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Patent 1340523 Summary

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(12) Patent: (11) CA 1340523
(21) Application Number: 581714
(54) English Title: ASSAY FOR IN VITRO MEASUREMENT OF AMINO-TELOPEPTIDES OF TYPE 1 COLLAGEN
(54) French Title: ESSAI POUR LA MESURE IN VITRO J'AMINE-TELOPEPTIDES DU COLLAGENE DE TYPE 1
Status: Expired
Bibliographic Data
(52) Canadian Patent Classification (CPC):
  • 167/37
  • 530/7.02
  • 195/1.112
(51) International Patent Classification (IPC):
  • C07K 14/78 (2006.01)
  • C07K 7/02 (2006.01)
  • G01N 33/53 (2006.01)
(72) Inventors :
  • EYRE, DAVID R. (United States of America)
(73) Owners :
  • WASHINGTON RESEARCH FOUNDATION (Not Available)
(71) Applicants :
  • WASHINGTON RESEARCH FOUNDATION (United States of America)
(74) Agent: MBM INTELLECTUAL PROPERTY LAW LLP
(74) Associate agent:
(45) Issued: 1999-05-04
(22) Filed Date: 1988-10-31
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): No

(30) Application Priority Data:
Application No. Country/Territory Date
118,234 United States of America 1987-11-06
03722 United States of America 1988-10-21

Abstracts

English Abstract



Methods of determining type I collagen degradation in vivo are provided by determining the presence or
concentration of a peptide in a body fluid, the peptide being an N-terminal type I collagen telopeptide
fragment containing a hydroxylysyl pyridinoline or lysyl pyridinoline cross-link. Kits comprising an
immunological binding partner which binds to a type I collagen telopeptide fragmentused to assay these
peptides in a body fluid are also described.


French Abstract

Des méthodes de détermination de la dégradation du collagène de type I in vivo sont fournies en déterminant la présence ou la concentration d’un peptide dans un fluide corporel, le peptide étant un fragment de télopeptide de collagène de type I N-terminal contenant une réticulation hydroxylysylpyridinoline ou lysylpyridinoline. Des trousses comprenant un partenaire de liaison immunologique qui se lie à un fragment de télopeptide de collagène de type I utilisé pour tester ces peptides dans un fluide corporel sont également décrites.

Claims

Note: Claims are shown in the official language in which they were submitted.



-18-
THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An assay for in vivo degradation of type I collagen, by determining in a sample of body
fluid the presence or concentration of a type I collagen telopeptide fragment comprising

Image


wherein
Image


is hydroxylysyl pyridinoline or lysyl pyridinoline and Gln is glutamine or wholly cyclized
pyrrolidone carboxylic acid, the assay comprising the steps of contacting the body fluid
sample with an immunological binding partner capable of binding to said telopeptide
fragment, and observing whether binding of the immunologic binding partner occurs in
the body fluid sample.

2. A kit for assaying in vivo degradation of type I collagen, comprising an immunological
binding partner which binds to a type I collagen telopeptide fragment comprising

Image


wherein
Image


is hydroxylysyl pyridinoline or lysyl pyridinoline, and Gln is glutamine or wholly
cyclized pyrrolidone carboxylic acid.


-19-
3. An isolated type I collagen fragment comprising


Image


wherein
Image


is hydroxylysyl pyridinoline or lysyl pyridinoline, and Gln is glutamine or wholly
cyclized pyrrolidone carboxylic acid.

Description

Note: Descriptions are shown in the official language in which they were submitted.


-1- 1340-~23




URINARY ASSAY FOR MEASURING BONE RESORPTION
This invention relates to a method for assaying bone resorption rates. More
specifically, it relates to a method for quantitating specific urinary cross-linking
amino acids, and peptide fragments that contain those amino acids derived from
degraded bone collagen.
Background of the Invention
Osteoporosis is the most common bone disease in man. Primary osteo-
porosis, 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 remodelling,
i.e., in the rates of synthesis and degradation of bone 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 peripheral bones. Twelve to 20% of the hipfractures are fatal because they cause severe trauma and bleeding, and half of the
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% 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 is available for measuring bone resorption rates in
patients or normal subjects. A major difficulty in monitoring the disease is thelack of a specific assay for measuring bone resorption rates.
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



....

1340~23
..
--2--

intake with output is tedious, unreliable, and can only indirectly appraise whether
bone mineral is being lost over the long term. Other methods currently availablefor assessing decreased bone mass and altered bone metabolism include quantita-
tive sc~nning radiometry at selected bone locations (wrist, calcaneus, etc.) and5 histomorphometry of iliac crest biopsies. The former provides a crude measure of
the bone mineral content at a specific site in a single bone. Histomorphometry
gives a semi-quantitative assessment of the balance between newly deposited boneseams and resorbing surfaces.
A urinary assay for the whole-body output of degraded bone in 24 hours
l0 would be much more useful. Mineral studies (e.g., calcium balance) cannot do this
reliably or easily. Since bone resorption involves degradation of the mineral and
the organic matrix, a specific biochemical marker for newly degraded bone
products in body fluids would be the ideal index. Several potential organic indices
have been tested. For example, hydroxyproline, an amino acid largely restricted
15 to collagen, and the principal structural protein in bone and all other connective
tissues, is excreted in urine. Its excretion rate is known to be increased in certain
conditions, notably Paget's disease, a metabolic bone disorder in which bone
turnover is greatly increased. For this reason, urinary hydroxyproline has been
used extensively as an amino acid marker for collagen degradation. Singer, F.R.,20 et al. (1978) In: Metabolic Bone Disease, Vol. II (eds. Avioli, L.V. and Krane, S.M.)
pp. 489-575, Academic Press, New York.
Goverde (U.S. Patent No. 3,600,132) discloses a process for determination of
hydroxyproline in body fluids such as serum, urine, lumbar fluid, and other
intercellular fluids in order to monitor deviations in collagen metabolism. In
25 particular, this inventor notes that in pathologic conditions such as Paget'sdisease, Marfan's syndrome, osteogenesis imperfecta, neoplastic growth in
collagen tissues, and in various forms of dwarfism, increased collagen anabolismor catabolism as measured by hydroxyproline content in biological fluids can be
determined. This inventor measures hydroxyproline by oxidizing it to a pyrrole
30 compound with hydlogen peroxide and N-chloro-E~-toluenesulphonamide followed
by colorimetric determination in E~-dimethyl-amino-benzaldehyde.
In the case of Paget's disease, the increased urinary hydroxyproline probably
comes largely from bone degradation, hydroxyproline, however, generally cannot
be used as a specific index. Much of the hydroxyproline in urine may come from
35 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



. ~ . . .

13qO~23


hydroxyproline. Furthermore, about 80% of the free hydroxyproline derived from
protein degradation is metabolized in the liver and never appears in the urine.
Kiviriko, K.I. (1970) Int. Rev. Connect. Tissue Res. 5, 93, and Weiss, P.H. and
Klein, L. (1969) J. Clin. Invest. 48, 1.
Hydroxylysine and its glycoside derivatives, both peculiar to collagenous
proteins, have been considered to be more accurate than hydroxyproline as
markers of collagen degradation. However, for the same reasons described above
for hydroxyproline, hydroxylysine and its glycosides are probably equally non-
specific markers of bone resorption. Krane, S.M. and Simon, L.S. (1981) Develop.Biochem. 22, 185.
In addition to amino acids unique to collagen, various non-collagenous
proteins of bone matrix such as osteocalcin, or their breakdown products, have
formed the basis of immunoassays aimed at measuring bone metabolism. Price,
P.A. et al. (1980) J. Clin. Invest. 66, 878, and Gundberg, C.M. et al. (1984) Meth.
Enzymol. 107, 516. However, it is now clear that bone-derived non-collagenous
proteins, though potentially a useful index of bone metabolic activity, are unlikely
on their own to provide quantitative measures of bone resorption. The
concentration in serum of osteocalcin, for example, fluctuates quite widely bothnormally and in metabolic bone disease. Its concentration is elevated in states of
high skeletal turnover, but it is unclear whether this results from increased
synthesis or degradation of bone. Krane, S.M., et al. (1981) Develop. Biochem. 22,
185, Price, P.A. et al. (1980) J. Clin. Invest. 66, 878, and Gundberg, C.M. et al.
(1984) Meth. Enzymol. 107, 516.
Collagen Cross-Linking
The polymers of most genetic types of vertebrate collagen require the
formation of aldehyde-mediated cross-links for normal function. Collagen alde-
hydes are derived from a few specific lysine or hydroxylysine side-chains by theaction of lysyl oxidase. Various di-, tri-, and tetrafunctional cross-linking amino
acids are formed by the spontaneous intra- and intermolecular reactions of thesealdehydes within the newly formed collagen polymers; the type of cross-linking
residue varies specifically with tissue type (see Eyre, D.R. et al. (1984) Ann. Rev.
Biochem. 53: 717-748). Two basic pathways of cross-linking can be differentiatedfor the banded (67nm repeat) fibrillar collagens, one based on lysine aldehydes, the
other on hydroxylysine aldehydes. The lysine aldehyde pathway dominates in adultskin, cornea, sclera, and rat tail tendon and also frequently occurs in other soft
connective tissues. The hydroxylysine aldehyde pathway dominates in bone,
cartilage, ligament, most tendons, and most internal connective tissues of the

13~0~23
--4--

body, Eyre, D.R. et al. tl974) vida supra. The operating pathway is governed by
whether lysine residues are hydroxylated in the telopeptide sites where aldehyderesidues will later be formed by lysyl oxidase (Barnes, M.J. et al. (1974) Biochem.
J. 139, 461). The chemical structure(s) of the mature cross-linking amino acids on
5 the 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 cross-
linking residues disappear as the newly formed collagen matures, suggesting thatthey are relatively short-lived intermediates (Bailey, A.J. et al. (1971) FEBS Lett.
l0 16, 86). Exceptions are bone and dentin, where the reducible residues persist in
appreciable concentration throughout life, in part apparently because the rapid
mineralization of the newly made collagen fibrils inhibits further spontaneous
cross-linking interactions (Eyre, D.R. (1981) In: The Chemistry and Biology of
Mineralized Connective Tissues (Veis, A. ed.) pp. 51-55, Elsevier, New York, and15 Walters, C. et al. (1983) Calc. Tiss. Intl. 35: 401-405).
Two chemical forms of 3-hydroxypyridinium cross-link have been identified
(Formula I and II). Both compounds are naturally fluorescent, with the same
characteristic excitation and emission spectra (Fujimoto, D. et al. (1977) Biochem.
Biophys. Res. Commun. 76, 1124, and Eyre, D.R. (1981) Develop. Biochem. 22,
20 50). These amino acids can be resolved and assayed directly in tissue hydrolysates
with good sensitivity using reverse phase HPLC and fluorescence detection. Eyre,D.R. et al. (1984) Analyt. Biochem. 137: 380-388.

FORMULA I FORMULA II
~C~NH2 ~cH~NH2

2 ~CH--CH2--CH~--0H 2 ~H-CH2 CH~oH'C00H
HOOC ~ Nl~ HOOC ~ +~J

C~H2 C~H2
CH-OH lH2
~H2 CIH2
C~H2 ~H2




,CH~ H2N~ ~COOH

hydroxylysyl pyridinoline (HP) lysyl pyridinoline (LP)

1340523


In growing animals it has been reported that these mature cross-links may be
concentrated more in an unmineralized fraction of bone collagen than in the
mineralized collagen (Banes, A.J., et al. (1983) Biochem. Biophys. Res. Commun.
113, 1975). However, other studies on young bovine or adult human bone do not
5 support this concept, Eyre, D.R. (1985) In: The Chemistry and Biology of
Mineralized Tissues (Butler, W.T. ed.) p 105, Ebsco Media Inc., Birmingham,
Alabama.
The presence of collagen hydroxypyridinium cross-links in human urine was
first reported by Gunja-Smith and Boucek (Gunja-Smith, Z. and Boucek, R.J.
lO (1981) Biochem J. 197: 759-762) using lengthy isolation procedures for peptides
and conventional amino acid analysis. At that time, they were aware only of the
HP form of the cross-link. Robins (Robins, S.P. (1982) Biochem J. 207: 617-620)
has reported an enzyme-linked immunoassay to measure HP in urine, having raised
polyclonal antibodies to the free amino acid conjugated to bovine serum albumin.l5 This assay is intended to provide an index for monitoring increased joint destruc-
tion that occurs with arthritic diseases and is based, according to Robins, on the
finding that pyridinoline is much more prevalent in cartilage than in bone colla-
gen. 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. (1986) Ann. Rheum. Diseases 45,
969-973). Robins' urinary index for 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. In
principal, this index could be used to measure whole body cartilage loss; however,
no information on bone resorption would be available. Indeed, it seems more
likely that Robin's assays were largely measuring the increased bone remodellingthat occurs in rheumatoid arthritis rather than cartilage destruction.
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, especially 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.
Summary of the Invention
A method for determining the absolute rate of bone resorption comprising
quantitating the concentration of peptide fragments having 3-hydroxypyridinium
cross-links derived from bone collagen resorption in a body fluid is provided. The

I3qO523

quantitating steps consists of contacting the body fluid with an immunological
binding partner specific to a peptide fragment having 3-hydroxypyridinium cross-links derived from bone collagen resorption. In one embodiment of the invention,the body fluid is optionally purified prior to the contacting step. This purification
5 step is selected from a number of standard procedures, including cartridge adsorp-
tion and elution, molecular sieve chromatography, dialysis, ion exchange, alumina
chromatography, hydroxyapatite chromatography, and combinations thereof.
The invention also encompasses other methods for quantitating the concen-
tration of peptide fragments having 3-hydroxypyridinium cross-links in a body
10 fluid. These methods include electrochemical titration, natural fluorescence
spectroscopy, and ultraviolet absorbance. Electrochemical titration may be
conducted directly upon a body fluid without further purification. However, whenthis is not possible due to excessive quantities of contaminating substances, the
body fluid is first purified prior to the electrochemical titration step. Suitable
15 methods for purification prior to electrochemical detection include dialysis, ion
exchange chromatography, alumina chromatography, molecular sieve
chromatography, hydroxyapatite chromatography, and ion exchange absorption and
elution.
Fluorometric measurement of a body fluid containing 3-hydroxypyridinium
20 cross-links is an alternative way of quantitating bone resorption. The
fluorometric 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 fluorometric assay. This
purification step consists of dialyzing an aliquot of urine against an aqueous
25 solution, thereby producing partially purified peptide fragments retained within
the nondiffusate. The nondiffusate is then lyophylized, dissolved in an ion pairing
solution and absorbed onto an affinity chromatography column. The
chromatography column is washed with a volume of ion pairing solution, and,
thereafter, the peptide fragments are eluted from the column with an eluting
30 solution. These purified peptide fragments are then hydrolyzed and the
hydrolyzate is resolved chromatographically. Chromatographic resolution is
conducted by either high-performance liquid chromatography or microbore high
performance liquid chromatography.
The invention includes a peptide fragment derived from bone collagen
35 substantially free from other human peptides obtained from a body fluid. The
peptide fragment may contain 3-hydroxypyridinium cross-links; in particular, lysyl
pyridinoline cross-links and hydroxylysyl pyridinoline cross-links.



. ,

1340~23
--7--

A specific peptide fragment having a 3-hydroxpyridinium cross-link derived
from the aminoterminal telopeptide domain of bone type I collagen has the
following amino acid sequence.

Asp-Glu-K-Ser-Thr-Gly-Gly.
Gln-Tyr-Asp-Gly- E~-Gly-Val-Gly


where K
1 0 K

is hydroxylysyl pyridinoline or lysyl pyridinoline and,
Gln is glutamine or wholly cyclized pyrrolidone carboxylic acid.
The invention also encompasses a peptide fragment containing
15 3-hydroxypyridinium cross-links derived from the carboxyterminal telopeptide
domain of bone type I collagen. These carboxyterminal telopeptide sequences are
cross-linked with either lysyl pyridinoline or hydroxylysyl pyridinoline. An
example of such a peptide sequence is represented by the formula:

20 Asp-Gly-Gln-Hyp-Gly-Ala
Hyp-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




is hydroxylysyl or lysyl pyridinoline.
The invention includes a fused cell hydrid which produces monoclonal anti-
bodies specific for the peptide fragment derived from bone collagen
having 3-hydroxypyridinium cross-links. The invention also includes monoclonal
antibodies produced by the fused cell hybrid including those antibodies coupled to
a detectable marker. Examples of detectable markers include enzymes,
chromophores, fluorophores, coenzymes, enzyme inhibitors, chemiluminescent
materials, paramagnetic metals, spin labels, and radio nucleotides. The invention
includes a test kit useful for quantitating the amount of peptide fragments having

-8- 13qO~23

3-hydroxypyridinium cross-links derived from bone collagen resorption found in abody fluid comprising the monoclonal antibody specific for peptide fragments
derived from bone collagen and containing 3-hydroxypyridinium cross-links. The
monoclonal antibodies of this test kit may be coupled to the detectable markers
5 described above.
Brief Description of the Drawings
FIGURE 1 is a graph of hydroxypyridinium residues in bone collagen versus
age.
FIGURE 2 is a graph of the ratio of hydroxylysyl pyridinoline (HP) to lysyl
l O pyridinoline (LP) versus age.
FIGURE 3a is a typical reverse phase HPLC natural fluorescence elution
profile of the aminoterminal telopeptides showing the location of the major
peptide fragment containing 3-hydroxypyridinium cross-links.
FIGURE 3b is a typical reverse phase HPLC natural fluoresence elution
l 5 profile of the carboxyterminal telopeptides showing the location of the major
peptide fragment containing 3-hydroxypyridinium cross-links.
FIGURE 4A is a typical reverse phase HPLC elution profile of natural
fluorescence for a hydrolysate of peptide fragments from normal human urine.
FIGURE 4B is a typical reverse phase HPLC elution profile of natural
20 fluorescence for a hydrolysate of peptide fragments from Paget's disease patent
urine.
Detailed Description of the Preferred Embodiments
This invention is based on the discovery that both lysyl pyridinoline (LP) and
hydroxylysyl pyridinoline (HP) peptide fragments derived from reabsorbed bone
25 collagen are excreted in the urine without being metabolized. The invention is
also based on the discovery that no other connective tissues contain significantlevels of LP and that the ratio of HP to LP in mature bone collagen remains
relatively constant over a person's lifetime.
FIGURE 1 compares the concentration of HP and LP in both cortical and
30 cancellous human bone with age. It is observed that the concentration of HP plus
LP cross-links in bone collagen reaches a maximum by age 10 to 15 years and
remains reasonably constant throughout adult life. Furthermore, the ratio of HP
to LP, shown in FIGURE 2, shows little change throughout life, remaining constant
at about 3.5 to 1. These baseline data demonstrate that the 3-hydroxypyridinium
35 cross-links in bone collagen remain relatively constant and therefore that body
fluids derived from bone collagen degradation will contain 3-hydroxypyridinium
cross-linked peptide fragments at concentrations proportional to the absolute rate
of bone resorption.



.. . .. . .. ........... .. . .. ..

9 13~0523

Since LP is the 3-hydroxypyridinium cross-link unique to bone collagen, the
method for determining the absolute rate of bone resorption, in its simplest form,
is based on quantitating the concentration of peptide fragments containing
3-hydroxypyridinium cross-links and preferably lysyl pyridinoline (LP) cross-links
in a body fluid. As used in this description and in the appended claims, by quanti-
tating is meant measuring by any suitable means, including but not limited to
spectrophotometric, gravimetric, volumetric, coulometric, immunometric,
potentiometric, or amperometric 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 synovial 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 is the body fluid selected,
the aliquot assayed be from a combined pool of urine collected over a fixed period
of time, for example, 24 hours. In this way, the absolute rate of bone resorption is
calculated for a 24 hour period. Alternatively, urinary peptides may be measuredas a ratio relative to a marker substance found in urine, such as creatinine. In this
way the urinary index of bone resorption would remain independent of urine
volume.
In one embodiment of the present invention, monoclonal or polyclonal anti-
bodies are produced which are specific to the peptide fragments containing lysylpyridinoline cross-links found in urine. Peptide fragments may be isolated from
the urine of any patient; however, it is preferred that these peptides are isolated
from patients with Paget's disease or hyperparathyroidism, due to the high
concentration of peptide fragments found in these patients.

ISOLATION OF URINARY PEPTIDES
Urine from patients with active Pag~t's disease is dialyzed in reduced
A porosity dialysis tubing (>3,500 Spectropore) at 4~C for 48h to remove bulk
solutes. Under these conditions the peptides of interest are largely retained. The
freeze-dried non-d~usate is then eluted (200 mg aliquots) from a column (90 cm x2.5 cm) of Bio-Gel P2 (200-400 mesh) in 10% acetic acid at room temperature. A
region of effluent that combines the cross-linked peptides is defined by measuring
the fluorescence of collected fractions at 297 nm excitation/395 nm emission, and
this pool is freeze-dried. Further resolution of this material is obtained on a
column of Bio-Gel P-4 (200-400 mesh, 90 cm x 2.5 cm) eluted in 10% acetic acid.
Two contiguous fraction pools are defined by monitoring the fluorescence of the

1340523
-10-

eluant above. The earlier fraction is enriched in peptide fragments having two
amino acid sequences that derive from the carboxyterminal telopeptide domain of
the ~I(I) chain of bone type I collagen linked to a third sequence derived from the
triple-helical body of bone type I collagen. These three peptide sequences are
5 cross-linked with 3-hydroxypyridinium. The overlapping later fraction is enriched
in peptide fragments having an amino acid sequence that derives from the
aminoterminal telopeptide domain of bone type I collagen linked through a
3-hydroxypyridinium cross-links. Individual peptides are then resolved from eachof the two fract~ ns obtained above by ion-exchange HPLC on a TSK DEAE-5-PW
A lo column (Bio Rad~7.5 cm x 7.5 mm) eluting with a gradient of NaCl (0-0.2M) in
0.02M Tris-HCl, pH 7.5 containing 10% (v/v) acetonitrile. The aminoterminal
telopeptide-based and carboxyterminal telopeptide-based cross-linked peptides
elute in a series of 3-4 peaks of fluorescence between 0.08M and 0.15M NaCl. Thecarboxyterminal telopeptide-based cross-linked peptides elute first as a series of
l 5 fluorescent peaks, and the major and minor aminoterminal telopeptide-based
cross-linked peptides elute toward the end of the gradient as characteristic
peaks. Each of these is collected, ~eeze-dried and chromatographed on a C-18
reverse phase HPLC column (Vydac~ 218TP54, 25 cm x 4.6 mm) eluted with a
gradient (0-10%) of acetonitrile: n-propanol (3:1 v/v) in 0.01M trifluoroacetic
20 acid. About 100-500 ug of individual peptide fragments containing 3-
hydroxypyridinium cross-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 stoichiometry
of recovered amino acids. Aminoterminal sequence analysis by Edman
25 degradation confirmed the basic core structures suspected from the sequences of
the known cross-linking sites in type I collagen and from the matching amino acid
compositions. The aminoterminal telopeptide sequence of the ~2(I) chain was
blocked from sequencing analysis due presumably to the known cyclization of the
aminoterminal glutamine to pyrrolidone carboxylic acid. A typical elution profile
30 of aminoterminal telopeptides obtained by the above procedure is shown in
FIGURE 3a. The rnajor peptide fragment obtained has an amino acid composition:
(Asx)2(Glx)2(Gly)5Val-Tyr-Ser-Thr, where Asx is the amino acid Asp or Asn and
Glx is the amino acid Gln or Glu. The sequence of this peptide is represented byFormula III below. Normal urine contains smaller amounts of the peptide
35 fragment represented by Formula III than the urine of Paget's disease patients.




. ~ .... ..

I34 0-52~

FORMULA m

Asp-Glu-K-Ser-Thr-Gly-Gly
Gln-Tyr-Asp-Gly-lK-Gly-Val-Gly
K

where K represents the HP or LP cross-linking amino acids, and
K




Gln represents glutamine or a wholly cyclized pyrrolidone carboxylic acid.
The carboxyterminal telopeptide-based cross-linked peptides resolved by
reverse phase HPLC as described above are shown in FIGURE 3b. As can be seen
from this figure, these peptides are further resolved into a series of
carboxyterminal telopeptides each containing the 3-hydroxypyridinium cross-
links. The major peptide, shown in FIGURE 3b, was analyzed as described above
and was found to have the amino acid composition:
5 4 Y)lo( s)2(Arg)2(Hyp)2(Ala)5. The sequence of this peptide is
represented by formula IV below. It is believed that the other carboxyterminal
telopeptide-based cross-linked peptides appearing as minor peaks in FIGURE 3b
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
immunogens as described below.

FORMULA IV
Asp-Gly-Gln-Hyp-Gly-Ala
Hyp-Glu-Gly-Lys
Gly-Asp-Ala-Gly-Ala- IK-Gly-Asp
Glu- K-Ala-His-Asp-Gly-Gly-Arg
Glu-K-Ala-His-Asp-Gly-Gly-Arg

where K represents the HP or LP cross-linking amino acids, and
K
Gln represents glutamine or a wholly cyclized pyrrolidone carboxylic acid.
Equivalents of the peptides represented by the above structures include
those cases where some variation in the urinary peptide structure accrues.



~ . . .

13~0523



Examples of variation include amino acld additions to the N and C termini of
Formulae 111 and IV as well as some terminal amino acld delctlons. Smallcr pcp-
tide fragments of the molecule represented by Formula IV derived from bone
readsorption are especially evident in urine. These are found in the minor peaks5 of the carboxytelopeptide fraction seen in Figure 3b and can be identiEied by
amino acid composition and sequence analysis. It is anticipated that antibodies
produced to the haptens represented by Formulae 111 and IV wlll cross re~ct withurinary peptides of slightly varied structure. In some siluations it may be
desirable to producc pntlcnt-specillc antlbodles lo lhe urhlury peplldcs derlvcd10 from bone resorption. In these cases the same procedure described above is
utilized to isolate urinary peptides whose structure may vary slightly from thatrepresented by Formulae 111 and IV.

IMMUNOLOGIC~L PROCI~I)URE ~OR INDI~XINC UONE I~ESORPTION
Immunological binding partners capable of specirically binding to peptide
fragments derived from bone collagen obtained from a physiological fluld can be
prepared by methods well known in Ihe nrt. Ihe prererred metllod for isolnllng
these peptide fragments is described above. Uy immunological bindlng parlners DSused herein is meant antibodies and antibody fragments.
130th monoclonal and polyclonal antibodies specifically binding the peptides
represented by Formulae 111 and IV and their equivalents are prepnred by methodsknown in the art. For example, Laboratory Techniques in Uiochelnlstry an(l
Molecular Biology, Campbell, A. M. (1986) Vo!. 13 Elsevier.
It is possible to produce antibodies to thc above pcplldes or their
25 equivalents as isolated. Ilowever, because the molecular weights of these peptide
fragments are 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 thc spcciflcity of thc nnti-
serum. Furthermore, not all hapten-protein conjugates are equally successful
immunogens. The selection of a protocol for binding the particular hapten to thecarrier protein therefore depends on the amino acid sequence of the urinary
35 peptide fragments selected. For example, if the urinary peptide fragment
represented by Formula 111 is selected, a preferred protocol would involve coupling
this hapten to keyhole limpet hemocyanin (KLH), or other suitable carrier, with



F '1

1340~23


carbodiimide. This would ensure that most of the hapten would be conjugsted
through the Gly carboxyterminus, thereby presenttng the preferred epitope,
namely Tyr and 3-hydroxypyridinium eross-link, to the primed vertebrate antlbodyproducing cells ~c.g., i3-lymphoeyte9).
Other urinary peptide fragments, depending on the source, may reguire
different binding protocols. Accordingly, a number of binding agents may be
suitably employed. These include, but are not limited to, carbodiimides, glutar-aldehyde, mixed anhydrides, as well as both homobifunctional and heterobifunc-
tional reagents (see for e~ample the Pierce 1986-87 catalog, Pierce Chemical Co.,
Rockford, IL). Preferred binding agents include carbodiimides and heterobifunc-
tional reagents such as m-Maleimidobenzyl-N-hydroxysuccinimide ester (MEIS).
Melhods ~or bincling the haplen to the eurrier molecule are known In tlle
art. See for example Laboratory Techniques in Biochemistry and Moleculur
Biology, Chard, T. (1987) Vol. 6, Partz Elsevier, N.Y.
Either monoclonal or polyclonal antibodies to the hapten-carrier molecule
immunogen can be produced. However, it is preferred tllnl monoclonal antibodies
(MAb) be prepared. For this reason it ;s preferred that immunization be carried
out in the mouse. Immunization protocols ror the mouse usually Include an
adjuvant. Examples of suitable protocols are described by Chard, T. (1987) vida
supra. Spleen cells from the immunized mouse are harvested and homogenized
and thereafter fused with cnncer cells in the presence of polyelhylene glyeol loproduce a fused cell hybrid whlch produces monoclonal antibo(lies specirie lo
peptide fragments derived from bone collagen. Examples of such peptide frag-
ments are represented by Formulae lll and IV above. Suitable cancer cells Include
myeloma, hepatoma, carcinoma, and sarcoma eells. Delailed descripttons of lhis
proeedure, ineluding screcning proloeols, proloeols ror growlng seleete(l 1lyl)rl(i
eells, and harvesting monoclonal antibodies produced by the selected hybrld cells
are provided in Galfre, G. and Milstein, C. (1981) Meth. Enzymol. 73, 1. /~
preferred preliminary screening protocol involves the use Or peptide fragments
derived from bone collagen resorption and containlng ~-hydroxypyrldlnlum croR~-
links in a solid phase radioimmunoassay.
Immunological binding partners, especially monoclonal antibodies, produced
by the above procedures, or equivalent procedures, are employed in various
immunometric assays to quantitate the concentration of peptide fragments having
3-hydroxypyridinium cross-links derived from bone collagen resorption in body
fluids. These immunometrie assays comprise a monoclonal antibody or antibody



~,

. .

-14- 13~0523

fragment coupled to a detectable marker. Examples of suitable detectable
markers include, but are not limited to, enzymes, coenzymes, enzyme inhibitors,
chromophores, fluorophores, chemiluminescent materials, paramagnetic metals,
spin labels, and radionuclides. Examples of standard immunometric methods
5 suitable for indexing bone resorption include, but are not limited to, enzyme
linked immunosorbent assay (ELISA) (Ingvall, E. (1981) Meth. Enzymol. 70), radio-
immunoassay (RIA), and "sandwich" Immuno radiometric assay (IRMA). In its
simplest form, these immunometric methods can be used to determine the
absolute rate of bone resorption by simply contacting a body fluid with the
l0 immunological binding partner specific to a peptide fragment having 3-hydroxy-
pyridinium cross-links derived from bone collagen resorption. It is preferred that
the immunometric assays described above be conducted directly on untreated body
fluids. Occasionally, however, contaminating substances may interfere with the
assay, necessitating partial purification of the body fluid. Partial purification
15 procedures include, but are not limited to, cartridge adsorption and elution, mole-
cular sieve chromatography, dialysis, ion exchange, alumina chromatography,
hydroxyapatite chromatography, and combinations thereof.
Test kits, suitable for use in accordance with the present invention, contain
monoclonal antibodies prepared as described above that specifically bind to pep-
20 tide fragments having 3-hydroxypyridinium cross-links derived from bone collagen
resorption found in a body fluid. It is preferred that the monoclonal antibodies of
this test kit be coupled to a detectable marker of the type described above.

ELECTROCHEMICAL PROCEDURE FOR INDE2~ING BONE RESORPTION
An alternative procedure for indexing bone resorption consists of measuring
a physical property of the peptide fragments having 3-hydroxypyridinium cross-
links. One such physical property suitable for indexing bone resorption 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 redox potential
30 suitable for detection of peptides containing the 3-hydroxypyridinium ring.
The 3-hydroxypyridinium ring, being a phenol, is subject to reversible o~dation,A and therefore the electrochemical detector (e.g., Model 5100A Coulochem old byesa 45 Wiggins Ave., Bedford, MA) is a highly desirable instrument suitable for
quantitating the concentration of urinary peptides derived from bone adsorption.35 Two basic forms of electrochemical detector are currently commercially
available: amperometric (e.g., BioAnalytical Systems) and coulometric (ESA, Inc.,
Bedford, MA 01730). Both are suitable for use in accordance with the present

~,,i-

_ _ _ _ _ ____ .

13~0~23
-15-

invention; however, the latter system is inherently more sensitive and thereforepreferred 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 analytical electrode to selectively oxidize or reduce5 interfering substances thereby greatly improving selectivity. Essentially, thevoltage of the analytical electrode is tuned to the redox potential of the sample
molecule, and one or more pre-treatment cells are set to destroy interferents inthe sample. In a preferred assay method, a standard current/voltage curve is
established for standard peptides containing lysyl pyridinoline or hydroxylysyl
l O pyridinoline in order to determine the proper voltage to set for optimal
sensitivity. This voltage is then modified, depending upon the body fluid, to
minimize interference from contaminants and optimize sensitivity.
Electrochemical detectors, and the optimum conditions for their use are known tothose skilled in the art. Complex mixtures of body fluids can often be directly
l 5 analyzed with the electrochemical detector without interference. Accordingly,
for most patients no pretreatment of the body fluid is necessary. In some cases,however, interfering compounds may reduce the reliability of the measurements.
In such cases, pretreatment of the body fluid (e.g., urine) may be necessary.
Accordingly, in an alternative embodiment of the invention, a body fluid is
20 first purified prior to electrochemically titrating the purified peptide fragments.
The purification step may be conducted in a variety of ways, including but not
limited to, dialysis, ion exchange chromatography, alumina chromatography,
hydroxyapatite chromatography, molecular sieve chromatography, or combinations
thereof. In a preferred purification protocol, a measured aliquot (25 ml) of a
25 24 hour urine sample is dialyzed in reduced porosity dialysis tubing to remove the
bulk of contaminating fluorescent solutes. The non-diffusate is then lyophilized,
redissolved in 1% heptafluorobutyric acid (HFBA), an ion pairing solution, and the
~4 peptides adsorbed on a Waters Sep-Pak C-18 cartridge. This cartridge is then
washed with 5 ml of 1% HFBA, and then eluted with 3 ml of 50% methanol in 1%
30 HFBA.
Another preferred method of purification consists of adsorbing a measured
aliquot of urine onto an ion-exchange adsorption filter and eluting the adsorption
filter with a buffered eluting solution. The eluate fractions containing peptidefragments having 3-hydroxypyridinium cross-links are then collected to be
35 assayed.
Still another preferred method of purification employs molecular sieve
chromatography. For example, an aliquot of urine is applied to a Bio-Gel P2 or

1340523

-16-

Sephadex~-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.
5 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
l0 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
(Coulochem Model 5100A) for maximum signal with pure HP. The detector is then
used to monitor the effluant from a C-18 HPLC column used to resolve the
l5 partially purified urinary peptides.

FLUOROMETRIC PROCEDURE FOR INDE~ING BONE RESORPTION
An alternative preferred method for quantitating the concentration of
peptide fragments having 3-hydroxypyridinium cross-links is to measure the
20 characteristic natural fluorescence of these peptide fragments. For those body
fluids containing few naturally occurring fluorescent materials other than the
3-hydroxypyridinium cross-links, fluorometric assay may be conducted directly
without further purification of the body fluid. In this case, peptide fragments are
resolved by HPLC and the natural fluorescence of the HP and LP amino acid
25 residues is measured at 395 nm upon excitation at 297 nm, essentially as described
by Eyre, D.R., et al., Analyl. Biochem. 137, 380 (1984).

It is preferred, in accordance with the present invention, that the fluoro-
metric assay be conducted on urine. Urine, however, usually contains substantial30 amounts of naturally occurring fluorescent contaminants that must be removed
prior to conducting the fluorometric assay. Accordingly, urine samples are firstpartially 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
35 samples or other body fluids can be hydrolyzed in 6M IlCl at about 108~C for
approximately 24 hours as described by Eyre, et al. (1984) vida supra. This process
hydrolyzes the amino acids connected to the lysine precursors of "tripeptide" HP
,~ ~
~ *Trade-mark

1340~23
-17-

and LP cross-links, producing the free HP and LP amino ucids represented by
Formulnc I nnd ll. Thcsc ~mnll "lrlpop(ldcs" nro tllcn rc.qolvcd by Iho tcchnl~ c~
described above, preferably by HPLC, and the natural fluorescence is measured
(Ex 297 nm, Em 390 nm).
S Optionally, the body fluid (preferably urine) is pnssed direclly lhrough D C-18
reverse phase affinity cartridge after ndding acetonitrile/metllnnol 5 to 1~% V/V.
The non-retentate is adjusted to 0.05-O.lOM with a cationic ion-pairing agent such
as tetr~butyl ammonium hydroxide and pns~cd througll n ~ec~nd C-t8 revelse
phase cartridge. The washed retentate, containing lluorescent peptides, from this
10 second cartri(Jge is eluted with acetonitrile:water (or melhallol:wnter), and dried,
and the fluorescent peptides are anslyzed by reverse phase IlPLC or microbore
IIPLC using an anionic ion-pairing ngent such as O.OlM lrlfluoroacellc acld In lhe
eluant. I',lternatively or included in thls peptide clean-up procedure, a moleculnr
sieve step employing a Bio-Gel P2 (Bio-Rad labs) or equivalent gel filtration
medium, can be used.
FIGURE 4A displays the elution profile resolved by reverse phase HPLC Or
natural fluorescencc for a hydrolysate of peptide fraglnents 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 disease, FIGURE 4B, are both approximately 4.5:1. This is slightly
higher than the 4:1 ratio found In bone Itself (Eyre et al., lD84). The higher ratio
found in urine indicates that a portion of the HP fraction in urine may come from
sources other than bone such as the diet, or other sources of collagen degradation;
Z5 i.e., cartilage catabolism. It is for this reason thst it is preferred that LP which
derives only from bone be used to provide an absolute index of bone r~sorption.
Ilowcver, in the absence of excessivc cnrtilagc degrndntion such ns in rheumntold
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 been described in conjunction with preferred embodi-
ments, one of ordinary skill after reading the loregoing specificatlon will be able
to effect various changes, substitutlons of eguivalents, and alterations to the
subject matter set forth herein. Hence, the invention can be practiced in ways
other than those specifically described herein.


Representative Drawing

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Administrative Status

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Administrative Status

Title Date
Forecasted Issue Date 1999-05-04
(22) Filed 1988-10-31
(45) Issued 1999-05-04
Expired 2016-05-04

Abandonment History

There is no abandonment history.

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $0.00 1988-10-31
Registration of a document - section 124 $0.00 1999-10-28
Registration of a document - section 124 $0.00 1999-11-09
Maintenance Fee - Patent - Old Act 2 2001-05-04 $100.00 2001-04-19
Maintenance Fee - Patent - Old Act 3 2002-05-06 $100.00 2002-04-25
Maintenance Fee - Patent - Old Act 4 2003-05-05 $100.00 2003-04-23
Maintenance Fee - Patent - Old Act 5 2004-05-04 $200.00 2004-04-21
Maintenance Fee - Patent - Old Act 6 2005-05-04 $200.00 2005-04-06
Maintenance Fee - Patent - Old Act 7 2006-05-04 $200.00 2006-04-07
Expired 2019 - Corrective payment/Section 78.6 $150.00 2007-01-19
Maintenance Fee - Patent - Old Act 8 2007-05-04 $200.00 2007-04-10
Maintenance Fee - Patent - Old Act 9 2008-05-05 $200.00 2008-04-10
Maintenance Fee - Patent - Old Act 10 2009-05-04 $250.00 2009-04-20
Maintenance Fee - Patent - Old Act 11 2010-05-04 $250.00 2010-04-14
Maintenance Fee - Patent - Old Act 12 2011-05-04 $250.00 2011-04-13
Maintenance Fee - Patent - Old Act 13 2012-05-04 $250.00 2012-04-11
Maintenance Fee - Patent - Old Act 14 2013-05-06 $250.00 2013-04-10
Maintenance Fee - Patent - Old Act 15 2014-05-05 $450.00 2014-04-09
Maintenance Fee - Patent - Old Act 16 2015-05-04 $450.00 2015-04-09
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
WASHINGTON RESEARCH FOUNDATION
Past Owners on Record
EYRE, DAVID R.
THE BOARD OF REGENTS OF THE UNIVERSITY OF WASHINGTON
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Cover Page 1999-05-14 1 16
Abstract 1999-05-04 1 17
Description 1999-05-04 17 962
Claims 1999-05-04 2 38
Drawings 1999-05-04 4 45
Fees 2002-04-25 1 32
Fees 2003-04-23 1 29
Fees 2001-04-19 1 36
Fees 2004-04-21 1 30
Prosecution-Amendment 2007-01-19 2 61
Correspondence 2007-02-21 1 12
Prosecution-Amendment 1989-01-23 1 33
Correspondence 1989-02-21 1 19
Correspondence 1988-11-17 1 42
Assignment 1988-10-31 2 103
Assignment 1989-06-06 1 36
Correspondence 1997-05-14 2 64
Correspondence 1997-07-09 1 19
Correspondence 1997-07-09 1 23
Prosecution-Amendment 1998-05-06 2 64
Prosecution-Amendment 1998-06-05 1 52
Correspondence 1999-02-11 1 42
Prosecution-Amendment 1999-02-11 2 66
Prosecution-Amendment 1998-06-30 1 34
Prosecution-Amendment 1998-04-14 2 46
Prosecution-Amendment 1998-04-03 2 56
Prosecution-Amendment 1998-03-11 2 43
Prosecution-Amendment 1997-02-14 2 116
Prosecution-Amendment 1995-11-06 2 47
Prosecution-Amendment 1995-08-04 2 109
Prosecution-Amendment 1995-01-19 5 195
Prosecution-Amendment 1994-07-16 3 149
Prosecution-Amendment 1991-07-17 2 45
Prosecution-Amendment 1991-05-08 1 58
Correspondence 1998-08-11 1 99
Prosecution-Amendment 1997-08-14 5 249