Note: Descriptions are shown in the official language in which they were submitted.
216935
METHODS OF DETECTING COLLAGEN DEGRADATION IN VIVO
This invention was made with U. S. government
support under grants AR37318 and AR36794 awarded by the
National Institutes of Health. The U. S. government has
certain rights in the invention. This application is a
divisional application of Serial No. 2,031,265 filed on
November 30th, 1990.
The present invention relates to methods for
detecting and monitoring collagen degradation in vivo. More
specifically, it relates to methods for quantitating cross-
linked telopeptides produced in vivo upon degradation of
collagen types II and III.
Three known classes of collagens have been described
to date. The class I collagens, subdivided into types I, II,
III, V, and XI, are known to form fibrils. These collagens
are all synthesized as procollagen molecules, made up of
N-terminal and C-terminal propeptides, which are attached to
the core collagen molecule. After removal of the propeptides,
which occurs naturally in vivo during collagen synthesis, the
remaining core of the collagen molecule consists largely of a
triple-helical domain having terminal telopeptide sequences
which are nontriple-helical. These telopeptide sequences
have an important function as sites of intermolecular cross-
linking of collagen fibrils extracellularly.
The present invention relates to methods of detecting
collagen degradation based on assaying for particular cross-
linked telopeptides produced in vivo upon collagen degradation.
- 1 -
62839-1685D
216935
In the past, assays have been developed for monitoring
degradation of collagen in vivo by measuring various
biochemical markers, some of which have been degradation
products of collagen. For example, bone turnover associated
with Paget's disease has been monitored by measuring small
peptides
- la -
62839-1685D
CA 02156935 2004-O1-28
-2-
containing hydroxyproline, which ere excreted in the urine following
degradation
of bone collagen. Russell et al., Metab. Eons Dis. and Ret. Rsa. 4 and 5, 255-
28Z
(1981); and Singer, F.R., et al., Metabolic Hone Disease, Vol. I1 (ads.
Avioli, L.Y.
and Kane, S.M.), 489-678 (1978), Academic Press, New York.
Other researchers have measured the crosa-iinking compound pyrid)noline in
urine sa an index of collagen degradation in joint disease. Bee, for
background and
for example, Wu and Eyre, Btochemi'try, Z3e1850 (1984); 8laek et al., Annals
of
the Rheumatic Diseases, 48:841-844 (18891; Robins et al.; Annals off' the
Rheumatic Diseases, 45:969-993 (198B); and Seibel et al., The Journal of
Rheumatology, 16:984 (1989). In contrast to the present invention, some prior
researohers have hydrolyzed peptides from body fluids and then looked for the
presence of individual hydroxypyridlnlum residues. None of these researchers
have
reported measuring a teiopeptfde containing a cross-link that is naturally
produced
tn vivo upon oollegen degradation, as in the present invention.
U.K. Patent application GB 2,206,843 reports that the degradation of type III
collagen in the body is quantitatively determined by measuring the
coneentratlon
of an N-terminal telopoptide from type Ill collagen in a body fluid. Irt this
reference, It is reported that cross-linked teIopeptide regions are not
desirable. In
fact, this reference reports that it is necessary to use a non-cross-linked
source of
ZO collagen to obtain the telopeptlde. The peptides of the present invention
are all
cross-linked. Collagen cross-links are discussed In greater detail below,
under the
heading "Collagen Cross-Linking."
There are a number of reports indicating that collagen degradation can be
measured by quantitating certain procollagen peptides. The present invention
involves telopeptides rather than propeptides, the two being distinguished by
their
location in the collagen molecule and the timing of their oleavage in vivo.
See
U.S. Patent 4,504,587; U.S. Patent 4,312,853; Pterard et al., Analytical
Biochemistry 141:129-I3B (1984); Niemela, Clin. Chem., 31/8:1301-1304 (1985);
and Rohde et al., European Journal of Clinical Inveattgation, 9:451-459
(I979).
U.9. Patent 4,7T8,T88 relates to a method of determining changes occurring
in artieular cartilage involving quantifying proteoglycan monomer or antigenic
fragments thereof in a synovial tluld sample. This patent does not relate to
deteoting cross-linked telopeptides derived from degraded collagen.
Dodge, J. C(in. Invest., 83:847-881 (1981) discloses methods for analyzing
type II collagen degradation utilizing a polyclonal antiserum that
specifically
reacts with unwound alpha-chains and cyanagen bromide-derived peptides of
human and bovine type II eollagens. The peptides involved are not cross-linked
telopeptides as in the present invention.
216935
Amino acid sequences of human type III collagen,
human proal(IT) collagen, and the entire preproal(III) chain
of human type III collagen and corresponding cDNA clones have
been investigated and determined by several groups of
researchers. See Loidl et al., Nucleic Acids Research
12:938.3-9394 (1984); Sangiorgi et al., Nucleic Acids Research,
1.3:2207-2225 (1985); Baldwin et al., Bioclrem. J., 262:521-528
(1989); and Ala-Kokko et al., Biochem. J., 260:509-516 (1989).
None of these references specifies the structures of
particular telopeptide degradation products that could be
measured to determine the amount of degraded fibrillar
collagen in vivo.
In spite of the above-described background
information, there remains a need for effective and simple
assays for determining collagen degradation in vivo. Such
assays could be used to detect and monitor disease states in
humans, such as osteoarth ritis (type II collagen degradation),
and various inflammatory disorders, such as vasculitis
syndrome (type III collagen degradation).
2Q Assays for type I collagen degradation can be
utilized to detect and assess bone resorption in vivo.
Detection of bone resorption may be a factor of interest in
monitoring and detecting diseases such as osteoporosis.
Osteoporosis is the most common bone disease in man. Primary
osteoporosis, with increased susceptibility to fractures,
results from a progressive net loss of skeletal bone mass. It
is estimated to affect 15-20 million individuals in the United
States. Its basis is an age-dependent imbalance in bone
- 3 -
62839-1685
215693
remodeling, i.e., lI1 the rates of synthesis and degradation of
tooue tissue.
About 1.2 million osteoporosis-related fractures
occur in the elderly each year including about 538,000
compression fractures of the spine, about 227,000 hip
fractures and a substantial number of early fractured
periptreral bones. Twelve to 200 of the hip fractures are
fatal because they cause severe trauma and bleeding, and half
of tt~e surviving patients require nursing home care. Total
costs from osteoporosis-related injuries now amount to at
least $7 billion annually (Barnes, O.M., Science, 236:914
(1987)).
Osteoporosis is most common in postmenopausal women
who, on average, lose 15°s of their bone mass in the 10 years
after menopause. This disease also occurs in men as they get
older and in young amenorrheic women athletes. Despite the
major, and growing, social and economic consequences of
osteoporosis, no method in available for measuring bone
resorption rates in patients or normal human subjects. A
2.0 major difficulty in monitoring the disease is the lack of a
specific assay for measuring bone resorption rates.
- 3a -
62839-1685
2156935
Methods for assessing bone mass often rely on measuring whole-body calcium
by neutron activation analysis or mineral mass in a given bone by photon
absorption techniques. These measurements can give only long-term impressions
of whether bone mass is decreasing. Measuring calcium balances by comparing
intake wtth output is tedious, unreliable and can only indirectly appraise
whether
bone mineral is being lost over the long term. Other methods currently
available
for assessing decreased bone mass and altered bone metabolism include
quantitative scanning radiometry at selected bone looations (wrist, calcaneus,
etc.) and histomorphometry of Iliac crest biopsies. The former provides a
crude
measure of the bone mineral content at a specific site in a single bone.
Hlstomorphometry gives a semi-quantitative assessment of the balance between
newly deposited bone seams and resorbing surfaces.
A urinary assay for the whole-body output of degrsded bone in 24 hours
would be much more useful. Mineral studies (e.g., calcium balance) cannot do
this
; 5 reliably or easily. Since bone resorptlon involves degradation of the
mineral and
the organic matrix, a specific biochemical marker for newly degraded bone
products in body fluids would be the (deal Index. Several potential organic
irldiQes
have been tested. For example, hydroxyproline, an amino acid largely
restricted
to collagen, and the principal structural protein In bone and all other
connective
2p tissues, is excreted 1n urine. its excretion rate is known to be increased
in certain
conditions, notably Paget's disease, a metabolic bone disocder in which bone
turnover is greatly increased, es pointed out above. For this reason, urinary
hydroxyproline has been used extensively as an amino acid marker for collagen
degradation. Singer, F.R., et al. (1978), cited hereinabove.
25 U.S. Patent No. 3,600,132 discloses a prncess foe determination of
hydroxyproline in body fluids such as serum, urine, Lumbar fluid and other
intercellular fluids in order to monitor deviations in collagen metabolism. (n
particular, this inventor notes that in pathologic conditions such as Pager's
disease, Marfan'a syndrome, osteogenesis imperfecta, neoplastic growth in
30 collagen tissues and in various forms of dwarfism, increased collagen
anabolism or
catabolism ae m~aaurvd by hydroxyproline content in biological fluids can be
determined. This inventor measures hydroxyproline by oxidizing it to a pyrrole
compound with hydrogen peroxide end N-ehloro-p-toluenesulphonamide followed
by colorimetric determination in p-dlmethyl-amino-benzaldehyde.
35 In the case of Paget's disease, the increased urinary hydroxyprvline
probably
comes largely from bone degradation; hydroxyproline, however, generally cannot
be used as a specific index. Much of the hydroxyproltnc In urine may come from
215693
-5-
new collagen synthesis (considerable amounts of the newly made protein are
degraded and excreted without ever becoming incorporated into tissue fabric),
and
from turnover of certain blood proteins as well as other proteins that contain
hydroxyproline. Furthermore, about 80Q6 of the free hydroxyproline derived
from
protein degradation la metabolized in the liver and never appears in the urine
Klviriko, K.I. Int. Rsv. Connect. ?tssua Res. 5s93 (1970), and Welss, P.H. and
Klein, L., J. Clin. lnvaat. 48t 1 (1969).
Hydroxylyaine and its glycoside derivatives, both peculiar to collagenous
proteins, have been aonaidered to be more accurate than hydroxypfoline as
markers of collagen degradation. However, for the same reasons described above
for hydsoxyproline, hydroxylysine and !ts glycosides are probably equally non-
specific markers of bone resorption. Krane, S.M. and Simon, L.S. Develop.
Btochsm., 22:185 (1981).
In addition to amino acids unique to collagen, various non-eollagenous
1 S proteins of bone matrix such as osteocaleln, or theft breakdown products,
have
formsd the basis of immunoassays aimed at measuring bone metabolism. Price,
P.A. et nl. J. Clip. Invsst., 68s 878 (1980), and Gundberg, C.M. et al., Meth.
Enzymol., 10T:518 (198!). However, it Is now clear that Donrderived non
collagenous prote(ns, though potentially a useful index o! bone metabolic
activity
are unlikely, on their own, to provide quantitative measures of bone
resorption.
The concentration In serum of osteocalein, for example, fluctuates quite
widely
both normally and in metabolic bone disease. its concentration is elevated in
states of high skeletal turnover but ft 1s unclear whether this results from
increased synthesis or degradation of bone. Krane, S.M., et al., Develop.
Biochsm., 2Zs185 (1981), Prlee, P.A. et al., J. Clip. invest., 66e878 (1980);
and
C3undberg, C.M. et al., Moth Enrymol., 107:516 (1984).
Collagen Croae-Linking
The polymers of most genetic types of vertebrate oollagen require the
formation of aldehyde-mediated cross-links for normal function. Collagen alde
3~ hydes are derived from a few specific lysine or hydroxylysine side-chains
by the
action of lyayl oxidnse. Various dl-, tri- and t~trafunetionel cross-linking
amino
acids are formed by the spontaneous intra- and intermolecular reactions of
these
aldehydes within the newly formed collagen polymers; the type of cross-linklng
residue varies specifically with tissue type (see Eyre, D.R. et al., Ann. Rev.
Btochsm., 5:717-748 (1984)).
Two basic pathways of cross-linking can be differentiated for the banded
($?nm repeat) fibrillar collagens, one based on lysine aldehydes, the other on
215693a
-s-
hydroxylysine aldehydes. The lysine aldehyde pathway dominates in adult skin,
cornea, sclera, and rat tall tendon and also frequently occurs in other soft
connective tissues. The hydroxylysine aldehyde pathway dominates in bone,
cartilage, ligement, most tendons and most internal eonnectivt tissues of the
body, Eyre, D.R. et al. (1974) vide supra. The operating pathway is governed
by
whether lysine residues are hydroxylated fn the telopeptide sites where
aldehyde
residues will later be formed by lysyl oxidase (Barnes, M.J. et al., Biochem.
J.,
139t4fi1 (1974)).
The chemical structures) of the mature cross-linking amino acids on the
t0 lysine aldehyde pathway are unknown, but hydroxypyridinium residues have
been
identified as mature products on the hydroxylysine aldehyde route. On both
pathways and in most tissues the intermediate, borohydride-reducible crass-
linking
residues disappear as the newly formed collagen matures, suggesting that they
ace
relatively short-lived intermediates (Bailey, A.J. et al., FEBS Lett., 16s8s
(1971)). Exceptions are bone and dentin, where the reducible residues persist
in
appreciable concentration throughout life, In part apparently because the
rapid
mineralization of the newsy made collagen fibrils inhibits further spontaneous
cross-linking interactions (Eyre, D.R., In: The Chemistry and Biology of
ldineralized Connective Tissues, (Veil, A. ed.) pp. 51-65 (1981), Elsevier,
New
York, and Waiters, C. et al., Calc. Tip. IntL, 35:401-405 (1983)).
Two chemical forms of 3-hydroxypyridinium cross-link have been identified
(Formula 1 and II). Both compounds are naturally fluoresetnt, with the same
characteristic excitation and emission spectra (Fujimoto, D. et al. Biochem.
Biophys. Ras. Common., 78:1124 (1977), and Eyre, D.R., Develop. Biochem.,
22:50
1981)). These amino acids can be resoived and assayed directly in tissue
hydrolysates with good sensitivity using reverse phase HPLC and fluorescence
detection. Eyre, D.R. et al., Analyta. Bioctt8m., 137:380-388 (1984). It
should be
noted that the present Invention involves quantitating particular peptides
rather
then amino acids.
215~~35
-7_
f~X
CXp-CB-XXZ g2~,~ CNI-CA-NHZ
_ CA-CXt CAZ Oil Cfl--CNZ-wCH2 Ol(
AOOC ~ ~ f100C ~
+~ I+~
x
i~r
A
I=
o ix~ iX
(xz I xz
CH Cll
/ \ / \
l~ N COOK A? N C00g
15 PORMULA 1 PORMULA U
In growing anima), it has been reported that these mature cross-links may
be concentrated more in an unmineralized fraotfon of bone collagen than in the
mineralized collagen (Bsnes, A.J., et al., Biochem. Biophys. Rea. Commun.,
20 113s1975 (1983). However, other studies on young bovine or adult human bone
do
not support this concept, Eyre, D.R., Ini The Chemistry and Biology of
Mineralized
Tissues (Butler, W.T. ed.) p. 105 (1985), l:bsco Media Inc., Htrmingham,
Alabama.
The presanee of oollagen hydroxypyrldinfum cross-links in human urine was
lirst reported by Gunja-Smith and Boucek (Gunja-Smith, Z. and Boucek, R.J.,
25 Btocherrr d., 197s759-782 (1981)) using lengthy isolation procedures for
peptides
and conventtonal amino acid analysis. At that time, they were aware only of
the
HP form of the erona-link. Robins (Robins, S.P., Biochem J., 209:619-620
(19821
has reported an enzyme-linked,mmunoassey to measure HP in urine, having raised
polyelonal antibodies to the free amino aold conjugated to bovine serum
nlbumin.
30 This assay is Intended to provide an index for monitoring increased Joint
destruc-
tion that occurs with arthritis diseases and is based, according to Robins, on
the
finding that pyridinoline is mueh,more prevalent !n cartilage than in bone
colla-
gen.
216935
In more recent work involving enzyme-linked
immunoassay, Robins reports that lysyl pyridinoline is
unreactive toward antiserum to pyridinoline covalently linked
to bovine serum albumin (Robins et al., Ann Rheum. Diseases,
45:969-973 (1986)). Robins' urinary index or cartilage
destruction is based on the discovery that hydroxylysyl
pyridinoline, derived primarily from cartilage, is found in
urine at concentrations proportional to the rate of joint
cartilage resorption (i.e., degradation). In principle, this
finder. could bP_ USP_d to measure whole body cartilage loss;
however, no information on bone resorption would be available.
A need therefore exists for a method that allows the
measurement of whole-body bone resorption rates in humans.
The most useful such method would be one that could be applied
to body fluids, esper_ially urine. The method should be
sensitive, i.e., quantifiable down to 1 picomole and rapidly
measure 24-hour bone resorption rates so that the progress of
various therapies (e. g., estrogen) can be assessed.
The present invention is based on the discovery of
the presence of particular cross-linked telopeptides in body
fluids of patients and normal human subjects. These
telopeptides are produced in vivo during collagen degradation
arrd remodeling. The term "telopeptides" is used in a broad
sense herein to mean cross-linked peptides craving sequences
that are associated with the telopeptide region of, e.g., type
II and type III collagens and which may have cross-linked to
them a residue or peptide associated with the collagen triple-
helical domain. Generally, the telopeptides disclosed herein
_ g _
62839-1685
215693
will have fewer amino acid residues than the entire
telopeptide domains of type II and type III collagens.
Typically, tire t,elopepl:ides of floe present invention will
comprise two peptides linked by a pyridinium cross-link and
further linked by a pyridinium cross-link to a residue or
peptide of the collagen triple-helical domain. Having
disclosed the structures of these telopeptides herein, it will
he appreciated by one of ordinary skill in the art that they
may also be produced other than vivo, e.g., synthetically.
These peptides will generally be provided in purified form,
e.g., substantially free of impurities, particularly other
peptides.
The present invention also relates to methods for
determining in vivo degradation of type II and type III
collagens. The methods involve quantitating in a fluid the
concentration of particular telopeptide that have a 3-
Irydroxypyridinium cross-link and that are derived from
collagen degradation. The methods disclosed in the present
invention are analogous to those for determining the
- 8a -
62839-1685
__ ._ __ _ ___
_g_
absolute rate of bone reaorption in vfvo. Those methods involved quantitatiri6
in a
body fluid the concentration of telopeptides having a 3-hydroxypyridinium
eross-
link derived from bone collagen reaorption.
In a representative assay, the patient's body fluid is contacted with an
immunologicai binding partner apenific to a telopeptide having a
3-hydroxypyridiniurn eroea-link derived from type II or type lII collagen. The
body
fluid may be used as is or purified prior to the contacting step. This
purification
step may be accomplished using a number of standard procedures, including
cartridge adsorption and elution, mol~cutar sieve chromatography, dialysis,
ion
exchange, alumlna chromatography, hydroxyapatIte chromatography, and
combinations thereof.
ether representative embodiments of quantitating the eoncentretion of
peptide fragments having a 3-hydrozypyridfniurn cross-link in a body fluid
include
eleetroehemical titration, natural fluorescence sp~etroacopy, and ultraviolet
absorbanoe. Eleotroohemical tltretion may be conducted directly upon a body
fluid without further purification. However, when this is not possible due to
excessive quantities of contaminating substances, the body fluid is first
purified
prior to the electrochemical titration step. Suitable methods for purification
prior to electrochemioal detection Include dialysis, ion ezehange
chromatography,
alumina chromatography, molecular sieve chromatography, hydroxyapatite
chromatography and ion exchange absorption and elution.
Fluorornetria measurement of a body fluid containing a 3-hydroxypyridinium
cross-link is an alternative way of quantitatlng collagen degradation (and,
hence,
bone resorption, if type I peptides are quantitated). Ths fluoromatric assay
can be
conducted directly on a body fluid without further purification. However, for
certain body fluids, particularly urine, it is preferred that purification of
the body
fluid be conducted prior to the fluorometric assay. Th(s purification stee
consist,
of dialyzing an aliquot of a body fluid such as urine against an aqueous
solution
thereby producing partially purtfled peptide fragment9 retained within the
nondfffusate (retentate). The nondiffusate is then lyophilized, dissolved in
an ion
pairing solution and adsorbed onto an affinity chromatography column. The
chromatography column la washed with a volume o! ton pairing solution and,
thereafter, the peptide fragments are eluted from the column with an eluting
solution. These purified peptide fragments may then be hydrolyzed and the
hydrolysate resolved ehromatographically. Chromatographic resolution may be
conducted by either high-performance liquid chromatography or microbore high
performance liquid chromatography.
216935
The invention includes peptides having structures
identical to peptides derived from collagen degradation,
substantially free from other human peptides, which may be
obtained from a body fluid. The peptides contain at least one
3-hydroxypyridinium cross-link, in particular, a lysyl
pyridinoline cross-link or a h yclroxylysyl pyridinoline cross-
link, and are derived from tile telopeptide region of type II
or type III collagen linked to one or more residues from a
triple-helical domain, typically by the action of endogenous
proteases and/or peptidases.
The structures of the type II and type III
l:elopeptides are disclosed below. Information on the type I
1:p1011epr:~C~P_S i.S alSO i11C1UdP_d.
Another aspect of the present invention involves
assays for the peptides described herein in which the
pyridinium rings are intact arid cleaved. Since it is
suspected that some cleavage of pyridinium rings occurs in
vivo, assays that detect both intact and cleaved pyridinium
rings may lead to more accurate assessments of collagen
degradation. In connection with this aspect of the present
invention, specific binding partners to the individual
peptides containing intact or cleaved pyridinium rings, may be
employed in the assays. Individual specific binding partners
that recognize both types of peptides (both intact and cleaved
pyrid~inium ring containing peptides) may be employed.
Alternatively, specific binding partners that discriminate
between peptides containing the intact pyridinium ring and
those in which the pyridinium ring is cleaved, could also be
- 10 -
62839-1685
2156935
used.
Structure of Cross-Licked Telopeptides Derived from
Type I Collagen
A specific telopeptide having a 3-hydroxypyridinium
cross-link derived from the N-terminal (amino-terminal)
telopeptide domain oL bone type I collagen has the following
amino acid sequence:
FORriULA III
Asp-Glu-K-Ser-Thr-Gly-Gly
Gln-Tyr-Asp-Gly-K-Gly-Val-Gly
K
where K
K
K
is liydroxylysyl pyridinoline or lysyl pyridinoline, and Gln is
glutamine or pyrrolidine carboxylic acid.
The invention also encompasses a peptide containing
at least one 3-hydroxypyridinium cross-link derived from the
C-terminal (carboxy-terminal) telopeptide domain of bone type
I collagen. These C-terminal telopeptide sequences are cross-
linked with either lysyl pyridinoline or hydroxylysyl
pyridinoline. An example of such a peptide sequence is
represented by the formula:
- loa -
62839-1685
21~693~
FoRriuLA zv
Asp-Gly-Gln-Fiyp-Gly-Ala
H yp-Glu-Gly-Lys
Gly-Asp-Ala-Gly-Ala-K-Gly-Asp
Glu-K-Ala-His-Asp-Gly-Gly-Arg
Glu-K-Ala-His-Asp-Gly-Gly-Arg
where K
K
K
is hydroxylysyl or lysyl pyridinoline.
The inventor has also discovered evidence of two
additional type I collagen telopeptides in body fluids, having
7_0 tire following structures:
FORriULA V
Gly-Glu-Hyp
Gly-Asp-Ala-Gly-Ala-K-Gly-Asp
Glu-K-Ala-His-Asp-Gly-Gly-Arg
Glu-K-Ala-His-Asp-Gly-Gly-Arg
30 and
- 11 -
62839-1685
2~~6935
FORMULA VI
K
Glu-K-Ala-His-Asp-Gly-Gly-Arg
Glu-K-Ala-His-Asp-Gly-Gly-Arg
These telopeptides may also be quantitated in body fluids in
accordance with the invention. The compounds of formula VI
are used as assays, kits and methods of the invention of the
parent application which also concerns binding partners to
such compounds and cells which produce such binding partners.
Structure of a Cross-Linked Telo eptide Derived from Type II
Collagen
A specific telopeptide having a hydroxylysyl
pyridinoline cross-link derived from the C-terminal telopeptide
domain of type II collagen has the following amino acid
sequence (referred to hereinbelow as the core peptide
structure):
FORMULA VII
17C
Glu-Hyl-Gly-Pro-Asp al(II)C-telopeptide
Glu-iyl-Gly-Pro-Asp al(II)C-telopeptide
Gly-Val-Hyl al(II)helical domain
87
wherein the cross-linking residue depleted as Hyl-Hyl-Hyl is
hydroxylysyl pyridinoline (HP), a natural 3-hydroxypyridinium
residue present in mature collagen fibrils of various tissues.
Amino-terminal telopeptides from type II collagen
have not been detected in body fluids, and it is suspected
that potential peptides derived from the N-terminal
telopeptide region of type II collagen are substantially
- 12 -
62839-1685D
216935
degraded in vivo, perhaps all the way to the free HP cross-
linking amino acid.
Structure of Cross-Linked Telopeptides Derived from Type III
Collagen
By analogy to the above disclosure, cross-linked
peptides that are derived from proteolysis of human type III
collagen may be present in body fluids. These peptides have
a core structure embodied in the following parent structures:
FORMULA VTII
Gln-Tyr-Ser-Tyr-Asp-Val-Hyl-Ser-Gly-Val al(III)N-telopeptide
Gln-Tyr-Ser-Tyr-Asp-Val-Hyl-Ser-Gly-Val al(III)N-telopeptide
Gly-Ala-Ala-Gly-Ile-Hyl-Gly-His-Arg al(III)helical domain
939
- 12a -
62839-1685D
216935
E04 EE2 EE82 McCarthyTetrault ili'_0i?0 11:06 014
-13-
and
FORMULA tR
Gly-tle-Gly-G1y-G1u-Hyl-Ala-G1y-Gly-Phe-A1a al(III)C-telopeptide
G1y-Ile-G1y-Gly-G1u-HI1-A1a-G1y-Gly-Phe-Ala al(III)C-telopeptide
G1y-Phe-Pro-G1y-Met-Hyl-G1y-His-Arg Q1(III} helical domain
96
where K
I
K
K
i~ hydroxylysyl or lysyl pyridinollnt, and
Gln is ~lutamine or pyrrolidine carboxylic acid.
A likely cross-linked pepttde derived from type IIl collagen in body fluids
has
the core struoture:
PORMULA 1~
Asp-Val-Hyl-Ser-Gly-Vat
Asp-Va1-Hyl-Ser-Gly-Vdl
Hlyl
chat is derived from two ol(IIl)H-telopeptide domnlna linked to an
hydroxylysyl
pyridlnoline residue (Hyl-Hyl-Hyl).
A second possible lraQmeni of the C-telopepttde cross-linking domain, based
on the collegen types I and II peptldea observed In urine, has the core
structure:
FORMULA 7C1
Glu-Hyi-A1a-G1y-Gly-Phe
Glu-Hlyl-Ala-Gly-Gly-Phe
Hy i
215693
E04 662 E682 McCarth~JTet~ault
-14-
ili30i90 11:06 015
Smaller and larger versions (differing by one to thrte amino acids on each
component chain) of these two peptides corresponding to the parent sequences
shown above (FORlHULAE VIII and I?Q may also be present end measurable in body
tlulds. Analogous smaller and larger versions of each of the peptides
disclosed
herein form pest of the present invention as well.
The Invention Qenerally includes all specific binding partners to the peptides
described herein. "Specific binding partners" era molecules that are capable
of
binding to the peptides of the prexnt Invention. Included within this farm are
fmmunologieal binding partners, such as antibodies (monoclonal and
polyalonal),
antigen-binding fragments of antibodies (e.g., Feb and F(ab72 tragmenb),
singte-
chain antigen-binding molecules, and the like, whether made by hybridoma or
rDNA technologies.
The Invention includes fused cell hybrids (hybridomas) that produce
monoclonal antibodies specific for the above-dtscrlbed collagen peptides
having
3-hydroxypyridtnlum cross-links (both with an intact pyridinium ring and one
that
has been cleeved).
The invention further Includes monoclonal antibodies produoed by the fused
cell hybrids, and those antibodies Ins will as binding fragments thereof,
e.g., Fab)
coupled to a detectable marker. Examples of dettetable markers include
enzymes, ohromophores, fluorophores, coenzymes, enzyme inhibitors,
cnemiluminesoent materials, paramagnetic metals, spin labels, and
radioisotopes.
Such specific binding partners may alternatively be coupled to one member of a
ligand-binding partner complex (a.g., avldin-biotin), in whioh case the
detectable
marker can be supplied bound to the complementary member of the complex.
c5 The invention aLo includes test kits useful for quantitating the amount of
peptides having 3-hydroxypyridinium cross-links derived from collagen
degradation
in a body fluid. The kits may include a specific binding partner to a peptide
derived from degraded oollagen as disclosed herein. The specittc binding
partner
of the test kits may be coupled to a detectable marker or a member of a ligand-
binding partner complex, as described show.
FIQURE 1 1t a depiction of type lI collagen and s proposal for the source of
telopeptides. It is not established whether the two talopeptldes shown come
from
one collagen molecule as depicted In F1QURE 1 or from two collagen molecules.
~5 FIGURE 2 shown relative fluorescence (297 nm exoitation= 390 nm emission)
versus fraction number (4 ml), obtained dur(ng moleoular sieve chromatographic
puriflcatton of cross-Linked telopepttdes. Cross-linked type I1 collagen
telopepttdes are contained In the fractions deaignatsd II.
CA 02156935 2004-O1-28
-15-
FIGURE 3A shows relative fluorescence (330nm excitation, 390nm emission)
versus
elution time of fractions during ion exchange HPLC (DEAF-SPW). Cross-linked
type II collagen
telopeptides are contained in the fraction designated IV.
FIGURE 3B shows absorbance (220nm) versus elution time in minutes for the same
S chromatogram.
FIGURE 4A shows relative fluorescence (297nm excitation, 390nm emission)
versus
elution time of fractions during reverse phase HPLC. Cross-linked type II
collagen telopeptides
are eluted as indicated. The fractions indicated by the bar (-) show evidence
by sequence and
composition analysis of the peptides indicated that retain or have lost the
gly (G) and pro (P)
residues.
FIGURE 4B shows absorbance (220nm) as a function of elution time during
reverse
phase HPLC.
FIGURE 5 compares the concentration of HP and LP in both cortical and
cancellous
human bone with age.
FIGURE 6 depicts the cross-link molar ratios of HP to LP as a function of age.
FIGURE 7A shows relative fluorescence (297nm excitation, >370nm emission) as a
function of elution volume during reverse phase HPLC separation of cross-
linked type I collagen
N-telopeptides.
FIGURE 7B shows relative fluorescence (297nm excitation, >370nm emission)
versus
elution volume during reverse phase HPLC separation of cross-linked type I
collagen
C-telopeptides.
FIGURE 8A shows relative fluorescence (297nm excitation, >380nm emission) as a
function of elution time for the cross-linked type I collagen telopeptides.
FIGURE 8B shows relative fluorescence (297nm excitation, >380nm emission) as a
function of elution time for the cross-linked type I collagen telopeptides.
FIGURE 9 shows results of binding experiments with the representative
monoclonal
antibody HB 10611 and: the P1 peptide (Formula III herein, open squares); an
a2 (I)
N-telopeptide (QYDGKGVGC, solid diamonds); and an al (I) N-telopeptide
(YDEKSTGGC,
solid squares).
FIGURE 10 shows a portion of the structure of the N-telopeptide region of
decalcified
human bone collagen. The F1 peptide (Formula III) is enclosed in a box; it
contains an epitope
that correlates with bone resorption.
''15695
-18-
ape II Collagen Telopeptldes
The core peptide structure of the type 1I collagen peptides may be found in
body fluids as a component of larger peptides that bear additional amino acids
or
S amino acid sequerrees on one or mare ends of the three peptide sequencts
Joined
by the HP residue. FIGURE 1 shows hog type ft collagen telopeptides, which are
linked to a triple-helical sequence, may be produced in vtvo lrom a human
source
using the proteolytle enzymes pepsin and trypatn. Smaller tra~mente that have
lost amino acids from the core peptide structure, particularly from the
helical
sequence, may also occur in booty fluids. Generally, additions or deletions of
amino acids from the core peptide structure will involve from 1 to about 3
amino
acids. Additional amino ealds will Qenerally be determined by the type It
collagen
telopeptide sequence that occurs naturally in vivo. As examples, peptides
having
the following structural
FORMULA Xll
Glu-Hyl-Gly-Pro-Asp-Pro-Leu
Glu-Hyl-Gly-Pro-Asp
G1y-Va1-Hy1
and
FORMULA XIII
Glu-Hyl-Gly-Pro-Asp-Pro
Glu-Hyl-Gly-Pro-Asp
Gly-yal-Hyl
can b~ isolated ehromato~raDhioally from urine, and another of strueturts
PORMULA ZIV
Glu-Hyl-Gly-Pro-ASp
Glu-Hyl-Gly-Pro-Asp
Val-Hlyl
~ms93~
-17-
may also be isolated. In addition, glycasylated variants of the core structure
and
its larger and smaller variants may occur in which a galactose residue or a
glucosyl galactose residue are attached to the side chain hydroxyl group of
the HP
- cross-linking residue. Each peak in the graph shown in Figures IA and lH may
correspond to a cross-linked fragment of particular structure that may bt
quantttated for purposes of the present invention.
These atructuree are consistent with their site of origin in human type lI
cohagen ilbrils at a molecular cross-linking site formed between two al(II)
C-telopeptides and residue B7 of a triple-helical domain, the known sequences
about which area
FORIiDLA JCV
17C
Gly-Leu-Gly-Pro-Arg-Glu-Hyl-G1y-Pro-Asp-Pro-Leu Human nl(II)
Gly-Leu-Gly-Pro-Arg-Glu-Hyl-Gly-Pro-AsD-Pro-Leu Human al(II)
Gly-Leu-Pro-Gly-Val-Hyl-Gly-His-Arg Human al(II)
87
The isolated peptide fragments represent the products of proteolytic
degradation of type lI collagen fibrils wfthtn the body. The core structure
containing the HP residue is relatively resLatant to further proteolysis and
provides a quantitative measure of the amount of type 11 collagen degraded.
Collagen type U fa present In hyaline cartilage of Joints in the adult
skeleton. Quantitation of the collagen type 11 telopeptides in a body fluid,
for
example by way of a monoclonal antibody that recognizes an epltop; in the
peptide structure, would provide a quantitative measure of whole-body
cartilage
dast~uetion oc remodeling. In a preferred embodiment, the present invention
involves an assay for cartilage tissue degradation fn humans based on
quantifying
the urinary ezoretfon rate of at least one member of this family of
telopeptides.
Sueh an assay could be used, for example, to:
(1) screen adult human subjects for those individuals having abnormally
high rates of cartilage destruction as an early diagnostic indicator of
osteoarthrltisi
(2) monitor the effects of pottntial antlarthrltle drug's on cartilage
metabolism In osteoarthritie and rheumatoid arthritic patlentsi or
~ms~J~
-18-
(3) monitor the progress of degenerative joint disease in patients with
osteoarthritis and rheumatoid arthritis and their responses to various
therapeutic
interventions.
Osteoarthritia is a degenerative disease of the artioulat(ng cartilages of
joints. In its early stages ft is largely non-inflammatory (l.e. distinct iron
rheumatoid arthritis). It is not a single disease but represents the later
stages of
joint failure that may cesult from various factors (e.g. genetic
predisposition,
meehanteal overuaage, jotnt malformation or a prior injury, eta.). Destruction
of
joint articular cartilage Is the central progressive feature of
oateoarthritis. The
incidence of asteoarthritts, based on rndiographio surveys, ranges from 496 in
the
18-24 year age group to 8596 in the T6-T9 year age group. At present the
disease
can only be die.gnoaed by pain e0d radiographic or other Imaging signs of
advanced
cartilage erosion.
The assays disclosed above may be used to deteot early evidenoe of
accelerated cartilage degradation fn mildly symptomatic patients, to monitor
disease progress In more advanced patients, and as a means of monitoring the
effects of drugs or other therapies.
In normal young adults (with mature skeletons) there is probably very little
degradation of cartilage collagen. A test that could measure fragments of
cartilage collagen fn the urine (and tn the blood and joint fluid) would be
very
useful for judging the "health" of cartilage in the whole body and in
individual
Joints. The type it collagen-specific peptide assays described above will
accomplish this. In the long term, such an assay could become a routine
diagnostic aerean for spotting those individuals whose joints are wearing
away.
They could be targeted early on for preventative therapy, for example, by the
nezt generation of so-aallad chondroprotectlve drugs now being evaluated by
the
major pharmaceutical companies who are all actively seeking better agents to
treat oeteoarthritis.
Other diseases In which joint cartilage is destroyed include: rheumatoid
arthcftts, juvenile rheumatoid arthritis, ankyloaing apondylltts, psoriatfe
arthritis,
Relter's syndrome, relapsing polychondritia, the low back pain syndrome, and
ocher
infectious corms of arthritis. The type II collagen-specific assays described
hccein
could be used to diagnose and monitor these diseases and evaluate their
response
to therapy, as disclosed above in conntetton with oeteoarthritis.
2156935
-19-
~e lII Collagen Telo eptidcs
As pointed out above, human type III collagen telopeptides that may be
present in body fluids are expected to have a core structure embodied in the
following parent structures:
FORb~IULA V>T1
Gln-Tyr-Ser-Tyr-Asp-V31-Hyl-Ser-Gly-Val nl(II1)N-telopeptide
Gin-Tyr-Ser-Tyr-Asp-Val-Hyl-Ser-Gly-Val al(III)N-telopeptide
G1y-A1a-Ala-Gly-Iie-Hyl-Gly-His-Arg al(III) helical domain
939
and
FORI4iUl(.A IR
Gly-I1e-Gly-Gly-Glu-Hyl-Ala-Gly-Gly-Phe-A1a a1(III)C-telopeptide
1$ Gly-Ile-Gly-Gly-Glu-Hlyl-Ala-Gly-Gly-Phe-Ala al(III)C-telopeptide
Gly-Phe-Pro-Gly-Met-Hyl-Gly-His-Arg al(III) helical domain
96
wherein Hy 1
Hy 1
Hyl
Is hydroxylyayl pyridinollne.
By analogy to the type II peptides, the type II1 collagen peptides may occur
In glycosylated for m s of the core structure. For exa m plc, galaetose
residues or
glucosylgalactose residues m ay be attached to the core structure, e.g. by w
ay of
hydroxyl eroupa.
The cross-linking residue of the type iIt collagen peptides Is depleted as a
3-hydroxypyridinium residue, hydroxylysyl pyridinoline. The type fI
telopeptlde
structures have been found to primarily have hydroxylyayl pyridinoline cross-
linking residues. However, whereas the type II collagen peptides are derived
from
the N-terminal telopeptide region of type I! collagen, the type III collagen
peptides may be derived from either the N-terminal or the C-terminal of type
lII
collagen, as long as at least one cross-linking residue is present.
Type III collagen Is present In many connective tissues in association with
type I collagen. It Is especially concentrated In vascular walls, in the skin
and in,
for example, the synovial membranes of joints where its accelerated turnover
might be observed in inflammatory joint diseases such as rheumatoid arthritis.
CA 02156935 2001-06-08
-2 0-
A specific assay for type !II collagen degradation by quantitating cross-
linked type III collagen peptides as disclosed above, can be used for
detecting,
diagnosing, and monitoring various inflammatory disorders, possibly with
particular application to the vaseulitia syndromes. In conjunotion with assays
for
measuring bone type i and cartilage tppe II collagen degradation rates, such
an
essay could be used as a differential diagnostic tool for patients with
various
degenerative and Inflammatory disorders that result in cvnnentive tissue
destruction or pathological processes.
Isolation of Type II and Type III Collagen Telopeptides
General Procedure:
Urine is collected form a normal adolescent during a rapid phase of skeletal
growth. Using a sequence of chromatographic steps that include but are not
limited to, adsorption on selective aertridges of a hydraphobie Interaction
support
and an ion-exchange support and molecular sieve, ion-exchange and reverse-
phase
HPLC column chromatography steps, individual peptides are isolated. The eross
tinked peptides containing HP (and LP) residues are d~teettd during column
chromatography by their natural fluorescence (Ex max 299 nm ~ pH 4, Bx max 330
nm, > pH 6; Em max 390 nm). An exemplary isolation procedure is provided In
the
Example below.
Specific Example:
Fresh urine (at 4°C) diluted 5 times with water and adjusted to
296 {v/v)
trifluoroacetie acid, passed through a C-18 hydrophobia binding cartridge
(waters
TM
25 C-18 9ep-pak prewetted with 8096 (v/v) aeetonitrile then washed with
water).
Retained peptides were washed with water then eluted with 3 ml of 2096 {v/v)
eaetonitrile, and this eluent was adjusted to 0,05 M NH4HC03, 1096 (v/v)
aeetonitrile by addition of an equal volume of 0.1 M NH4HC03. This solution
was
TM
passed through a QMA-Sep-pak (Waters), which was washed with 10 ml of
O.1M NeCi, 2096 (vlv) acetonitrile followed by 10 ml of water and the p~ptldo~
were then eluted with 3 ml of 196 (v/v) trifluoroaeetl~ acid and dried by
Spesd-VacM
(Savant).
Peptides were fractionated in three chromatographic steps. The first step
TM
was rooleeular sieve chromatography on a column of Hio-Gel P-10 (Bio Rad Labs,
2.5 cm X 90 cm) eluted by 1096 (v/v) acetic acid, monitoring the effluent far
HP
fluorescence as shown in FIGURE 2. 1n FIGURE 2, the Y-axis is the relative
fluorescence emi3sion at 390nm (29T nm eROitation), and the X-axis is the
fraction
CA 02156935 2001-06-08
-21-
number. The fraction size rues 4 ml. The fractions indicated as 1I are
enriched in
the cross-linked collagen type II telopeptides. The cross-linked collagen type
I
telopeptides are contained in the fractions indicated as III and IY. Fractiens
spanning pool II (enriched in the type II collagen cross-linked peptides) were
5 combined; freeze-dried and fractionated by ien-exchange column
chromatography
on a DEAE-HPLC column (TSK-DEAE-SPW, 7.5mm X 7.Smm, Hlo-Rad Labs),
equilibrated with 0.02 M Tcis/IiCI, 1096 (v/v) acetonitrile, pH T.S and eluted
with a
gradient of 0-0.5M NaCl in the same buffer as shown in FIGURL 2.
FIQURE 3A plots relative fluorescence emission at 390nm (330 nm
10 excitation) versus elution time. The cross-linked collagen type II
telopeptides are
found primarily in the segment indicated as IV. FIGURE 3f3 plots absorbance at
220nm as a function of elution tims in minutes. Pool IV contains the type II
collagen arose-linked peptides. individual peptides were then resolved from
pool
IV by reverse phase HPLC on a C-18 column (Aquapore RP-300,M25em IC d.Bmm,
15 Srownlee Lebs), eluting with a grgdient of 0-309b (v/v) aeetonitrile in
0.196 (vlv)
trifluoroacetie acid. F1QURE 4A shows a plot of relative fluorescence
intensity
at 390 nm (29? nm excitation) as a function of elution time. The peaks
associated
with particular peptides are indicated in FIGURE 4A. FIGURE ~1H shows the
relative absorbanee at 220nm as a function of time.
20 Cross-linked peptide fragments of type Ill collagen containing HP eross-
iinking residues may be isolated by a similar combination of steps from the
urine
of normal growing subjects or, for example, from the urine of patients with
inflammatory disorders of the vaseulature.
Typo i Collas~,en Telo aptides
25 This aspect of the invention is based on the discovery that both lysyl
pyridinoline (LP) and hydroxylysyl pyridinoline (HP) peptide fragments (i.e.,
telopeptides, as used herein) derived from reabsorbed bone collagen are
excreted
in the urine without being metabolized. The Invention is also based on the
discovery that no other eonn~ctive tissues contain significant levels of LP
and
30 that the ratio of HP to LP In mature bone collagen remains relatively
constant
over a person's lifetime.
FIGURE 5 compares the concentration of HP and LP in both cortical and
caneellous human bone with age. It is observed that the eoncentratton of HP
plus
LP cross-links in bane collagen reaohes a maximum by age 10 to 15 years and
35 remains reasonably constant throughout adult life. Furthermore, the ratio
of HP
to LP, shown in FIGURE 6, shows little change throughout life, remaining
constant
at about 3.5 to 1. These baseline data demonstrate that the 3-
hydroxypyTidinium
-22-
cross-links fn bone collagen remains relatively constant and therefore that
body
fluids derived from bone collagen degradation will contain 3-hydroxyoyridinium
cross-linked peptide fragments at concentrations proportional to the absolute
rate
of bone resorption.
Since LP is the 3-hydroxypyridinium cross-link unique to bone collagen, the
method for determining the absolute rate of bone reaorptlon, in its simplest
form,
is based on quantitating the concentration of peptide fragments containing
3~hydroxypyridinlum cross-links and preferably lysyl pyridinoline (LF) cross-
links
in a body fluid.
As used in this description and in the appended claims pith respect to type I,
It, or III telopeptides, by "quantttattng" la meant measuring by any suitable
means,
including but not limited to spectrophotometrte, gravimetrte, volumetric,
coulometric, immunornetric, potenttometric, or amperometrlc means the
concentration of peptide fragments containing 3-hydroxypyridinium cross-links
in
an aliquot of a body fluid. Suitable body fluids include urine, serum, and
synovlal
fluid. The preferred body fluid is urine.
Since the concentration of urinary peptides will decrease as the volume of
urine increases, it Is further preferred that when urine !s the body fluid
selected,
the aliquot assayed be from a combined pool of urine collected over a fixed
period
ZD of time, for example, 24 hours. in this way, the absolute rate of bone
resorptton
or collagen degradation is calculated for a 24 hour period. Alternatively,
urinary
peptides may be measured as a ratio relative to a marker substance found in
urine
such as ereatinine. In this way the urinary index of collagen degradation and
bone
resorption would remain independent of urine volume.
In one embodiment of the present invention, monoclonal or polyclonal anti-
bodies ere produced which are specific to the peptide fragments containing
lysyl
pyridlnoiine cross-links found in a body fluid such as urine. Type 1
telopeptlde
fragments may be isolated from a body fluid of any patient, however, it is
preferred that these peptides are isolated from patients with Paget's disease
or
3D from rapidly growing adolescents, due to their high concentration of type 1
pepttde
fragment. Type lI and type III telopeptldes may be isolated from a Dody fluid
of
any patient but may be more easily obtained from patients suffering from
diseases
involving type II or type III collagen degradation or from rapidly growing
adolescents.
CA 02156935 2004-O1-28
-23-
Isolation of Type ( Collagen Tslopeptidea
Urine from patients with active Paget's disease is dialyzed in reduced
porosity dialysis tubing (<3,500 mol. wt. cut off Spectropore) at 4°C
for 48h to
remove bulk solutes. Under these conditions the peptides of interest ere
largely
retained. The freeze-dried non-diffusate is then eluted (200 mg aliquots) from
a -
TM
column (90.cm x 2.b em) of Blo-eel P2 (200-400 mesh) in 1096 acetic acid at
room
temperature. A region of elfluent that ~ombinea the cross-linked peptides la
defined by measuring the fluoreacenae of collected fractions at 297 nm
exoltation/395 ntn emission, and this pool is freeze-dried. Further resolution
of
TM
this materiel is obtained on a column of 8to-Oel P-d (200-400 mesh, 90 cm x
2.5
em) eluted in 1096 acetic acid.
Two contiguous bastion pools are defined by monitoring the fluorescence of
the eluant above. Tha earlier fraction is enriched in peptide fragments having
two
amino acid sequences that derive from the C-terminal teloDeptide domain of the
0l(1) chain of bone type 1 coilagen linked to a third sequence derived from
the
triple-helical body of bone type I collagen. These three peptide sequences are
cross-linked with 3-hydroxypyridinium. The overlapping later fraction is
enriched
in peptide fragments having an amino acid sequence that is derived from the
N-terminal telopeptida domain of bone type 1 collagen linked through a 3-
hydroxy
pyrldinium cross-links.
Individual peptides are then resolved from each of the two fractions obtained
above by ion-exchange HPLC on a TSK DEAE-S-PW column (Bio Rad 9.5 cm z 7.5
mm) eluting with a gradient of NaCl (0-0.2M) in 0.02M Tris-HCI, pH 7.5
containing 10916 (v/v) a~etonitrUe. The N-terminal telopeptide-based and
C-terminal telopeptide-based cross-ltnked peptides elute in a series of 3-4
peaks
o! fluorescence between 0.08M and O.iSM NaCI. The C-terminal telopeptlde-
based cross-linked peptides elute first es a series of fluorescent peaks, and
the
major and minor N-terminal telopeptide-based cross-linked peptides elute
towards
the end of the gradient ae oharaeteristic peaks. Each of these is collected,
freeze-dried and ehromatographed on a C-18 revera~ phase HPLC column (vydae
218TP54, 25 em x 4.6 mm) eluted with a gradient (0-1096) of eaetonitrile:
n-propanol (3:1 v/v) in O.O1M trifluoroacetic acid. About 100-600 a g of
individual
peptide fragments containing 3-hydroxypyridinlum arose-links can be Isolated
by
this procedure from a single 24h collection of Paget's urine.
Amino acid compositions of the major isolated peptides confirmed purity and
molecular sizes by the whole number stoieMometcy of recovered amino acids.
N-terminal sequence analysis by Edman degradation confirmed the bade core
~15~935
-24-
structures corresponding to the sequences of the known cross-linking sites in
type
I collagen and from the matching amino acid eomposittons. The N-terminal
telopeptide sequence of the a2(I) chain was blocked from sequencing analysts
due
presumably to the known cycllzation of the N-terminal glutamine to pyrrolldone
carborylic acid.
A typical elution profile of N-terminal telopeptidee obtained by the above
procedure is shown in FIGURE ?A. The major peptide fragment obtained has an
amino acid composition: (Aax)z(Glx)2(Gly)SVaI-Tyr-Sar-Thr, whtre Asx is the
amino acid Asp or Asn and Glx i9 the amino acid Gln or Giu. The sequence of
this
peptide is represented by Formula III below.
The C-terminal telopeptide-based cross-linked peptides resolved Dy reverse
phase HPLC as described above arc shown in FIGURE 78. As can be seen from
this figure, these peptides are further resolved into a series of C-terminal
telopeptides each containing the 3-hydroxypyrfdi.nium cross-links. The major
1 ~ peptide, shown in FIGURE 7H, was analyzed as described above and was found
to
have the amino acid composition: (Asp)5(Glu)4(Gly)10(His)Z(Arg)2(Hyp)2(Ala)6.
The sequence of this peptide is represented by formula IV below. It is
believed
that the other C-terminal telopeptlde-based cross-linked peptides appearing as
minor peaks In FIGURE 7B represent additions and deletions of amino acids to
the
structure shown in Formula IV. Any of the peptides contained within these
minor
peaks are suitable for use as immunogena as described below.
FORMULA iII
Asp-Glu-K-Ser-Thr-Gly-G1y
Gln-Tyr-Asp-Gly-i-Gly-11a1-Gly
K
FORMOLA IV
Asp-G1y-Gin-Hyp-G1y-A1a
Hyp-Glu-Gly-Lys
Gly-ASp-A1a-Gly-Ala-K-Gly-Asp
G1u-K-Ala-H1s-Asp-Gly-Gly-Arg
3~
G1u-K-Ala-His-Asp-G1y-G1y-Arg
~1~693~
-2 5-
Tt(1D illTl.l V
Hyp-61u-G1y
G1y-Asp-A1a-Gly-Ala=i-Gly-Asp
G1u-K-A1a-His-Asp-G1y-G1y-Ar9
G1u-K-A1a-His-Asp-Gty-Gly-Arg
and
FORMULA VI
K
Glu-K-Ald-His-Asp-Gly-Gly-Arg
Glu-K-Ala-His-Asp-Gly-Gly-Arg
where K
K
K
represents the HP or LP cross-links and Gln represents glutamine or
pyrrolidone
carboxylic acid.
Equivalents of the peptides represented by the above structures, in terms of
their presence in a body fluid due to collagen degradation, Include those
Cases
where there is some variation in the peptide structure. Examples of such
variation include 1-3 amino said additions to the N and C termini as well as 1-
3
terminal amino acid deletions. For example, a peptide corresponding to
Formula tit, but having a tyrosine residue attached to the amino terminus of
the
N-terminal aspartate residue has been detected In relatively minor quantities
In
human urine. Smaller peptide fragments of the molecule represented by Formula
IV derived from bone resorption are especially evident In urine. These are
found
in the minor peaks of the C-terminal telopeptide fraction seen in Figure 7H
and
can be identified by amino acid composition and sequence analysis.
Examples of Procedures for Quantitating Peptides
A. lmmunological Procedure For Quantitating Peotides
lmmunological binding partners capable of specifically binding to peptide
fragments derived from bone collagen obtained from a physiological fluid can
be
prepared by methods well known in the art. The preferred method for isolating
these peptide fragments Is described above. By irnmunological binding partners
as
21~6~3~
used herein is meant antibodies and antibody fragments capable
of binding to a telopeptide.
Both monoclonal and polyclonal antibodies
specifically binding the peptides disclosed here in and their
equivalents are prepared by methods known in the art. For
example, Campbell, A. M. Laboratory Techniques in Biochemistry
and Molecular Biolocty, Vol. 13 (1986). Elsevier. It is
possible to produce antibodies to the above peptides or their
equivalents as isolated. However, because the molecular
weights of these peptide fragments are generally less than
5,000, it is preferred that the hapten be conjugated to a
carrier molecule. Suitable carrier molecules include, but are
not limited to, bovine serum albumin, ovalbumin,
thyroglobulin, and keyhole limpet hemocyanin (KLH). Preferred
carriers are thyroglobulin and KLH.
It is well known in the art that the orientation of
the hapten, as it is bound to the carrier protein, is of
critical importance to the specificity of the antiserum.
Furthermore, not all hapten-protein conjugates are equally
successful immunogens. The selection of a protocol for
binding the particular hapten to the carrier protein therefore
depends on the amino acid sequence of the urinary peptide.
fragments selected. For example, if the peptide represented
by Formula III is selected, a preferred protocol involves
coupling this hapten to keyhole limpet hemocyanin (KLH), or
other suitable carrier, with glutaraldehyde. An alternative
protocol is to couple the peptides to KLH with a carbodiimide.
These protocols help to ensure that the preferred epitope
- 26 -
62839-1685
215~9~5
(discussed below under the heading "Characteristics of a
Preferred epitope") are presented to the primed vertebrate
antibody producing cells (e. g., B lymphocytes).
Other peptides, depending on the source, may require
different binding protocols. Accordingly, a number of binding
agents may be suitably employed. These include, but are not
limited to, carbodiimides, glutaraldehyde, mixed anhydrides,
as well as both homobifunctional and heterobifunctional
reagents (see for example the Pierce 1986-87 catalog, Pierce
Chemical Co., Rockford, IL). Preferred binding agents include
carbodiimides and heterobifunctional reagents such as m-
Maleimidobenzyl-N-hydroxysuccinimide ester (MBS).
Methods for binding the hapten to the carrier
molecule are known in the art. See for example, Chard, T.,
Laboratory Techniaues in Biochemistry and Molecular Biology,
Vol. 6 (1987) Partz Elsevier, N.Y..
Either monoclonal or polyclonal antibodies to the
hapten-carrier molecule immunogen can be produced. However,
it is preferred that monoclonal antibodies
- 26a -
62839-1685
. ~ _ __
21~693~
-27-
(MAb) be prepared. For this reason it is preferred that immunization be
carried
out in the mouse. Immunization protocols for the moue usually include an
adjuvant. Examples of suitable protocols arc described by Chard, T. (1987)
olds
supra. Spleen calls from the immunized mouse are harvested and homogenised
and thereafter fused with cancer cello in the presence of polyethylene glycol
to
produce a fused cell hybrid which produces monoclonal antibodies apecif is to
peptide fragments derived from collagen. Examples of ouch peptides are
represented by the formulas given above. Suitable cancer cells include
myeloma,
hepatoma, carcinoma, and sarcoma cells. Detailed deacriptiona of this
procedure,
including screening protocols, protocols for growing selected hybrid cells and
harvesting monoclonal antibodies produced by the selected hybrid cells are
provided tn Galfre, G. and Milstein, C., Meth. Enzymol., 731 (1981). A
preferred
prelimtnnry screening protocol Involves the use of peptide fragments derived
from
bone collagen resorption and containing 3-hydroxypyridinium cross-links in a
solid
1 S phase radtolmmunoasaay. A specific example describing a preferred
monoclonal
antibody is provided below.
The monoclonal antibodies or other immunologicnl binding partners used In
connection with the present are preferably specific for a particular type of
collagen telopeptide. For example, assays for the type II or type III collagen
degradation telopeptides should preferably be able to distinguish between the
type I, type II, and type III peptides. However, In some cases, such
selectivity will
not be neaeasary, for example, if it is known that a patient is not suffering
degradation of one type of collagen but is suspected of suffering degradation
from
the assayed type of collagen. Because of the differences In amino acid
sequences
between the type I, type II, and type III families of telopeptides, cross-
reactivity
should not occur to a significant degree. Indeed, hybridomas can be selected
for
during the screening of splenocyte fusion clones that produce monoclonal
antibodies specific for the cross-linked telopeptide of interest (and lack
affinity
for those of the other two collagen types). Based on the differences in
sequence
of the isolated peptide structures, such specificity is entirely feasible.
Peptide
fragments of the parent types I, II and IlI collagens, suitable for such
hybridoma
screening, can be prepared from human bone, cartilage and other tissues end
used
to screen clones from mice Immunized appropriately with the individual cross-
linked peptide antigens isolated from body fluid.
. _ 215693 __ __ __ __ ___
-28-
Immunologieal binding partners, especially monoclonal antibodies, produced
by the above procedures, or equivalent procedures, are employed In various
immunometric assays to quantitate the concentration of the peptides having
3-hydroxypyridinium cross-links described above. These immunometric assays
preferably comprise a monoclonal antibody or antibody fragment coupled to a
detectable marker. Examples of suitable detectable marker: include but are not
limited to: enzymes, coenzymes, enzyme inhibitor:, ohromophorea, fluorophores,
chemiluminescent materials, paramagnetic metals, spin labels, .and
radionuelides.
Examples of standard immunometric methods suitable for quantitating the
telopeptides include, but are not limited to, enzyme linked immunoaorbent
assay
(ELIBA) (lngvall, E., M~th En,rymol., TO (1981)), radio-immunoassay (RIA), and
"sandwich" immunoradiometric away (IRMA).
In its simplest form, these lmmunometric methods can be used to determine
the absolute rate of bone resorptton or collagen degradation by simply
contacting
a body fluid with the immunological binding partner specific to a collagen
telopeptfde having a 3-hydroxypyridinium cross-link.
It is preferred that the immunometrie assays described above be conducted
directly on untreated body fluids (e.g. urine, blood, strum, or synovial
fluid).
Occasionally, however, contaminating substances may interfere with the assay
necessitating partial purification of the body fluid. Partial puritfeatton
procedures include, but are not limited to, cartridge adsorption and elution,
mole-
cular sieve chromatography, dlalysi~, ion exchange, elumina chromatography,
hydroxyapatite chromatography and combinations thereof.
Test kits, suitable for use in accordance with the present invention, contain
specific binding partners such as monoclonal antibodies prepared as described
above, that apeaifically bind to peptide fragments derived from collagen
degradation found in a body fluid. It is preferred that the specific binding
partners of this test kit be coupled to a detectable marker of the type
described
above. Test kits contalntng a panel of two ar more speclfia binding partners,
particularly immunologieal binding partners, are also contemplated. Each
immunological binding part~tar in such a test kit will preferably not cross-
react
substantially with a telopeptide derived from another type of collagen. For
example, an irnmunological binding partner that binds specifically with a type
lI
collagen telopeptide should preferably not cross-react with either a type I or
type
III collagen telopeptide. A small degree (e.g., 5-1096) of cross-reactivity
may be
tolerable. Other test kits may contain a first speclfte Dlnding partner to a
collagen-derived telopeptide having a cross-link containing a pyridinium ring
CA 02156935 2004-O1-28
-29-
(which may be OH-substituted), and a second specific binding partner to a
telopeptide
having the same structure as the first telopeptide except that the pyridinium
ring has been
cleaved, such as photolytically.
(1) Monoclonal Antibody Production
S The following is an example of preparation of a monoclonal antibody against
a peptide
immunogen based on Formula III above.
A fraction enriched in the peptide of Formula III (indicative of bone collagen
degradation) was prepared from adolescent human urine using reverse phase and
molecular
sieve chromatography. The peptide was conjugated to keyhole limpet hemocyanin
(KLH)
with glutaraldehyde using standard procedures. Mice (Balb/c) were immunized
subcutaneously
with this conjugate (50-70 fig), first in complete Freund's adjuvant, then
boosted (25 ~tg) at
3 weekly intervals in incomplete Freund's adjuvant intraperitoneally. After
test bleeds had
shown a high titer against the Formula III peptide (referred to herein as P1)
conjugated
to bovine serum albumin (BSA) using an ELISA format, selected mice were
boosted with
a low dose (S pg) of the immunogen in sterile PBS Intravenously. Three days
later, cells from
the spleens of individual mice were fused with mouse myeloma cells using
standard
hybridoma technology. The supernatants of hybridoma clones growing in
individual wells of
96-well plates were screened for reactive monoclonal antibodies, initially
using a crude P1
preparation conjugated to BSA. After formal cloning by limiting dilution, the
antibodies
produced by individual hybridomas were characterized against a panel of
screening antigens
using ELISA analysis. These antigens were the P1 (Formula III) and P2 (Formula
VII) peptides
conjugated to BSA. An inhibition assay was used in which P1 conjugated to BSA
was plated
out in the plastic walls, and antibody was pre-incubated with a solution of
the potential
antigen. A secondary antibody (goat anti-mouse IgG conjugated to horseradish
peroxidase, HRP) was used for color development using an appropriate
substrate. A
desirable monoclonal antibody with high binding affinity for the P1 peptide
was identified.
When used as an ascites fluid preparation, the antibody worked in an
inhibition assay with
optimal color yield at 2 million-fold dilution (which indicates a binding
constant in the
range of 10-9 to 10-11 M-1, most likely about 10'10 M-1). In an ELISA format,
the antibody
was able to detect and measure P 1 present in normal human urine without any
concentration or
clean-up steps. The hybridoma that produces this preferred monoclonal antibody
has been
deposited at the American Type Culture Collection (ATCC), 12301 Parklawn
Drive, Rockville,
Maryland 20852, under accession number HB 10611. This hybridoma is designated
below as
1H11; the monoclonal antibody it produces is designated below as MAb-1H11.
21~6~~5
-30-
Sandwich assays were also shown to work using the P1-specific monoclonal
antibody and a polyclonal antle~rum rained in rabbits egainat conjugated P1.
Either P1-specific monoclonal antibodies, polyelonal antiserum, binding
fragments
thereof, or the like can be used to bind specifically to P1 from urine, in a
detectable manner using3tandard ELISA and other immunoaaaay protocols.
(II) Charaeteriatiea of a Preferred Epitope
The epitope recognized by the antibody MAb-1H11 is embodied in the
structure of P 1. The epitope is raco~nlzed in pure P 1 and in certain larger
peptides that contained the P1 structure (e.g., P1 attached to a tyrosine
residue
via the N-terminal aspartate residue of P1). The epitope includes chemical
features of both of the two telopeptide sequences embodied in the structure of
peptide P1. Peptides synthesized to match the human al (I) and a2 (I) N-
telopeptide sequences, with the addition of a C-terminal cyateine for coupling
to
bovine serum albumin (i.e., YDEK9TGGC and 6~YDGKGVaC), were riot recognized
1 S by MAb-1H11. Thin was shown by ELI$A using the free peptides competing
against plated-out P1 (see FIGURE 9) or directly as binding partners
conjugated to
HSA and plated out. Referring to FIGURE 9, the absorbanee at a = 450 nm of a
detectable marker Is plotted aQainat the concentration of fret P1 peptide. As
the
amount of free P1 inereasee, the amount of det~etable marker bound to
immobilized (plated-out) Pl diminishes. In comparison, the a2(I) and al(I)
N-telopeptides demonstrate little if any significant competitive binding with
MAb-1H11.
In addition, a larger form of P1 bearing a tyrosine residue on the N-terminal
aspartie acid was recovered Prom urine by affinity binding to MAb-1H11, but in
lower yield than P1. Other slightly iarger peptides bearing the P1 epitope
were
also recovered but In even smaller amounts.
The antibody was not seleetlv~ for the nature of the cross-link in P1, i.e.,
whether hydroxylysyl pyridinoline (HP) or lysyl pyridinoline (LP). Hoth HP-
containing and LP-containing forma were bound, apparently with equal affinity,
judging by the analysis of peptides isolated Crom urine by an affinity column
consisting of MAb-1H11 coupled to agaroee.
The free cross-linking amino acids, HP and LP, either made by acid
hydrolysis from bone colleQen or as present naturally in urine were not
recognized
by MAb-1H11. After photolytie opening of the 3-pyTidinol ring in peptide P1
with
UV light (long UV wavelengths), speeifiQ antibody binding was also unaffected,
presumably because the individual peptides remained cross-linked to each
other.
The epitope recognized by MAb-1H11, therefore, fs made up of at least a
CA 02156935 2004-O1-28
combination of chemical and conformational features embodied
i.n the two telopepti.de sequences shown boxed in FIGURE 10,
t:oget:lrer with sterir_ features imposed by the trivalent cross-
linking amino acid that links them. The a2 (I) N telopeptide
sequence, QYDGK, is a particularly significant part of the
epitope.
Tlre fact that the epitope recognized by MAb-1H11 does
not depend on an intact pyridiniurn ring is an unexpected
discovery. If ring-opening occurs either in vivo or even in
vitro under routine handling conditions, as appears likely, .
then a quantitative assay of the subject peptides) having
ini.~ct-. pyr:idi.ni.~.rm rings will underestimate the amount of_ bone
resort>taon. Preliminary ol:rservations indicate that
degradation of pyridinium rings in the subject peptides
appears to occur particularly in urine and/or in urine
samples, even if refrigerated. Accordingly, an assay based on
tine present disclosure i_s expected to be comparatively more
accurate. Two embodiments are envisioned: a single specific
binding partner is employed that recognizes both closed and
open-ringed embodiments of the targeted peptides) or two
sper_ific binding partners are employed, which differentiate
between tire closed-and open-ringed epitopes, respectively.
Specific binding partners that discriminate between open and
closed ring forms of the targeted peptides may be obtained by
incorporating an appropriate screening step into the standard
procedures for obtaining such specific binding partners. For
example, to obtain a monoclonal antibody that binds
specifically to an open ring from of the P1 peptide, a library
- 31 -
CA 02156935 2004-O1-28
-31 a-
of candidate monoclonal antibodies can be screened for their ability to bind
to P1 having an
opened pyridinoline ring (e.g., by ultraviolet light irradiation) and their
inability to bind to P1
having an intact pyridinoline ring.
Recent results have shown the following:
Using conditions that had been shown to completely destroy HP and LP (as
evidenced by loss of fluorescence of characteristic fluorescent peaks on RP-
HPLC) either
as the free amino acids or insoluble peptides and intact protein chains, a
preparation of PI
was irradiated (long wavelength setting - Mineralight UV SL-25 lamp, Ultra-
Violet
Products, Inc., San Gabriel, California).
This solution was assayed for binding to mAB 1H11, using a control solution of
exactly the same material not irradiated. The results of an ELISA with 1H11
showed
essentially no loss of binding to P1, implying that ring cleavage had not
affected the
epitope significantly. Under the conditions of UV Irradiation (pH9), cleavage
of a single
bond to open the ring rather than a double cleavage to eliminate the ring
nitrogen and its
side-arm would be expected.
Further experiments showed that the epitope resides in human bone collagen but
is
exposed and bound by MAb-1H11 only after extensive proteolysis. Thus, peptides
produced from decalcified human bone collagen by bacterial collagenase were
bound by MAb-
1 H 11 and shown to be derived from the N-telopeptide to helix site shown in
FIGURE 10. One
form contained the hexapeptide GIKGHR (in place of the non-telopeptide K arm
in P1), which is
clearly derived from al (I) residues 928-933. Another form embodied an
equivalent but
distinct hexapeptide that was derived from the a2 (I) chain. Fragments of
human bone
collagen solubilized by pepsin, CNBr, or trypsin were not recognized by MAb-
1H11, either in an
ELISA format when used as competitive inhibitors or on a Western blot after
SDA-
polyacrylamide electrophoresis, indicating that these solubilizing agents do
not produce the
epitope recognized by MAb-1 H 11.
B. Electrochemical Procedure For Assaying For Peptides
An alternative procedure for assaying for the above-described peptides
consists of
measuring a physical property of the peptides having 3-hydroxypyridinium cross-
links.
One such physical property relies upon electrochemical detection. This method
consists of
injecting an aliquot of a body fluid, such as urine, into an electrochemical
detector
poised at a
2156935
redox potential suitable for detection of peptides containing
the 3-hydroxypyridinium ring. The 3-hydroxypyridinium ring,
being a phenol, is subject to reversible oxidation and
tl~erefor_e the electrochem.ir_al detector (e. g., l4odel 5100A
Coulochem sold by Esa
- 31b -
62839-1685
CA 02156935 2004-O1-28
-32-
45 Wlggins Ave., Bedford, MA) is a highly desirable instrument suitable for
quantitating the concentration of the present peptides. Two basic forms of
electrochemical detector are currently commercially available= amperometrie
(e.g., HioAnalytical Systems) and coulometrie (ESA, lnc., Bedford, MA 01730).
S Both are suitable for use in accordance with the present invention, however,
the
latter system is inherently more sensitlva and therefore preferred since
complete
oxidation or reduction of the analyzed molecule in the column effluent is
achieved. In addition, screening or guard electrodes can be placed "upstream"
from the analytlcnl electrode to selectively oxidize or reduce interfering
substances thereby greatly improving selectivity. Essentially, the voltage of
the
analytfeal electrode is tuned to the redox potential of the sample molecule,
and
one or more pretreatment dells are set to destroy interferents in the sample.
In a preferred essay method, a standud ourrent/voltage curve is established
for standard peptides containing lysyl pyridlnoline or hydroxylysyl
pyrldinoline in
order to determine the proper voltage to set for optimal sensitivity. This
voltage
is then modified depending upon the body fluid, to mlntmize Interference from
contaminants and optimize sensitivity. Electroehernieel detectors, and the
optimum conditions for their use art known to those skilled in the art.
Complex
mixtures of body fluids can olten be directly analyzed with the
eleetrachemlcal
detector w(thout interference. Accordingly, for most patients no pretreatment
of
the body fluid is necessary. In same cases however, interfering compounds may
reduce the reliability of the measurements. In such cases, pretreatment of the
body fluid (e.g., urine) may be necesanry.
Accordingly, in an alternative embodiment of the invention, a body fluid is
first purified prior to electrochemically tltratfng the purified peptide
fragments.
The purification step may be conducted in a variety of ways including but not
limited to dialyei3, ion exchange chromatography, alumina chromatography,
hydroxyapatite chromatography, molecular sieve chromatography, or combinations
thereof. In a protected purification protocol, a measured aliquot (25 ml) of a
24 hour urine sample is dialy2ed in reduced porosity dialysis tubing to remove
the
bulk of contaminating fluorescent solutes. The non-diffusate is then
lyophilized,
redlssolved in 196 heptafluorobutyrie acid (HFHA1, an ion pairing solution,
and the
peptides adsorbed on a Waters Sep-1?ak C-18 cartridge. This cartridge is then
washed with 5 ml of 196 HFBA, and then eluted with 3 mi of 5096 methanol in
196
HFHA.
Another preferred method of puriftcatfon consists of adsorbing a measured
aliquot of urine onto an ion-exchange adsorption filter and eluting the
adsorption
CA 02156935 2004-O1-28
filter with a buffered eluting solution. The eluate fractions
containing peptide fragments having 3-hydroxypyridinium cross-
links are then collected to be assayed.
Still another preferred method of purification
employs molecular sieve chromatography. Fvr example, an
nH
aliquot of urine is applied to a Bio-Gel-P2 or Sephadex G-20
column and the fraction eluting in the 1000-5000 Dalton range
is collected. It will be obvious to those skilled in the art
that a combination of the above methods may be used.to purify
or partially purify urine or other body fluids in order to
isolate the peptide fragments having 3-hydroxypyridinium
cross-links. The purified or partially purified peptide
fragments obtained by the above procedures may be subjected to
additional purification procedures, further processed or
assayed directly in the partially purified state. Additional
purification procedures include resolving partially purified
peptide fragments employing high performance liquid
chromatography (HPLC) or microbore HPLC when increased
sensitivity is desired. These peptides may.then be
quantitated by electrochemical titration.
A preferred electrochemical titration protocol
consists of tuning the redox potential of the detecting cell
Of the electrochemical detector (Coulocl~emMl~todel 5100A) for
maximum signal with pure HP. The detector is then used to
monitor the effluent from a C-18 HPLC column used to resolve
the partially purified peptides.
C. Fluorometric Procedure For Quantitatinq Peptides
An alternative preferred method for quantitating the
- 33 -
z156~3~
COIIGPIrtratlOn of peptides having 3-hydroxypyridinium cross-
links as described herein i.s to measure tl~e characteristic
natural fluorescence Uf these peptides. For those body fluids
containing few naturally occurring fluorescent materials other
tl~ac~ the 3-lrydroxypyridinium cross-links, fluorometric assay
may be r_onducted directly without further purification of the
uody fluid. In this Case, the peptides are resolved by FiPLC
and the natural fluorescence of the HP and LP amino acid
residues is measured at 395 nm upon excitation at 297 nm,
esse«tially as described by Eyre, D.R., et al., Analyte,
B Lowlrem. 13'7 : 38U ( 1984 ) .
It is preferred, in accordance with the present
lrlVP_Iltion, that the fluorometric assay be conducted on urine.
()rive, however, usually contains substantial amounts of
~iaturally occurring fluorescent contaminants that must be
removed prior to conducting the fluorometric assay.
llccordingly, urine samples are first
- 33a -
62839-1685
r~
~I~~~3
-34-
partially purified as described above for electrochemical detection. This
partially
purified urine sample can then be fluorometrically assayed as described above.
Alternatively, the HP and LP cross-linked peptides in the partially purified
urine
samples or other body fluids can be hydrolyzed in 6M HCl at about 108°C
for
approximately 24 hours as described by Eyre, et al. (1984) vide auk. This
process
hydrolyzes the amino acids connected to the lysine precursors of "tripeptide"
HP
and LP cross-links, producing the free HP and LP amino acids represented by
Formulae I and lt. These small "tripeptides" are then resolved by the
techniques
described above, preferably by HPLC, and the natural fluorescence is measured
(Ex 297 nm, Ex 390 nm).
Optionally, the body fluid (preferably urine) is passed directly through a C-
18
reverse phase affinity cartridge after adding acetonltrtle/methanol 5 to 1096
V/V.
The non-retentatt is adjusted to 0.05-O.lOM with a cationic ion-pairing agent
such
as tetrabutyl ammonium hydroxide and passed through a second C-18 revere
phase cartridge. The washed retentate, containing fluorescent peptides, from
this
second cartridge is eluted with acetonitrile:water (or methanol:water), dried
and
fluoresoent peptides are analyzed by reverse phase HPLC or microbore HPLC
using an anionic ion-pairing agent such as O.O1M trifluoroncttie said !n the
eluant.
FIGURE 8A displays tht elution profile resolved by reverse phase HPLC of
natural fluorescence for a hydrolysate of peptide fragments from normal human
urine. Measurement of the integrated area within the envelope of a given
component is used to determine the concentration of that component within the
sample. The ratio of HP:LP found in normal human urine and urine from patients
having Paget's dia~ase, FIGURE 88, are both approximately 4.5:1. This is
slightly
higher than the 4:1 ratio found In bone itself (Eyre, et al., 1984). The
higher ratio
found in urine indioatea that a portion of the HP traction in urine may come
from
source' other than bone, such as the diet, or other sources of collagen
degradation, i.e., cartilage catabolism. it fs for this reason that it is
preferred
that LP which derives only from bone be used to provide an absolute Index of
Done
resorption. However, Sn the absence of excessive cartilage degradation such as
in
rheumatoid arthritis or in cases where bone is rapidly being absorbed, HP or a
combination of HP plus LP may be used as an index of bone resorption.
While the invention has bets described in conjunction with preferred embodi
ments, one of ordinary skill after reading t"~e foregoing specification will
be able
to effect various changes, substitutions of equivalents, and alterations to
the
subject matter set forth herein. Renee, the invention can be practiced In ways
other than those specifically described herein, It is therefore intended that
the
21~693~
-35-
protection granted by Letters Patent hereon be limited only by the appended
claims and equivalents thereof.
