U.S. patent application number 09/976784 was filed with the patent office on 2002-08-22 for organ and biological tissue preservation cold storage solution.
This patent application is currently assigned to Pike Laboratories, Inc.. Invention is credited to Mar Arrington, Ben O?apos, Polyak, Maximilian.
Application Number | 20020115634 09/976784 |
Document ID | / |
Family ID | 22904780 |
Filed Date | 2002-08-22 |
United States Patent
Application |
20020115634 |
Kind Code |
A1 |
Polyak, Maximilian ; et
al. |
August 22, 2002 |
Organ and biological tissue preservation cold storage solution
Abstract
Cold storage solutions for the preservation of organs and
biological tissues prior to implantation, including a cellular
energy production stimulator under anaerobic conditions, an
anti-inflammatory agent, and an oxygen free radical scavenger.
Inventors: |
Polyak, Maximilian;
(Glenmoore, PA) ; Arrington, Ben O?apos;Mar; (East
Stroudsbrug, PA) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS LLP
1701 Market Street
Philadelphia
PA
19103
US
|
Assignee: |
Pike Laboratories, Inc.
|
Family ID: |
22904780 |
Appl. No.: |
09/976784 |
Filed: |
October 12, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60240023 |
Oct 13, 2000 |
|
|
|
Current U.S.
Class: |
514/45 ; 435/1.3;
514/12.2; 514/15.1; 514/262.1; 514/5.9; 514/60; 514/61 |
Current CPC
Class: |
A01N 1/02 20130101; A01N
1/0226 20130101 |
Class at
Publication: |
514/45 ; 435/1.3;
514/60; 514/61; 514/262.1; 514/3 |
International
Class: |
A01N 001/02; A61K
031/715; A61K 038/28; A61K 031/519 |
Claims
What we claim is:
1. An organ or biological tissue preservation aqueous cold storage
solution comprising: a cellular energy production stimulator under
anaerobic conditions; an anti-inflammatory agent; and an oxygen
free radical scavenger.
2. The cold storage solution of claim 1 wherein the cellular energy
production stimulator comprises insulin.
3. The cold storage solution of claim 1 wherein the
anti-inflammatory agent comprises dexamethasone.
4. The cold storage solution of claim 1 wherein the oxygen free
radical scavenger comprises superoxide dismutase.
5. The cold storage solution of claim 4 wherein the superoxide
dismutase conjugates to polyethylene glycol.
6. The cold storage solution of claim 1 further comprising
potassium lactobionate, KH.sub.2PO.sub.4, MgSO.sub.4, and
raffinose.
7. The cold storage solution of claim 1 further comprising
adenosine, allopurinol and pentastarch.
8. The cold storage solution of claim 1 further comprising NaCl and
KOH.
9. The cold storage solution of claim 1 wherein the cellular energy
production stimulator comprises about 4-100 U/L insulin, the
anti-inflammatory agent comprises about 4-24 mg/L dexamethasone,
and the oxygen free radical scavenger comprises about 1,000-100,000
U/L superoxide dismutase, further comprising: about 50-l50 mM
potassium lactobionate; about 10-40 mM KH.sub.2PO.sub.4; about 2-8
mM MgSO.sub.4; about 10-50 mM raffinose; about 1-20 mM adenosine;
about 1-10 mM allopurinol; and about 40-60 g/L pentastarch.
10. The cold storage solution of claim 1 wherein the cellular
energy production stimulator comprises about 20-60 U/L insulin, the
anti-inflammatory agent comprises about 6-16 mg/L dexamethasone,
and the oxygen free radical scavenger comprises about 5,000-50,000
U/L superoxide dismutase, further comprising: about 75-125 mM
potassium lactobionate; about 20-30 mM KH.sub.2PO.sub.4; about 3-7
mM MgSO.sub.4; about 20-40 mM raffinose; about 2-10 mM adenosine;
about 1-5 mM allopurinol; and about 45-55 g/L pentastarch.
11. The cold storage solution of claim 1 wherein the cellular
energy production stimulator comprises about 40 U/L insulin, the
anti-inflammatory agent comprises about 8 mg/L dexamethasone, and
the oxygen free radical scavenger comprises about 25,000 U/L
superoxide dismutase, further comprising: about 100 mM potassium
lactobionate; about 25 mM KH.sub.2PO.sub.4; about 5 mM MgSO.sub.4;
about 30 mM raffinose; about 5 mM adenosine; about 1 mM
allopurinol; and about 50 g/L pentastarch.
12. The cold storage solution of claim 1 further comprising sterile
water.
13. A preserved organ or biological tissue comprising at least one
of a cadaveric organ and tissue within the cold storage solution of
claim 1 in at least one of a deep hypothermic condition and
physiological condition.
14. The preserved organ or biological tissue of claim 13 wherein
the cold storage solution is infused into vasculature of at least
one of a cadaveric organ, living donor organ, and tissue.
15. The preserved organ or biological tissue of claim 13 wherein
the deep hypothermic condition comprises a temperature of about
2-10.degree. C.
16. The preserved organ or biological tissue of claim 13 wherein
the physiological condition comprises a temperature of about
37.degree. C.
17. The preserved organ or biological tissue of claim 13 wherein
the cold storage solution is cooled to below 10.degree. C.
18. The preserved organ or biological tissue of claim 13 wherein
any precipitates in the cold storage solution are removed prior to
use.
19. An organ or biological tissue preservation aqueous cold storage
solution comprising: about 1,000-100,000 U/L superoxide dismutase;
about 50-150 mM potassium lactobionate; about 10-40 mM
KH.sub.2PO.sub.4; about 2-8 mM MgSO.sub.4; about 10-50 mM
raffinose; about 1-20 mM adenosine; about 1-1 mM allopurinol; about
40-60 g/L pentastarch; about 4-100 U/L insulin; about 4-24 mg/L
dexamethasone; and about 700-900 mL sterile water.
20. A method for preserving an organ or biological tissue
comprising: flushing at least one of a cadaveric organ and tissue
with a cold storage solution, having a cellular energy production
stimulator under anaerobic conditions, an anti-inflammatory agent,
and an oxygen free radical scavenger; allowing the flushed at least
one of a cadaveric organ and tissue to be enveloped in the cold
storage solution; and storing the at least one of a cadaveric organ
and tissue in the cold storage solution in at least one of a deep
hypothermic condition and physiological condition.
21. The method of claim 20 wherein the flushing comprises: infusing
the solution into vasculature of the at least one of a cadaveric
organ and tissue; and exsanguinating the at least one of a
cadaveric organ and tissue.
22. The method of claim 20 wherein the storing comprises: replacing
blood in vasculature of the at least one of a cadaveric organ and
tissue with the solution.
23. The method of claim 20 further comprising: replacing the
solution with at least blood to return the at least one of a
cadaveric organ and tissue to a normothermic condition.
24. The method of claim 20 further comprising: cooling the solution
to below 10.degree. C.; inspecting the cooled solution for
precipitates; and removing any precipitates by filtration.
25. A method of preparing an organ or biological tissue
preservation cold storage solution comprising: providing a solution
with sterile water; adding potassium lactobionate, potassium
phosphate, raffinose, adenosine, allopurinol, pentastarch, insulin
and dexamethasone to the solution; and mixing superoxide dismutase
into the solution.
26. The method of claim 25 further comprising: mixing the solution
until all components are dissolved.
27. The method of claim 25 further comprising: infusing the
pentastarch under pressure through a dialyzing filter; and
conjugating the superoxide dismutase to polyethylene glycol.
Description
REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/240,023 filed on Oct. 13, 2000, entitled "Organ
and Biological Tissue Preservation Cold Storage Solution," which is
incorporated herein by reference.
FIELD OF INVENTION
[0002] The invention relates to the field of organ and biological
tissue preservation. In particular, the invention relates to cold
storage solutions for the preservation of organs and biological
tissues for implant.
BACKGROUND OF INVENTION
[0003] It is believed that the ability to preserve human organs for
a few days by cold storage after initial flushing with an
intracellular electrolyte solution or by pulsatile perfusion with
an electrolyte-protein solution has allowed sufficient time for
histo-compatibility testing of donor and recipient. It is also
believed that preservation by solution or perfusion has also
allowed for organ sharing among transplant centers, careful
preoperative preparation of the recipient, time for preliminary
donor culture results to become available, and vascular repairs of
the organ prior to implantation.
[0004] It is believed that the 1990's has been a decade
characterized by increasing waiting times for cadaveric organs. In
renal transplantation, the growing disparity between available
donors and patients on the waiting list has stimulated efforts to
maximize utilization of cadaveric organs. An obstacle that may
arise in the effort to increase utilization is that maximal
utilization may require transplantation of all available organs,
including extended criteria donor organs. However, by extending the
criteria for suitability of donor organs, transplant clinicians may
risk a penalty with respect to graft function, diminishing the
efficiency of organ utilization if transplanted organs exhibit
inferior graft survival. Consequently, interventions that both
improve graft function and improve the ability of clinicians to
assess the donor organ may be crucial to achieving the goal of
maximizing the efficiency of cadaveric transplantation.
[0005] The mechanisms of injuries sustained by the cadaveric renal
allograft during pre-preservation, cold ischemic preservation and
reperfusion are believed to be complex and not fully understood.
However, it is believed that there exists ample evidence to suggest
that many of the injurious mechanisms occur as a result of the
combination of prolonged cold ischemia and reperfusion (I/R).
Reperfusion alone may not be deleterious to the graft, since
reperfusion after short periods of cold ischemia may be
well-tolerated, but reperfusion may be necessary for the
manifestation of injuries that originate during deep and prolonged
hypothermia. It is suggested that four major components of I/R
injury that affect the preserved renal allograft begin during cold
ischemia and are expressed during reperfusion. These include
endothelial injury, leukocyte sequestration, platelet adhesion and
increased coagulation.
[0006] Hypothermically-induced injury to the endothelium during
preservation may lead to drastic alterations in cytoskeletal and
organelle structures. During ischemic stress, profound changes in
endothelial cell calcium metabolism may occur. These changes may be
marked by the release of calcium from intracellular depots and by
the pathological influx of calcium through the plasma membrane.
Hypothermic preservation may disrupt the membrane electrical
potential gradient, resulting in ion redistribution and
uncontrolled circulation of Ca++. The depletion of ATP stored
during I/R may compromise ATP-dependent pumps that extrude Ca++
from the cell and the energy intensive shuttle of organelle
membranes, causing a dramatic elevation of intracellular free
Ca++.
[0007] Alterations in cytosolic Ca++ concentration may disrupt
several intracellular functions, many of which may result in
damaging effects. Unregulated calcium homeostasis has been
implicated in the development of endothelial and parenchymal injury
and is believed to be a fundamental step in the sequelae of steps
leading to lethal cell injury. Among the most significant damaging
effects of increased cytosolic Ca++ are believed to be the
activation of phospholipase A1, 2 and C, the cytotoxic production
of reactive oxygen species by macrophages, the activation of
proteases that enhance the conversion of xanthine dehydrogenase to
xanthine oxidase, and mitochondrial derangements.
[0008] Solutions for preserving organs are described in U.S. Pat.
Nos. 4,798,824 and 4,879,283, the disclosures of which are
incorporated herein in their entirety. One such solution is
Viaspan.RTM. cold storage solution, which may be used for
hypothermic flushing and storage of organs. Despite such solutions,
it is believed that there remains a need for organ and tissue
preserving solutions that allow for static storage and
preservation, while demonstrating superior quality preservation of
organ and tissue viability and function.
SUMMARY OF THE INVENTION
[0009] The invention provides an organ and tissue preserving
solution for static storage preservation that demonstrates superior
quality preservation when compared to existing preserving media, in
terms of organ and tissue viability and function. The organ and
biological tissue preservation aqueous cold storage solution
includes a cellular energy production stimulator under anaerobic
conditions, an anti-inflammatory agent, and an oxygen free radical
scavenger.
[0010] The invention also provides a preserved organ or biological
tissue, including a cadaveric organ or tissue within a cold storage
solution of the invention in a deep hypothermic condition or
physiological condition.
[0011] The invention also provides a method for preserving an organ
or biological tissue. The method includes flushing a cadaveric
organ or tissue with a cold storage solution of the invention,
allowing the flushed cadaveric organ or tissue to be enveloped in
the cold storage solution, and storing the cadaveric organ or
tissue in the cold storage solution in a deep hypothermic condition
or physiological condition.
[0012] The invention further provides a method of preparing an
organ or biological tissue preservation cold storage solution. The
method includes providing a solution with distilled water or
deionized water, adding potassium lactobionate, potassium
phosphate, raffinose, adenosine, allopurinol, pentastarch, insulin
and dexamethasone to the solution, and mixing superoxide dismutase
into the solution.
DETAILED DESCRIPTION OF THE INVENTION
[0013] In accordance with the invention, the organ and biological
tissue preservation cold storage solution includes a cellular
energy production stimulator under anaerobic conditions, an
anti-inflammatory agent, and an oxygen free radical scavenger. The
organ and biological tissue preservation cold storage solution is
intended for infusion into the vasculature of cadaveric and living
donor organs for transplantation. Once infused, the donor organs
are exsanguinated and blood is replaced by the solution in the
native vasculature of the organs to return the organs to a
normothermic condition. The solution may be used under deep
hypothermic conditions or physiological conditions. The solution
remains in the vasculature of the organ, as well as envelopes the
entire organ during the period of cold ischemia. This method of
preservation allows for the extended storage of organs, tissues,
and all biological substances. When the organ or tissue is returned
to normothermic conditions, the solution is replaced with blood or
other physiologic media. Variations of this solution may also be
used for machine perfusion preservation. The cold storage solution
of the invention may be used in the same manner and for the same
tissues and organs as known storage solutions.
[0014] A cold storage solution of the invention includes a cellular
energy production stimulator under anaerobic conditions. Insulin,
which is a polypeptide hormone, is the primary hormone responsible
for controlling the uptake, utilization, and storage of cellular
nutrients. Insulin stimulates transport of substrates and ions into
cells, promotes translocation of proteins between cellular
compartments and activates and inactivates specific enzymes.
[0015] A cold storage solution of the invention also includes an
anti-inflammatory agent. Dexamethasone, which is a steroid
analogue, is used for its anti-inflammatory properties.
Dexamethasone has a prolonged plasma half-life and pronounced
growth-suppressing properties.
[0016] A cold storage solution of the invention also contains an
oxygen free radical scavenger. One example, superoxide dismutase,
is known for its potent free oxygen radical scavenging properties.
Preferably, the superoxide dismutase is conjugated to polyethylene
glycol so that its half-life is extended by a factor of about 100
times. Superoxide dismutase is a potent scavenger of several
classes of free oxygen radicals during cold ischemia and upon
reperfusion. When conjugated to polyethylene glycol, superoxide
dismutase can remain active for several hours in the cold storage
solution of the invention.
[0017] According to a preferred embodiment of the invention, an
organ and biological tissue preservation cold storage solution
containing superoxide dismutase in the preserving solution
significantly improves vascular resistance, vascular flow, and
calcium efflux during the organ preservation period. The inhibition
of calcium efflux over time in kidneys preserved by the proposed
solution suggests that, in addition to vasoactive effects, an
additional cytoprotective and cryoprotective effect may also be
important in ameliorating ischemic injury. These improvements are
substantiated ultrastructurally by improved appearance of
mitochondria in proximal tubular cells compared to mitochondria
from kidneys not exposed to the proposed solution.
[0018] A cold storage solution of the invention may also contain
components that are typically used in known cold storage solutions.
See, U.S. Pat. Nos. 4,798,824 and 4,879,283. For example, other
components that may be utilized in the solution include: potassium
lactobionate, which is an impermeant anion that reduces cell
swelling, KH.sub.2PO.sub.4, which provides acid-base buffering and
maintains the pH of the solution, MgSO.sub.4, which stabilizes
cellular and organelle membranes, and raffinose, which is a complex
sugar that reduces cell swelling and provides energy stores for
metabolically stressed cells. In addition, adenosine, which is a
precursor to ATP synthesis, allopurinol, which is also a free
oxygen radical scavenger, and pentastarch, which is an oncotic
supporter, may be added to the solution. NaCl and KOH may also be
used for acid-base buffering and maintenance of the pH of the
solution. In a preferred embodiment, the organ or biological tissue
preservation cold storage solution includes, but is not limited
to:
1 TABLE 1 COMPOSITION AMOUNT IN 1 LITER Potassium Lactobionate
50-150 mM KH.sub.2PO.sub.4 10-40 mM MgSO.sub.4 2-8 mM Raffinose
10-50 mM Adenosine 1-20 mM Allopurinol 1-10 mM Pentastarch 40-60
g/L Insulin 4-100 U/L Dexamethasone 4-24 mg/L Superoxide Dismutase
1,000-100,000 U/L Sterile Water 700-900 mL
[0019] In a more preferred embodiment, the organ or biological
tissue preservation cold storage solution includes, but is not
limited to:
2 TABLE 2 COMPOSITION AMOUNT IN 1 LITER Potassium Lactobionate
75-125 mM KH.sub.2PO.sub.4 20-30 mM MgSO.sub.4 3-7 mM Raffinose
20-40 mM Adenosine 2-10 mM Allopurinol 1-5 mM Pentastarch 45-55 g/L
Insulin 20-60 U/L Dexamethasone 6-16 mg/L Superoxide Dismutase
5,000-50,000 U/L Sterile Water 700-900 mL
[0020] In a most preferred embodiment, the organ or biological
tissue preservation cold storage solution includes, but is not
limited to:
3 TABLE 3 COMPOSITION AMOUNT IN 1 LITER Potassium Lactobionate 100
mM KH.sub.2PO.sub.4 25 mM MgSO.sub.4 5 mM Raffinose 30 mM Adenosine
5 mM Allopurinol 1 mM Pentastarch 50 g/L Insulin 40 U/L
Dexamethasone 8 mg/L Superoxide Dismutase 25,000 U/L Sterile Water
800 mL
[0021] A cold storage solution of the invention may be prepared by
combining the components described above with sterile water, such
as distilled and/or deionized water. For example, to prepare the
organ and biological tissue preservation cold storage solution,
approximately 700-900 mL, or preferably about 800 mL, of sterile
water is poured into a one liter beaker at approximately room
temperature. Although a one liter beaker is used in this example,
any other container of any size may be used to prepare the
solution, where the component amounts would be adjusted
accordingly. In the most preferred embodiment, the following are
added, in any order, to the solution and each is mixed until
dissolved in the solution: approximately 100 mol/L of potassium
lactobionate, approximately 25 mol/L of potassium phosphate,
approximately 30 mol/L of raffinose, approximately 5 mol/L of
adenosine, approximately 1 mol/L of allopurinol and approximately
50 g of modified pentastarch. The modified pentastarch is a
fractionated colloid mixture of 40-60 kDaltons in diameter and is
modified by infusing the pentastarch under 3 atm of pressure
through a dialyzing filter with a bore size of about 40-60
kDaltons. Approximately 40 U of insulin and approximately 8mg/L
dexamethasone are also added to the solution. Then, in a second
step, approximately 25,000 U of superoxide dismutase, which is
conjugated to polyethylene glycol, is added to the solution. The
first and second step may also be reversed.
[0022] The invention also provides a method for preserving an organ
or biological tissue. The method flushes a cadaveric organ or
tissue with a cold storage solution of the invention, allows the
cadaveric organ or tissue to be enveloped in the cold storage
solution, and then stores the cadaveric organ or tissue in the cold
storage solution in a deep hypothermic condition or physiological
condition. Additional cold storage solution may be added to ensure
adequate preservation of the organ or tissue. Preferred
temperatures range from about 2-10.degree. C. in the deep
hypothermic condition and are about 37.degree. C., or room
temperature, in the physiological condition. In one embodiment, the
cold storage solution is first cooled to below 10.degree. C.using
an ice bath or other cooling means known in the art. It is typical
to inspect the cooled solution for any precipitates which may be
removed by filtration prior to use. Alternatively, the organ or
tissue to be preserved may be placed in the solution and then
cooled.
[0023] The invention further provides a preserved organ or
biological tissue comprising a cadaveric organ or tissue within a
cold storage solution of the invention in a deep hypothermic
condition or physiological condition. As discussed above, preferred
temperatures range from about 2-10.degree. C. in the deep
hypothermic condition and are about 37.degree. C., or room
temperature, in the physiological condition.
[0024] The invention is further explained by the following examples
of the invention as well as comparison examples. In all of the
examples, kidneys were procured from heart-beating donors and
preserved in a laboratory by cold storage preservation. Although
kidneys were used in the examples, any organ or biological tissue
may be preserved in the cold storage solution. Randomization was
accomplished as an open labeled, sequential analysis. All agents
were added immediately prior to vascular flush.
[0025] Data Collected
[0026] The following donor, preservation, and postoperative
recipient outcome data were collected for either Example 1 or 2:
donor age (D age, years), final donor creatinine (D Cr, mg/dL),
donor intraoperative urine output (U/O, mL), cold ischemic time
(CIT, hours), perfusion time (PT, hours), perfusate [Na+] (mM/100
g), perfusate [C 1-] (mM/100g), perfusate [K+] (mM/100 g),
perfusate [Ca++] (mM/100 g), perfusate pH, renal flow during MP
(FL, mL/min/100 g), renal resistance during MP (RES,
mmHg/(mL/min/100 g), recipient age (R age, years), recipient
discharge creatinine (R Cr, mg/dL), initial length of recipient
hospital stay (LOS, days), immediate graft function (IF, %) defined
as urine production exceeding 200 mL during the first 24
post-operative hours, delayed renal allograft graft function (DGF,
%) defined as the need for dialysis within the first 7 days
post-transplant, and present function (3 Mo or 1 Yr, %) defined as
3 month or one year post-operative graft status.
[0027] Method of Preservation
[0028] All cold stored kidneys subject to the above criteria were
flushed and cold stored at 4.degree. C. in 1L of either University
of Wisconsin (UW) solution (Viaspan.RTM. cold storage solution,
Dupont Pharma, Wilmington, Del.) or the Cold Organ Storage
(Perfusion) solution (OPS) of the present invention. The University
of Wisconsin solution, which is also the Control-UW solution, is
described in U.S. Pat. Nos. 4,798,824 and 4,879,283.
[0029] Statistical Analysis
[0030] All data are reported as mean values .+-.SEM unless
otherwise noted. Paired and unpaired student's t-tests were used
where appropriate. All statistical analyses were performed by
Statview 4.5 software (Abacus Concepts, Berkeley, Calif.)
EXAMPLE 1
[0031] Comparison of selected donor, preservation, and outcome
variables by method and type of organ preservation solution
(mean+/-SEM)
[0032] n=number of recipients
[0033] ns=not significant
4 p value University (unpaired OPS of Wisconsin Student's (n = 152)
(n = 160) t-test) Donor characteristics Donor age (y) 44.1 42.1 ns
Final serum creatinine (mg/dl) 1.1 0.9 ns Preservation
characteristics Cold ischemic time (h) 23 24 ns Outcome
characteristics Delayed graft function (%) 17 32 0.03 1 yr. graft
survival (%) 98 91 0.04
EXAMPLE 2
[0034] Comparison of selected donor, preservation, and outcome
characteristics by type of organ preservation solution--cold
storage formulation (mean +/-SEM)
[0035] SOD=superoxide dismutase-polyethylene glycol (25,000
units/L)
[0036] n=number of recipients
5 p value SOD (n = 48) Control-UW (unpaired (Embodiment solution
student's of Table 3) (n = 86) t-test) Donor Characteristics Donor
age (y) 39.2 +/- 10 44.0 +/- 12 0.8 Final serum creatinine (mg/dl)
1.1 +/- 0.6 0.9 +/- 0.5 0.65 Intraoperative urine 250 +/- 80 200
+/- 80 0.45 output (ml) Preservation characteristics Cold ischemic
time (h) 26 +/- 4 23 +/- 4 0.59 Outcome characteristics Immediate
function (%) 79 +/- 5 77 +/- 5 Delayed grant function (.degree.) 20
+/- 4 22 +/- 5 3 month function (%) 91 +/- 4 87 +/- 5
[0037] While the invention has been disclosed with reference to
certain preferred embodiments, numerous modifications, alterations,
and changes to the described embodiments are possible without
departing from the sphere and scope of the invention, as defined in
the appended claims and their equivalents thereof. For example,
although the detailed description may refer, at times, to only
organs, the terms "organs" and "organ" encompass all organs,
tissues and body parts that may be transplanted. Accordingly, it is
intended that the invention not be limited to the described
embodiments, but that it have the full scope defined by the
language of the following claims.
* * * * *