthritis in mammals and, more particularly, relates to the enteral 
administration of compositions comprising notochord and/or notochord 
extracts. The invention preferably uses sturgeon notochord, collagen 
derived from sturgeon notochord, or mixtures thereof to suppress the 
clinical manifestations of arthritis. The invention also relates to 
enteral compositions that contain notochord and its structural components 
to suppress and/or treat arthritis in mammals. 
BACKGROUND OF INVENTION 
Arthritis, and particularly rheumatoid arthritis (RA), is a painful and 
often crippling disease that initially results in swollen and inflamed 
joints, but often progresses to deform or completely destroy joints. This 
is a result of the body mistakenly attacking its own cartilage. Cartilage 
is a specialized kind of connective tissue which is found in human adults 
in three forms: hyaline or glossy cartilage; elastic cartilage; and 
fibrocartilage. Hyaline cartilage is the type found in the ventral ends of 
ribs, in joints, and in the walls of the larger respiratory passages. It 
is the hyaline cartilage that provides a low friction surface to prevent 
bone from rubbing on bone during motion. As arthritis progresses, 
cartilage is damaged and bone may also start to erode. This results in 
severe pain and ultimate destruction of the joint itself. 
Arthritis is a group of diseases affecting joints and the component 
tissues. Several types of arthritis are recognized, and these can be 
divided into several groups by their clinical course or pathological 
manifestations. The most common form of arthritis is Osteoarthritis (OA). 
Osteoarthritis is mainly caused by mechanical damage to the joints, either 
by repetitive use of particular joints as seen in athletes and physical 
laborers, or by overloading structural joints as seen in the knee joints 
of obese individuals. 
The second most common form of the disease is RA, which is a chronic 
multisystem disease of unknown cause. RA is characterized by chronic 
inflammation of the synovium associated with considerable erosion of both 
cartilage and bone, particularly in and around the joints. RA is currently 
understood as an autoimmune disease in which the pathological process 
appears to start by the presentation of an unknown 
"rheumatoid"self-antigen by an antigen presenting cell. Studies addressing 
family history indicate a genetic predisposition wherein a particular 
amino acid sequence in the third hypervariable region of the HLA-DR 
molecule is a major genetic element conveying susceptibility to RA. See 
Lipsky P.E., "Rheumatoid Arthritis "in Harrisons 'Principles of Internal 
Medicine, 13th ed. McGraw-Hill, Inc., New York, N.Y. 
The T cell receptor on CD4 + T cells, which form the target of the antigen, 
also plays an important role in the inflammatory process. The presentation 
of the antigen causes the activation of CD4 + T cells, with the consequent 
secretion of cytokines such as interleukin-2 (IL-2) and 
interferon-.gamma.(IFN-.gamma.). These cytokines induce clonal expansion 
of the T cells and activation of the cytokine network. These cytokines 
trigger the production of endothelial adhesion molecules (such as ICAM-1) 
whose expression in rheumatoid synovium enhances the activation of 
inflammatory cells in the joints. See Vitali C, Sciuto M, Bombardieri S., 
"Immunotherapy in Rheumatoid Arthritis: A Review", Int J Art Organs 
1993:16;196 -200. 
The modern therapy for arthritic conditions begins with nonsteroidal 
anti-inflammatory drugs such as aspirin, anthranilic acid, and ibuprofen; 
and more aggressive therapies involve disease-modifying antirheumatic 
drugs, such as D-penicillamine, methotrexate, and sulfasalazine. However, 
these treatments are often deficient in their efficacy and tolerability, 
causing a wide range of serious side effects. More severe forms of the 
disease may even require surgery. 
(A) Oral Tolerance 
Novel therapies for treating arthritis include immune response modifiers, 
gene therapy, enzyme inhibitors, monoclonal antibodies and dietary 
therapy. Dietary therapy for arthritis has received a great deal of 
publicity over the years. Although scientific basis, at present, for 
dietary remedies is still in doubt, there are valid reasons for 
considering whether dietary management can successfully modify disease 
activity as we better understand its etiology and pathology. "Oral 
tolerance"is a long recognized method to induce peripheral immune 
response. It was first described by Wells in 1911 as a state in which 
systemic anaphylaxis in guinea pigs was prevented by previous feeding of 
hen's egg proteins. See Wells H, "Studies on the Chemistry of Anaphylaxis 
III. Experiments with Isolated Proteins, Especially Those of Hen's Eggs", 
J Infect Dis 1911:9:147 -151. 
Particularly, oral tolerance is thought to be an ideal candidate to 
consider as a treatment of RA because of the etiology of RA as an 
autoimmune disease. Orally administered autoantigens have shown activity 
in several experimental autoimmune models including experimental 
autoimmune encephalomyelitis, uveitis, myasthenia, diabetes, and collagen- 
and adjuvant-induced arthritis. See Weiner HL, Friedman A, Miller A, 
Khoury S.J., Al-Sabbagh A, Santos L, Sayegh M, Nussenblatt R.B., Trentham 
D.E., Hafler DA. "Oral Tolerance: Immunologic Mechanisms and Treatment of 
Animal and Human Organ-Specific Autoimmune Diseases by Oral Administration 
of Autoantigens",Annu Rev Immunol 1994;12:809 -837. 
The mechanism of how oral tolerance works is, at this time, unclear. The 
primary mechanisms by which an orally administered antigen induces 
tolerance are believed to be via the generation of active suppression or 
clonal anergy. 
Collagen-induced arthritis (CIA) in experimental animals is the best known 
animal model for human RA. See Durie F.H., Fava R.A., Noelle R.J., 
"Collagen-Induced Arthritis as a Model of Rheumatoid Arthritis"See Clin 
Immu and Immupath 1994; 73:11 -18 and Staines N.A., Wooley P.H., "Collagen 
Arthritis --What Can it Teach Us?"Brit J Rheum 1994;33:798 -807. It was 
first described by Trentham in 1977, see Trentham DE, Townes A.S., Kang 
A.H. "Autoimmunity to Type II Collagen: An Experimental Model of 
Arthritis", J Exp Med 1977;146:857 -868, and has been demonstrated to 
resemble human RA sufficiently to now be recognized as an important 
experimental tool. It is generally induced in susceptible strains of 
experimental animals (such as mice and rats) by immunization with 
heterologous type-II collagen (CII) isolated from a heterologous species. 
See Courtenay J.S., Dallman M.J., Dayyan A.D., Martin A, Mosedale 
B,"Immunization Against Heterologous Type II Collagen Induced Arthritis in 
Mice",Nature 1980;283 -665. In a susceptible strain of mice (DBA/1), 
immunization with CII initiates a combined humoral and cellular immune 
response targeted to joint tissues, where the antigen is predominantly 
located. Differences between the animal model and the human RA include: 
(1) the model is an induced state and therefore does not occur 
spontaneously, as in humans; 
(2) the model lacks many extra-articular manifestations of the human RA 
including subcutaneous nodules and pulmonary fibrosis; and (3) the 
induction of the disease is of rapid onset in the model, which is 
different from humans in that it typically takes years. Nevertheless, CIA 
is the best available animal model for human RA. 
Intragastric administration of soluble Type II collagen (CII) prior to 
immunization with CII has been shown to suppress the incidence of CIA in 
DBA/1 Lac J mice, and WA/KIR rats. See Nagler-Anderson C, Bober L.A., 
Robinson M.E., Siskind G.W., Thorbecke G.J.,"Suppression of the Type II 
Collagen-Induced Arthritis by Intragastric Administration of Soluble Type 
II Collagen",Proc Natl Acad Sci USA 1986;83:7443-7446 and Thompson HSG, 
Harper N, Devan D, Staines N.A., "Suppression of CIA by Oral 
Administration of Type II Collagen: Changes in Immune and Arthritic 
Responses Mediated by Active Peripheral Suppression"Autoimmunity 
1993;16:189 -199. 
Adjuvant-induced arthritis in Lewis rats was also shown to be suppressed by 
oral administration of soluble CII. See Zhang Z.J., Lee C.S.Y., Lider O, 
Weiner H, "Suppression of Adjuvant Arthritis in Lewis Rats by Oral 
Administration of Type II Collagen", J Immunol 1990;145:2489 -2493. The 
type of immunogen as well as the type of toleragen seems to be very 
important in exerting their effect in inducing and protecting the animal. 
Cartilage is produced by cells called chondrocytes which synthesize and 
deposit around themselves a matrix of macromolecules that are known as 
collagen and proteoglycans. A remarkable function of cartilage tissue is 
that it replenishes itself in response to mechanical forces placed upon 
it. 
A number of collagen types have been identified which provide the tough 
connective character of cartilage. The proteoglycans consist mainly of the 
high molecular weight molecules known as glycosaminoglycans (GAG) which 
include hyaluronic acid and chondroitin sulfate. GAGs were previously 
known as mucopolysaccharides. An overview of GAG's and their application 
in OA therapy is presented by Paroli et al. in "A Pharmacological Approach 
to Glycosaminoglycans"Drugs Expth. Clin. Res.VII (1) 9 -20 (1991). 
A review of the various types of collagen can be found in Protein Profile, 
Vol. 1, 1994, pages 550 -571; P. Sheterline, Ed. A review of the capacity 
of the immune system to discriminate between self and non-self structures 
and a discussion of how the immune system normally interacts with 
cartilage and how such interactions can lead to arthritis is set forth by 
Holmdahl et al. in an article entitled "Autoimmune Recognition of 
Cartilage Collagens", Annals of Medicine 25:251 -264, 1993. Type II 
collagen may be the best known oral toleragen for arthritis, however, 
there may exist other potential toleragens derived from non-cartilaginous 
tissues, such as vitreous humor, neural tubes and neural retina. 
U.S. Pat. No. 5,529,786 to Moore, discloses the use of animal tissue 
containing a therapeutic amount of CII for the treatment of RA in humans. 
This patent describes the animal tissue as preferably being chicken 
cartilage obtained from chickens less than about one year of age. Other 
animal tissues disclosed are bovine cartilage, the vitreous humor of eyes 
and a variety of other animals. 
U.S. Pat. No. 4,473,551 to Schinitsky discloses a composition for the 
treatment of inflammatory disorders (such as RA, OA, acne, psoriasis and 
the like) which comprises animal cartilage and glucosamine. This patent 
describes the synergistic effect of a glucosamine and cartilages from 
whatever source derived, including shark and other marine dwellers, 
cattle, hogs, chicken and the like. 
U.S. Pat. No. 5,075,112 to Lane discloses the use of finely divided shark 
cartilage for inhibiting tumor growth, arthritis, in particular RA, and 
inflammatory diseases with a vascular component. This patent does not 
suggest nor equate shark cartilage with any other mammal or avian 
cartilage. 
U.S. Pat. No. 5,399,347 to Trentham et al. discloses the use of a highly 
purified component from cartilage, whole CII protein, for the treatment of 
RA. 
EP0254289B1 by Koepff et al. discloses the treatment of arthritis through 
the administration of enzymatically hydrolyzed collagen from animal skins, 
animal bones, refined connective tissue or gelatin (Type I collagen) 
having an average molecular weight of from 10 to 80 KD. 
Arthritis affects an estimated 40,000,000 people (15% of the population) in 
the United States. With an increasing survival time in the population, 
arthritis constitutes one of the greatest medical, social and economical 
problems in existence. The present invention furthers the state of the art 
of aritis treatments and offers several advantages to presently accepted 
therapies. 
(B) Notochord 
As will be discussed and demonstrated below, notochord is a unique tissue 
to primitive groups of Osteichthyes, such as sturgeon and lamprey. 
Notochord appears in the post-gastrulation embryo as a very specialized 
mesoderm. In vertebrates, notochord serves as a core around which 
mesodermal cells gather to form the vertebrae (i.e., the notochord is the 
precursor of the vertebral column), but it disappears by the end of the 
embryonic stage. In the most primitive chordates, however, the notochord 
is retained as a primitive substitute for a vertebral column. Sturgeon and 
lamprey maintain a significant amount of notochord tissue in their 
vertebral column even in the adult stage. Regular mesoderm gives rise to 
the connective tissues of the body, such as hyaline cartilage and CII 
within. In fact, sturgeon notochord collagen may be a precursor form of 
CII, but it certainly is not CII. Notochord and cartilage are 
evolutionarily, developmentally, functionally, and anatomically different 
from each other. These differences in characteristics will be demonstrated 
below. The present invention relates to the use of intact and whole 
sturgeon notochord as well as its structural and chemical components 
(i.e., collagen derived from the notochord) for the treatment of RA and 
OA. 
Support for the Applicant's position that notochord is different from 
cartilage can be found in an article by Miller and Mathews entitled, 
"Characterization of Notochord Collagen", published in Biochemical and 
Biophysical Research Communications, Vol. 60, No.1, 1974 and in an article 
published by Mathews entitled, "Comparative Biochemistry of Chondroitin 
Sulphate-Proteins of Cartilage and Notochord", published in Biochem 
J,(1971), 125, 37-46. These publications discuss the characterization of 
sturgeon notochord by chromatographic properties, amino acid composition, 
carbohydrate content, cyanogen bromide cleavage products of the component 
.alpha.-chain, the molecular parameters of tryptic-chymotryptic 
hydrolysates of chondroitin sulfate-protein, and the fraction of 
chondroitin sulfate-protein in a caesium chloride density gradient. These 
analyses point out some of the chemical similarities, but more 
importantly, they evidence the distinct chemical and structural 
differences between cartilage and notochord. 
The following Tables 1 and 2 are from the Mathews (1971) publication, 
supra, and highlight the differences of notochord collagen from ox 
cartilage and sturgeon cartilage. 
TABLE 1 
______________________________________ 
Molecular Parameters of Tryptic-Chymotryptic Hydrolysate of 
Chondroitin Sulfate-Protein and Products after Various Treatments 
Molecular 
TREATMENT 
Tissue Parameter 
None NaOH Papain 
Pronase 
______________________________________ 
Ox cartilage 
.eta.! 102 60 60 96 
Mn 43000 21000 21800 -- 
Mv 56000 27000 27000 51000 
Sturgeon cartilage 
.eta.! 215 135 130 200 
Mn 50000 25400 23000 -- 
Mv 66000 35000 34000 61000 
Sturgeon notochord 
.eta.! 38 25 23 33 
Mn 10000 5900 7100 -- 
Mv 15000 8100 7400 12000 
______________________________________ 
Mn viscosity molecular weight, was estimated from relationship .eta.! = 
3.1 .times. 10.sup.-2 Mv.sup.0.74 for chondroitin sulphate A and .eta.! 
5.8 .times. 10.sup.-2 M.sup.0.74 for chondroitin sulphate C. (Mathews, 
1971). 
From Table 1, it is apparent notochord is a unique tissue, even when 
compared to cartilage from the same fish. 
TABLE 2 
______________________________________ 
Fractionation of Chondroitin Sulphate-Protein in a 
Caesium Chloride Density Gradient 
Protein in Chondroitin 
Pro- Chondroitin 
Sulphate-Protein from 
Source Zone tein* sulphate* 
each Zone (%) 
______________________________________ 
Ox cartilage 
A 10 0 -- 
B 20 &lt;5 -- 
C 70 &gt;95 10.4 
Sturgeon cartilage 
A 10 0 -- 
B 10 &lt;4 -- 
C 80 &gt;96 7.0 
Sturgeon Notochord 
A 40 6 69 
B 40 30 30 
C 20 64 21 
______________________________________ 
The volumes and average densities at 20.degree. C. of zones were: A, 3 ml 
1.42; B, 5 ml, 1.47; C, 4 ml, 1.54. 
Protein in chondroitin sulphateprotein from each zone was calculated from 
amino acid analysis. (Mathews, 1971) 
*percentage of total

DISCLOSURE OF THE INVENTION 
The invention is generally directed to the enteral administration of 
notochord and extracts of notochord for the treatment of arthritis, 
specifically RA. The preferred source of notochord is sturgeon. Sturgeon 
notochord is readily available, efficacious and distinct from chicken and 
bovine cartilage. Sturgeon are presently raised in captivity for their 
flesh and, consequently, the quality (i.e., lack of pollutants) of the 
notochord can be controlled. One advantage in the use of sturgeon 
notochord and the extracts derived therefrom is the ease in combining the 
material with nutritional components that are also efficacious in the 
suppression of RA. A further advantage is that the intact sturgeon 
notochord is not subject to rigorous regulatory approval, as a drug would 
be. 
As used herein and in the claims, the term "extracts of notochord"means the 
components that can be extracted from notochord using sutable extraction 
techniques, and include, for example, notochord collagens and GAG 
(including glucosamine and chondroitin sulfate). 
There is disclosed a composition for oral consumption by mammals comprising 
at least one component selected from the group consisting of notochord, 
extracts of notochord and mixtures thereof. The composition may be in the 
form of a tablet, a pill, a capsule, a liquid (i.e., aqueous or oil 
suspension), a prepared food or food items, a nutritionally complete 
enteral formula or in the form of a nutritional supplement. 
The compositions of the invention may contain from 10 .mu.g to 1000 mg of 
notochord or notochord extract per gram of composition. When the 
composition is in the form of a liquid (i.e., aqueous suspension or oil 
suspension), the notochord component may comprise from 10 .mu.g to 700 gms 
per liter; more preferably 10 .mu.g to 500 gms per liter and most 
preferably 50 .mu.g to 500 mg per liter. In a preferred embodiment, the 
liquid composition contains notochord collagen at a concentration of 10 
.mu.g to 10.5 gms per liter; 10 gms to 20 gms of glucosamine per liter; or 
8 gms to 16 gms of chondroitin sulfate per liter. 
In a further embodiment, the composition is in the form of a tablet, 
capsule or pill which contains from 1.0 .mu.g to 2.0 gms of notochord or 
an extract of notochord. In a more preferred embodiment, the pill or 
tablet contains from 50 .mu.g to 500 mg of the notochord and/or notochord 
extracts. 
There is further disclosed a method for treating or reducing the incidence 
of the symptoms of arthritis, specifically RA in mammals, which comprises 
orally administering, to the mammal, a composition comprising at least one 
component selected from the group consisting of notochord, extracts of 
notochord and mixtures thereof in an amount and for a time effective to 
alleviate such symptoms. 
In the method of the present invention, the amount of notochord consumed 
per day for a 70 kg human can range from 10 .mu.g to 700 gms per day. More 
preferred, the dosage of notochord can range from 10 .mu.g to 11 gms per 
day and most preferred from 10 .mu.g to 1 gm per day. Collagen, as an 
extract from notochord, can be administered to a 70 kg human at from 1 
.mu.g to 1.05 gms per day. More preferred is the administration of 1 .mu.g 
to 100 mgs of collagen purified from notochord per day. The most preferred 
dosage range of collagen purified from notochord is 1 .mu.g to 10 mgs per 
day. Glucosamine purified from notochord is typically administered at a 
level of 1000 mgs to 2000 mgs per day per 70 kg human. The chondroitin 
sulfate purified from notochord is administered at a level of from 800 mgs 
to 1600 mgs per day. Those skilled in the art will appreciate that the 
rate of administration of the notochord containing composition will vary 
with many factors and that optimal dosage levels can be derived without 
undue experimentation. 
The composition and method of the present invention are also applicable to 
non-human animals, such as dogs and horses. Many domesticated animals 
suffer from RA and they would benefit from the discovery set forth herein. 
The dosage levels for the 70 kg human set forth in the paragraph above can 
be converted to a dose range per kilogram of body weight, and that range 
can be used for animals. For example, the amount of notochord consumed per 
day for a 20 kg dog can range from 2.9 .mu.g to 200 gms per day. 
There is still further disclosed a process for the treatment of RA which 
comprises orally ingesting therapeutic quantities of notochord that has 
been removed from an animal of the Osteichthyes order or Acipenseridae 
family, preferably the Acipenser genus, and comminuted under clean or 
sterile conditions. The notochord is preferably treated with a sterilizing 
agent or by a sterilizing process prior to storage and ingestion. The oral 
administration of the notochord or a component thereof can be accomplished 
with other edible components such as in a nutritional supplement, a 
prepared food or in a nutritionally complete formula. 
The three main species of sturgeon presently reared in the aquafarming 
industry are white sturgeon (Acipenser transmontanus), Italian sturgeon 
(Acipenser naccarii) and Siberian sturgeon (Acipenser baeri). Sturgeon is 
a cartilaginous fish of the family Acipenseridae, having no hard bones. 
Sturgeon are widely distributed in the north temperate zone of the planet. 
Sturgeon is one of the oldest creatures found on earth, not having changed 
in the last 300 million years. White sturgeon, the largest freshwater fish 
in North America, can reach up to 2,000 lbs. Sturgeon, once sought after 
for caviar production, have recently become appreciated for the quality of 
their flesh. White sturgeon are now farm raised, indoors, in recycled well 
waters and are available from numerous commercial sources (e.g., Stolt Sea 
Farm California, L.L.C., Elverta, Calif. under the trade name of 
Belusa.RTM. White Sturgeon). 
While the notochord of sturgeon are preferably disclosed herein for the 
treatment/reducing of arthritis, it is contemplated that the notochord of 
other chordates would also be useful. Chordates are animals of the phylum 
chordata and have, at least at some stage of their development, a 
notochord, a dorsally situated central nervous system, and gill clefts. 
The sturgeon notochord and extracts thereof described in this invention 
have been shown by the inventors to be effective in treating 
experimentally induced arthritis in mice. As discussed above, the mice and 
rat models of arthritis are accepted in the medical community as highly 
predictive of efficacy in humans. 
EXAMPLE I 
Comparative Testing of Various Collagen-Containing Compositions 
The chemical heterogenicity of sturgeon notochord from other 
collagen-containing tissues such as sturgeon cartilage, chicken cartilage 
and bovine cartilage is based on variations in hydroxylation, amino acid 
constitutents, glycosylation patterns, cross linking, conformation and the 
like. The following experiments (Experiments 1.1 to 1.4) were conducted to 
compare sturgeon notochord, chicken sternum cartilage and the collagen 
purified from each for amino acid profile, simulated gastric fluid 
digestion, trypsin digestion and hexosamine release. 
EXPERIMENT 1.1: Amino Acid Profile 
In this Experiment, the amino acid composition of sturgeon notochord was 
compared to the amino acid composition of chicken sternum cartilage. The 
samples of the sturgeon notochord were obtained from Stolt Sea Farm 
California, L.L.C. of Elverta, Calif. and the chicken cartilage was 
obtained from Tyson Farms, Inc. of Springdale, Ark. 
An equal weight of each sample was frozen with liquid nitrogen and 
pulverized. The samples were hydrolyzed to amino acids in 22 hours at 
110.degree. C in 6M HCl. The amino acids were separated by ion exchange 
chromatography, derivatized with ninhydrin and determined with a 
calorimeter. The amino acid profile was determined using a Beckman 6300 
model amino acid analyzer. The results of this analysis are set forth in 
Table 3 as residues per 
TABLE 3 
______________________________________ 
Comparative Amino Acid Composition: Chicken 
Cartilage vs. Sturgeon Notochord (residues/100) 
Chicken Sturgeon 
Amino Acid Cartilage Notochord % Difference 
______________________________________ 
Aspartic acid 6.06 5.95 -2% 
Threonine 3.3 2.93 -11% 
Serine 3.45 4.31 25%* 
Glutamic acid 9.72 10.23 5% 
Proline 10.05 10.92 9% 
Glycine 25.6 28.55 12% 
Alanine 9.35 8.21 -12% 
Cysteine 0.39 0.16 -59%* 
Valine 3.12 2.76 -12% 
Methionine 1.52 1.43 -6% 
Isoleucine 1.94 1.98 2% 
Leucine 4.23 4.09 -3% 
Tyrosine 0.63 0.84 33%* 
Phenylalanine 2.01 1.48 -26%* 
Histidine 1.17 0.96 -18%* 
Lysine 2.75 1.92 -30%* 
Arginine 5.57 4.95 -11% 
4-Hydroxyproline (HPro) 
7.81 6.16 -21%* 
5-Hydroxylysine (HLys) 
1.53 2.16 41%* 
Protein 58% 55% 
______________________________________ 
*Deemed most significant; Tryptophan was not determined 
From Table 3, it can be seen that the protein content on a dry weight basis 
is about the same for each sample, but the frequencies of serine, 
phenylalanine, lysine, 4-hydroxyproline (HPro), and 5-hydroxylysine (HLys) 
are most notably different. 
In addition, the amino acid composition of collagen isolated from sturgeon 
notochord and bovine CII (bovine type II collagen from Sigma) was compared 
to the amino acid composition of chicken CII (chicken type II collagen 
from Sigma). The sturgeon notochord collagen was isolated as described in 
Example V, below. The results of this analysis are set forth in Table 4 as 
residues per 100. 
TABLE 4 
__________________________________________________________________________ 
Comparative Amino Acid Composition (Residues per 100) 
% Difference 
% Difference 
Between 
Between Chicken CII 
Chicken CII 
Bovine CII 
Chicken CII 
Sturgeon 
and Sturgeon 
Sigma Lot 
Sigma Lot 
and Notochord 
Notochord 
#104H40111 
#72H3799 
Bovine CII 
Collagen 
Collagen 
__________________________________________________________________________ 
Aspartic acid 
4.51 4.57 1% 4.58 2% 
Threonine 
2.67 2.21 -17% 2.42 -9% 
Serine 2.22 2.52 14% 3.64 64% 
Glutamic acid 
8.59 8.56 -0% 9.45 10% 
Proline 11.32 12.38 9% 12.55 
11% 
Glycine 32.79 33.87 3% 31.35 
-4% 
Alanine 9.79 10.26 5% 8.63 -12% 
Cysteine 
0 0 0 
Valine 1.94 1.97 2% 1.94 0% 
Methionine 
1.17 1.09 -7% 1.07 -9% 
Isoleucine 
1.04 1.1 6% 1.52 46% 
Leucine 2.7 2.55 -6% 3.49 29% 
Tyrosine 
0.25 0.1 -60% 0.28 12% 
Phenylalanine 
1.42 1.39 -2% 1.25 -12% 
Histidine 
0.96 0.35 -64% 0.71 -26% 
Lysine 1.61 1.88 17% 1.62 1% 
Arginine 
4.98 4.89 -2% 5.07 2% 
4-Hydroxyproline 
9.8 8.6 -12% 7.56 -23% 
(HPro) 
5-Hydroxylysine 
2.24 1.73 -23% 2.89 29% 
(HLys) 
__________________________________________________________________________ 
Tryptophan was not determined. 
Table 4 summarizes the amino acid composition of purified chicken CII, 
bovine CII and sturgeon notochord collagen. The amino acid profiles of 
purified chicken CII and purified bovine CII are similar, however, there 
are substantial differences between purified chicken CII and purified 
sturgeon notochord collagen (i.e., serine, isoleucine and leucine). 
TABLE 5 
______________________________________ 
Amino Acid Ratio Analysis 
Cumulative 
Absolute % Point 
Differences in 
HLys/ Amino Acid 
HLys/HPro 
Gly Content.sup.1 
______________________________________ 
Tissue 
Chicken Cartilage 
0.20 0.06 
Sturgeon Notochord 
0.35 0.08 228.sup.2 
Purified Collagen 
Chicken CII 0.23 0.07 
Bovine CII 0.20 0.05 126.sup.3 
Sturgeon Notochord Collagen 
0.38 0.09 263.sup.3 
______________________________________ 
.sup.1 Absolute differences in amino acid composition between two tissues 
(or collagen) were calculated based on the values from Table 3 and 4 
except for cysteine, tyrosine, and histidine as these amino acids are at 
less than 1% which could distort the percent differences between two 
tissues. 
.sup.2 Compared against chicken cartilage. 
.sup.3 Compared against chicken CII. 
One skilled in the art in collagen chemistry will quickly appreciate the 
importance of HPro (for providing sites for cross-linking within the 
collagen) and HLys (for providing binding sites for glycans, glycosamines 
and the like which likely impact toleragenicity) and their relative levels 
(as this ratio is a major determinant of collagen types). Thus, 
differences in the levels of these amino acids are significant. In 
addition, the HLys to HPro ratio in cod type I collagen and bovine type 
III collagen is 0.11 and 0.07, respectively (data not shown). 
Furthermore, the cumulative absolute percentage point differences between 
the collagens is greater betwen chicken CII and sturgeon notochord 
collagen (263 percentage points) than between chicken CII and bovine CII 
(126 percentage points). This means that bovine CII is closer to chicken 
CII in chemical composition than sturgeon notochord collagen is to chicken 
CII. Likewise, the difference in the amino acid composition between 
chicken cartilage and sturgeon notochord was 228 percentage points, 
indicating that they contain very different collagen from each other. 
These findings imply that sturgeon notochord and the collagen found within 
it are distinctly different from chicken cartilage and from type II 
collagen ("CII") in general. 
As noted previously, these differences raise considerable doubt that 
sturgeon notochord would function in the same manner as chicken cartilage 
or chicken CII in a CIA model of RA. Experiments 1.2 to 1.4 reinforce the 
differences between notochord and other tissues. 
EXPERIMENT 1.2: Gastric Fluid Digestion 
In this Experiment, pulverized preparations of chicken cartilage and 
sturgeon notochord were suspended in USP Simulated Gastric Fluid (United 
States Pharmacopeia, 23, 1994, page 2053) and incubated at 37.degree. C 
for 3 hours. A portion of each digest was filtered at specified intervals 
and the filtrates analyzed for HPro and HLys which are considered markers 
for collagen. The filtrates were also analyzed by size exclusion 
chromatography. The size exclusion chromatograms were generated on 
Hewlett-Packard Model 1090M instrument with Shodex Protein Column KW-803; 
8 .times.300 mm; 7 .mu.m particle size; Waters P/N 35946 and a 20 .mu.L 
Injection. A Mobile Phase of 600 volumes of water, 400 volumes of 
acetonitrile and 0.8 volumes of trifluoroacetic acid was used at ambient 
temperature at a Flow Rate of 0.3 mL/minute for 60 minutes. Detection was 
by U.V. absorbance at 214 nm. 
Table 6 sets forth the collagen solubilization (measured as concentration 
of HPro and HLys versus time) in the digestion fluid after filtering 
through a 0.45 .mu.m polysulfone membrane. 
TABLE 6 
______________________________________ 
Simulated Gastric Fluid Digestion Comparison 
Sturgeon Notochord vs. Chicken Sternum Cartilage 
4-Hydroxy- 
Tissue Digestion time 
proline* 5-Hydroxylysine* 
______________________________________ 
Chicken Cartilage 
0 minutes 0 0 
45 minutes 58 54 
90 minutes 73 65 
180 minutes 
80 80 
Sturgeon Notochord 
0 minutes 0 0 
45 minutes 84 82 
90 minutes 92 93 
180 minutes 
102 100 
______________________________________ 
*% of total HPro and HLys present in the undigested tissue 
Table 6 demonstrates that in simulated gastric fluid, notochord is 
solubilized at a rate that significantly exceeds the rate of chicken 
cartilage solubilization. Without being bound to a particular theory, it 
is believed that notochord is more readily solubilized by virtue of its 
less developed matrix of chondroitin sulphate chains, proteoglycans and 
polypeptides. Further, this data would suggest that notochord could 
potentially have an advantage over cartilage as an enterally administered 
toleragen since it is more readily solubilized by gastric juices. If 
notochord is more readily solubilized, it theoretically may reduce 
required dosages and/or expedite therapeutic impact. 
The size exclusion chromatograph indicated for 0-time digests a peak at 
about 43 minutes for chicken cartilage which is essentially missing for 
notochord. At 45 minutes of digestion, the notochord and chicken cartilage 
digests begin to diverge substantially. For example, there are substantial 
peaks between about 30 minutes and 40 minutes for the notochord digest 
which are either missing or greatly reduced in the chicken cartilage 
digest. Most interesting is a peak at about 43 minutes wherein the 
notochord peak is about one fifth the size of the chicken cartilage peak. 
Similar differences also appear for the 90 and 180 minute digestion. This 
Experiment further demonstrates the substantial and significant 
differences between the sturgeon notochord and chicken cartilage. 
EXPERIMENT 1.3: Trypsin Digestion 
Pulverized preparations of chicken cartilage and notochord were suspended 
in 0.05M TRIS buffer, pH 7.5, containing trypsin (Sigma Type XIII) at 1 
mg/14 mg of dry tissue, and incubated at 37.degree. C. for 4 hours. Each 
digest was filtered and the filtrates were "peptide mapped"by Reversed 
Phase HPLC using a Hewlett-Packard Model 1090M with Vydac C18 Protein and 
Peptide; 4.6 .times.250 mm; 5 .mu.m particle size column (The Separations 
Group; P/N 218TP54) and two mobile phases: Mobile Phase A was 0.08% 
trifluoroacetic acid in water and Mobile Phase B was 0.08% trifluoroacetic 
acid in acetonitrile. Injection volume of 25 .mu.L flowed at 0.8 mL/minute 
at 40.degree. C. for 90 minutes according to the following elution 
gradient. Detection was by U.V. absorbance at 214 nm and at 280 nm. 
______________________________________ 
Gradient Elution Program 
Time in Minutes 
% Mobile Phase B 
______________________________________ 
0 5 
5 5 
65 35 
67 55 
70 55 
75 5 
90 stop 
______________________________________ 
A significant number of qualitative and quantitative differences were 
evidenced between the notochord and cartilage digests which indicates that 
the trypsin was cleaving the tissues at different locations. This fact 
indicates that the amino acid sequences are significantly different from 
each other in these tissues. More specifically, at 214 nm detection and at 
about 17 minutes, the chicken cartilage produced a peak that was more than 
twice the peak for sturgeon notochord at the same time. At 280 nm 
detection, the cartilage produced a peak at about 50 minutes that was at 
least an order of magnitude greater than the corresponding peak for 
notochord. 
EXPERIMENT 1.4: Hexosamine Release 
In this Experiment, trifluoroacetic acid (TFA) digests of chicken cartilage 
and sturgeon notochord were conducted to evaluate the galactosamine and 
glucosamine content of each tissue. Galactosamine and glucosamine are 
monomer components of GAG copolymer synthesis. Hexosamine contents may 
relate to an immunological or toleragen potential of a tissue. 
Pulverized preparations of each tissue were hydrolyzed and the 
galactosamine and glucosamine released by each tissue was measured by HPLC 
as AQC (6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate) derivatives. The 
hydrolysis was conducted for 14 hours at 120.degree. C. in 1.35 M TFA. The 
galactosamine concentrations and their chondroitin sulfate-equivalents 
(chondroitin sulfate is a copolymer of N-acetyl-galactosamine sulfate and 
glucuronic acid), as well as the glucosamine concentrations and their 
hyaluronic acid equivalents (hyaluronic acid is a copolymer of 
N-acetyl-glycosamine and glucuronic acid) are presented in Table 7. The 
hexosamines released by TFA hydrolysis were separated and detected by HPLC 
using a Hewlett-Packard Model 1090M and a Brownlee Spher-5 RP-8; 4.6 
.times.250 nmn; 5 .mu.m particle size column (Alltech P/N 141033) and two 
mobile phases: Mobile Phase A was 0.15 M sodium acetate, 0.019M 
triethylamine, 10 mg/L disodium EDTA; pH 5.0; and Mobile Phase B was 70 
volumes A+30 volumes acetonitrile. Injection volume of 20 .mu.L flowed at 
0.6 mL/minute at 40.degree. C. for 50 minutes according to the following 
elution gradient. Detection was by U.V. absorbance at 248 nm. 
______________________________________ 
Gradient Elution Program: 
Time in Minutes 
% Mobile Phase B 
______________________________________ 
0 0 
5 23 
25 23 
27 100 
32 100 
35 0 
50 stop 
______________________________________ 
This experiment is one means of comparing the proteoglycan content of each 
tissue which is an approximate measure of its character as an 
immunogen/toleragen. The data presented in Table 7 clearly illustrate the 
difference in the two tissues. 
TABLE 7 
______________________________________ 
Hexosamine Release Comparison 
Sturgeon Notochord vs. Chicken Sternum Cartilage 
Hexosamine Released per gram of Dry Tissue* 
Chicken 
Saccharide Cartilage (mg) 
Sturgeon Notochord (mg) 
______________________________________ 
Galactosamine 55 114 
Glucosamine 105 19 
Chondroitin SO.sub.4 equivalents 
147 303 
Hyaluronic Acid equivalents 
234 42 
______________________________________ 
*Hydrolysis 14 hours at 120.degree. C. in 1.35M TFA 
In conclusion, the data presented in Experiments 1.1 to 1.4 evidences a 
number of significant differences between chicken cartilage and sturgeon 
notochord: (1) amino acid composition; (2) peptides from hydrolysis based 
on simulated gastric fluid digestion; (3) amino acid sequence based on 
trypsin digestion; and (4) proteoglycan content. In addition, collagen 
purified from sturgeon notochord was significantly different from bovine 
CII. 
In spite of the above demonstration that sturgeon notochord and its 
collagen is qualitatively and quantitatively different from chicken 
cartilage and CII found within, the present inventors believed that 
investigating notochord efficacy was warranted as sturgeon notochord is 
highly digestible, low in cost, and is available from a highly controlled 
environment. The following Examples compare the activity of sturgeon 
notochord and collagen derived therefrom against various 
collagen-containing tissues, including chicken cartilage. All following 
examples were conducted in accordance with the current guidelines for 
animal welfare. 
EXAMPLE II: Protective Effect 
2.1 Protective Effect of Sturgeon Notochord 
These Examples were conducted to investigate the effectiveness of sturgeon 
notochord in protecting mice from collagen-induced arthritis (CIA). Female 
DBA/1Lac J strain mice, approximately 6 -8 weeks of age, were obtained 
from Jackson Laboratories of Bar Harbor, Me. Three groups of 20 mice per 
group were acclimated to their surroundings for 7 days. A standard mouse 
chow (Purina Certified Mouse Chow #5015; 11% by wt. fat; collagen-free) 
and water were provided for ad libitum consumption to all animals. 
On Days -10, -7, -5 and -2 prior to immunization, mice were sensitized by 
one of three test compositions; bovine serum albumin (BSA, a 
tolerogenically inert protein in CIA), cod skin gelatin, or sturgeon 
notochord. Bovine serum albumin (a protein derived from non-connective 
tissue) and cod skin gelatin (a protein derived from another connective 
tissue containing Type I collagen of a fish) served as negative controls. 
Sturgeon notochord was obtained from farm raised sturgeon and pulverized 
in liquid nitrogen. The level of each test composition was equilibrated to 
a 300 .mu.g protein dose based on protein (including collagen) content. 
Test articles were dissolved/suspended in 0.01M acetic acid and 0.3 ml was 
administered per dose to each mouse with a ball tipped feeding needle. 
______________________________________ 
(mg/ 
Group Treatment dose) 
______________________________________ 
1 BSA (91.7% protein, 0% collagen) 
0.33 
2 Cod skin gelatin (93.6% protein, 93.6% Type I collagen) 
0.32 
3 Sturgeon notochord (10% protein, 8.5% collagen) 
3.53 
______________________________________ 
On Day 0, the mice were immunized at the base of the tail with 100 .mu.g of 
bovine CII which was emulsified in Complete Freund's Adjuvant (CFA). On 
Day 7, a second booster dose of 100 .mu.g bovine CII in CFA was 
administered by the same route. On Day 14, the mice were injected 
subcutaneously with 100 .mu.g of lipopolysaccharide (LPS). This protocol 
represents a mild induction of CIA. 
Mice were inspected daily for the onset of CIA, characterized by erythema 
and edema. Limbs were clinically evaluated and graded on a scale of 0 to 3 
(0, absence of arthritis; 1, mild swelling and erythema; 2, swelling and 
erythema of both tarsus and ankle; 3, ankylosis and bone deformity). Mice 
that did not develop arthritis were marked negative for arthritis. All 
mice were euthanized by CO.sub.2 on Day 50. 
On Days 20, 28, 40 and 50, the group fed with sturgeon notochord 
consistently had fewer animals with CIA than those fed BSA or cod skin 
gelatin (Table 8). The % onset calculated based on the Day 50 data showed 
that 78% and 79% of mice fed BSA and cod skin gelatin, respectively, had 
CIA whereas only 58% of mice fed sturgeon notochord evidenced CIA. 
Furthermore, the median day of CIA onset for the group fed sturgeon 
notochord was delayed by 13 days compared to the negative control groups. 
The mean severity score was highest for the group fed cod skin gelatin 
followed by the BSA-fed group, and the sturgeon notochord-fed group had 
the lowest severity score. The data herein suggest that sturgeon notochord 
is effective in delaying and attenuating CIA compared to BSA or cod skin 
gelatin. 
TABLE 8 
__________________________________________________________________________ 
# of median 
mean 
animals animals with arthritis 
% day of 
severity 
Treatment 
dead 
alive 
Day 20 
Day 28 
Day 40 
Day 50 
onset* 
onset 
score** 
__________________________________________________________________________ 
BSA 2 18 12 12 12 14 78 17 2.05 
Cod skin 
1 19 14 14 14 15 79 17 2.40 
gelatin 
Sturgeon 
1 19 9 10 1 11 58 30 1.99 
notochord 
__________________________________________________________________________ 
*Calculated based on the Day 50 data 
**Based on Day 20, 29, 34, 43 and 50 data which allowed the most data 
points for all groups. 
2.2 Protective Effect of Sturgeon Notochord - Stronger Induction of CIA 
To evaluate the effectiveness of sturgeon notochord in protecting mice from 
CIA, this experiment was conducted in a manner similar to Example 2.1 
except that the immunization protocol was slightly modified. The 
experimental protocol employed in this experiment represents a stronger 
induction of CIA compared to that of Example 2.1. 
The mice were DBA/1 Lac J strain supplied by Jackson Laboratories of Bar 
Harbor, Me. All mice were female and about 6 -8 weeks of age. Three groups 
of 15 -18 mice per group were acclimated to their surroundings for 7 days. 
A standard mouse chow and water were provided for ad libitumconsumption. 
On Days - 10, -7, -5 and -2 prior to immunization, mice were sensitized by 
one of three test samples: 1) vehicle; 2) cod skin gelatin; and 3) 
sturgeon notochord. Cod skin gelatin (Type I collagen) was used as a 
negative control. Sturgeon notochord was obtained from farm-raised 
sturgeon and pulverized in liquid nitrogen as previously described. The 
level of each test composition was equilibrated to a 300 .mu.g collagen 
per dose. Test compositions were dissolved/suspended in 0.01M acetic acid 
and 0.3 ml was administered to each mouse with a ball tipped feeding 
needle. 
______________________________________ 
(mg/ 
Group Treatment dose) 
______________________________________ 
1 Vehicle (0.01M acetic acid) 0 
2 Cod skin gelatin (93.6% protein, 93.6% Type I collagen) 
0.32 
3 Sturgeon notochord (10% protein, 9.5% collagen) 
3.16 
______________________________________ 
On Day 0,the mice were immunized at the base of the tail with 100 
.parallel.g of bovine CII which was emulsified in CFA. On Day 14, five 
animals from each group were bled retroorbitally and anti-type II collagen 
antibody titer in the serum was analyzed by an ELISA method to confirm 
that the animals were primed. On Day 21, the mice were injected 
subcutaneously with 50 .mu.g of LPS. 
TABLE 9 
______________________________________ 
Day 14 Type II Collagen Antibody Titer - 1:1000 Dilution 
Mean Antibody 
% Change from 
Treatment n Titer* Control 
______________________________________ 
Vehicle 5 0.89 .+-. 0.27 
-- 
Cod skin gelatin 
4 0.57 .+-. 0.23 
-37 
Sturgeon notochord 
5 0.22 .+-. 0.05 
-76 
______________________________________ 
*The average value for nonimmunized animals was 0.03. 
The CII antibody titer for the vehicle-fed immunized mice increased to 0.89 
.+-.0.27 compared to the average CII antibody titer for non-immunized mice 
of 0.03. This indicates that animals were primed by CII immunization. On 
the other hand, those mice tolerized with sturgeon notochord showed 
significant reduction (-76%) in the CII antibody titer, which suggests 
that their immune system was de-sensitized compared to the vehicle-fed 
group. These findings are in agreement with the antibody data of 
Nagler-Anderson et al. "Suppression of Type II Collagen-Induced Arthritis 
by Intragastric Administration of Soluble Type II Collagen", Proc. Natl 
Acad, SciU.S.A., 1986:83;7443 -7446 and the clinical findings set forth in 
Table 10. Although there was some reduction over the vehicle control, the 
reduction seen in the cod skin gelatin fed group was not significant. 
Mice were inspected daily for the onset of CIA, characterized by erythema 
and edema. Limbs were clinically evaluated and graded on a scale of 0 to 6 
(0, absence of arthritis; 0.5, one or more digits swollen or only the paw 
swollen; 1, entire paw swollen; 2, entire paw severely swollen; 3, mild 
deformity after inflammation subsides; 4, severe deformity; 5, mild 
ankylosis with partial loss of joint function in the paw; 6, severe 
ankylosis with total loss of joint finction in the paw). All mice were 
euthanized by CO.sub.2 on Day 35. 
TABLE 10 
__________________________________________________________________________ 
% of incidence 
severity score* 
Treatment 
n D25 
D28 
D35 
D25 D28 D35 
__________________________________________________________________________ 
Vehicle 
18 94 89 83 4.67 .+-. 0.64.sup.ab 
7.14 .+-. 1.09 
10.00 .+-. 1.66.sup.a 
Cod Skin 
Gelatin 
18 89 89 94 5.53 .+-. 0.57.sup.a 
8.61 .+-. 0.85 
10.67 .+-. 1.18.sup.a 
Sturgeon 
15 87 87 93 3.73 .+-. 0.68.sup.b 
6.40 .+-. 1.14 
5.83 .+-. 1.30.sup.b 
notochord 
__________________________________________________________________________ 
*Values in columns with different superscript letters represent 
significant differences with P &lt; 0.05. 
The % incidence of CIA was higher across all treatments compared with 
Example 2.1, confirming the harsh nature of the CIA induction protocol 
employed in this Example. Nevertheless, the group fed sturgeon notochord 
had significantly lower (P&lt;0.05) severity scores than those fed vehicle or 
cod skin gelatin. These findings are consistent with the results of 
Example 2.1 which indicate that sturgeon notochord is effective in 
attenuating CIA. 
EXAMPLE III 
Sturgeon Notochord vs. Chicken Cartilage 
This experiment was conducted to compare the effectiveness of chicken 
cartilage and sturgeon notochord in protecting mice from CIA. This 
Experiment was conducted in a manner similar to Example II. The treatment 
groups were as follows: 
______________________________________ 
Group Treatment (mg/dose) 
______________________________________ 
1 vehicle 0 
(0.01M acetic acid) 
2 vehicle + chicken cartilage 
2.21 
(17% protein, 13.6% CII) 
3 vehicle + sturgeon notochord 
3.16 
(10% protein, 9.5% collagen) 
______________________________________ 
Group 1 seved as the control. Chicken cartilage (xiphoid cartilage on the 
sternum) was obtained from a commercial source and was pulverized in 
liquid nitrogen and used for Group 2. Sturgeon notochord as described 
previously was used for Group 3. The dose level (300 .mu.g/dose) was 
selected based on previous data. 
The sensitization procedure consisted of dissolving/suspending each sample 
in 0.01M acetic acid and 0.3 ml was administered to each mouse with a ball 
tipped feeding needle on Days -10, -7, -5 and -2 prior to immunization. On 
Day 0, the mice were immunized at the base of the tail with 100 .mu.g of 
bovine CII which was emulsified in CFA. On Day 21, the mice were injected, 
subcutaneously, with 50 .mu.g of LPS. 
Body weights for each mouse were recorded on Day 0, 21, 28 and 35. Mice 
were inspected daily for the onset of CIA, which was characterized by 
erythema and edema. Limbs were clinically evaluated and graded on a scale 
of 0 to 6, as previously described. 
The data presented in Table 11 demonstrates that sturgeon notochord is 
efficacious in attenuating CIA. In fact, the sturgeon notochord fed group 
was the only group that was significantly (p&lt;0.05) different from the 
control group in the severity score. There was a numerical improvement in 
the severity score by chicken cartilage, but it was not signifantly 
different from the control group. 
TABLE 11 
______________________________________ 
Tolerizing Effect of Chicken Cartilage and Sturgeon Notochord 
on Mouse Developing CIA - Day 35 
Severity 
% 
% Score Change 
% Wt. 
inci- Mean .+-. 
From Change 
Treated Groups 
n dence SB Control 
From Day 21 
______________________________________ 
1: 0.01M acetic acid 
18 83 10.0 .+-. 1.7.sup.a 
-- -3 
2: Chicken Cartilage 
14 86 6.6 .+-. 1.6.sup.a,b 
-34 -10 
3: Sturgeon Notochord 
15 93 5.8 .+-. 1.3.sup.b 
-42 +3 
______________________________________ 
.sup.a,b Numbers with different alphabetical superscripts are 
significantly different from each other at a value of P &lt; 0.05. 
Furthermore and most importantly, Table 11 surprisingly evidences that the 
only group to gain weight was the sturgeon notochord group. The chicken 
cartilage actually evidenced a weight loss of 10%. When animals are 
subjected to trauma or sick from diseases, they typically reduce their 
feed intake which results in weight loss as seen herein in the chicken 
cartilage group. Thus, this data further supports the inventors'belief 
that sturgeon notochord is not at all similar to chicken cartilage and in 
fact may be better. 
EXAMPLE IV 
Dose Response 
To evaluate the dose response of sturgeon notochord in protecting mice from 
CIA, this experiment was conducted in a manner similar to Examples II and 
III. The mice were DBA/1 Lac J strain supplied by Jackson Laboratories of 
Bar Harbor, Maine. All mice were female and about 6 -8 weeks of age. Eight 
groups of 10 -15 mice per group were acclimated to their surroundings for 
7 days. A standard mouse chow and water were provided for ad libitum 
consumption to all animals. 
On Days -10, -7, -5 and -2 prior to immunization, mice were sensitized by 
sturgeon notochord. A group fed only vehicle was used as the negative 
control and a group fed with an effective dose of dexamethasone (0.1 mg/kg 
per os) was used as the positive control. Sturgeon notochord was obtained 
from farm raised sturgeon and pulverized in liquid nitrogen. The dose 
level of sturgeon notochord was equilibrated to 1, 3, 10, 30, 100 or 300 
.mu.g of collagen per dose based on the protein and collagen content. 
Sturgeon notochord was dissolved/suspended in 0.01M acetic acid and 0.3ml 
was administered to each mouse with a ball tipped feeding needle. 
______________________________________ 
Group Treatment Dose 
______________________________________ 
1 Vehicle (0.01M acetic acid) 
2 Dexamethasone 0.1 mg/kg p.o. 
3 Sturgeon notochord (10% protein, 9.5% 
0.0105 mg 
collagen) 
4 Sturgeon notochord 0.032 mg 
5 Sturgeon notochord 0.105 mg 
6 Sturgeon notochord 0.316 mg 
7 Sturgeon notochord 1.053 mg 
8 Sturgeon notochord 3.160 mg 
______________________________________ 
On Day 0, the mice were immunized at the base of the tail with 100 .mu.g of 
bovine CII which was emulsified in CFA. On Day 21, the mice were injected 
subcutaneously with 50 .mu.g of LPS. 
Mice were inspected daily for the onset of CIA, characterized by erythema 
and edema. Limbs were clinically evaluated and graded on a scale of 0 to 6 
as described in Example III. 
Dexamethasone (an agent known to reduce CIA) significantly reduced both % 
of incidence and severity score (Table 12). The % incidence of CIA was 
also reduced in all groups fed sturgeon notochord, regardless of the dose 
level (except Day 35 at 3.160 mg/dose group). Severity score was also 
reduced in all sturgeon notochord fed groups in a dose-dependent manner on 
Day 26. Moreover, the group fed 1.053 mg sturgeon notochord per dose 
significantly (P&lt;0.05) reduced the severity score on Day 35 (a 50% 
reduction) and a 20% reduction in % incidence. These findings are 
consistent with the results of previous Examples that sturgeion notochord 
is effective in attenuating CIA. 
TABLE 12 
__________________________________________________________________________ 
Dose Study 
% of incidence 
severity score 
Treatment 
n D26 
D28 
D35 
D26 D28 D35 
__________________________________________________________________________ 
Vehicle 15 100 
100 
100 
4.3 .+-. 0.5 
6.2 .+-. 0.8 
8.8 .+-. 0.9 
Dexamethasone 
10 40 50 50 1.0 .+-. 0.4* 
1.1 .+-. 0.4* 
1.1 .+-. 0.4* 
Sturgeon notochord 
10 80 89 80 3.5 .+-. 0.8 
5.2 .+-. 1.3 
6.8 .+-. 1.7 
(0.0105 mg) 
Sturgeon notochord 
10 70 70 70 3.2 .+-. 1.0 
4.7 .+-. 1.5 
6.7 .+-. 2.1 
(0.032 mg) 
Sturgeon notochord 
9 89 89 89 2.7 .+-. 0.8 
3.4 .+-. 1.2 
5.2 .+-. 1.4 
(0.105 mg) 
Sturgeon notochord 
9 67 78 78 2.8 .+-. 1.1 
4.3 .+-. 1.1 
5.9 .+-. 2.1 
(0.316 mg) 
Sturgeon notochord 
10 80 80 80 2.5 .+-. 0.9 
3.6 .+-. 1.4 
4.4 .+-. 1.8* 
(1.053 mg) 
Sturgeon notochord 
10 90 89 100 
2.3 .+-. 0.4 
4.4 .+-. 1.0 
6.6 .+-. 1.7 
(3.160 mg) 
__________________________________________________________________________ 
*(P &lt; 0.05) from the vehicle fed Control. 
The inventors continued to observe four selected groups (vehicle control 
group, sturgeon notochord 0.105 mg/dose group, sturgeon notochord 1.053 
mg/dose group, and dexamethasone group) until Day 79. Severity scores 
plotted against days post-sensitization are found in FIG. 1. 
From the data generated, it is evident that the vehicle-fed control group 
showed a normal disease progression while dexamethasone was able to 
significantly suppress the severity score. The suppressive effect of 
sturgeon notochord lasted to Day 79 in the group fed 1.053 mg/dose. The 
group fed with 0.105 mg/dose also showed similar effect up to Day 70. 
EXAMPLE V 
Sturgeon Collagen 
This experiment was conducted to show the effect of collagen isolated from 
sturgeon notochord in protecting mice from CIA. This Experiment was 
conducted in a manner similar to Examples II, III, and IV. 
The mice were DBA/1 Lac J strain supplied by Jackson Laboratories of Bar 
Harbor, Me. All mice were female and about 6 -8 weeks of age. Three groups 
of 10 -15 mice per group were acclimated to their surroundings for 7 days. 
Standard mouse chow and water were provided for ad libitum consumption to 
all animals. 
On Days - 10, -7, -5 and -2 prior to immunization, mice were sensitized by 
notochord collagen. Sturgeon notochord was obtained from farm raised 
sturgeon and collagen was extracted and purified by the method described 
in Eyre and Muir, "The Distribution of Different Molecular Species of 
Collagen in Fibrous, Elastic and Hyaline Cartilages of the Pig" Biochem 
J(1975) 151;595 -602. Notochord collagen was dissolved/suspended in 0.01M 
acetic acid and 0.3 ml was administered to each mouse with a ball tipped 
feeding needle. The dose levels tested were 30 or 100 .mu.g per dose per 
animal. 
______________________________________ 
Group Treatment Collagen Dose 
______________________________________ 
1 Vehicle (0.01M acetic acid) 
2 Sturgeon notochord collagen (60% collagen) 
30 .mu.g 
3 Sturgeon notochord collagen (60% collagen) 
100 .mu.g 
______________________________________ 
On Day 0, the mice were immunized at the base of the tail with 100 .mu.g of 
bovine CII which was emulsified in CFA. On Day 21, the mice were injected 
subcutaneously with 50 .mu.g of LPS. 
Mice were inspected daily for the onset of CIA, characterized by erythema 
and edema. Limbs were clinically evaluated and graded on a scale of 0 to 6 
as previously described. Table 13 sets forth the results from this 
experiment. 
TABLE 13 
______________________________________ 
% weight 
change 
% incidence 
severity score 
(D35 vs. 
Treatment n D25 D35 D25 D35 D21) 
______________________________________ 
Vehicle 10 100 100 3.2 .+-. 0.4 
6.4 .+-. 1.2 
0 
Sturgeon 10 100 100 2.3 .+-. 0.7 
3.7 .+-. 1.2 
+4 
notochord 
collagen (30 .mu.g) 
Sturgeon 9 89 89 3.1 .+-. 0.6 
5.0 .+-. 1.4 
+4 
notochord 
collagen (100 .mu.g) 
______________________________________ 
Severity scores were reduced in both groups fed sturgeon notochord 
collagen. The group fed 30 .mu.g showed a 42% reduction in the severity 
score on Day 35 compared to the vehicle-fed control group. The group fed 
100 .mu.g showed a decrease in the percentage of incidence. This is 
consistent with previously published data by Zhang Z.J., C.S.Y Lee, O 
Lider, HL Weiner. "Suppression of Adjuvant Arthritis in Lewis Rats by Oral 
Administration of Type II Collagen", J Immuno(1990), 145; 2489 -2493. It 
was also observed that both groups fed with sturgeon notochord collagen 
evidenced an increase in body weight. 
Industrial Applicability 
The medical community is constantly searching for improved treatments for 
arthritis. This invention provides for the use of notochord and its 
extracts (i.e., notochord collagen) for oral administration to lessen the 
onset of arthritis and to reduce the intensity of the disease. Sturgeon 
notochord, is particularly suited for inclusion into nutritional formulas 
and supplements. Further, sturgeon notochord is inexpensive and of 
abundant and reliable supply. Various modifications may be made in the 
present invention without departing from the spirit or scope thereof, as 
will be readily apparent to those skilled in the art.