Ex Parte NG et al

Patent Trial and Appeal Board

Feb 27, 2019

14664243 (P.T.A.B. Feb. 27, 2019)

UNITED STA TES p A TENT AND TRADEMARK OFFICE
APPLICATION NO. FILING DATE FIRST NAMED INVENTOR
14/664,243 03/20/2015
23599 7590 03/01/2019
MILLEN, WHITE, ZELANO & BRANIGAN, P.C.
2200 CLARENDON BL VD.
SUITE 1400
ARLINGTON, VA 22201
LEONGL.NG
UNITED STATES DEPARTMENT OF COMMERCE
United States Patent and Trademark Office
Address: COMMISSIONER FOR PATENTS
P.O. Box 1450
Alexandria, Virginia 22313-1450
www .uspto.gov
ATTORNEY DOCKET NO. CONFIRMATION NO.
SIMANDI-0008-COl 1908
EXAMINER
SHAFER, SHULAMITH H
ART UNIT PAPER NUMBER
1647
NOTIFICATION DATE DELIVERY MODE
03/01/2019 ELECTRONIC
Please find below and/or attached an Office communication concerning this application or proceeding.
The time period for reply, if any, is set in the attached communication.
Notice of the Office communication was sent electronically on above-indicated "Notification Date" to the
following e-mail address(es):
docketing@mwzb.com
PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
BEFORE THE PATENT TRIAL AND APPEAL BOARD
Ex parte LEONG L. NG, ANDREAS BERGMANN, JOACHIM STRUCK,
NILS MORGENTHALER, and JANA PAP AS SOTIRIOU
(APPLICANT: BRAHMS GMBH)
Appeal2018-002919 1
Application 14/664,243
Technology Center 1600
Before DONALD E. ADAMS, ULRIKE W. JENKS, and RYAN H. FLAX,
Administrative Patent Judges.
ADAMS, Administrative Patent Judge.
DECISION ON APPEAL
This Appeal under 35 U.S.C. § 134(a) involves claims 1, 4---6, 8, 11-
17, 21, and 22 (App. Br. 1-2).2 Examiner entered a rejection under 35
U.S.C. § 101. We have jurisdiction under 35 U.S.C. § 6(b).
We AFFIRM.
1 Appellants identify "B.R.A.H.M.S. GmbH" as the Real Party in Interest
(see Appellants' August 15, 2017 Appeal Brief(App. Br.) 1).
2 Pending claims 7, 9, 10, and 18-20 stand withdrawn from consideration
(see App. Br. 2).
Appeal2018-002919
Application 14/664,243
STATEMENT OF THE CASE
Appellants' disclosure
relates to a method for the diagnosis and/or risk stratification of
adverse events in post-myocardial infarction patients, whereby
a determination of the marker pro-adrenomedullin (pro ADM)
or a partial sequence or fragment thereof or contained in a
marker combination (panel, cluster) is carried out on a patient
who is to be examined.
(Spec. ,r 2.) Appellants' claim 1 is representative and reproduced below:
1. A method for the in vitro diagnosis and/or risk stratification
of an adverse event in post-myocardial infarction patients,
comprising determining the level of MR-pro-adrenomedullin
consisting of the SEQ ID NO: 2 from a post-myocardial
infarction patient, wherein a level of more than O. 73 nmol/L of
the pro-adrenomedullin correlates with a greater risk of an
adverse event in said post-myocardial infarction patient, and
wherein said adverse event is at least one of recurrent
myocardial infarction, heart failure and death.
(App. Br. 10.)
Ground of rejection before this Panel for review:
Claims 1, 4---6, 8, 11-17, 21, and 22 stand rejected under 35 U.S.C.
§ 101.
ISSUE
Does the preponderance of evidence of record support Examiner's
finding that Appellants' claimed invention is directed to patent ineligible
subject matter?
ANALYSIS
An invention is patent-eligible if it claims a "new and useful process,
machine, manufacture, or composition of matter." 35 U.S.C. § 101. The
2
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Application 14/664,243
Supreme Court, however, has long interpreted 35 U.S.C. § 101 to include
implicit exceptions: "Laws of nature, natural phenomena, and abstract ideas
are not patentable." Alice Corp. Pty. v. CLS Bankint'l, 573 U.S. 208,216
(2014).
In determining whether a claim falls within an excluded category, we
are guided by the Supreme Court's two-step framework, described in Alice.
Id. at 217 ( citing Mayo Collaborative Servs. v. Prometheus Labs., Inc., 566
U.S. 66 (2012)). In accordance with Alice's framework, we first determine
what concept the claim is "directed to." See Alice, 573 U.S. at 219 ("On
their face, the claims before us are drawn to the concept of intermediated
settlement, i.e., the use of a third party to mitigate settlement risk.").
If the claim is "directed to" a patent-ineligible concept, e.g., a law of
nature, natural phenomenon, or abstract idea, we tum to the second step of
the Alice and Mayo framework, where we must examine the elements of the
claim to determine whether it contains an inventive concept sufficient to
ensure that the patent in practice amounts to significantly more than a patent
upon the ineligible concept itself. See Alice, 573 U.S. at 215-16.
On January 7, 2019, the United States Patent and Trademark Office
published the 2019 Revised Patent Subject Matter Eligibility Guidance in
the Federal Register in which the Office revised its examination procedure.
84 Fed. Reg. 50 ("Revised Guidance"). Under the Revised Guidance, we
first determine whether the claim recites:
( 1) any judicial exceptions, including certain groupings of
abstract ideas (i.e., mathematical concepts; certain methods of
organizing human activity such as a fundamental economic
practice; or mental processes); and
(2) additional elements that integrate the judicial exception into
a practical application (see MPEP § 2106.05(a}-(c), (e}-(h)).
3
Appeal2018-002919
Application 14/664,243
Only if a claim (1) recites a judicial exception and (2) does not integrate that
exception into a practical application, do we then determine whether the
claim:
(3) adds a specific limitation beyond the judicial exception that
is not a "well-understood, routine, conventional activity" in the
field (see MPEP § 2106.05(d)); or
(4) simply appends well-understood, routine, conventional
activities previously known to the industry, specified at a high
level of generality, to the judicial exception.
(Revised Guidance.)
On this record, Appellants' claim 1 is directed to a method of
diagnosing or stratifying risk of myocardial infarction, heart failure, and
death in post-myocardial infarction patients by determining whether the
level of MR-pro-adrenomedullin, consisting of a particular sequence, from a
post-myocardial infarction patient is above a threshold that correlates with a
greater risk of at least one of recurrent myocardial infarction, heart failure,
and death in a post-myocardial infarction patient (see Final Act. 5; Ans. 4--5;
cf App. Br. 10). Stated differently, Appellants' claimed method is directed
to a law of nature, specifically, the naturally existing correlation between the
level of MR-pro-adrenomedullin consisting of Appellants' SEQ ID NO: 2
and an increased risk of at least one of recurrent myocardial infarction, heart
failure, and death (see Final Act. 5; Ans. 4--5; see also App. Br. 3 ("The
natural principle at issue here is that there is a relation between
adrenomedullin levels and certain cardiac physiological conditions")).
Appellants' dependent claim 4 combines the correlation of Appellants' claim
1 with an additional correlation between a threshold level of NT-proBNP
and a risk of at least one of recurrent myocardial infarction, heart failure, and
4
Appeal2018-002919
Application 14/664,243
death (see App. Br. 10). 3 See Mayo, 566 U.S. at 74 ("The patent claims at
issue here set forth processes embodying researchers' findings that identified
these correlations with some precision").
Appellants contend, however, that their claimed invention is limited to
the application of the relationship between adrenomedullin levels and certain
cardiac physiological conditions (App. Br. 3). In this regard, Appellants
contend that their claims are limited with respect to the "patient class,"
"specified single portion of the adrenomedullin sequence, i.e., the MR-pro-
adrenomedullin consisting of the specified SEQ ID NO: 2," "a previously
unknown threshold value," and "physiological condition being specified as
recurrent myocardial infarction, heart failure or death" (id.; Reply Br. 2; see
also App. Br. 6 ("[T]he Final Office action does not point out any specific
teachings in the art to suggest that such elements were known in the art in
combination"); App. Br. 6-7).
We are not persuaded by Appellants' contentions regarding patient
class, threshold value, and physiological condition (see App. Br. 4--5; see
also Reply Br. 2-3). To the contrary, the correlation between Appellants'
patient class, threshold value, and physiological condition is the natural
principle at issue on this record (see Final Act. 5; Ans. 4--5). On this record,
the correlation recited in Appellants' claim 1 "exists in nature apart from any
human action" and "is an ineligible natural law." Athena Diagnostics Inc. v.
Mayo Collaborative Servs., No. 2017-2508, 2019 WL 453489, at *4 (Fed.
Cir. Feb. 6, 2019) ("[T]he correlation between the presence of naturally-
3 We note that Appellants' claim 5 depends from and further limits
Appellants' claim 1 to require that the level of at least one additional marker
that is different from pro-adrenomedullin, i.e., C-reactive protein("CRP"), is
determined for a reason unspecified by the claim.
5
Appeal2018-002919
Application 14/664,243
occurring MuSK autoantibodies in bodily fluid and MuSK-related
neurological diseases" that "exists in nature apart from any human action ...
is an ineligible natural law"); see also Mayo, 566 U.S. at 76-77 ("[T]he
patents effectively claim natural laws or natural phenomena-namely the
correlations between thiopurine metabolite levels and the toxicity and
efficacy of thiopurine drug dosages-and so are not patentable," because
"[t]he relation is a consequence of the ways in which thiopurine compounds
are metabolized by the body----entirely natural processes. And so a patent
that simply describes that relation sets forth a natural law").
For the foregoing reasons, we find that Appellants' claims recite a
judicial exception and do not integrate that exception into a practical
application. Therefore, we determine whether Appellants' claims add a
specific limitation beyond the judicial exception that is not a "well-
understood, routine, conventional activity" or simply appends well-
understood, routine, conventional activities previously known to the
industry, specified at a high level of generality, to the judicial exception (see
Revised Guidance).
As Examiner explains, Bergmann[4J supports a finding that the steps
recited in Appellants' independent claim 1 "of providing a sample from a
patient and determining the levels of pro-adrenomedullin [are] well
understood and routine[]" in this art (Final Act. 6; see Ans. 5---6). In this
regard, we note that Bergmann discloses that Appellants' SEQ ID NO: 2,
which Bergmann identifies as SEQ ID NO: 3, "gives highly significant
measurement results ... not only [for] sepsis diagnostics but also to cardiac
4 Bergmann et al., WO 2004/090546 Al, Oct. 21, 2004 (machine translated
Jan. 31, 2019). All reference to Bergmann refer to the machine translation.
6
Appeal2018-002919
Application 14/664,243
diagnostics" (Bergmann 11:17-19). Thus, we are not persuaded by
Appellants' contentions regarding Appellants' SEQ ID NO: 2 (see App. Br.
3--4; see also Reply Br. 2). Bergmann also discloses a multi-parameter
diagnostic, for use in the field of, inter alia, cardiac diagnostics, wherein in
addition to detecting adrenomedullin levels, the levels of at least one
additional marker, such as proBNP and/or CRP, are detected for use in
diagnostically significant correlations (see Bergmann 11:20-12:3; see also
Final Act. 6 (Examiner additionally finds that the limitations of Appellants'
"dependent claims are also well-understood and routinely conducted in the
art or are simply a recognition of the naturally-occurring correlation"); cf
App. Br. 7-9; Reply Br. 3--4).
For the foregoing reasons, we find that Appellants' claimed invention
simply appends well-understood, routine, conventional activities previously
known to the industry, specified at a high level of generality, to the judicial
exception.
To be complete, we note that where Appellants' "claims are deemed
only to disclose patent ineligible subject matter under the Mayo framework,
as they are in this case, preemption concerns are fully addressed and made
moot." Ariosa Diagnostics, Inc. v. Sequenom Inc., 788 F.3d 1371, 1379
(Fed. Cir. 2015); see also Athena, 2019 WL 453489, at *6 ("Preemption is
sufficient to render a claim ineligible under § 101, but it is not necessary").
CONCLUSION
The preponderance of evidence of record supports Examiner's finding
that Appellants' claimed invention is directed to patent ineligible subject
matter. The rejection of claim 1 under 35 U.S.C. § 101 is affirmed. Claims
4---6, 8, 11-1 7, 21, and 22 are not separately argued and fall with claim 1.
7
Appeal2018-002919
Application 14/664,243
TIME PERIOD FOR RESPONSE
No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a).
AFFIRMED
8
Application/Control No. Applicant(s)/Patent Under
Reexamination
14/664,243 Ng, et al.,
Notice of References Cited
Examiner Art Unit
Shulamith Shafer 1647
Page 1 of 1
U.S. PATENT DOCUMENTS
*
Document Number Date
Name CPC Classification US Classification Country Code-Number-Kind Code MM-YYYY
A US-
B US-
C US-
D US-
E US-
F US-
G US-
H US-
I US-
J US-
K US-
L US-
M US-
FOREIGN PATENT DOCUMENTS
*
Document Number Date
Country Code-Number-Kind Code MM-YYYY Country Name CPC Classification
N
0
p
Q
R
s
T
NON-PATENT DOCUMENTS
* Include as applicable: Author, Title Date, Publisher, Edition or Volume, Pertinent Pages)
u Bergman et al., WO 2004/090546 A1, published Oct. 21, 2004 (Translation of Description)
V
w
X
*A copy of this reference 1s not being furnished with this Office action. (See MPEP § 707.05(a).)
Dates in MM-YYYY format are publication dates. Classifications may be US or foreign.
U.S. Patent and Trademark Office
PT0-892 (Rev. 01-2001) Notice of References Cited Part of Paper No. 20170303
Notice
This translation is machine-generated. It cannot be guaranteed that it is intelligible, accurate,
complete, reliable or fit for specific purposes. Critical decisions, such as commercially relevant or
financial decisions, should not be based on machine-translation output
DESCRIPTION W02004090546
The invention relates to methods for the determination of a midregional partial peptide of the
proadrenomedullin (mid-proAl'v1), in particular the determination of the proAl'v1 (45-92) The
invention relates to methods for the determination of a midregional partial peptide of the
proadrenomedullin (mid-proAM), in particular the determination of the proAM (45-92). Teilpeptids
in biological fluids for the purposes of medical diagnostics, in particular in sepsis diagnostics, but
also z. For example, in cancer diagnostics and cardiac diagnostics or in general in the context of
the diagnosis of such disease states, in which a determination of the peptide adrenomedullin (AM)
provides diagnostisch'relevante results. The determinations according to the invention are carried
out in particular by means of immunoassays of a type in which a labeled antibody is used
(sandwich assay, competitive assay according to the SPAL Tor SPART principle).
In this description, the term "diagnosis" is generally used as a simplifying generic term, which also
includes prognostics/ early prognosis and therapy-accompanying
including running controL
The peptide adrenomedullin (AM) was first reported in 1993 as a novel 52 amino acid hypotensive
peptide isolated from a human phenochromocytoma by Kitamura et al. (see. 18; Fi~Jures refer to
the attached literature list). In the same year. The cDNA encoding a precursor peptide of 185
amino acids and the complete amino acid sequence of this precursor peptide has also been
described (19, SEQ ID NO: 1 ). The precursor peptide, the u. a. comprising a signal sequence of
21 amino acids at the N-terminus is termed "preproadrenomedullin" (pre-proAM). In the present
description all stated amino acid positions normally refer to the 185 amino acid pre-proAM which
has the sequence according to SEQ ID NO: 1, unless something else results from the concrete
31-01-2019 1
context of the text
The peptide adrenomedullin (AM) is a 52 amino acid peptide (SEQ ID NO: 2) comprising amino
acids 95 to 146 of pre-proAM, from which it is formed by proteolytic cleavage. Of the peptide
fragments formed during the cleavage of the pre-proAM, only a few fragments have hitherto been
characterized in more detail, namely the physiologically active peptides adrenomedullin (AM) and
"PAMP", a peptide from the 20 amino acids (22-41 ), which follow the 21 amino acids of the signal
peptide in the pre-proAM.
Both AM and PAMP have also been used to detect and investigate physiologically active
subfragments.
The discovery and characterization of AM in the year. In 1993, intensive research and a flood of
publications were launched, the results of which have recently been summarized in various review
articles, with the present description drawing particular attention to the number of "Peppers"
published in an AM.
tides "(Peptides 22 (2001 )), in particular (12) and (2). Another review is (3). In the previous
scientific studies u. a. found that AM can be considered as a multifunctional regulatoiy peptide. It
is released into the circulation in a glycine-extended inactive form (5). There is also a specific
binding protein for AM (11 ), which probably also modulates the effect of AM.
The physiological effects of AM, as well as of PAMP, which were the focus of previous studies,
were the blood pressure-influencing effects. Thus, AM is an effective vasodilator, whereby the
hypotensive effect can be assigned in particular to peptide segments in the C-terminal part of the
AM. By contrast, peptide sequences from the N-terminus of AM show hypertensive effects (cf. B.
(6) ).
It has also been found that the o. G. Formed from the pre-proAM. Another physiologically active
peptide PAMP also shows a hypotensive effect, even if it seems to have a different mechanism of
action than AM (see next to the o. Review articles (2) and (3) 'also (8), (9) or (14) and EP O 622
458 A2).
31-01-2019 2
It has also been found that the levels of AM that can be measured in the circulation and other
biological fluids are significantly above the levels found in healthy controls in a variety of disease
states. Thus, in patients with congestive heart failure, myocardial infarction, kidney disease,
hypertensive disease, diabetes mellitus, AM levels are significantly, albeit to varying degrees,
elevated in the acute phase of shock and sepsis and septic shock (see e.g. B. (2), chapter 7, and
the literature cited thereon). Also the PAMP
Concentrations are elevated in some of the disease states mentioned, although the plasma levels
are relatively low compared to AM ((2), page 1702).
It is also known that in sepsis or septic shock unusually high concentrations of AM are observed
(see (2) and (4), (1), (13), (15) and (16)). The findings are related to the typical hemodynamic
changes that are typical features of the course of the disease in patients with sepsis and other
serious conditions such as: B. SIRS are known.
Although it is believed that AM and PAMP are formed from the same precursor peptide, pre-
proAM (SEQ ID NO: 1 ), in which the amino acid sequences corresponding to these peptides are
present as partial peptides in equimolar amounts the concentrations of AM or PAMP apparently
different
That's nothing unusual. Thus, the measurable concentrations of various degradation products of
one and the same Vorlauferpeptids z. B. be different because they are the result of different
competing degradation pathways, the z. B. at different disease states lead to a different
fragmentation of a precursor peptide and thus to different degradation products. Certain partial
peptides contained in the precursor peptide can thereby be formed or not formed as free peptides,
and / or different peptides are formed in different ways and in different amounts. Even if only a
single degradation pathway is used to process a precursor peptide, and thus all degradation
products originate from one and the same precursor peptide and must have arisen primarily in
equimolar amounts, the stationary concentrations of various partial peptides and measurable in
biological fluids can Fragments are very different, namely z.
B. when individual of them are formed at different speeds and / or in the
different biological fluid. have individual stabilities (lifetimes), or if they are removed from the
31-01-2019 3
circulation due to differential clearance mechanisms and / or different rates of clearance.
Thus, in connection with the formation of AM, it can be assumed that in addition to AM and PAMP,
other peptide fragments must be formed during the proteolytic processing of pre-proAM. However,
there are no data in the scientific literature on the occurrence and stability of such other possible
fragments to find, and although for research purposes z. 8. from the company. Phoenix
Pharmaceuticals, Inc., such pre-proAM peptide fragments corresponding peptides and
radioimmunoassays (RIA) are offered commercially for their determination.
Based on findings gained with the occurrence of the pro- hormone procalcitonin in sepsis (cf. EP 0
656 121 B1 ), and based on the hypothesis that in sepsis possibly other normally unobservable
prohormones could be detected in the circulation of sepsis patients, the Applicant was using a
commercially available RIA with an antibody which binds to amino acids 45-92 of the pre-proAM,
but not to sequences of mature AM, an origenic assay for the detection of proadrenomedullin in
sera from sepsis patients. The results, which are described in the publication WO 00/22439, prove
an increased concentration compared to healthy control persons of a provisionally as
Proadrenomedullin designated analyte. However, the increase measured was only on the order of
about twice the normal value; So it was relatively minor. In the light of literature data reporting
increased AM levels on the order of 12 times normal in sepsis, the observed one appeared
Increasing to approximately twice the normal value for the proAM immunoreactivity assayed with
the assay used would be less appealing to determine this "proAM immunoreactivity" in the context
of sepsis diagnostics rather than AM. Whether propadrenomedullin (22-185 or 22-146) was
actually measured in the described experiment, or whether the pro-adrenomedullin
immunoreactivity measured in the manner described was due to one or more other species
occurring in the patient samples was based the measured findings are not decidable.
As part of its extensive research and development work on biomarkers that may be of clinical
value for sepsis diagnostics, and in particular with regard to the goal of multiparameter
determination (simultaneous determination of multiple biomarkers) to improve the diagnosis of
sepsis The applicant also considered the determination or additional determination of AM
increased in sepsis. However, it was found that reliable determination of AM was not readily
possible, yielding results that would allow easy comparison of measurement results beyond the
limits of the individual research work. The data from most research has been obtained with RIAs
based on competition of AM with a labeled marker peptide around a common AM binding site of
an antibody. The respective RIAs were often individual developments, and various antibodies and
peptides were used, which made a quantitative interassay comparison of the measurement results
31-01-2019 4
obtained more difficult (cf.
B. 10). In addition, recent research has shown that various forms of AM (with or without C-terminal
~Jlycine residue) exist, to which different forms. Activities (see (2) and z. B. (5)). The discovery of a
binding protein (cf.
11) for AM fuiiher complicates the situation-both the presence or absence of glycine residue,
as well as the lacking or existing complexation of AM by its binding protein can unpredictably
influence the determination of AM depending on the particular assay. These. Circumstances put
high demands on a valid immunoassay for AM that is suitable for routine testing: For such an
assay, suitable antibodies must be found that bind to those AM areas that are not occupied by the
binding protein-if such areas exist at all. Or, a preliminary step of the release and separation of the
binding protein from the AM must be performed, and the impact of such a step on the stability of
the AM and / or the available readings is difficult to predict. The fact that apart from the complete
AM physiologically also different AM partial peptides are found and appear to play a role in the
overall physiological situation further complicates the creation of a valid immunoassay and the
comparability of readable measurements from the literature.
The Applicant has therefore set itself the task of creating a valid, routine-suitable measurement
method, which compared to the o. G. Interference of a direct measurement of AM is largely
insensitive and can provide reliable levels for the physiological production of AM and / or its
precursors in various disease states, particularly sepsis or other disease states where elevated
levels of AM are found.
This object is achieved according to the invention in that for diagnostic purposes not AM or
another of the previously investigated pre-proAM partial peptides is determined, but a midregional
partial peptide which contains the amino acids 42-95 des'preproAM (SEQ ID NO: 3), wherein the
Determination is particularly preferably carried out with an immunoassay, which is carried out with
a labeled antibody.
Claim 1 represents the core of the present invention.
Advantageous and currently preferred embodiments of the invention can be found in the
31-01-2019 5
dependent claims.
To solve the task, a determination method too. which causes the formation of AM or its precursors
or by-products in various disease states, in particular sepsis, but also, for. As cardiac diseases,
high blood pressure diseases or cancers or other diseases in which elevated levels of AM can be
observed reliably detected, was on the one hand to the result described in WO 00/22439 one
increased in sepsis, despite the rather unpromising results be linked "proadrenomedullin
immunoreactivity". On the other hand, complementary extensive clinical studies have been carried
out by measuring sepsis of sepsis, cancer and cardiac patients with various new assays that
surprisingly provided readings with significantly improved validity.
The investigations carried out and the most significant results of these investigations are explained
in more detail below, with reference to figures.
1 shows the results of the measurement of mid-proAM in sera of 109 healthy normal persons,
compared with the results of the measurement of 110 sera from secrets and of 20 sera from
patients with polytrauma. All measurements were made with a SPAL T assay, as described in
more detail in the experimental section. Remarkably, in contrast to the case of procalcitonin and
other inflammatory markers, only the values for the sepsis patients (high concentrations of about
550 pmol / I and 550 fmol / ml, respectively, compared to values of 33 pmol / I for healthy persons)
were increased, not however the
Values for polytrauma patients.
FIG. 2 shows the results of the measurement of mid-proAM in sera from 274 healthy normal
persons, from 267 septins, from 20 patients with heart diseases and 49 cancer patients with a
sandwich assay, as described in more detail in the experimental section.
In connection with the investigations which led to the present invention, the question of the nature
of the species measured in the various diseases, or of the choice of a particularly suitable species
for AIVl / proAM measurements, came to the fore , Since RIAs are in principle not veiy suitable for
providing valuable insights into this question, and because RIAs for various reasons also
appeared less promising for the intended development goal of creating a valid assay for routine
determinations, new assays had to be developed first, with immunoassays of a type in which
labeled antibodies could be used.
31-01-2019 6
In the context of the investigation of whether the increased values for a propadrenomedullin
immunoreactivity in sepsis measured in accordance with WO 00/22439 actually reflect increased
levels of proadrenomedullin in the samples examined, a sandwich assay was first developed that
was largely specific due to the assay design for proadrenomedullin (22-146 and 22-185,
respectively) was able to recognize neither AM nor pre-proAM partial peptides that did not contain
AM.
In this sandwich assay, two different antibodies were used that specifically recognized the amino
acid sequence of the peptide (69-86: peptide region SPCD19, SEQ ID NO: 4) and a peptide (129-
147, C-terminal AM peptide).
The standard material was the recombinant whole
Proadrenomedullin (22-185), which had been calibrated in a commercial AM competitive assay.
When measuring sera from normal healthy individuals and from sepsis patients, this sandwich
assay did not yield any elevated readings above the detection limit of approximately 40 pg / ml
(results not shown). From these findings, it was concluded that the increased proAl'v1
immunoreactivity found in sepsis was not due to the presence of the proadrenomedullin peptide in
the samples.
To further examine the question of whether the relatively few (approx.
2
times), or whether these measurements may have involved artifacts attributable to the commercial
RIA used, another assay was developed based on the so-called SPAL T Principle was based. In
such an assay, competition between a solid phase (SP) competitor for the analyte ("antigen" = A)
and the analyte is shared around shared binding sites of a labeled antibody that is in the reaction
fluid. In the present case, the antibody was labeled with a luminescent tracer (LT) (cf.
Experimental part). The presence of the analyte, or the occupation of binding sites of the antibody
by competing binding partners from the sample, is shown to reduce the binding of the labeled
31-01-2019 7
antibody to the solid phase.
The solid phase-bound competitor used in the SPAL T assay described was the solid-phase-bound
peptide (69-86: peptide region SPCD19, SEQ ID NO: 4), as antibody a labeled anti-SPCD19
sheep antibody formed against this peptide and recognizing this peptide (affinity-purified; see.
Experimental part). Served as standard
Dilutions of the peptide SPCD19 in normal horse serum. The detection limit was approx. 50 pmol /
L The determinations each included 100 Al samples (resp. Standard) and 100 yl tracers were
incubated at 4 ° C in SPsS-9-peptide coated Polysorb tubes, followed by washing with 4 x 1 ml of
standard wash solution from Applicants' LUYlltest and then measuring in a luminometer.
Surveying sepsis sera with this SPAL T assay found a drastic demarcation of sepsis against
normal healthy individuals. At a detection limit of about 1 ng / ml, sera from secrets were found to
average about 19,000 pg / ml. The clear distinction between sera from secrets and healthy was
very surprising in view of the rather slight increase in the preliminary experiments using a
commercial RIA (WO 00/22439).
The clinical measurements with the mentioned SPAL T assay have been extended. The results of
the expanded study are graphically summarized in Fi~Jure 1, with reference being made to the
above discussion of Figure 1.
Theo. G. positive results with SPAL T assays showed that (i) the "proAM immunoreactivity"
measured in sepsis sera could not be applied to the actual presence of proadrenomedullin, but (ii)
a corresponding measurement of an analyte with an amino acid sequence from a middle one
Section of the pre-proAM, more precisely, the mid-proAM according to SEQ ID NO: 3, was able to
clearly distinguish septic from normal persons.
Building on these results, the research was deepened in two directions: 1. The pre-proAl\!1 species
actually occurring in the circulation should be identified and, if necessary, theirs
Suitability to be studied as a biomarker for routine measurements.
31-01-2019 8
2. At the same time, the extent to which the measurement of these species provides diagnostically
valuable measurement results should be deepened.
The results are described in more detail below with reference to the experimental part. They can
be summarized as follows: 1. In the circulation (serum, plasma), there is a significantly higher
concentration, and a readily reproducible measurability, a peptide which contains or consists of the
amino acids 45-92 of the pre-proAM (SEQ ID NO: 3) and referred to in this application as mid-
proAM.
2. The measurement of sera patients with an assay that specifically measures this mid-proAM
provides measurement results that not only allow a clear distinction between septinics and normal
individuals, but also other diseases in connection with clinical findings cardiovascular and cancers
that are associated with increased AIVl production.
The method thus relates in particular to the determination of the mid-proAIVl in the circulation of a
patient, in particular using plasma samples.
In the following, certain general aspects of preferred embodiments of the invention are explained
in more detail, and further selected test results are explained in more detail.
For the practice of the invention, an assay
preferred format in which is worked with labeled antibodies, for. For example, an assay that
operates on the competitive SPAL T principle described above (although other labels, e.g.
Radioactive as a SPART assay).
However, particularly preferred are non-competitive sandwich assays, e.g. The type used for the
more in-depth studies and will be described in more detail below.
Non-competitive sandwich immunoassays (bi-side immunoassays) have a number of advanta~Jes
over competitive immunoassays, including that they can be better designed as solid phase assays
(heterogeneous assays) in which handleability can be more robust higher sensitivity and are also
better suited for automation and series measurement.
31-01-2019 9
In addition, compared to competitive immunoassays that use only one type of antibody, they can
also provide additional information by using sandwich immunoassays only for molecules or
Recognize peptides where both binding sites for the antibodies used for sandwich formation are
present on the same molecule.
The antibodies may in principle be any suitable monoclonal and / or polyclonal antibodies, but
currently affinity-purified polyclonal antibodies are preferred.
Particularly preferred. one of the antibodies obtained by immunizing an animal. in particular sheep,
with an antigen which contains a synthetic peptide sequence which has the amino acids 69-86 of
the pre-proAM and an additional cysteine residue at the N-terminus (SEQ ID NO: 4), The other
antibody may, for. B. can be obtained in accordance with an antigen containing a synthetic peptide
sequence, the
Amino acids 83-94 (peptide region PSR13; SEQ ID NO: 5) of the pre-proAM with an additional
cysteine residue at the N-terminus. The antibodies obtained using said synthetic peptides which
collectively sense a gapless midregional portion of the proAM sequence recognize only binding
sites in the range of o.g. mid-proAM (amino acids 45-92), more specifically in the region of amino
acids 60-92 of the pre-proAM.
In a preferred embodiment the method is performed as a heterogeneous sandwich immunoassay
in which one of the antibodies is attached to any solid phase, for example the walls of coated test
tubes (e.g. B. of polystyrene; "Coated Tubes"; CT) or to microtiter plates, for example made of
polystyrene, or immobilized on particles, for example magnetic particles, while the other antibody
carries a residue which represents a directly detectable label or allows a selective linkage with a
label and the detection of the formed sandwich Structures serves.
A delayed or subsequent immobilization using suitable solid phases is also possible.
In principle, all marking techniques which can be used in assays of the type described can be
used, to which markings with radioisotopes, enzymes, fluorescence, chemiluminescence or
bioluminescence labels and directly optically detectable color labels, such as, for example, gold
atoms and dye particles, as used in particular for so-called. Point of Care (POC) or rapid tests are
31-01-2019 10
used. In the case of heterogeneous sandwich immunoassays, the two antibodies may also have
parts of a detection system of the type described below in connection with homogeneous assays.
It is therefore within the scope of the present invention to design the method accordin~J to the
invention also as a rapid test.
The inventive method can also be configured as a homogeneous method in which the sandwich
complexes formed from the two antibodies and the mid-proAl'v1 to be detected remain suspended
in the liquid phase.
In such a case, it is preferred to label both antibodies with portions of a detection system which,
when both antibodies are integrated into a single sandwich, will enable signal generation or signal
initiation. Such techniques can be designed in particular as fluorescence enhancement or
fluorescence quenching detection methods. A particularly preferred such method involves the use
of paired detection reagents such as those described in US-A-4,822, 733, EP-B 1-180492 or EP-
B 1-539 477 and the prior art cited therein. They allow a measurement which selectively detects
only reaction products containing both labeling components in a single immune complex, directly
in the reaction mixture. As an example, the TRACE (Time Resolved Amplified Cryptate Emission)
brand or CRYPTON offered technology, the teachings of o. G. Registrations implemented.
It has surprisingly been found that the determination according to the invention of the mid-proAl'v1
(SEQ ID NO: 3) gives highly significant measurement results. As stated below, this statement
applies not only to sepsis diagnostics but also to cardiac diagnostics and cancer diagnostics.
It is assumed that the determination method according to the invention can be carried out
particularly advantageously also in the context of a so-called multi-parameter diagnostics, both in
the field of cardiac diagnostics and sepsis and cancer diagnostics. Other specific parameters are,
for example, the cardiac parameters ANP, BNP, proANP or proBNP or sepsis parameters, which
may be, for example, B. are selected from the group consisting of anti-gan glioside antibodies, the
proteins procalcitonin, CA 125, CA
19 -9, S1 OOB. SIOOA proteins, LASP-1, soluble cytokeratin fragments, especially CYFRA 21. TPS
and/ or soluble cytokeratin 1 fragments (sCYIF), the peptides inflammin and CHP, other peptide
prohormones, the glycine N-acyltransferase (GNAT), carbamoyl phosphate synthetase 1 (CPS.1)
and the C-reactive protein (CRP) or fragments thereof. In the case of the aforementioned
31-01-2019 11
multiparameter determinations, it is provided to determine the measurement results for a plurality
of parameters simultaneously or in parallel, and z. This can be evaluated, for example, by means
of a computer program which also uses diagnostically significant parameter correlations.
In the following, the invention will be described by a description of the preparation of the preferred
assay components, the peiiormance of a preferred embodiment of a sandwich-type assay and the
results of mid-proAM determinations, obtained using such an assay, in EDTA plasmas of control
subjects. and sepsis, hemi and cancer patients.
Furthermore, the identification of the actually determined circulating proAM partial peptide is
described.
Experimental part Materials and Methods 1. Peptide Syntheses Derived from the known amino
acid sequence of human preproadrenomedullin (SEQ ID NO: ·1 ), a first region (pos. 69-86: peptide
region SPCD19; SEQ ID NO: 4) and a second region (Pos 83-94: peptide region PSRn; SEQ ID
NO: 5). In each case supplemented by an N-terminal cysteine residue, both regions were
chemically synthesized as soluble peptides by standard methods, purified by means of
Mass spectrometry and reversed phase HPLC were quality-controlled and lyophilized in aliquots
(JERINI AG, Berlin, Germany). The amino acid sequences of the peptides are as follows: Peptide
SPCD19: CRPQDMKGASRSPEDSSPD (SEQ ID NO: 4) Peptide PSR13: CSSPDMRI RVKR
(SEQ ID NO: 5) In addition, the entire mid-proAM (corresponding to Pos. 45-92; SEQ ID NO: 3)
synthesized: ELRMSSSYPTG LADVKAG PAQTLI RPQDM KGASRSPEDSSPDAARI RV (SEQ ID
NO: 3) 2. Conjugation and Immunization Using MBS (m-Maleimidobenzoyl-N-hydroxysuccinimide
ester), the above peptides SPCD19 and PSR13 is conjugated to the carrier protein KLH (keyhole
limpet hemocyanin) (seep.
Working Instructions "NHS-Esters-Maleimide Crosslinkers" Company PIERCE, Rockford, IL,
USA). Sheep were immunized with these conjugates according to the following scheme: each
sheep initially received 100 µ~J of Ag conjugate (by mass based on the peptide portion of the
conjugate) and then each week 50 µg of Ag conjugate (by mass relative to the peptide portion of
the conjugate), From the fourth month after initiation of immunization, 700 ml of blood were
collected per sheep per week and antiserum was collected therefrom by centrifugation.
Conjugations, immunizations and recovery of antisera were performed by MicroPharm,
Carmarthenshire, UK.
31-01-2019 12
3.
Purification of the Antibodies In a one-step procedure, the antisera raised from the fourth month
after the immunization, peptide-specific antibodies such as
follows prepared.
For this purpose, the o. G.
Peptides SPCD19 and PSR13 coupled to Sulfolink gel (s. Working Instructions "Sulfolink Kit"
Company PIERCE, Rockford, IL, USA). In each case, 5 mg of peptide per 5 ml of gel were offered
for coupling.
The affinity purification of peptide-specific antibodies from sheep antisera to both peptides was
carried out as follows: The peptide columns were initially washed three times alternately with 10 ml
each of elution buffer (50 mM citric acid, pH 2.2) and binding buffer (100 mM sodium phosphate, 0
, 1 % Tween, pH 6.8). 100 ml of the sheep antisera were filtered over 0.2 µm and mixed with the
column material present.
For this, the gel was rinsed quantitatively with 10 ml of binding buffer from the column. The
incubation was carried out overnight at room temperature while panning. The batches were
transferred quantitatively to empty columns (NAP 25, Pharmacia, deflated).
The runs were discarded. Subsequently, protein-free (protein content of the wash eluate <0.02
A280 nm) was washed with 250 ml of binding buffer. On. the washed columns were added to
elution buffer and fractions of 1 ml were collected. The protein content of each fraction was
determined by the BCA method (seep. Work Instructions Company PIERCE, Rockford, IL, USA).
Fractions with protein concentrations> 0.8 mg I ml were pooled. After protein determination of the
pools by the BCA method, yields of 49 mg for the anti-SPCD19 antibody (affinity-purified,
polyclonal) and 60 mg for the anti-PSR13 antibody (affinity-purified, polyconal) were obtained.
4.
Labeling Via a NAP-5 gel filtration column (Pharmacia), 500 I of the purified anti-SPCD19 antibody
(see above) in 1 ml
31-01-2019 13
100
mM potassium phosphate buffer (pH 8.0) re-buffered according to the instructions. The protein
concentration determination of the antibody solution gave a value of 1.5 mg / mL
For the chemiluminescent labeling of the antibody, 67 µI of the antibody solution were mixed with
10 µI of MA70-acridinium-NHS ester (1 mg/ ml, from HOECHST Behring) and incubated for 15
minutes at room temperature. Then, 423 µI of 1 M glycine was added and incubated for a further
10 minutes. Subsequently, the labeling mixture was buffered via a NAP-5 gel filtration column
(Pharmacia) in 1 ml of eluent A (50 mM potassium phosphate, 100 mM NaCl, pH 7.4) according to
the instructions, and freed from low molecular weight constituents. A gel filtration HPLC (column:
Waters Protein Pak SW300) was carried out for the separation of last residues of labels not bound
to antibodies. The sample was applied and chromatographed with solvent A at a flow rate of 1 ml /
min. With a flow-through photometer, the wavelengths 280 nm and 368 nm were measured. The
absorption ratio of 368 nm / 280 nm as a measure of the degree of labeling of the antibody was
0.10 at the peak. The monomeric antibody-containing fractions (retention time 8-10 min) were
collected and dissolved in 3 ml of 100 ml'v1 sodium phosphate, 150 mlVl NaCl, 5% Bovine Serum
Albumin, 0.
1 % Sodium azide, pH 7.4.
The labeled antibody was used in a sandwich immunoassay on the one hand, as will be described
in more detail below, but on the other hand also in the SPAL T assay already described.
5.
Coupling To create the solid phase of a sandwich immunoassay, irradiated 5 ml polystyrene tubes
(Greiner) were coated with purified anti-PSR13 antibody as follows: The antibody was added in 50
mlVl Tris, 100 mlVl NaCl, pH 7.8
diluted to a concentration of 6.6 yg / ml.
300 µI of this solution were pipetted into each tube. The tubes were incubated at 22 C for 20
31-01-2019 14
hours. The solution was sucked off. Then, each tube was filled with 4.2 ml of 10 mM sodium
phosphate, 2% Karion FP, 0. 3 Bovine serum albumin. pH 6.5 filled. After 20 hours, the solution
was filtered with suction.
Finally, the tubes were dried in a vacuum dryer.
The described labeling and immobilization procedures were also performed in substantially the
same manner with the other antibody, respectively, to obtain an "inverse" sandwich assay.
Provisions analogous to those described below using such "inverse" labeled / immobilized
immunoassay yielded substantially identical results and therefore are not described separately.
6.
Implementation and Evaluation of the Sandwich Immunoassay An assay buffer of the following
composition was prepared: 100 mM sodium phosphate, 150 mM NaCl, 5% bovine serum albumin,
0. ·1 k unspecific. Sheep lgG, 0. 1 k sodium azide, pH 7.4 The standard material used was a
chemically synthesized mid-proAM (SEQ ID NO: 3). This peptide was serially diluted in normal
horse serum (SIGMA). Concentrations according to the weight of peptide were ascribed to the
standards thus prepared.
Test samples were. EDTA plasmas of apparently healthy, of patients with sepsis and of patients
with cardiac and with cancers.
In the test tubes were 10 y1 standards or Samples and 200 Al assay buffers containing 1 million
RLU (relative light units) of MAYO-labeled anti-SPCD·19 antibody, pipetted. It was incubated for two
hours at 22 C with shaking. Then, 4 times with 1 ml of washing solution (0. 1 % Tween 20) per
tube, drained, and the chemiluminescence bound to the tube measured in a luminorneter
(BERTHOLD, LB952T, BRAHMS AG base reagents).
Using the software MultiCalc (Spline Fit), the midregional proadrenomedullin concentrations of the
samples were read on the standard curve. The results are graphically summarized in FIG.
7.
31-01-2019 15
Performance and Evaluation of the SPAL T Immunoassay The solid-phase-bound competitor used
in the described SPAL T assay was the solid phase-bound peptide SPCD19 (peptide region 69-86,
SEQ ID NO: 4 ), which had been bound to the walls of Polysorb tubes. The antibody used was the
labeled anti-SPCD19 sheep antibody (affinity-purified) obtained as described above under 1 to 4.
Dilutions of the peptide SPCD19 in normal horse serum served as standard.
The determinations were each 100 yl sample (or.
Standard) and 100 yl tracers at 4 C in the SPCD19-peptide coated Polysorb tubes were incubated
overnight, after which washed with 4 x 1 ml of standard washing solution from the Applicant's
LUMltest and then measured in the luminometer.
The results of a series of measurements obtained with this assay are shown in FIG.
8.
Identification of the measured in the described assays
For the enrichment of the analyte, which is different from the one described in o. g.
Assays used antibodies detected, three individual sepsis plasmas were fractionated directly
analytically via a C18 reversed phase HPLC, which was eluted by means of a linear acetonitrile
gradient.
1 ml fractions were collected and dried. The fractions were taken in assay buffer and the SPCD19
immunoreactivity of the individual fractions was determined. certainly. For this purpose, an anti-
SPCD19 antibody (see above under 3.) was immobilized on the walls of a Polysorb tube, and the
competition of sample (fraction) and luminescence-labeled SPCD19 for this antibody was
determined.
In such an analysis it was found that in all sepsis plasmas the greatest immunoreactivity was
found in the same fraction (fraction 22).
31-01-2019 16
For further identification of the measured analyte, 7 sepsis sera about 3 ml were pooled (final
volume 22 ml). Using a Carbolink column with an anti-SPCD19 antibody, the pooled sera were
subjected to affinity purification and the acidic eluate was fractionated as above via C18 reversed
phase HPLC. Fraction 22 was dried and analyzed by mass spectrometry.
In a direct mass spectrometric analysis, the molar mass of the isolated analyte was a value of
approx. 5146 Dalton determined. This value corresponds to the molecular weight of a proAM
fragment containing the amino acids of positions 45-92, d. H. of the mid-proAM (the theoretical
value is 5146, 72 daltons, assuming that the two methionine residues present are oxidized).
In a MALDI-TOF analysis of the tryptic digest of the isolated fraction 22, monoisotopic masses (M
+ H +) u. a. Identified peptide fragments corresponding to the amino acids of positions 79-89 ,. 75-
89, 61-74 and 61-78 of preproAl\!L The molecular mass data and the mass spectrometric analysis
of the tiyptic degradation together prove that the peptide contained in the isolated fraction is the
peptide designated as mid-proAM (45-92) (SEQ ID NO: 3). Its genesis can be explained by
proteolytic processing of the original pre-proAM translation product by signal peptidase,
prohormone convertase (cleavage between basic amino acids), and amino and carboxypeptidase
(cleavage of basic amino acids) (see the analogous scheme for procalcitonin degradation in 20)).
9.
Stability test To check whether mid-proAM measurement has problems due to insufficient stability
of the mid-proAM in a sample or Measuring solution must be calculated, 20 sepsis sera fresh each
and measured after a 3-day storage at room temperature. The results are summarized in the table
below. They show that after 3 days storage, the immunoreactivity was almost unchanged. This
proven stability of the mid-proAM is a great advantage from a handling point of view for
diagnostics.
Table 1 Patient? mid-proAM [nmol / 1] mid-proAM [nmol i I] change day= 0 day= 3 1 6.2 6, 1 98,
8% 2 3.3 3, 2 98, ·1 % 3 2.2 2, ·1 97, 0% 4 1, 6 1, 5 95, 4% 5 1, 1 1, 0 92, 7% 6 1,3 1, 2 9 $, 7% 7
1,9 2, 1 109, 6 % 8 2.6 2, 7 102, 8% 9 2,8 2, 7 96, 4% 10 3, 1 3, 1 99, 9% 11 4,6 4, 9 106, 3% 12
5,8 5 , 9 102, 1 % 13 3,6 3, 4 95, 2% 14 4,2 4, 6 110, 7% 15 3,0 2, 4 80, 0% 16 1,2 1, 3 105, 5%
31-01-2019 17
'17 1.5 ·1, 5 102, 2% 18 1.7 1, 8 '103, 4% 19 2.0 1, 8 89, 5% 20 2.1 2, 0 94, 1 % Average= 98, 8%
In summary, it can be said that a determination of mid-proAM, e.g. B. using an SPCD19 antibody,
against a determination of z. B. AM has numerous advantages: A determination of the mid-proAM
is not subject to any known limitations due to the existence of a binding protein, fragmentation and
a weak concentration dynamics.
The analyte mid-proAM also has good stability, i. H. a very small loss of immunoreactivity when
stored at room temperature, which is a great practical advantage for routine diagnostic assays.
Extremely favorable dynamics are observed, and it is not expected to be specific to sepsis.
It is therefore to be assumed that a measurement of mid-proAIVl can generally have advantages in
all clinical pictures for which AM concentration increases are described, and in particular a
determination in the context of sepsis, cardiac and cancer diagnostics appears to be
advantageous.
Literature list 1. K. Ehlenz et al. : "High levels of circulating adreno-medulline in severe illness:
Correlation with C-reactive protein and evidence against adrenal medulla as site of origin", Exp.
Clin. Endocrinol. Diabetes (1997) 105, 156-162 2. Tanenao Eto: "A review of the biological
properties and clinical implications of adrenomedullin and proadreno-medulline N-terminal peptide
(PAM P), hypotensive and vasodilating peptides", Peptides (2001) 22, 1669-1711 3. Joy Patricia
Hinson et al. : "Adrenomedullin, a multi- functional regulatory peptide", Endocr. Rev. (2000) 21 (2),
138-167 4. Yukio Hirata et al. : "Increased circulating adrenomedullin, a novel vasodilatory
peptide, in sepsis", J.
Clin. Endocrinol. IVletab. (1996) Vol. 81, No. 4, 1449-1453 5. K. Kitamura et al. : "The intermediate
form of glycine-extended adrenomedullin is the major circulating molecular form in human
plasma", Biochem. Biophys. Res.
Commun. (1998) 244 (2), 551-555 6. Kazuo Kitamura et al.: "Adrenomedullin (11-26): a novel
endogenous hypertensive peptide isolated from bovine adrenal medulla", Peptides (2001) 22,
1713-1718 7. M. Kohno et al. : "Plasma adrenomedullin concentrations in essential hypertension",
Hypertension (1996) 27 ('1 ), 102-107
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8. K. Kuwasako et al. : "Purification and characterization of PAMP-12 (PAMP-20) in porcine
adrenal medulla as a major endo~Jenously biologically active peptide", FEBS Lett (1997) 414 (1 ),
105-1109. K. Kuwasako et al. : "Increased plasma proadrenomedullin N-terminal 20 peptides in
patients with essential hyper-tension", Ann. Clin. Biochem. (1999) 36 (Pt 5), 622-628 10. Lynley K.
Lewis et al. : "Adrenomedullin (1-52) measured in human plasma by radioimmunoassay: plasma
concen- tration, adsorption, and storage", Clin. Chem. (1998) 44 : 3,57·1-577 11. Beets Pio et al. :
"Complement factor H is a serum-binding protein for adrenomedullin, and the resulting complex
modulates the bioactivities of both partners", J. Biol. Chem. (2001) Vol. 276, No. 15, 12292-12300
12. Kazuhiro Takahashi: "Adrenomedullin: from a pheochromocytoma to the eyes", Peptides
(2001) 22, 1691 13. Yoshio Tomoda et al. : "Regulation of adrenomedullin secretion from cultured
cells", Peptides (2001) 22, 1783-1794 14.
T. Tsuruda et al. : "Secretion of proadrenomedullin N-terminal 20 peptide from cultured neonatal
rat cardiac cells", Life Sci. (2001) 69 (2), 2:39-245 15. Shiro Ueda et al. : "Increased plasma levels
of adreno-medulline in patients with systemic inflammatory response syndrome", Am. J. Respir.
Grit Care Med.
(1999) Vol. 160, 132-136 16. Ping Wang: "Adrenomedullin and cardiovascular responses in
sepsis", Peptides (2001) 22, 1835-1840
17. H. Washimine et al. : "Plasma concentration of human adrenomedullin in patients on
hemodialysis", Clin.
Nephrol. (1995) 44 (6), 389-393 18. K. Kitamura et al. : "Adrenomedullin: A Novel Hypotensive
Peptides Isolated from Human Pheochromocytoma"; Biochem. Biophys. Res. Commun. 192 : 55:3-
560 (1993) 19. K. Kitamura et al. : "Cloning and Characterization of cDNA Encoding a Precursor
for Human Adrenomedullin"; Biochem. Biophys. Res. Commun. 194: 720-725 (1993) 20. Meisner
M., (2000) "Procalcitonin", Georg Thieme Verlag, ISBN 3-13-105473-5, S. 22
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