U.S. patent application number 14/290752 was filed with the patent office on 2014-11-27 for modified two-component gelation systems, methods of use and methods of manufacture.
This patent application is currently assigned to Abbott Cardiovascular Systems Inc.. The applicant listed for this patent is Abbott Cardiovascular Systems Inc.. Invention is credited to Shubhayu Basu, Gene Michal.
Application Number | 20140348800 14/290752 |
Document ID | / |
Family ID | 39417624 |
Filed Date | 2014-11-27 |
United States Patent
Application |
20140348800 |
Kind Code |
A1 |
Michal; Gene ; et
al. |
November 27, 2014 |
MODIFIED TWO-COMPONENT GELATION SYSTEMS, METHODS OF USE AND METHODS
OF MANUFACTURE
Abstract
A bioscaffolding can be formed from a mixture of gel components
of different gelation systems. For example, a bioscaffolding can be
formed by mixing at least two different components of at least two
different two-component gelation systems to form a first mixture
and by mixing at least two different components (other than the
components that make up the first mixture) of the at least two
different two-component gelation systems to form a second mixture.
A treatment agent, such as a cell type or a growth factor, can be
added to either the first mixture or the second mixture. In some
embodiments, the treatment agent is not added to either mixture.
The first mixture can be co-injected with the second mixture to
form a bioscaffolding in an infarct region for treatment
thereof.
Inventors: |
Michal; Gene; (San
Francisco, CA) ; Basu; Shubhayu; (Anaheim,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Abbott Cardiovascular Systems Inc. |
Santa Clara |
CA |
US |
|
|
Assignee: |
Abbott Cardiovascular Systems
Inc.
Santa Clara
CA
|
Family ID: |
39417624 |
Appl. No.: |
14/290752 |
Filed: |
May 29, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11561328 |
Nov 17, 2006 |
8741326 |
|
|
14290752 |
|
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Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
C12Y 304/21005 20130101;
A61K 38/363 20130101; A61L 27/3873 20130101; A61K 33/14 20130101;
A61P 7/00 20180101; A61K 33/20 20130101; A61K 38/1858 20130101;
A61P 9/10 20180101; A61K 35/28 20130101; A61K 33/14 20130101; A61K
33/20 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
38/4833 20130101; A61L 27/26 20130101; A61L 27/52 20130101; A61K
38/1858 20130101; A61K 2300/00 20130101; A61K 38/00 20130101; A61K
35/28 20130101 |
Class at
Publication: |
424/93.7 |
International
Class: |
A61K 35/28 20060101
A61K035/28; A61K 38/48 20060101 A61K038/48; A61K 38/18 20060101
A61K038/18; A61K 38/36 20060101 A61K038/36 |
Claims
1. A method comprising: combining a first component of a first
two-component gel system with a first component of a second
two-component gel system to form a first mixture; combining a
second component of a first two-component gel system with a second
component of a second two-component gel system to form a second
mixture; and adding a treatment agent to one of the first mixture
and the second mixture.
2. The method of claim 1, further comprising simultaneously
delivering the first mixture and the second mixture to a
post-myocardial infarct region.
3. The method of claim 1, wherein the treatment agent is a cell
type selected from the group consisting of localized cardiac
progenitor cells, mesenchymal stem cells, bone marrow derived
mononuclear cells, adipose stem cells, embryonic stem cells,
umbilical cord blood derived stem cells, smooth muscle cells and
skeletal myoblasts.
4. The method of claim 1, wherein the treatment agent is selected
from the group consisting of vasoendothelial growth factor,
fibroblast growth factor, Del 1, hypoxia inducing factor, monocyte
chemoattractant protein, nicotine, platelet derived growth factor,
insulin-like growth factor 1, transforming growth factor,
hepatocyte growth factor, estrogens, follistatin, proliferin,
prostaglandin E1 and E2, tumor necrosis factor, interleukin 8,
hematopoietic growth factors, erythropoietin, granulocyte-colony
stimulating factors and platelet-derived endothelial growth
factor.
5. The method of claim 1, wherein the first component of the first
two-gel component system is one of an alginate-collagen, an
alginate-laminin, an alginate-elastin, an
alginate-collagen-laminin, an alginate-hyaluronic acid, an alginate
and a self-assembled peptide.
6. The method of claim 1, wherein the first component of the second
two-component gel system is one of fibrinogen, a fibrinogen
derivative, a fibrinogen conjugate, sodium hyaluronate and a
self-assembled peptide.
7. The method of claim 1, wherein the second component of the first
two-component gel system is one of sodium chloride, calcium
chloride, barium chloride, strontium chloride and sucrose.
8. The method of claim 1, wherein the second component of the
second two-component gel system is one of sodium chloride, calcium
chloride, barium chloride, strontium chloride, thrombin and
sucrose.
9. The method of claim 1, wherein the first component of the first
two-component gel system and the second component of the second
two-component gel system is mixed in a one-to-one ratio.
10. The method of claim 1, wherein (a) the first component of the
first two-component gel system is an alginate-collagen solution and
the first component of the second two-component gel system is a
fibrinogen solution and (b) the second component of the first
two-component gel system is a thrombin solution and the second
component of the second two-component gel system is a 2.0% mass per
volume calcium chloride solution.
11. The method of claim 10, further comprising human mesenchymal
stem cells suspended in one of the first mixture or the second
mixture.
12. The method of claim 10, wherein the concentration of
alginate-collagen is in a range from between 0.5% mass per volume
to 1.0% mass per volume.
13. The method of claim 10, wherein the concentration of fibrinogen
is in a range from between 5.0% mass per volume to 7.0% mass per
volume.
14. The method of claim 10, wherein the concentration of thrombin
is in a range from between 9.5% mass per volume to 10.0% mass per
volume.
15. The method of claim 10, wherein the alginate-collagen and
fibrinogen are mixed in a one-to-one ratio.
16. The method of the claim 10, wherein the thrombin and calcium
chloride are mixed in a one-to-one ratio.
17. The method of claim 1, wherein (a) the first component of the
first two-component gel is one of alginate-collagen or fibrinogen
and the first component of the second two-component gel is a
self-assembled peptide and (b) the second component of the first
two-component gel is thrombin and the second component of the
second two-component gel is 2.0% mass per volume calcium
chloride.
18. The method of claim 17, further comprising platelet derived
growth factor combined with the self-assembled peptide in one of
the first mixture or the second mixture.
19. The method of claim 17, wherein the self-assembled peptide is
selected from the group consisting of RAD 16-II (SEQ ID NO: 1),
MAX-1 (SEQ ID NO: 2) and EAK16-II (SEQ ID NO: 3).
20. The method of claim 2, wherein the first mixture and the second
mixture is delivered with a dual bore delivery device.
21. The method of claim 2, wherein the first mixture and the second
mixture is delivered by one of (a) percutaneously or endoscopically
using a dual bore catheter or (b) in an open chest procedure using
a dual bore syringe.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of co-pending U.S. patent
application Ser. No. 11/561,328, filed Nov. 17, 2006, the
disclosure of which is incorporated herein by reference.
FIELD OF INVENTION
[0002] Post-myocardial infarction treatments and compositions.
SEQUENCE LISTING
[0003] An electronic copy of the Sequence Listing entitled
"5618P5128D_SeqList_ST25" is incorporated herein by reference. This
Sequence Listing consists of [SEQ ID NOs: 1-3].
BACKGROUND OF INVENTION
[0004] Ischemic heart disease typically results from an imbalance
between the myocardial blood flow and the metabolic demand of the
myocardium. Progressive atherosclerosis with increasing occlusion
of coronary arteries leads to a reduction in coronary blood flow.
"Atherosclerosis" is a type of arteriosclerosis in which cells
including smooth muscle cells and macrophages, fatty substances,
cholesterol, cellular waste product, calcium and fibrin build up in
the inner lining of a body vessel. "Arteriosclerosis" refers to the
thickening and hardening of arteries. Blood flow can be further
decreased by additional events such as changes in circulation that
lead to hypoperfusion, vasospasm or thrombosis.
[0005] Myocardial infarction (MI) is one form of heart disease that
often results from the sudden lack of supply of oxygen and other
nutrients. The lack of blood supply is a result of a closure of the
coronary artery (or any other artery feeding the heart) which
nourishes a particular part of the heart muscle. The cause of this
event is generally attributed to arteriosclerosis in coronary
vessels.
[0006] Formerly, it was believed that an MI was caused from a slow
progression of closure from, for example, 95% then to 100%.
However, an MI can also be a result of minor blockages where, for
example, there is a rupture of the cholesterol plaque resulting in
blood clotting within the artery. Thus, the flow of blood is
blocked and downstream cellular damage occurs. This damage can
cause irregular rhythms that can be fatal, even though the
remaining muscle is strong enough to pump a sufficient amount of
blood. As a result of this insult to the heart tissue, scar tissue
tends to naturally form.
[0007] Various procedures, including mechanical and therapeutic
agent application procedures, are known for reopening blocked
arteries. An example of a mechanical procedure includes balloon
angioplasty with stenting, while an example of a therapeutic agent
application includes the administration of a thrombolytic agent,
such as urokinase. Such procedures do not, however, treat actual
tissue damage to the heart. Other systemic drugs, such as
ACE-inhibitors and Beta-blockers, may be effective in reducing
cardiac load post-MI, although a significant portion of the
population that experiences a major MI ultimately develop heart
failure.
[0008] An important component in the progression to heart failure
is remodeling of the heart due to mismatched mechanical forces
between the infarcted region and the healthy tissue resulting in
uneven stress and strain distribution in the left ventricle. Once
an MI occurs, remodeling of the heart begins. The principle
components of the remodeling event include myocyte death, edema and
inflammation, followed by fibroblast infiltration and collagen
deposition, and finally scar formation from extra-cellular matrix
(ECM) deposition. The principle component of the scar is collagen
which is non-contractile and causes strain on the heart with each
beat. Non-contractility causes poor pump performance as seen by low
ejection fraction (EF) and akinetic or diskinetic local wall
motion. Low EF leads to high residual blood volume in the
ventricle, causes additional wall stress and leads to eventual
infarct expansion via scar stretching and thinning and border-zone
cell apoptosis. In addition, the remote-zone thickens as a result
of higher stress which impairs systolic pumping while the infarct
region experiences significant thinning because mature myocytes of
an adult are not regenerated. Myocyte loss is a major etiologic
factor of wall thinning and chamber dilation that may ultimately
lead to progression of cardiac myopathy. In other areas, remote
regions experience hypertrophy (thickening) resulting in an overall
enlargement of the left ventricle. This is the end result of the
remodeling cascade. These changes also correlate with physiological
changes that result in increase in blood pressure and worsening
systolic and diastolic performance.
SUMMARY OF INVENTION
[0009] A bioscaffolding can be formed from a mixture of gel
components of different gelation systems. In some embodiments, a
bioscaffolding can be formed by mixing at least two different
components (which do not gel upon mixing) of at least two different
two-component gelation systems to form a first mixture and by
mixing at least two different components (other than the components
that make up the first mixture and which do not gel upon mixing) of
the at least two different two-component gelation systems to form a
second mixture. A treatment agent, such as a cell type or a growth
factor, can be added to either the first mixture or the second
mixture. The first mixture can then be co-injected with the second
mixture to form a bioscaffolding in an infarct region for treatment
thereof. The first and second mixtures can be co-injected with a
dual-lumen delivery device, which can include, but are not limited
to, a dual syringe, a dual-needle left-ventricle injection device,
a dual-needle transvascular wall injection device and the like.
[0010] In some embodiments, a bioscaffolding can be formed by
mixing at least two different gelation components (which do not gel
upon mixing) to form a first mixture. A treatment agent, such as a
cell type or a growth factor, can be added to the first mixture.
The first mixture can then be co-injected with a second gelation
component to form a bioscaffolding on or within an infarct region
for treatment thereof. The first mixture and the gelation component
can be co-injected with, a dual-lumen delivery device, which can
include, but are not limited to, a dual syringes a dual-needle
left-ventricle injection device, a dual-needle transvascular wall
injection device and the like.
BRIEF DESCRIPTION OF DRAWINGS
[0011] FIGS. 1A-1B illustrate the progression of heart damage once
the build-up of plaque in an artery induces an infarct to
occur.
[0012] FIG. 2 illustrates an embodiment of a dual bore delivery
device.
[0013] FIGS. 3A-3B illustrate an alternative embodiment of a dual
bore delivery device.
[0014] FIGS. 4A-4C illustrate a second alternative embodiment of a
dual bore delivery device.
DETAILED DESCRIPTION
[0015] FIGS. 1A-1B illustrate the progression of heart damage once
the build-up of plaque induces an infarct to occur. FIG. 1A
illustrates a site 10 where blockage and restricted blood flow can
occur from, for example, a thrombus or embolus. FIG. 1B illustrates
resultant damage area 20 to the left ventricle that can result from
the lack of oxygen and nutrient flow carried by the blood to the
inferior region left of the heart. The damage area 20 will likely
undergo remodeling, and eventually scarring, resulting in a
non-functional area.
[0016] Bioscaffoldings formed of two components and applied in situ
to the left heart ventricle can be used to treat post-myocardial
infarction tissue damage. "Bioscaffolding" and "two-component
gelation system" and "gelation system" are hereinafter used
interchangeably. Examples of two-component gelation systems
include, but are not limited to, alginate construct systems, fibrin
glues and fibrin glue-like systems, self-assembled peptides and
combinations thereof. Each component of the two-component gelation
system may be co-injected to an infarct region by a dual-lumen
delivery device. Examples of dual-lumen delivery devices include,
but are not limited to, a dual syringe, dual-needle left-ventricle
injection devices, dual-needle transvascular wall injection devices
and the like.
[0017] In some embodiments, at least one cell type may be
co-injected with at least one component of the two-component
gelation system to an infarct region. In some embodiments, the
cells may be mixed with at least one component of the two-component
gelation system before the two-components are co-injected to the
infarct region. Examples of cell types, include, but are not
limited to, localized cardiac progenitor cells, mesenchymal stem
cells, bone marrow derived mononuclear cells, adipose stem cells,
embryonic stem cells, umbilical cord blood derived stem cells,
smooth muscle cells or skeletal myoblasts.
[0018] In some applications, the two-component gelation system
includes a fibrin glue. Fibrin glue consists of two main
components, fibrinogen and thrombin. Fibrinogen is a plasma
glycoprotein of about 340 kiloDaltons (kDa) in its endogenous
state. Fibrinogen is a symmetrical dimer comprised of six paired
polypeptide chains, alpha, beta and gamma chains. On the alpha and
beta chains, there is a small peptide sequence called a
fibrinopeptide which prevent fibrinogen from spontaneously forming
polymers with itself. In some embodiments, fibrinogen is modified
with proteins. Thrombin is a coagulation protein. When combined in
equal volumes, thrombin converts the fibrinogen to fibrin by
enzymatic action at a rate determined by the concentration of
thrombin. The result is a biocompatible gel which gelates when
combined at the infarct region. Fibrin glue can undergo gelation
between about 5 to about 60 seconds. Examples of other fibrin
glue-like systems include, but are not limited to, Tisseel.TM.
(Baxter), Beriplast P.TM. (Aventis Behring), Biocol.RTM. (LFB,
France), Crosseal.TM. (Omrix Biopharmaceuticals, Ltd.), Hemaseel
HMN.RTM. (Haemacure Corp.), Bolheal (Kaketsuken Pharma, Japan) and
CoStasis.RTM. (Angiotech Pharmaceuticals).
[0019] In some applications, the two-component gelation system
includes self-assembled peptides. Self-assembled peptides generally
include repeat sequences of alternating hydrophobic and hydrophilic
amino acid chains. The hydrophilic amino acids are generally
charge-bearing and can be anionic, cationic or both. Examples of
cationic amino acids are lysine and arginine. Examples of anionic
amino acids are aspartic acid and glutamic acid. Examples of
hydrophobic amino acids are alanine, valine, leucine, isoleucine or
phenylalanine Self-assembled peptides can range from 8 to 40 amino
acids in length and can assemble into nanoscale fibers under
conditions of physiological pH and osmolarity. In sufficient
concentration and over time, the fibers can assemble into an
interconnected structure that appears macroscopically as a gel.
Self-assembled peptides typically undergo gelation between several
minutes to several hours. Examples of self-assembled peptides
include, but are not limited to: AcN-RARADADARARADADA-CNH.sub.2
(SEQ ID NO: 1) (RAD 16-II) wherein R is arginine, A is alanine, D
is aspartic acid, and Ac indicates acetylation;
VKVKVKVKV-PP-TKVKVKVKV-NH.sub.2 (SEQ ID NO: 2) (MAX-1) wherein V is
valine, K is lysine and P is proline; and
AcN-AEAEAKAKAEAEAKAK-CNH.sub.2 (SEQ ID NO: 3) wherein A is alanine,
K is lysine and E is glutamic acid (EAK 16-II). Self-assembled
peptides show good cytocompatibility, as represented by cell
adhesion, cell migration and proliferation.
[0020] In some applications, the two-component gelation system is
an alginate construct system. One component may be an alginate
conjugate (or alginate alone) which can include alginate and a
protein constituent. The second component may be a salt. Examples
of alginate conjugates can include, but are not limited to,
alginate-collagen, alginate-laminin, alginate-elastin,
alginate-collagen-laminin and alginate-hyaluronic acid in which the
collagen, laminin, elastin, collagen-laminin or hyaluronic acid is
covalently bonded (or not bonded) to alginate. Examples of salts
which can be used to gel the alginate constructs include, but are
not limited to, calcium chloride (CaCl.sub.2), barium chloride
(BaCl.sub.2) or strontium chloride (SrCl.sub.2).
[0021] In one embodiment, the alginate construct is
alginate-gelatin. The molecular weight of the gelatin may be in the
approximate range of 5 kDa to 100 kDa. The relatively low molecular
weight of gelatin offers processing advantages in that it is more
soluble and has lower viscosity than hydrogels of higher molecular
weight. Another advantage of gelatin is that it contains from 1 to
4 RGD (arginine-glycine-aspartic acid peptide sequence) sites per
molecule. RGD is a common cell adhesion ligand and would increase
the retention of cells within the infarct zone where the
bioscaffolding is formed. The cells retained by the RGD sites may
be cells co-injected with the bioscaffolding components or
dispersed throughout a component of the system.
[0022] The gelatin may be a porcine gelatin or a recombinant human
gelatin. The porcine gelatin is a hydrolyzed type 1 collagen
extracted from porcine skin. In one embodiment, the molecular
weight of the porcine gelatin is approximately 20 kDa. The human
gelatin is produced by bacteria using human genetic material. The
human recombinant gelatin is equivalent to the porcine gelatin but
may reduce the likelihood of an immune response when injected into
an infarct region of a human subject.
[0023] Alginate is a linear polysaccharide derived from seaweed and
contains mannuronic (M) and gluronic acid (G), presented in both
alternating blocks and alternating individual residues. It is
possible to use some of the carboxyl groups of the alginate as
sites to graft useful cell adhesion ligands, such as collagen,
laminin, elastin and other peptide fragments of the ECM matrix,
forming an alginate conjugate, because alginate does not have RGD
groups for cell retention.
[0024] An alginate-gelatin conjugate is valuable because it
combines characteristics of alginate with characteristics of
gelatin, which include, but are not limited to, RGD sites and
immunocompatibility. Characteristics of alginate include rapid,
almost instantaneous gelation, and an inflammation stimulating
effect. The alginate-gelatin conjugate can be formed of
approximately 1% to 30% and more particularly approximately 10% to
20% gelatin (either porcine or human recombinant) and approximately
80% to 90% alginate. The relatively lower proportion of
alginate-gelatin conjugate is used to retain gelation capacity once
combined with pure alginate because the alginate carboxyl groups of
alginate that cause the gelation may be bound up in the
alginate-gelatin conjugate.
[0025] Two-component gelation systems exhibit different
characteristics relative to one another including, but not limited
to, pore size, storage modulus and gelation time. The gelation
system behaves as a sieving media and therefore includes small
pores. "Pore size" refers to small, vacuous openings within the
gel. "Storage modulus" refers to the strength or the stiffness of
the material upon gelation. Storage modulus can be measured by a
rheometric instrument. "Gelation time" refers to the kinetics of
gelation. Alginate constructs can gel within about 1 second, while
fibrin glues can gel between about 5 seconds and about 60 seconds.
Self-assembled peptides typically undergo gelation between several
minutes to several hours.
[0026] In embodiments in which cells are co-injected with the
two-component gelation system, or mixed with one component before
combining the two components, the gelation system can exhibit
different characteristics relative to one another relating to the
cells. Such characteristics can include, but are not limited to,
morphology of the cells, cell survivability, encapsulation
efficiency and/or cell concentration. "Morphology" refers to the
physical structure of the cells. In the case of hMSC, the natural
morphology is a flattened spindle-shaped morphology. "Cell
survivability" is the amount of time that the cells remain viable
within the gel post-injection. "Encapsulation efficiency" refers to
the fraction of the initial number of cells in suspension that are
entrapped within the gel. "Cell concentration" is the encapsulation
efficiency divided by the volume of gel formed.
[0027] A characteristic which affects the encapsulation efficiency
is the difference in viscosity (.eta.) of the two components. If
the difference in viscosity between the two components of the
gelation system is large, then the encapsulation efficiency is high
only when cells are in the high viscosity component. However, if
the viscosity of each individual component is lowered without
compromising the gelation kinetics, the encapsulation efficiency
increases dramatically. For a catheter-based delivery system, low
viscosity components are very helpful for successful delivery. A
successful application of the two components (which are in solution
before delivery) can be dependent upon low viscosity of the
individual components.
Modified Gelation Systems
[0028] In some embodiments, a bioscaffolding can be formed from a
mixture of gel components of different gelation systems. For
example, a bioscaffolding can be formed by mixing at least two
different components (which do not gel upon mixing under standard
cath lab process conditions) of at least two different
two-component gelation systems to form a first mixture, and, by
mixing at least two different components (other than the components
that make up the first mixture and which do not gel upon mixing
under standard cath lab process conditions) of the at least two
different two-component gelation systems to form a second mixture.
"Gel" refers to the phase change from a liquid to a solid upon the
combination of two different components or two different mixtures.
A treatment agent, such as a cell type or a growth factor, can be
added to either the first mixture or the second mixture. The first
mixture can then be co-injected with the second mixture to form a
bioscaffolding in an infarct region for treatment thereof. In some
embodiments, a bioscaffolding can be formed by mixing at least two
different gelation components (which do not gel upon mixing under
standard cath lab process conditions) to form a first mixture. A
treatment agent, such as a cell type or a growth factor, can be
added to the first mixture. The first mixture can then be
co-injected with a gelation component to form a bioscaffolding on
an infarct region for treatment thereof. In some embodiments, the
treatment agent can be co-injected with the first mixture or the
gelation component without first interdispersing the treatment
agent within the first mixture or the gelation component.
[0029] In some embodiments, an alginate construct system can
include an alginate-gelatin solution as a first component and a
calcium chloride solution as a second component. In some
embodiments, human mesenchymal stems cells (hMSC) are suspended in
one component of the gelation system. hMSC are thought to be
capable of both self renewal and differentiation into bone,
cartilage, muscle, tendon and fat. hMSC also give rise to a variety
of mature cell types via a step-wise maturation process called
mesengenesis. The natural morphology of hMSC is elongated and
spindle shaped. The gelatin provides RGD sites for cellular
adhesion i.e. adhesion of hMSC. Alginate construct systems exhibit
rapid gelling kinetics. When combined, alginate-gelatin and calcium
chloride gel to form a bioscaffolding in less than 1 second. The
resulting gel has a storage modulus of approximately 1 kiloPascal.
In application, cell survivability has been observed up to at least
12 days. Encapsulation efficiency is approximately 99%. However,
the small pore size of alginate construct systems, which is from
about 2 nm to about 500 nm, can lead to low cell spreadability as
observed by the round morphology of the hMSC cells over time. "Cell
spreading" refers to the naturally occurring morphology of cells.
Advantages of alginate construct systems include, but are not
limited to, enhanced immune response (a controlled foreign body
response) to effect positive remodeling of the injured myocardium,
and immunoprotectivity, by shielding via its small pore size, the
encapsulated cells from this enhanced immune response (protected
from host immune response), instantaneous gelation kinetics,
substantial or complete non-adherence to a needle when injected,
and long term cell viability. Furthermore, alginate construct
systems degrade slowly (at least 8 weeks in vivo).
[0030] Fibrin glue can include fibrinogen (modified or not modified
with protein constituents) as a first component and thrombin as a
second component. In some embodiments, human mesenchymal stems
cells (hMSC) are suspended in one component of the gelation system.
Fibrin glue systems exhibit fast gelling kinetics, but not as rapid
as alginate construct systems. When combined, fibrinogen and
thrombin gel to form a bioscaffolding in about 5 seconds to about
10 seconds. The resulting gel has a storage modulus of
approximately 3 kiloPascals which is higher than that of alginate
construct systems. A higher storage modulus may improve mechanical
reinforcement at the infarct region. In application, cell
survivability has been observed up to at least 12 days. The pore
size of fibrin glue systems is from about 1.5 microns to about 2.5
microns and can lead to high cell spreadability of hMSC cells. That
is, hMSC cells have been observed to have an elongate and stellate
morphology which is more natural to their endogenous state when
compared to the morphology observed in alginate construct systems
alone. Advantages of fibrin glue include, but are not limited to,
material strength, promotion of angiogenesis, good
cytocompatibility (compatible with cell growth), good cell
morphology and high cell proliferation in fibrinogen.
[0031] The following is a table comparing experimental data and
characteristics of alginate construct and fibrin glue systems:
TABLE-US-00001 TABLE 1 1% Alginate-collagen 100% Tiseel .TM.
Characteristic in 2% CaCl.sub.2 (no dilution) Pore size 200-500 nm
1.5-2.5 .mu.m Storage modulus 1 kPa 3 kPa Gelation kinetics 1
second 5-10 seconds Encapsulation 99.26 .+-. 0.61 93.71 .+-. 0.61
efficiency Morphology spherical elongated, spindle-shaped within 24
hours Cell 12 days to 6 months at least 12 days survivability
Degradation time 8 weeks 2 weeks in vivo
[0032] In some embodiments, a bioscaffolding is formed from mixing
components of at least two gelation systems. For example, a first
component of a first two-component gel and a first component of a
second two-component gel can be combined to form a first mixture. A
second component of a first two-component gel and a second
component of the second two-component gel can be combined to form a
second mixture. Cells can be suspended within either the first
mixture or the second mixture. When the two mixtures are combined,
a bioscaffolding including at least some advantageous
characteristics of both gelation systems can be realized. In some
embodiments, a bioscaffolding can be formed by mixing at least two
different gelation components to form a first mixture. When the
first mixture is combined with a gelation salt, a bioscaffolding
including at least some advantageous characteristics of the
individual components can be realized. It should be appreciated
that a number of different combinations of gelation components can
be mixed together in different ratios to accentuate various
advantageous characteristics of the individual gelation systems.
Furthermore, the concentration of the individual components, either
singly or combined, can influence certain characteristics of the
bioscaffolding, such as viscosity and encapsulation efficiency.
Alginate Construct Systems and Fibrin Glues
Experiment 1
[0033] hMSC were suspended in a 1% alginate-collagen solution at
1.43.times.10.sup.7 cells/mL. 200 microliters of the
alginate-collagen solution was combined with 200 microliters of a
thrombin solution containing 40 mM CaCl.sub.2. Encapsulation
efficiency of the resulting gel was measured at 99.16.+-.0.007%.
Encapsulation efficiency was measured as the difference between the
initial number of cells in the suspension and the number of cells
remaining in the supernatant divided by the initial number of cells
and expressed as a percentage. On day 3, the cells exhibited a
round morphology. On day 7 and 12, the cells continued to exhibit a
round morphology. Cells were analyzed by a molecular probe
LIVE/DEAD.RTM. Assay available from Invitrogen, Inc. The viscosity
of the suspension component was measured at approximately 33
centipoise (cp).
Experiment 2
[0034] hMSC were suspended in a thrombin solution containing 40 mM
of CaC12 at a concentration of 2.12.times.10.sup.7 cells/mL. A 1%
solution of alginate-collagen was combined with the thrombin
solution in a 1:1 ratio (2004:2004). Encapsulation efficiency of
the resulting gel was measured at 26.47.+-.5.5%. The viscosity of
the suspension component was measured at approximately 7 cp.
Experiment 3
[0035] A fibrin glue kit Tisseel.TM. including modified fibrinogen
and thrombin was obtained from Baxter. hMSC were suspended in a
thrombin solution at a concentration of 2.96.times.10.sup.7
cells/mL. 200 microliters of the thrombin solution containing cells
was combined with the fibrinogen solution in a 1:1 ratio.
Encapsulation efficiency of the resulting gel was measured at
93.71.+-.3.48%. On day 3, the cells exhibited an elongated, spindle
shaped morphology. On day 7 & 12 the cells continued to exhibit
similar elongated and spindle shaped morphology and there was
visible but no quantitative evidence of cell proliferation. Cells
were analyzed by a molecular probe LIVE/DEAD.RTM. Assay available
from Invitrogen, Inc. The viscosity of the suspension component was
measured at approximately 7 cp.
Experiment 4
[0036] A fibrin glue kit Tisseel.TM. including modified fibrinogen
and thrombin was obtained from Baxter. Fibrinogen was reconstituted
as directed in accompanying instructions and then further diluted
with water to half of its original concentration. hMSC were
suspended in the reconstituted, full strength thrombin solution at
a concentration of 2.96.times.10.sup.7 cells/mL. 200 microliters of
the thrombin solution containing hMSC was combined with the diluted
fibrinogen solution in a 1:1 ratio. Encapsulation efficiency of the
resulting gel was measured at 98.07.+-.1.6%. The viscosity of the
suspension component was measured at approximately 7 cp.
Experiment 5
[0037] A fibrin glue kit Tisseel.TM. including modified fibrinogen
and thrombin was obtained from Baxter. Fibrinogen was reconstituted
as directed in accompanying instructions and then further diluted
with water to half of its original concentration. hMSC were
suspended in the diluted fibrinogen solution at a concentration of
2.96.times.10.sup.7 cells/mL. 200 microliters of the diluted
fibrinogen solution containing hMSC was combined with the thrombin
solution in a 1:1 ratio. Encapsulation efficiency of the resulting
gel was measured at 97.12.+-.1.73%. The viscosity of the suspension
component was measured at approximately 5 cp.
Experiment 6
[0038] A fibrin glue kit Tisseel.TM. including modified fibrinogen
and thrombin was obtained from Baxter. Fibrinogen was reconstituted
as directed and then further diluted with water to half of its
original concentration. The diluted fibrinogen solution was mixed
with a 0.5% alginate-collagen solution in a 1:1 ratio to form a
first mixture. hMSC were suspended in a thrombin solution (which
contains 40 mM CaCl.sub.2) at a concentration of
2.96.times.10.sup.7 cells/mL. 200 microliters of the first mixture
was combined with the thrombin in a 1:1 ratio. Encapsulation
efficiency of the resulting gel was measured at 91.39.+-.6.78%. The
viscosity of the suspension component was measured at approximately
7 cp.
Experiment 7
[0039] A fibrin glue kit Tisseel.TM. including modified fibrinogen
and thrombin was obtained from Baxter. Fibrinogen was reconstituted
as directed and then further diluted with water to half of its
original concentration. The diluted fibrinogen solution was mixed
with a 0.5% alginate-collagen solution in a 1:1 ratio to form a
first mixture. hMSC were suspended in the first mixture at a
concentration of 5.51.times.10.sup.7 cells/mL. The first mixture
was combined with the second mixture comprising thrombin and a 2%
CaCl.sub.2 solution in a 1:1 ratio. Encapsulation efficiency of the
resulting gel was measured at 99.42.+-.0.12%. The viscosity of the
suspension component was measured at approximately 6 cp.
[0040] Table 2 summarizes the results of a table of experimental
data showing the various characteristics of combinations of
alginate construct and fibrin glue systems:
TABLE-US-00002 TABLE 2 First Second Component Viscosity component/
component/ Encap- in which of first second sulation cells component
mixture mixture Efficiency suspended (centipoise) 1 1% Thrombin
99.16 .+-. 0.007 alginate- 33.28 .+-. 2.3 alginate- containing
collagen collagen 40 mM CaC1.sub.2 2 1% Thrombin 26.47 .+-. 5.5
thrombin 7.32 .+-. 2.6 alginate- containing collagen 40 mM
CaCl.sub.2 3 Un- Thrombin 93.71 .+-. 3.48 thrombin 7.32 .+-. 2.6
diluted containing fibrinogen 40 mM CaCl.sub.2 4 Di- Thrombin 98.07
.+-. 1.6 thrombin 7.32 .+-. 2.6 luted containing fibrinogen 40 mM
CaCl.sub.2 5 Di- Thrombin 97.12 .+-. 1.73 fibrinogen 5.4 .+-. 2.8
luted containing fibrinogen 40 mM CaC1.sub.2 6 Di- Thrombin 91.39
.+-. 6.78 thrombin 7.32 .+-. 2.6 luted containing fibrinogen + 40
mM 0.5% - CaCl.sub.2 alginate collagen 7 Diluted Thrombin 99.42
.+-. 0.12 5% 6.5 .+-. 3.2 fibrinogen + containing fibrinogen + 0.5%
40 mM 0.5% alginate- CaC1.sub.2 alginate- collagen collagen
[0041] In one embodiment, a bioscaffolding can be formed by mixing
components of more than one gelation system. For example,
alginate-gelatin (of an alginate construct system) can be mixed
with fibrinogen (of a fibrin glue system) in a 1:1 ratio to form a
first mixture. Thrombin (of a fibrin glue system) already contains
40 mM calcium chloride (which is required to gel an alginate
construct system) to form a second mixture. Experiments 6 and 7 may
represent such a combination. hMSC can be added to an individual
component or to the first or second mixture.
[0042] Experiment 3 illustrates that, in a standard two-component
gelation system which includes a first component (alginate) or a
second component (thrombin) only, if the hMSC are suspended in a
low viscosity component, i.e., thrombin (.eta.=7.32.+-.2.6),
encapsulation efficiency is very low (E=26.47.+-.5.5%) (i.e.,
alginate-collagen). However, note that in these experiments, the
viscosity of the other component is high and there is a wide
mismatch between the two viscosities e.g., the viscosity of 1%
alginate-collagen is 33.2%+2.3%. Further, alginate has faster
gelation kinetics than the other systems. In experiments 6 and 7
however, even though the hMSC were suspended in a low viscosity
component or mixture, the resulting gel showed high encapsulation
efficiency. It is theorized that this result is due to the
viscosity of the other component being low as well and that there
was not much of a mismatch in viscosities between the two
components, thus allowing good mixing. Further, the mixed systems
gel slower allowing more cell diffusion from the lower viscosity
component to the gel. Thus, it has been shown that even if the
individual components in which the hMSC is suspended have low
viscosities (before gelation), the resulting gel can still exhibit
a high encapsulation efficiency (after gelation). This is highly
advantageous, as lower viscosity may result in a more successful
delivery with a catheter-based delivery system because, for
example, such a system requires less force for injection into the
infarct region and higher flow rates can be achieved. Additionally,
lower viscosity solutions may result in less residual within the
lumens of a delivery system. The combination also may provide a
high encapsulation efficiency while allowing hMSC to achieve a
natural state of morphology, i.e., elongated and spindle shaped.
Thus, such a bioscaffolding may include at least some advantageous
characteristics of the individual components comprising the
two-component gelation systems.
[0043] In another embodiment, a bioscaffolding can be formed by
mixing alginate-gelatin with sodium-hyaluronate and gelled with
calcium chloride. The hyaluronate will be immobilized by chain
entanglement and provide attachment ligands for stem cells bearing
the CD44 receptor, e.g., human mesenchymal stem cells. Appropriate
formulations can have 50% to 99% of a 0.5% to 1.0% solution of
alginate-gelatin combined with 1% to 50% of a 0.05% to 1% solution
of sodium hyaluronate (Genzyme Biosurgery, MA). The mixture can be
gelled by the addition of an equal volume of a 0.5% to 1.5% of
calcium chloride dihydrate.
[0044] In another embodiment, a bioscaffolding can be formed by
mixing reconstituted lyophilized peptide (with buffer) with
alginate and gelled with calcium chloride. Examples of peptides
include self-assembled peptides, such as RAD 16-II (SEQ ID NO: 1),
MAX-1 (SEQ ID NO: 2) and EAK16-II (SEQ ID NO: 3). These peptides
gel when exposed to conditions of physiological or greater
osmolarity, at neutral pH. However, gelation kinetics are
relatively slow, ranging from minutes to hours. Introduction of
these peptides as a sole scaffold would result in an intercalated
structure, at best, due to the slow gelation. Addition of alginate
can provide rapid gelation kinetics, thus preventing dissipation of
the peptide into tissue.
[0045] In some embodiments, a self-assembled peptide (SAP)
component can be mixed with a growth factor to form a first
mixture. Examples of self-assembled peptides include RAD 16-II (SEQ
ID NO: 1), MAX-1 (SEQ ID NO: 2) and EAK16-II (SEQ ID NO: 3).
Examples of growth factors in include, but are not limited to,
isoforms of vasoendothelial growth factor (VEGF), fibroblast growth
factor (FGF, e.g. beta-FGF), Del 1, hypoxia inducing factor (HIF
1-alpha), monocyte chemoattractant protein (MCP-1), nicotine,
platelet derived growth factor (PDGF), insulin-like growth factor 1
(IGF-1), transforming growth factor (TGF alpha), hepatocyte growth
factor (HGF), estrogens, follistatin, proliferin, prostaglandin E1
and E2, tumor necrosis factor (TNF-alpha), interleukin 8 (11-8),
hematopoietic growth factors, erythropoietin, granulocyte-colony
stimulating factors (G-CSF) and platelet-derived endothelial growth
factor (PD-ECGF). The first mixture can be combined with a
component of a two-component gelation system, such as an alginate
construct system, a fibrin glue or a polymer system, or any
combination thereof. In some embodiments, human mesenchymal stem
cells can be added to the system.
[0046] In one embodiment, the self-assembled peptide is RAD 16-II
(SEQ ID NO: 1) and the growth factor is PDGF or a derivative
thereof and combines to form a first mixture. It has been shown
that PDGF mediates cardiac microvascular endothelial cell
communication with cardiomyocytes and it is anticipated that an
application of PDGF can restore damaged endothelial PDGF-regulated
angiogenesis and enhance post-ischemic neovascularization in an
infarct region. When combined with RAD 16-II (SEQ ID NO: 1), PDGF
binds to RAD 16-II (SEQ ID NO: 1) through weak molecular
interactions. In some applications, PDGF remains viable for
approximately fourteen days when combined with RAD 16-II (SEQ ID
NO: 1) when applied to an infarct region. However, it is
anticipated that the slow kinetics of the self-assembled peptide,
i.e., minutes to hours, will cause significant leakage, backflow
and dissipation before the peptide can form a nanofiber
bioscaffolding at the infarct region.
[0047] In some embodiments, the first mixture (comprising RAD 16-II
(SEQ ID NO: 1) and PDGF) can be combined with any one component of
fibrin glue or an alginate construct. The rapid kinetics of both
fibrin glue and alginate constructs can counteract the slow
kinetics of the SAP-PDGF construct. It is therefore anticipated
that leakage and dissipation at the infarct region can be reduced
by combining the SAP-PDGF construct with any one component of
fibrin glue or an alginate construct.
Delivery Systems
[0048] Devices which can be used to deliver modified or combined
components of the gelation systems include, but are not limited to,
dual-needle left-ventricle injection devices, dual-needle
transvascular wall injection devices and dual syringes. Methods of
access to use the minimally invasive (i.e., percutaneous or
endoscopic) injection devices include access via the femoral artery
or the sub-xiphoid. "Xiphoid" or "xiphoid process" is a pointed
cartilage attached to the lower end of the breastbone or sternum,
the smallest and lowest division of the sternum. Both methods are
known by those skilled in the art.
[0049] FIG. 2 illustrates an embodiment of a dual syringe device
which can be used to deliver the compositions of the present
invention. Dual syringe 400 can include first barrel 410 and second
barrel 420 adjacent to one another and connected at a proximal end
455, distal end 460 and middle region 465 by plates 440, 445 and
450, respectively. In some embodiments, barrels 410 and 420 can be
connected by less than three plates. Each barrel 410 and 420
includes plunger 415 and plunger 425, respectively. Barrels 410 and
420 can terminate at a distal end into needles 430 and 435,
respectively, for extruding a substance. In some embodiments,
barrels 410 and 420 can terminate into cannula protrusions for
extruding a substance. Barrels 410 and 420 should be in close
enough proximity to each other such that the substances in each
respective barrel are capable of mixing with one another to form a
bioscaffolding in the treatment area, e.g., a post-infarct
myocardial region. Dual syringe 400 can be constructed of any metal
or plastic which is minimally reactive or completely unreactive
with the formulations described in the present invention. In some
embodiments, dual syringe 400 includes a pre-mixing chamber
attached to distal end 465.
[0050] In some applications, first barrel 410 can include a first
mixture of a modified two-component gelation system and second
barrel 420 can include a second mixture of a modified two-component
gelation system according to any of the embodiments described
previously. A therapeutic amount of the resulting gel is between
about 25 .mu.L to about 200 .mu.L, preferably about 50 .mu.L. Dual
syringe 400 can be used during, for example, an open chest surgical
procedure.
[0051] FIGS. 3A-3B illustrate an embodiment of a dual-needle
injection device which can be used to deliver the compositions of
the present invention. Delivery assembly 500 includes lumen 510
which may house delivery lumens, guidewire lumens and/or other
lumens. Lumen 510, in this example, extends between distal portion
505 and proximal end 515 of delivery assembly 500.
[0052] In one embodiment, delivery assembly 500 includes first
needle 520 movably disposed within delivery lumen 530. Delivery
lumen 530 is, for example, a polymer tubing of a suitable material
(e.g., polyamides, polyolefins, polyurethanes, etc.). First needle
520 is, for example, a stainless steel hypotube that extends a
length of the delivery assembly. First needle 520 includes a lumen
with an inside diameter of, for example, 0.08 inches (0.20
centimeters). In one example for a retractable needle catheter,
first needle 520 has a needle length on the order of about 40
inches (about 1.6 meters) from distal portion 505 to proximal
portion 515. Lumen 510 also includes auxiliary lumen 540 extending,
in this example, co-linearly along the length of the catheter (from
a distal portion 505 to proximal portion 515). Auxiliary lumen 540
is, for example, a polymer tubing of a suitable material (e.g.,
polyamides, polyolefins, polyurethanes, etc.). At distal portion
505, auxiliary lumen 540 is terminated at a delivery end of second
needle 550 and co-linearly aligned with a delivery end of needle
520. Auxiliary lumen 540 may be terminated to a delivery end of
second needle 550 with a radiation-curable adhesive, such as an
ultraviolet curable adhesive. Second needle 550 is, for example, a
stainless steel hypotube that is joined co-linearly to the end of
main needle 520 by, for example, solder (illustrated as joint 555).
Second needle 550 has a length on the order of about 0.08 inches
(0.20 centimeters). FIG. 3B shows a cross-sectional front view
through line A-A' of delivery assembly 500. FIG. 3B shows main
needle 520 and second needle 550 in a co-linear alignment.
[0053] Referring to FIG. 3A, at proximal portion 515, auxiliary
lumen 540 is terminated to auxiliary side arm 560. Auxiliary side
arm 560 includes a portion extending co-linearly with main needle
520. Auxiliary side arm 560 is, for example, a stainless steel
hypotube material that may be soldered to main needle 520
(illustrated as joint 565). Auxiliary side arm 560 has a co-linear
length on the order of about, in one example, 1.2 inches (3
centimeters).
[0054] The proximal end of main needle 520 includes adaptor 570 for
accommodating a substance delivery device. Adaptor 570 is, for
example, a molded female luer housing. Similarly, a proximal end of
auxiliary side arm 560 includes adaptor 580 to accommodate a
substance delivery device (e.g., a female luer housing).
[0055] The design configuration described above with respect to
FIGS. 3A-3B is suitable for introducing modified two-component gel
compositions of the present invention. For example, a gel may be
formed by a combination (mixing, contact, etc.) of a first mixture
of a modified two-component gelation system and a second mixture of
a modified two-component gelation system. Representatively, a first
mixture may be introduced by a one cubic centimeters syringe at
adaptor 570 through main needle 520. At the same time or shortly
before or after, a second mixture may be introduced with a one
cubic centimeter syringe at adaptor 580. When the first and second
components combine at the exit of delivery assembly 500 (at an
infarct region), the materials combine (mix, contact) to form a
bioerodable gel.
[0056] FIGS. 4A-4C illustrate an alternative embodiment of a
dual-needle injection device which can be used to deliver
two-component gel compositions of the present invention. In
general, the catheter assembly 600 provides a system for delivering
substances, such as modified two-component gel compositions, to or
through a desired area of a blood vessel (a physiological lumen) or
tissue in order to treat a myocardial infarct region. The catheter
assembly 600 is similar to the catheter assembly described in
commonly-owned, U.S. Pat. No. 6,554,801, titled "Directional Needle
Injection Drug Delivery Device", which is incorporated herein by
reference.
[0057] In one embodiment, catheter assembly 600 is defined by
elongated catheter body 650 having proximal portion 620 and distal
portion 610. Guidewire cannula 670 is formed within catheter body
(from proximal portion 610 to distal portion 620) for allowing
catheter assembly 600 to be fed and maneuvered over guidewire 680.
Balloon 630 is incorporated at distal portion 610 of catheter
assembly 600 and is in fluid communication with inflation cannula
660 of catheter assembly 600.
[0058] Balloon 630 can be formed from balloon wall or membrane 635
which is selectively inflatable to dilate from a collapsed
configuration to a desired and controlled expanded configuration.
Balloon 630 can be selectively dilated (inflated) by supplying a
fluid into inflation cannula 660 at a predetermined rate of
pressure through inflation port 665 (located at proximal end 620).
Balloon wall 635 is selectively deflatable, after inflation, to
return to the collapsed configuration or a deflated profile.
Balloon 630 may be dilated (inflated) by the introduction of a
liquid into inflation cannula 660. Liquids containing treatment
and/or diagnostic agents may also be used to inflate balloon 630.
In one embodiment, balloon 630 may be made of a material that is
permeable to such treatment and/or diagnostic liquids. To inflate
balloon 630, the fluid can be supplied into inflation cannula 660
at a predetermined pressure, for example, between about one and 20
atmospheres. The specific pressure depends on various factors, such
as the thickness of balloon wall 635, the material from which
balloon wall 635 is made, the type of substance employed and the
flow-rate that is desired.
[0059] Catheter assembly 600 also includes at least two substance
delivery assemblies (not shown; see FIGS. 4B-4C) for injecting a
substance into a myocardial infarct region. In one embodiment,
substance delivery assembly 605a includes needle 615a movably
disposed within hollow delivery lumen 625a. Delivery assembly 605b
includes needle 615b movably disposed within hollow delivery lumen
625b (not shown; see FIGS. 4B-4C). Delivery lumen 625a and delivery
lumen 625b each extend between distal portion 610 and proximal
portion 620. Delivery lumen 625a and delivery lumen 625b can be
made from any suitable material, such as polymers and copolymers of
polyamides, polyolefins, polyurethanes and the like. Access to the
proximal end of delivery lumen 625a or delivery lumen 625b for
insertion of needle 615a or 615b, respectively is provided through
hub 635 (located at proximal end 620). Delivery lumens 625a and
625b may be used to deliver first and second mixtures of a modified
two-component gel composition to a post-myocardial infarct
region.
[0060] FIG. 4B shows a cross-section of catheter assembly 600
through line A-A' of FIG. 4A (at distal portion 610). FIG. 4C shows
a cross-section of catheter assembly 600 through line B-B' of FIG.
4A. In some embodiments, delivery assemblies 605a and 605b are
adjacent to each other. The proximity of delivery assemblies 605a
and 605b allows each mixture of the modified two-component gelation
system to rapidly gel when delivered to a treatment site, such as a
post-myocardial infarct region.
[0061] From the foregoing detailed description, it will be evident
that there are a number of changes, adaptations and modifications
of the present invention which come within the province of those
skilled in the part. The scope of the invention includes any
combination of the elements from the different species and
embodiments disclosed herein, as well as subassemblies, assemblies
and methods thereof. However, it is intended that all such
variations not departing from the spirit of the invention be
considered as within the scope thereof.
Sequence CWU 1
1
3116PRTArtificial SequenceSelf-assembled peptides 1Arg Ala Arg Ala
Asp Ala Asp Ala Arg Ala Arg Ala Asp Ala Asp Ala 1 5 10 15
220PRTArtificial SequenceSelf-assembled peptides 2Val Lys Val Lys
Val Lys Val Lys Val Pro Pro Thr Lys Val Lys Val 1 5 10 15 Lys Val
Lys Val 20 316PRTArtificial SequenceSelf-assembled peptides 3Ala
Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys 1 5 10
15
* * * * *