U.S. patent application number 11/149515 was filed with the patent office on 2005-12-08 for delivery of compounds with rehydrated blood cells.
This patent application is currently assigned to UNC-Chapel Hill. Invention is credited to Fischer, Thomas, Nichols, Timothy C., Read, Marjorie S..
Application Number | 20050272161 11/149515 |
Document ID | / |
Family ID | 34279762 |
Filed Date | 2005-12-08 |
United States Patent
Application |
20050272161 |
Kind Code |
A1 |
Nichols, Timothy C. ; et
al. |
December 8, 2005 |
Delivery of compounds with rehydrated blood cells
Abstract
Fixed-dried blood cells carrying an active agent are described,
along with methods of making the same, methods of using the same,
and compositions containing the same. The blood cells may be red
blood cells or blood platelets.
Inventors: |
Nichols, Timothy C.; (Chapel
Hill, NC) ; Fischer, Thomas; (Hillsborough, NC)
; Read, Marjorie S.; (Durham, NC) |
Correspondence
Address: |
MYERS BIGEL SIBLEY & SAJOVEC
PO BOX 37428
RALEIGH
NC
27627
US
|
Assignee: |
UNC-Chapel Hill
|
Family ID: |
34279762 |
Appl. No.: |
11/149515 |
Filed: |
June 10, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11149515 |
Jun 10, 2005 |
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10845045 |
May 13, 2004 |
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60533059 |
Dec 29, 2003 |
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60471005 |
May 16, 2003 |
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Current U.S.
Class: |
436/63 |
Current CPC
Class: |
A01N 1/02 20130101; A61P
7/02 20180101; Y10S 977/905 20130101; A61K 31/00 20130101; A61K
45/06 20130101; A61K 31/7088 20130101; A01N 1/0215 20130101; A61K
47/6901 20170801; A61K 35/18 20130101; A61P 31/12 20180101; A61K
35/19 20130101; A61P 7/04 20180101; A61K 31/00 20130101; A61K 38/02
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
436/063 |
International
Class: |
G01N 033/48 |
Goverment Interests
[0002] This invention was made with government support from the
Department of Defense and the National Institutes of Health under
Grant Numbers 1-P20-DE123474, 1-P60-DE 130789, and 1 R21 EB002863.
The United States Government has certain rights to this invention.
Claims
1-48. (canceled)
49. A method of delivering an active agent to a site of interest,
comprising: providing fixed-dried blood cells carrying said active
agent, said fixed-dried blood cells selected from the group
consisting of fixed-dried red blood cells, fixed-dried blood
platelets, and combinations thereof; rehydrating said fixed-dried
blood cells in a pharmaceutically acceptable carrier to provide a
pharmaceuticaly acceptable composition comprising rehydrated
fixed-dried blood cells carrying said active agent; and then
administering said pharmaceutical composition to a subject so that
an effective amount of said active agent is delivered to said site
of interest;
50. The method of claim 49, wherein said fixed-dried blood cells
comprise mammalian fixed-dried blood cells.
51. The method of claim 49, wherein said fixed-dried blood cells
comprise human fixed-dried blood cells.
52. The method of claim 49, wherein said subject is a mammalian
subject.
53. The method of claim 49, wherein said subject is a human
subject.
54. The method of claim 49, wherein said active agent is coupled to
said fixed-dried blood cells.
55. The method of claim 49, wherein said active agent is contained
within said fixed-dried blood cells.
56. The method of claim 49, wherein said fixed-dried blood cells
comprise aldehyde-fixed dried blood cells, and wherein said site of
interest is a vascular injury.
57. The method of claim 49, wherein said rehydrated fixed-dried
blood cells are rehydrated fixed-dried blood platelets which:
adhere to thrombogenic surfaces; undergo shape change upon adhering
to a thrombogenic surface; lead to the formation of a hemostatic
plug upon adhering to a thrombogenic surface; and release their
granular contents.
58. The method of claim 49, wherein said fixed-dried blood cells
are fixed-dried red blood cells.
59. The method of claim 49, wherein said active agent comprises an
antiviral agent.
60. The method of claim 49, wherein said active agent comprises a
blood coagulation protein.
61. The method of claim 49, wherein said active agent comprises a
blood anti-coagulation protein.
62. The method of claim 49, wherein said active agent comprises a
nucleic acid.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 60/533,059, filed Dec. 29, 2003, and
U.S. Provisional Patent Application Ser. No. 60/471,005, filed
filed May 16, 2003, the disclosures of which are incorporated by
reference herein in their entirety.
FIELD OF THE INVENTION
[0003] The present invention concerns methods and compositions for
delivering active agents to a subject in need thereof and more
particularly to fixed-dried blood cells that carry an active agent
and that are useful to deliver active agents to a site of
interest.
BACKGROUND OF THE INVENTION
[0004] Platelets have been recognized for decades as potential
tools for carrying therapeutics to sites of vascular injury.
However, the practical utility of platelets as therapeutic delivery
vehicles has been limited because platelets must be freshly
isolated, modified with the therapeutic, and then infused in a
short time-span. The utility of cryopreserved platelets and
normally liquid-stored for therapeutic delivery is limited by
storage lesion and, in the case of frozen platelets, the need to
remove cryopreservatives. Hence the practical application of
cryopreserved platelets, as well as preserved blood cells in
general, has been limited.
SUMMARY OF THE INVENTION
[0005] A first aspect of the present invention is fixed-dried blood
platelets, red blood cells (RBCs,) or combinations thereof carrying
a heterologous compound, active agent or compound of interest,
along with compositions comprising, consisting of or consisting
essentially of the same. Such platelets, RBCs or combinations
thereof are preferably mammalian blood cells such as human blood
platelets and RBCs. The compound or active agent may be associated
with the platelets and/or RBCs in any manner, such as coupling or
by being contained within the platelets and/or RBCs. The platelets
and/or RBCs may be aldehyde-fixed, and in one embodiment the
platelets are characterized in that they adhere to thrombogenic
surfaces; undergo shape change upon adhering to a thrombogenic
surface; lead to the formation of a hemostatic plug upon adhering
to a thrombogenic surface; and release their granular contents. The
active agent or compound to be delivered may be an antiviral agent
such as a nucleoside analog antiviral agent (for example
ribavirin), a blood coagulation protein such as Factor VII, a
nucleic acid (e.g., DNA, RNA), a detectable compound such as a
detectable protein or peptide, etc.
[0006] A second aspect of the present invention is a pharmaceutical
composition comprising, consisting of or consisting essentially of,
for example, from 0.01 or 0.1 to 99.9 or 99.99 percent by weight of
a pharmaceutically acceptable carrier (e.g., an aqueous or
nonaqueous carrier; a solid, liquid or gel carrier, etc.); and, for
example, from 0.01 or 0.1 to 99.9 or 99.99 percent by weight of
fixed-dried blood cells carrying an active agent as described
herein, the fixed-dried blood cells optionally rehydrated in the
pharmaceutically acceptable carrier.
[0007] A further aspect of the present invention is a method of
making fixed-dried blood cells for delivering a compound of
interest or an active agent to a subject in need thereof,
comprising: providing fixed blood cells (typically mammalian cells
and preferably human cells, particularly RBCs, platelets, and
combinations thereof) carrying the active agent; associating the
active agent to the fixed blood cells; and drying the fixed blood
cells (e.g., by freeze-drying or lyophilization) to produced
fixed-dried blood cells carrying the active compound. The method
may further comprise the step of rehydrating the fixed-dried blood
cells in a pharmaceutically acceptable carrier to provide a
pharmaceuticaly acceptable composition comprising rehydrated
fixed-dried blood cells carrying the active agent. The associating
step may be carried out by any suitable means, such as by coupling
the active agent to the cells or introducing the active agent into
the cells. The cells may be aldehyde-fixed platelets, and in one
embodiment the cells are platelets that are characterized in that,
upon reconstitution, they: adhere to thrombogenic surfaces; undergo
shape change upon adhering to a thrombogenic surface; lead to the
formation of a hemostatic plug upon adhering to a thrombogenic
surface; and release their granular contents.
[0008] A further aspect of the present invention is a method of
delivering a compound of interest to a thrombogenic surface (e.g.,
in a subject, or to a tissue in vitro for diagnostic or compound
testing purposes), comprising: providing fixed-dried blood cells
carrying the compound of interest as described herein; rehydrating
the fixed-dried blood cells in an acceptable carrier (e.g., a
pharmaceutically or physiologically acceptable carrier) to provide
a composition comprising rehydrated fixed-dried blood cells
carrying the active agent; and then administering the
pharmaceutical composition (e.g., to the subject, to the tissue in
vitro) so that an effective amount of the compound of interest is
delivered to a thrombogenic surface. The subject is typically a
mammalian subject such as a human subject, and the compound of
interest may be a diagnostic or therapeutic agent.
[0009] A further aspect of the present invention is a method of
delivering a compound of interest to macrophages of the RES in a
subject. The subject is typically a mammalian subject such as a
human subject, and the compound of interest may be a diagnostic or
therapeutic agent.
[0010] A further aspect of the present invention is a method of
delivering an active agent to a site of interest (e.g., to a
subject or to a tissue in vitro), comprising: providing fixed-dried
blood cells carrying the active agent as described herein;
rehydrating the fixed-dried blood cells in a pharmaceutically
acceptable carrier to provide a pharmaceuticaly acceptable
composition comprising rehydrated fixed-dried blood cells carrying
the active agent; and then administering the pharmaceutical
composition to the site of interest (e.g., to a subject or to a
tissue in vitro), so that an effective amount of the active agent
is delivered to said site of interst. The effective amount of
active agent may be less than 15% (15 percent) of endogenous
platelet equivalent. The subject is typically a mammalian subject
such as a human subject, and the active agent may be a diagnostic
or therapeutic agent. The cells may be aldehyde-fixed cells, and
the subject may for example be afflicted with a vascular
injury.
[0011] The foregoing and other objects and aspects of the present
invention are explained in greater detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows the proton NMR Spectra of nucleoside
copolymers.
[0013] FIG. 2 shows the fluorescent microscopy of polymer-modified
RL platelets.
[0014] FIG. 3 shows ristocetin aggregation of control and polymer
modified RL platelets.
[0015] FIG. 4 schematically illustrates the preparation of
Platelet/AAV.about.Lac Z reporter conjugates.
[0016] FIG. 5 shows flow cytmometric analysis of rFVIIa-FITC bound
to RL platelets.
[0017] FIG. 6 shows that rFVIIa binding to RL:platelets is
concentration dependent.
[0018] FIG. 7 shows that rFVIIa is active on the surface of RL
platelets.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0019] The present invention is explained in greater detail below.
This description is not intended to be a detailed catalog of all
the different ways in which the invention may be implemented, or
all the features that may be added to the instant invention. For
example, features illustrated with respect to one embodiment may be
incorporated into other embodiments, and features illustrated with
respect to a particular embodiment may be deleted from that
embodiment. In addition, numerous variations and additions to the
various embodiments suggested herein will be apparent to those
skilled in the art in light of the instant disclosure which do not
depart from the instant invention. Hence, the following
specification is intended to illustrate some particular embodiments
of the invention, and not to exhaustively specify all permutations,
combinations and variations thereof.
[0020] Subjects to be treated with the methods and compositions of
the present invention include both human subjects and animal
subjects for veterinary and drug development purposes. Animal
subjects are, in general, mammalian subjects, including but not
limited to pig, sheep, cow, horse, goat, cat, dog, mouse and rat
subjects.
[0021] "Thrombogenic surface" as used herein refers to any natural
or artificial thromgobenic surface, including but not limited to
wound tissue, blood vessel plaques such as atherosclerotic plaques,
activated endothelium due to local or systemic inflammation,
vessels, surfaces or tissues that are rendered thrombogenic in a
subject as a toxic side-effect due to administration of anticancer,
antineoplastic or antiproliferative agent, surfaces of foreign or
implanted items in the subject, including metals, polymers, etc.,
as found in stents, catheters, biomedical implants such as
pacemakers and leads, orthopedic implants such as artificial
joints, etc. A compound may be administered to a thrombogenic
surface for any purpose, such as for therapeutic purposes or
diagnostic purposes (e.g., imaging or detection of a thrombogenic
surface by any suitable means such as radioimaging, tissue biopsy,
implant removal and determination of whether the delivered compound
is found on the implant surface, etc.).
[0022] "Platelets" utilized in carrying out the present invention
are, in general, of animal, and preferably mammalian, origin (e.g.,
pig, sheep, cow, horse, goat, cat, dog, mouse, rat, human, etc.).
Platelets may be derived from the same species into which the
platelets are introduced, or from a different species from which
the platelets are introduced. In one embodiment, platelets are
harvested from a subject, used to prepare the active agents
described herein, and after being so prepared are administered at a
later time back to the same subject from which the platelets were
harvested.
[0023] "Red blood cell" as used herein includes any erythrocyte of
mammalian origin (e.g., pig, sheep, cow, horse, goat, cat, dog,
mouse, rat, human, etc.). Red blood cells may be derived from the
same species into which the red blood cells are introduced, or from
a different species from which the red blood cells are introduced.
In one embodiment, red blood cells are harvested from a subject,
used to prepare the active agents described herein, and after being
so prepared are administered at a later time back to the same
subject from which the red blood cells were harvested.
[0024] "Fixed blood cells" herein refers to blood cells, which may
be platelets, red blood cells, or mixtures thereof, which have been
chemically treated with at least one chemical compound that is
incorporated into at least a portion of the cells to structurally
stabilize and/or extend the shelf-life of the cells.
[0025] "Fixed-dried blood cells" herein refers to blood cells,
which may be platelets, red blood cells, or mixtures thereof, which
have been fixed, and additionally have had water removed therefrom
by any suitable technique such as drying, dehydrating, lyophilizing
or freeze-drying, etc., to further structurally stabilize and/or
extend the shelf-life thereof.
[0026] "Rehydrated fixed-dried blood cells" refers to a fixed-dried
blood cells, which may be platelets, red blood cells, or mixtures
thereof, which has been contacted to or combined with an aqueous
solution so that water is taken up into the intracellular
space.
[0027] "Blood coagulation protein" as used herein includes any
suitable blood coagulation protein, including but not limited to
Factor VII, Factor IX, Factor X, as well as coagulation proteins
that generate Factor VII or FVIIa, such as Factor XII or Factor
XIIa, or Factor X or Factor Xa, Protein C, Protein S, and
Prothrombin. Such proteins may be natural or synthetic and include
proteins containing minor modifications of the naturally occurring
protein (i.e., analogs). Where naturally occurring the protein may
be of any species of origin, preferably mammalian or human as
described herein, and in one embodiment is of the same species of
origin as the subject to which it is administered.
[0028] "Blood anti-coagulation protein" as used herein includes any
suitable blood anti-coagulation protein, including but not limited
to activated protein C, protein S, heparin and heparinoids, etc.
Such proteins may be natural or synthetic and include proteins
containing minor modifications of the naturally occurring protein
(i.e., analogs). Where naturally occurring the protein may be of
any species of origin, preferably mammalian or human as described
herein, and in one embodiment is of the same species of origin as
the subject to which it is administered.
[0029] "AAV" as used herein refers to "adeno-associated virus".
[0030] The term "treat" as used herein refers to any type of
treatment that imparts a benefit to a patient afflicted with a
disease, including improvement in the condition of the patient
(e.g., in one or more symptoms), delay in the progression of the
disease, etc.
[0031] The term "pharmaceutically acceptable" as used herein means
that the compound or composition is suitable for administration to
a subject to achieve the treatments described herein, without
unduly deleterious side effects in light of the severity of the
disease and necessity of the treatment.
[0032] Applicants specifically intend that the disclosures of all
United States patent references cited herein be incorporated by
reference herein in their entireties.
[0033] 1. Platelets.
[0034] Platelets may be fixed in accordance with known techniques,
such as described in U.S. Pat. Nos. 4,287,087; 5,651,966;
5,902,608; 5,891,393; and 5,993,084. In general, such methods
involve providing blood platelets (e.g., human, mammalian);
contacting said human platelets to a fixative (e.g., an aldehyde)
for a time sufficient to fix said platelets; removing said fixative
from said platelets; and then drying said platelets to produce
fixed-dried blood platelets. In a preferred embodiment, the
contacting step is carried out for a time sufficient to kill said
microorganisms. In a preferred embodiment, the contacting step is
carried out for a time insufficient to cause said platelets to lose
the capability, upon reconstitution, to; (i) adhere to thrombogenic
surfaces; (ii) not adhere to non-thrombogenic surfaces; (iii)
undergo shape change (spreading) upon adhering to a thrombogenic
surface; (iv) adhere to one another to form a hemostatic plug upon
adhering to a thrombogenic surface; and (v) release their granular
contents.
[0035] More particularly, fixed-dried blood platelets for use in
the present invention may be fixed with a compound selected from
the group consisting of formaldehyde, paraformaldehyde and
glutaraldehyde, with paraformaldehye currently preferred. In
general, washed platelets may be fixed by incubating them,
typically at room temperature, for up to 60 minutes in a solution
of up to 1.8% aldehyde. An alternative technique is to fix
platelets by incubating the platelets in a permanganate solution
(e.g., sodium permanganate, potassium permanganate). In general,
washed platelets may be prepared by this technique by incubating
them for from 5 to 20 minutes in from 0.001 to 1 g/dL of KMnO.sub.4
or NaMnO.sub.4 solution, more preferably by incubating them for
from 5 to 15 minutes in from 0.005 to 0.5 g/dL of KMnO.sub.4 or
NaMnO.sub.4 solution,
[0036] Blood platelet preparations for use in preparing
pharmaceutical formulations should be essentially free of
extraneous matter, particularly lysed blood platelets which would
present free thrombogenic agents to a patient administered the
preparation. Hence, care must be taken to sufficiently fix the
platelets (preferably without destroying the viability thereof, as
indicated by the characteristics set forth above) prior to drying,
as undue lysis will otherwise occur during the drying step. For
example, platelet preparations suitable for use in preparing human
pharmaceutical formulations preferably show, on reconstitution of
109 platelets in one milliliter of solution, less than
10.times.10.sup.6 microparticles (the fragmentary remains of lysed
platelets) per milliliter, and preferably show less than 150
International Units (IU) per liter of lactate dehydrogenase in the
supernatant after resuspension and pelleting (where 2200 IU per
liter represents total lysis of 10.sup.9 cells in 1
milliliter).
[0037] Drying of platelets after fixation may be carried out by any
suitable means, but is preferably carried out by lyophilization.
Care should be taken to stabilize the platelet preparation prior to
drying as an unacceptable level of platelet lysis may otherwise
occur. Stabilization may be carried out by suspending the platelets
in a solution containing a suitable water replacing molecule (or
"stabilizer"), such as albumin or trehalose, and then drying the
solution. In one embodiment, from 0.1 to 20 percent by weight
albumin is employed, more preferably from 1 to 10 percent by weight
albumin is employed, and most preferably from 5 to 10 percent by
weight albumin is employed. For administration to a subject, the
albumin in the preparation should be of the same species as the
subject (e.g., human albumin). In the alternative, the preparation
may be dried with albumin of a different species, the albumin
separated from the platelets on reconstitution, and albumin of the
same species added back to the reconstituted preparation for
administration to the subject, but care should be taken to remove
all non-species specific albumin as it may be antigenic in the
subject being treated. Once dried, the platelets may be coupled or
associated with a compound to be delivered to produce an "active
agent" of the present invention, as described further below.
[0038] 2. Lyophilized RBCs.
[0039] Cross-linked and lyophilized RBCs of the present invention
may be prepared with bifunctional cross-linking reagents that are
homo or heteromeric with reactive the following reactive moeties:
aldehydes, ketones, hydrazides, N-hydroxysulfosuccinimides,
N-hydroxysuccinimides, maleimides, imidoesters, active halogens,
pyridyl-disulfides, isocyanates, nitrobenzoyloxysuccinimides,
nitrobenzenes, imidoesters, photo-activatable azidophenyls and
azidopolyaromatics, as well as zero-spacer carbodimide catalysts.
Multi (poly) functional reagents are also considered, as are
combinations of two or more cross-linkers, either serially reacted
with RBCs for reacted together with RBCs.
[0040] RBCs are obtained from mammalian blood with standard
phlebotomy, apheresis or exsangination methods according to
approved IACCOC protocols. RBCs are freed from plasma platelets,
leukocytes and plasma proteins my differential centrifugation, and
then treated with chemical cross-linkers. The reaction of the RBCs
with the cross-linkers are in general carried out for defined
periods of time at temperatures between 20.degree. C. and
37.degree. C. at pre-determined concentration of RBCs. As discussed
in greater detail below, care must be taken to sufficiently fix the
platelets or undue lysis will be measured upon rehydration of the
lyophilized product. The cross-linking step can be carried out in
the presence of antioxidants and free-radical scavengers, and the
cross-linking reaction can be quenched by adding compounds that
contain primary amines. After cross-linking, the RBCs are removed
from excess cross-linker and reaction products with differential
centrifugation, chromatography and/or dialysis.
[0041] Freezing of RBCs after cross-linking may be carried out over
a wide range of cooling rates at ambient or hyperbaric pressures.
If RBCs (with zero or reduced concentrations of cross-linkers) are
frozen into the high-pressure phase states of ice (e.g., ice
II/III) samples are preferably isothermally pressurized and then
isobarically cooled to under -120.degree. C., the point at which
ice II/III is metastable.--RBCs can be frozen in the presence of
"stabilizer" small molecules (e.g., glycerol), proteins (e.g.,
albumin) and polymers (e.g., PEG) which substitute for water in the
ice crystal matrix. The perfered "stabilizer" is PEG 8,000 at a
final concentration of 1% (w.v). The type and level of "stabilizer"
must be infusible as rehydrated. Lyophilization is carried out from
temperatures below 0.degree. C., preferably 40.degree. C. if the
RBCs were frozen at ambient pressure for ice I, and near or less
than -120.degree. C. for molecular distillation from the ice II/III
phase states.
[0042] The chemical modification of RBC membranes with
cross-linkers imparts a "foreign" nature to the cells with respect
to recognition by the reticuloendothelial system and thrombogenic
with respect to contact activation of platelets. The surface
membrane is thus occluded by covalently attaching polymers that
sterically coat the cell membrane. Polymers, particularly
water-soluble polymers, that may be used to carry out the present
invention are, in general, naturally occurring polymers such as
polysaccharides, or synthetic polymers such as polyalkylene oxides
such as polyethylene glycols (PEG), polyalkylene glycols,
polyoxyethylated polyols, polyvinylpyrrolidone, polyacrylates such
as polyhydroxyethyl methacrylate, polyvinyl alcohols, and
polyurethane. The polymers may be linear, branched or dendrimeric
and may be substituted or unsubstituted. The polymers may, as noted
above, be hydrophilic, lipophilic, or both hydrophilic and
lipophilic. Polymers are covalently attached through the membrane
through reactive chemical functions that include, but are not
limited to, aldehydes, ketones, hydrazides,
N-hydroxysulfosuccinimides, N-hydroxysuccinimides, maleimides,
imidoesters, active halogens, pyridyl-disulfides, isocyanates,
nitrobenzoyloxysuccinimides, nitrobenzenes, imidoesters,
photo-activatable azidophenyls and azidopolyaromatics, as well as
zero-spacer carbodimide catalysts. The preferred polymer is PEG
5,000 with a terminal aldehyde for covalent attachment to surface
lysines via Schift's base formation. We have found that most of the
washing and fixation steps for a larger-scale red cell preparation
can advantageously be performed in a closed system by utilizing an
appliance called the IBM 2991 Cell Washer, which was originally
designed for blood banks to facilitate washing of frozen red cell
units to remove cryoprotectant agents like DMSO or glycerol just
prior to transfusion. The advantage to employing this device for
our purposes in preparing freeze-dried red blood cells is that it
provides an aseptic environment for the multiple steps of washing
and fixation which would otherwise require handling of the red
cells in an open container, and the Cell Washer induces less shear
stress to resuspend the packed red cells after each step than would
be experienced with a resuspension method by hand. As an example,
we introduce into the Cell Washer processing bag a volume of
150-200 mL of a suspension of leukodepleted red blood cells at a
cell count of 3-4.times.10.sup.9/mL and attach the tubing harness
as described in the operating instructions. Then we introduce a
volume of 200-250 mL phosphate washing buffer containing 0.1% BSA
sterilly through the tubing harness and perform wash cycle #1. At
the end of the agitation and spinning period programmed into the
2991, the supernatant washing fluid is automatically expressed out
to waste and fresh buffer is introduced thru the harness for a
total of three washes. After the spinning step of the third wash, a
fixation solution containing 0.05% glutaraldehyde in Hank's
buffered salt solution [HBSS] is introduced for a timed incubation
of 20 minutes at room temperature before spinning, and then three
more washing steps are performed with the phosphate buffer. At this
point the fixed, washed red cell suspension is removed from the
2991 processing bag and handled in vials and bottles for the
bulking and freeze-drying steps.
[0043] 3. Compounds to be Delivered.
[0044] Examples of compounds that may be coupled to platelets to
produce active agents of the present invention include, but are not
limited to, nucleic acids such as RNA, DNA, proteins or peptides
(enzymes, antibodies, etc,), viruses, bacteria, small organic
compounds (e.g., monomers), synthetic and semisynthetic polymers,
nanoparticles, chelated metals and ions, etc. Such compounds may
have any suitable function or activity depending upon the
particular object of the treatment or method, including but not
limited to antimicrobial, antibacterial, or antiviral activity;
blood coagulation or anti-coagulation activity; reporter or
detectable activity, etc. Additional examples of compounds that may
be coupled to platelets to produce active agents of the present
invention include, but are not limited to, vasoactive, antioxidant,
antithrombotic, anticarcinogenic, antiatherogenic, antimitotic,
antiangiogenic, and antiproliferative compounds. It will be
appreciated that all such compounds can be contained within a
vesicle, micelle or other particle which is in turn associated with
or coupled to the platelet.
[0045] Antiviral compounds. In one embodiment of the present
invention, the compound carried by the platelets is an antiviral
compound. Any suitable antiviral compound can be used, including
but not limited to sialic acid analogues, amantadine, rimantadine,
zidovudine, vidarabine, idoxuridine, trifluridine, foscarnet, etc.
In one embodiment the antiviral compound is a purine nucleoside
analog, examples of which include but are not limited to acyclovir,
didanosine, ribavirin, ganciclovir, and vidarabine, and antisense
nucleosides, RNA and DNA.
[0046] In one embodiment of the invention, platelets carrying
antiviral compounds are administered to patients afflicted with a
viral infection in an amount sufficient to treat the viral
infection. In one embodiment the patient is infected with a
hemorrhagic fever viruses, such as a virus of the Filoviridae,
Arenaviridae, Bunyaviridae or Flaviviridae families.
[0047] Blood coagulation and anticoagulation proteins. The compound
to be delivered may be a drug coagulation protein, such as Factor
VII, Factor IX, Factor X, Factor XII, Protein C, Protein S,
Prothrombin, anticoagulation proteins such as activated protein C,
protein S, heparin and heparinoids, and pro- or anti-coagulation
proteins of reptile or insect origin, and others.
[0048] Such compounds are known. For example, Factor VII or Factor
VIIa which may be utilized in carrying out the present invention
(this term including modified Factor VII or Factor VIIa or Factor
VII analogs which retain the blood coagulation activity of Factor
VII) is described in, among others, U.S. Pat. Nos. 6,461,610;
6,132,7306,329,176; 6,183,743; 6,186,789; 5,997,864; 5,861,374;
5,824,639; 5,817,788; 5,788,965; 5,700,914; 5,344,918; and
5,190,919. In one embodiment, recombinant human Factor VIIa is
preferred.
[0049] In one embodiment of the invention, platelets carrying blood
coagulation proteins are administered to a subject afflicted with a
wound in an amount effective to promote blood coagulation at the
wound and/or healing of the wound.
[0050] Nucleic Acids. Nucleic acids to be carried by platelets of
the present invention may encode a detectable protein or peptide
such as Lac-Z, beta-glucuronidase, horseradish peroxidase, a
fluorescent protein such as green fluorescent protein, etc.
[0051] In one embodiment of the invention, platelets carrying
nucleic acids encoding a detectable protein are administered to a
subject in an amount effective to express the detectable protein in
atherosclerotic tissue or plaques in blood vessels to thereby
produce an improved animal model of atherosclerosis. Such animals
(which preferably are animals that, by diet and/or breeding are
susceptible to atherosclerosis) may be administered a putative
antiatherogenic compound, and/or antiatherogenic diet, and then
compared to a control animal that has not been administered the
putative anti-atherogenic compound and/or anti-atherogenic diet,
and the extent of atherogenic plaque formation in experimental
animals versus control animals compared by visualizing plaques
through expression of the detectable protein.
[0052] In other embodiments of the invention, the nucleic acid may
encode a therapeutic protein or peptide. Examples include, but are
not limited to, nucleic acids encoding an anti-atherogenic protein
or peptide such as DNA encoding the ras binding domain (RBD) of the
ras effector protein RGL.sub.2 (e.g., to inhibit proliferation),
DNA encoding endothelial nitric oxide sythetase varienats (e.g., to
inhibit platelet function), DNA encoding the NF-.kappa.B
super-repressor (e.g., to inhibit inflammatory processes).
[0053] 4. Associating Compounds with Platelets.
[0054] Compounds to be delivered may be associated with platelets
by any suitable technique, including but not limited to: (1)
directly chemically coupling the compound to be delivered to the
platelet surface membrane; (2) conjugating the compound to be
delivered to a polymer that is in turn coupled to the platelet's
internal membrane; (3) incorporating the compound to be delivered
into unilamellar or multilamellar phospholipid vesicles that are in
turn internalized into the platelets; (4) absorbing or
internalizing the compound to be delivered into nanoparticles,
e.g., buckminsterfullerene, that are in turn internalized into the
platelets; (5) coupling the compound to be delivered to proteins
that are internalized for trafficking to alpha granules in the
platelets; (6) coupling the compound to be delivered to proteins
(or other macromolecules) or particles that are phagocitized by the
platelets; (7) hydrophobically partitioning the compound to be
delivered into membranes; (8) physically entrapping the compound to
be delivered in the platelet intracellular space through pores that
are formed with electrophoration, complement treatment, lytic
protein exposure, etc; (9) adsorbing the compound to the exterior
surface of the cell by non-covalent physical or chemical
adsorption, that are in turn internalized into the platelets.
[0055] Cross-linking chemistry for preparing compound-platelet
conjugates. Compound-platelet conjugates in which platelets are
coupled to proteins, antiviral compounds or the like may be
prepared with homo- or hetero-bifunctional cross-linking reagents
that can contain, but are not limited to, the following reactive
moeties: aldehydes, ketones, hydrazides,
N-hydroxysulfosuccinimides, N-hydroxysuccinimides, maleimides,
imidoesters, active halogens, pyridyl-disulfides, isocyanates,
nitrobenzoyloxysuccinimides, nitrobenzenes, imidoesters,
photo-activatable azidophenyls and azidopolyaromatics, as well as
zero-spacer carbodimide catalysts. Multi (poly) functional reagents
with on or more of these moieties are also considered, as are
combinations of two or more cross-linkers, either serially reacted
or reacted in concert. The cross-linking step can be carried out in
the presence of antioxidants and free-radical scavengers, and the
cross-linking reaction can be quenched by adding compounds that
contain primary amines. After cross-linking, excess reagent can be
removed with methods that include but are not limited to with
differential centrifugation, chromatography and/or dialysis.
[0056] Viral encapsidation. In the case of nucleic acids to be
delivered, the nucleic acid may be encapsidated or enclosed within
a viral capsid or particle, which viral capsid or particle may in
turn be conjugated or coupled to the platelets. One suitable virus
into which the nucleic acid may be encapsidated is the
adeno-associated virus (AAV). The AAV virus is known and a nucleic
acid of interest to be delivered may be encapsidated or packaged
therein in accordance with known techniques. See, e.g., U.S. Pat.
Nos. 6,548,286; 6,491,907; 6,489,162; 6,458,587; 6,410,300;
6,268,213; 6,204,059; 6,093,570; 6,057,152; 6,040,183; 5,869,305;
5,863,541; 5,773,289; 5,753,500; 5,478,745; 5,436,146; and
5,139,941. In addition to AAV, it will be appreciated that other
viruses, including but not limited to such as adenoviruses,
lentiviruses, hepatitus viruses, herpesviruses, can also be used to
encapsidate a nucleic acid for association with a platelet to carry
out the present invention.
[0057] Once the nucleic acid of interest is encapsidated or
packaged in a viral particle, viral particles may be associated
with or coupled to platelets in accordance with known
techniques.
[0058] In general, the compound to be delivered is coupled to or
associated with the platelets so that each platelet carries, or has
associated therewith, at least 1,000, and more preferably at least
10,000, individual molecules of the compound to be delivered.
[0059] Once prepared, the platelets with associated compound to be
delivered comprise an "active agent" which may be stored in frozen
form, refrigerated, or at room temperature (depending upon the
shelf life required) for subsequent reconstitution and use.
[0060] 5. Reconstitution and Administration of Active Agents.
[0061] Pharmaceutical formulations of the present invention may
simply comprise dried (preferably lyophilized) blood cells carrying
the active agent, pyrogen-free and sterile in a sterile aseptic
package. Albumin may be included, as noted above. Pharmaceutical
formulations may also comprise a platelet preparation of the
present invention reconstituted in a pharmaceutically acceptable
carrier. Additional agents, such as buffers, preservatives, and
other therapeutically active agents, may also be included in the
reconstituted formulation. See, e.g., U.S. Pat. No. 4,994,367 (the
disclosure of which is incorporated herein by reference). The
amount of blood cells and pharmaceutical carrier is not critical
and will depend upon the particular application of the blood cells,
whether or not they have been rehydrated, etc., but in general will
range from 1 or 10 percent by weight blood cells up to 90 or 99 or
even 99.9 percent by weight blood cells, and from 0.01, 1 or 10
percent by weight of pharmaceutically acceptable carrier up to 90
or even 99 percent by weigh pharmaceutically acceptable carrier. In
some embodiments of fixed-dried blood cells that have not been
rehydrated the composition may consist essentially of or consist
entirely of the fixed-dried blood cells, free of any particular
carrier.
[0062] The fixed-dried blood cells described above may be
formulated for administration in a pharmaceutical carrier in
accordance with known techniques. See, e.g., Remington, The Science
And Practice of Pharmacy (9.sup.th Ed. 1995). In the manufacture of
a pharmaceutical formulation according to the invention, the
fixed-dried blood cells may be admixed with, inter alia, an
acceptable carrier. The carrier must, of course, be acceptable in
the sense of being compatible with any other ingredients in the
formulation and must not be deleterious to the patient. The carrier
may be a solid (including powders), and is preferably formulated
with the blood cells and packaged as a unit-dose formulation, for
example, a bottle of lyophilized powder which may be reconstituted
by addition of an aqueous solution.
[0063] For rehydrating the blood cells, any aqueous carrier which
rehydrates the platelets so that they possess the characteristics
enumerated above and are suitable for intravenous injection may be
used (e.g., sterile, pyrogen free, physiological saline solution).
For example, an aqueous carrier may be injected into a bottle
containing a pharmaceutical formulation of the invention in the
form of a lyophilized powder, the contents aggitated if necessary,
and then the pharmaceutical formulation in the form of an aqueous
suspension of rehydrated fixed-dried blood cells withdrawn from the
bottle and administered by injection into a patient.
[0064] The compounds of the invention are preferably administered
internally, e.g., orally or intravenously, in the form of
conventional pharmaceutical compositions, for example in
conventional enteral or parenteral pharmaceutically acceptable
excipients containing organic and/or inorganic inert carriers, such
as water, gelatin, lactose, starch, magnesium stearate, talc, plant
oils, gums, alcohol, Vaseline, or the like. The pharmaceutical
compositions can be in conventional solid forms, for example,
tablets, dragees, suppositories, capsules, or the like, or
conventional liquid forms, such as suspensions, emulsions, or the
like. If desired, they can be sterilized and/or contain
conventional pharmaceutical adjuvants, such as preservatives,
stabilizing agents, wetting agents, emulsifying agents, buffers, or
salts used for the adjustment of osmotic pressure. The
pharmaceutical compositions may also contain other therapeutically
active materials. The pharmaceutical compositions of the invention
can be made using conventional methods know in the art of
pharmaceutical manufacturing.
[0065] Reconstituted pharmaceutical formulations of the present
invention are typically administered to human patients by
pareneteral administration (e.g., intravenous injection,
intraarterial injection). The amount of the pharmaceutical
formulation administered will vary depending upon the weight and
condition of the patient, but will typically range from 20 to 350
milliliters in volume, and from 1.times.10.sup.9 to
3.times.10.sup.9 platelets per milliliter (and more preferably from
2.times.10.sup.9 to 3.times.10.sup.9 platelets per milliliter) in
concentration. Pharmaceutical formulations may be packaged in a
sterile, pyrogen free container to provide these volumes and
dosages as a unit dose. The particular route of administration is
not critical and will depend upon the condition being treated, with
topical administration or application into penetrating wound sites
also being utilized for such injuries and their corresponding
treatments.
[0066] Three applications, involving wound site imaging,
coagulation factor delivery and antiviral delivery, are among those
considered in the following discussion.
[0067] a) RL platelets for Wound Localization with Magnetic
Resonance Imaging--
[0068] RL platelets can be loaded with paramagnetic nanoparticles
that can function as MRI contrast agents when the rehydrated cells
localize to the sites of vascular injury. Two recent advances have
provided the tools to prepare RL platelet-MRI contrast agent
formulations. First, infusable gadolinium (Gd)-chelates have been
approved by the FDA (e.g., Magnevist.TM.) and are proving useful,
as a relaxation contrast agent, for the evaluation of vascular
pathologies (e.g., Rofsky and Adelman, 2000). A wide range
chemically activated of Gd-chelators for attachment to primary
amines on macromoleculas are available, and have lead to the
development of research probes for specific angiographic imaging
applications (see Artemov, 2003, for a review). For example, fibrin
clots have been localized with Gd-anti-fibrin antibody probes for
the detection of vulnerable plaques (Flacke et al., 2001).
[0069] Secondly, major advances have been made in the preparation
of magnetic nanoparticles (see Pankhurst et al, 2003, for a
review). Of particlar importance is the synthesis of iron oxide
(maghemite and magnetite) nanoparticles with narrow size
distribution (Chatterjee et al, 2003). Furthermore, methods for the
modification of iron oxide particles with polymers for the
attachment of macromolecules have been developed (Chatterjee et al.
2003) and used to isolate blood cells in magnetic fields (Chen, et
al., 2000). The RL platelet represents a unique platform for
targeting iron oxide nanoprobe fabrication and Gd-chelates to sites
of vascular injury.
[0070] b) RL Platelet-Coagulation Factor Conjugates for Hemostasis
in Coagulopathic Conditions--
[0071] The tethering of coagulation factors (such as FVIIa) to the
surface of RL platelets and/or loading of a-granules etc) with
coagulation factors (FXIIIa for clot stabilization, caged thrombin
for photoactivation (Arroyo et al., 1997)), is a logical strategy
for delivering replacement factors to vascular wound sites in
hemophiliacs that have circulating antibody inhibitors to standard
replacement therapeutics (refractory hemophiliacs) as well as
patients with other types of coagulopathies. Recombinant FVIIa has
been hypothesized to provide hemostasis through two mechanisms that
are operant on the platelet surface. First, the ability of
recombinant factor VIIa (rFVIIa) to provide hemostasis in
hemophilia patients (Hedner and Glazer, 1992) has been theorized to
involve the direct activation of factor Xa on the surface of
platelets (e.g., see Monroe et al., 1997 and 2000), thus
"bypassing" the need for factors 1.times. and VIII. This "bypass"
activity of the rec FVIIa might be the functional basis for the
utility of the Novo Nordisk product as a hemostatic agent in
hemophilia A and B. Secondly, in the presence of normal levels of
coagulation factors, rFVIIa might augment hemostatic mechanisms by
activating factor IX on the platelet surface. Thus, rFVIIa
mediation of thrombin generation on the platelet surface might
compensate for a lack of platelets in thrombocytopenia or a lack of
proper platelet function in thrombasthenia. The hypothesis that
rFVIIa can play a "compensating" role for platelet function(s) is
supported by the clinical observation that rFVIIa can improve
hemostasis in patients that are thrombocytopenic (e.g., Poon et
al., 2000, Kristensen et al., 1996), thromboasthenic (e.g., Al
Douri et al., 2000) or are those affected by both platelet defects,
such as in trauma (e.g., Dutton et al., 2003). However, high-doses
of rFVIIa are frequently required for hemostasis, perhaps due to
the low (Kd.about.90 nM) affinity of the coagulation factor for the
platelet surface (Monroe et al., 1997). We thus hypothesize that
the direct chemical tethering of rFVIIa to the platelet surface
will improve the therapeutic effectiveness (index) of the
recombinant protein as a hemostatic agent to stop active
bleeding
[0072] c) Antiviral Delivery with Lyophilized Platelets--
[0073] RL platelets and RL RBCs hold promise for delivering
antiviral therapeutics to macrophages that are infected with the
single strand RNA viruses that cause hemorrhagic fevers and
hepatitis. This is important because the macrophages of the
reticuloendothelial system (RES) are involved in the initial stage
of viral infection, as well as macrophages at sites of vascular
injury in the later acute-hemorrhagic phase of infection. Viruses
of the Arenaviridea (e.g., Lassa fever virus), Filoviridae (e.g.,
Ebola and Marburg viruses), Bunyaviridae (e.g., Rift Valley virus)
and Flaviviridae (e.g., Yellow fever virus) families cause
viral-induced cellular damage to vascular tissues that result in
hemorrhage. Similarly, hepatitis C virus (a Flaviviridae family
member) propagation is frequently associated with
bleeding-intensive hepatic surgeries. Ribavirin, as a
broad-spectrum antiviral RNA mutagen, holds promise for the
treatment of these hemorrhage-associated viruses. However, adverse
toxicities have limited the clinical use of this ribonucleoside as
an antiviral chemotherapeutic. We seek to increase the therapeutic
efficacy of ribavirin by using RL platelets to deliver the
ribonucleoside. The intrinsic hemostatic function of RL platelets
will thus concentrate the ribavirin in the microenvironment of the
virus for increased chemotherapeutic efficacy; in RES and vascular
wound site macrophages.
[0074] The present invention is described in greater detail in the
following non-limiting Examples.
EXAMPLES 1-5
Attachment of Ribavirin to Platelets
[0075] These examples describe methods for the preparation and
characterization of reconstituted platelets having ribavirin and
rFVIIa coupled thereto.
EXAMPLE 1
Synthesis of Ribavirin-Polylysine Polymers
[0076] Ribavirin is chemically phosphorylated for ribavirin
monophosphate (RMP) as detailed by Yoshikawa et al., Tetrahedron
Lett. 50, 5065-5068 (1967)). Ribavirin monophosphate (RMP) is
coupled to polylysine via a pH-sensitive phosphoramide linkage in
accordance with the procedure of Di Stefano and Fiume (Trends in
Glycosci. and Glycotech. 50, 461-472 (1997)) or a simplified
procedure is based on the formation of an imidazole-ribavirin
adduct (see Chu et al., Nuc. Acids. Res. 11, 6513-6529 (1983)). The
conjugation chemistry for ribonucleosides polymer synthesis was
tested with uracil rather than ribavirin. We do not anticipate that
the difference in nitrogenous base structure between uracil and
ribavirin will have a large effect on the synthesis.
[0077] Polylysine (<mw>=205 kDa for .about.1,400 residues
lysine/molecule) was reacted with FITC (fluorescein isothiocyanate)
and SANPAH (N-succinimidyl-6-[4'-azido-2'-nitrophenylamino]
hexanoate) to respectively provide a fluorescent label and a
photoactivatable moiety for the covalent attachment of the final
product to the platelets. Uracil was coupled to the lysine
side-arms by first activating UMP with imidazole with EDAC
(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) as a catalysis.
The activated UMP.about.imidazole complex was then added to the
FITC/SANPAH-modified lysine chain to form phosphoamide bonds. Based
on peak sizes from proton NMR analysis (.alpha.-carbon proton vs.
nitrogenous base protons, see FIG. 1, "Proton NMR Spectra of
Nucleoside Copolymer" 12% of the lysine residues were coupled to
UMP. The final average composition of the 1400 residue copolymer
was 168, 20 and 4 lysine side-arms modified respectively with
uracil, SANPAH and FITC, leaving .about.1200 lysine residues
unmodified.
[0078] In a separate series of reactions, epsilon amino groups of
polylysine (Sigma-Aldrich, <MW>=200,000 kDa) are modified
with FITC to provide a fluorophoric label and with the
heterobifunctional cross-linker SANPAH (Pierce) to provide a moiety
for the photochemical attachment to platelets in the next step (b).
FITC and SANPAH are attached to the polylysine for one FITC
moiety/.about.300 lysine residues and one SANPAH moiety/.about.30
lysine residues. RMP is imidazolated and then coupled to the FITC,
SANPAH polylysine to form the phosphoramide bond for the ribavirin
copolymer as detailed by Chu et. al. (supra). The goal is to
saturate the lysine residues with RMP residues. Proton and .sup.13C
NMR are performed to characterize the extent of polymer
modification.
[0079] The pH sensitivity of the phosphoamide bond (see Fume et
al., Pharm. Acta. Helv. 137-139 (1988)) is verified by incubating
the ribavirin copolymer in buffers with pHs varying between 2.4 and
7.4 for one hr. After the incubation, pHs are adjusted to
neutrality and dialyzed vs. PBS to remove free RMP. The samples ds
examined with proton NMR to determine the extent of hydrolysis. The
rate of hydrolysis should increase as the pH is lowered from
neutrality.
[0080] We choose to test the conjugation chemistry for
ribonucleosides polymer synthesis with uracil rather than ribavirin
because of the temporary unavailability of the antiviral
immediately before and during the military operation in Iraq. We do
not anticipate that the difference in nitrogenous base structure
between uracil and ribavirin will have a large effect on the
synthesis.
EXAMPLE 2
Attachment of Ribavirin-Polylysine Polymers for Ribavirin-Loaded
Lyophilized Platelets
[0081] The procedure for preparing lyophilized platelets involves
four steps: Removal of platelets from excess plasma proteins, mild
paraformaldehyde cross-linking to stabilize cellular structures,
removal of platelets from unreacted cross-linker and lyophilization
(Read et al, U.S. Pat. No. 5,651,966). After removal of the excess
paraformaldehyde, the platelets are mixed with varying
concentrations of the ribavirin polymer and exposed to visible
light to activate the SANPAH moieties for the formation of covalent
linkage with the platelet surface. The platelets are then
lyophilized with standard procedures. Platelets are prepared with
different amounts of ribavirin by varying the concentration of
ribavirin-copolymer in the coupling step. The ability of the
ribonucleoside copolymer to covalently couple to RL platelets was
studied by incubating the cells with the delivery polymer in
visible-spectrum light to photoactivate the SANPAH moiety for
covalent coupling. Unreacted copolymer was removed with
centrifugational washing, and then the platelets were examined with
fluorescent microscopy. FIG. 2, "Fluorescent Microscopy of
Polymer-Modified RL Platelets", demonstrates that he
fluorescent-labeled ribonucleoside copolymer associated with the
cells. Based on the average fluorescent intensity of the platelets,
each cell was covalently labeled with 24,800 copolymers.
EXAMPLE 3
Characterization of Ribavirin-Platelets
[0082] Ribavirin-platelets prepared in accordance with example 2
above are rehydrated with normal saline and then subject to
analysis to verify chemical nature of the copolymer attachment, the
functionality of the platelets and the ability of the
ribavirin-platelets to release the ribonucleoside.
[0083] Surface density and cellular distribution of the
ribavirin-polylysine polymer. Ribavirin-loaded platelets are
subjected to liquid scintillation counting, and then the amount of
attached [.sup.3H]ribavirin-copolymer per platelet calculated from
the specific activity of the radiolabel. Flow cytometry may also be
performed to assess the relationship between fluorescence intensity
and the number of copolymers per platelet. The cellular
localization of the [.sup.3H]ribavirin-copolymer may be ascertained
with confocal microscopy from the fluorescence of the FITC
moiety.
[0084] The chemical integrity of the ribavirin units is verified by
placing the ribavirin-platelets in low pH buffer to hydrolyze the
nucleoside. Samples will then be subjected to TLC analysis (as
detailed in Fischer et al, Brit. J Haem. 111, 167-175 (2000)) to
determine if the [.sup.3Hhydrolysis product co-elutes with
[.sup.3H]ribavirin monophosphate standards.
[0085] The activation response of ribavirin-platelets. Platelets
loaded with increasing amounts of [.sup.3H]ribavirin-copolymer are
subjected to the same type of analysis that was utilized to
characterize reconstituted lyophilized platelets prepared for
transfusion purposes. Briefly, the morphology of activated and
unactivated ribavirin-platelets is examined with scanning and
transmission electron microscopy. vWf-mediated adhesion may be
investigated with ristocetin aggregation. The surface density of
phosphatidylserine, p-selectin, activated GPIIb/IIIa and fibrinogen
may be ascertained with flow cytometry. These procedures may be
used to to confirm the maximum degree of copolymer modification
that can be obtained without unduly adversely affecting hemostatic
function and hence optimize any particular preparation
protocol.
[0086] Endocytosis of ribavirin-platelets by macrophages for drug
release and antiviral activity. This analysis utilizes a tissue
culture-based splenic macrophage phagocytosis system that has been
detailed elsewhere (Fischer et al., Art. Cell. Blood Subs. 1 mm.
Biotech. 29, 439-451 (2001)). The antiviral activity of
ribavirin-RL platelets may be assessed by infecting the rat splenic
macrophages with the Adames strain of Punta Toro virus, since it
can be handled under level 2 biosafety conditions, is known to
infect rodent macrophages, causes a lethal hepatic necrosis in
infected mice, and is sensitive to the antiviral effects of
ribavirin in vivo and in vitro. Alternatively these studies can
optionally be adapted to mouse macrophage cultures, which are known
to be highly permissive for Punta Toro virus replication.
[0087] The [.sup.3H]ribavirin-copolymer-RL platelets are incubated
with the macrophages with gentle rocking at 37.degree. C. Samples
are withdrawn as a function of time and subjected to two types of
analysis to respectively follow a) platelet internalization for
viral inactivation and b) ribavirin metabolism.
[0088] Platelet phagocytosis and viral inactivation. These studies
employ fluorescent microscopic and flow cytometric analysis to
identify macrophages (PE fluorescence from anti-macrophage-PE) and
ribavirin-RL platelet phagocytosis (from
[.sup.3H]ribavirin-copolymer FITC fluorescence). The cell mixtures
are incubated with anti-macrophage-PE conjugate (MCA-342, Serotec)
to label the macrophages, then flow cytometry and confocal
fluorescence microscopy are performed to follow the internalization
and phagocytic processes. Flow cytometry may be be relied upon to
provide quantitative data, while confocal microscopy may be
performed to examine the distribution of the ribavirin-platelets
that are docked to and/or internalized by macrophages.
[0089] A subset of macrophages treated with ribavirin-RL platelets
or control platelets may be infected in triplicate with Punta Toro
virus at a multiplicity of infection of 10 for one hour at
37.degree. C. At this point, the cells are washed 3 times with room
temperature growth medium, and maintained in growth medium at 37
degrees C. After completing the washes, a sample of growth medium
is removed from each well for analysis of viral levels by plaque
assay. Additional samples are removed at four-hour intervals to
determine levels of virus within the supernatant. Viral titers will
be assessed by plaque assays on Vero cells, which are permissive
for Punta Toro virus infection and have been used previously to
titer Punta Toro virus levels by plaque assay. Alternatively, cells
are infected with Punta Toro virus prior to treatment with
ribavirin-RL or control platelets. This will assess whether
rivavirin-RL platelets can control previously established
infections. As a positive control for these studies, groups of
macrophages are infected with Punta Toro virus in triplicate. These
cultures are then treated with ribavirin (not loaded on platelets)
at a concentration ranging from 4-10 micrograms per ml, a
concentration range that has previously been shown to inhibit Punta
Toro virus replication in vitro. Ribavirin-RL platelets are
considered to exhibit antiviral effect if they decrease viral
titers in treated macrophage cultured to levels that are comparable
to those observed with conventional ribavirin treatment.
[0090] Ribavirin metabolism. The time course of
[.sup.3H]ribavirin-monopho- sphate release and metabolism is
examined by lysing cell mixtures with TX-100 and then resolving
free nucleosides with TLC analysis (Fischer et al., 2000, supra).
This information is used to verify the release of
[.sup.3H]ribavirin monophosphate from the copolymer as well as the
conversion to [.sup.3H]ribavirin-diphosphate and triphosphate.
EXAMPLE 4
Characteristics of Platelets
[0091] Preferably platelets with at least 100,000 ribavirin
copolymers per cell that function with 90% efficiency in the
ristocetin aggregation analysis are obtained by the procedures
described above (or modifications thereof which will be readily
apparent to those skilled in the art). These criteria are chosen
for two reasons. First, a 10% reduction in the ristocetin in vitro
hemostasis parameter has an insignificant effect on in vivo
hemostatic function of RL platelets as judged by the ability to
correct bleeding in thrombocytopenic rabbits. Second, the delivery
of 100,000 ribavirin-copolymer molecules on a single platelet
represents .about.3.times.10.sup.8 ribavirin moieties (or
.about.5.times.10.sup.-.su- p.16 moles of ribavirin). If a single
macrophage (with an intracellular volume of .about.10.sup.3
um.sup.3) internalizes a single ribavirin-platelet, the
intracellular concentration of ribavirin functions will be
.about.5.times.10.sup.-19 moles/um.sup.3 or
.about.5.times.10.sup.-4 moles/liter (500 uM). This is a
concentration that is well above the range of ribavirin (10-100 uM)
that exhibits antiviral activity in tissue culture (see e.g. Crotty
et al., J. Mol. Med. 80, 86-95 (2002)).
[0092] The in vitro metrics that are most correlative with in vivo
hemostatic efficacy for RL platelets are related to GPIb-von
Willebrand factor mediated adhesion. We thus utilized
ristocetin-mediated aggregation for the preliminary functional
characterization of the ribonucleoside-labeled RL platelets. FIG.
3, "Ristocetin Aggregation of Control and Polymer Modified RL
Platelets" demonstrates that after rehydration, the labeled cells
retained .about.70% of functionality as judged by the initial slope
and extent of aggregation curves.
EXAMPLE 5
Internalization of Ribavirin in Platelets
[0093] In an alternative embodiment of the foregoing, the ribavirin
is modified for platelet internalization. For example, this is
carried out by coupling the short chain ribavirin polylysine
copolymers to IgG or fibrinogen for alpha granule uptake.
Alternatively, latex nanoparticles are loaded with ribavirin for
platelet endocytosis, and then cells will processed for
lyophilization.
EXAMPLES 6-8
Lyophilized Platelets for Delivery of Nucleic Acids
[0094] These examples describe methods for using rehydratable,
lyophilized (RL) platelets to deliver nucleic acids to sites of
vascular injury for drug development, diagnostic, or therapeutic
purposes. The nucleic acid may encode any protein or peptide of
interest, such as a reporter protein or peptide for use in
diagnostic or drug screening purposes.
[0095] RL platelet/AAV.about.Lac Z conjugates are prepared as
depicted in FIG. 4, "Preparation of Platelet/AAV.about.Lac Z
Reporter Conjugates" herein. The procedure is a variation of the
procedure described in Read et al., U.S. Pat. No. 5,651,966.
EXAMPLE 6
Attachment of anti-AAV Antibodies to Platelets
[0096] Preparation of platelets for antibody attachment. Fresh
porcine platelets are isolated with differential centrifugation (to
obtain platelet-rich plasma) and Sepharose CL2B chromatography (to
free platelets from plasma proteins) in accordance with known
techniques (Read et al., 1Proc. Natl. Acad. Sci. USA 92, 397-401
(1995)). The cells are then be stabilized with paraformaldehyde and
washed with centrifugation to remove unreacted cross-linker. These
steps will employ known protocols that have been detailed elsewhere
(Read et al., supra).
[0097] Modification and attachment of anti-AAV antibody to
platelets. Anti-AAV capsid protein VP 1 polyclonal antibody
(Research Diagnostics Inc, Pleasant Hill, NJ, ALS24107) polyclonal
antibody (to residues 278-289 on the capsid protein) is reacted
with N-succinimidyl 3-[2-pyridylthio]propionate (SPDP) to form
lysine-imide linkages between the cross-linker and the antibody in
accordance with known techniques (see Carlsson et al., Biochemistry
J 173, 723-737 (1978)). Excess cross-linker is removed with sizing
chromatography, and then the thiol-activated antibody is incubated
for 2 hr with the aldehyde stabilized platelets prepared as above
for attachment of the antibody to the platelet surface via
sulfhydryl moieties. The reaction stoichiometry is 10,000
anti-capsid antibodies per platelet. Unbound antibody is removed
with two centrifugation washes.
[0098] Attachment of AAV.about.Lac Z to platelets for lyophilized
platelet/AAV.about.Lac Z conjugates. AAV.about.-Lac Z is prepared
with "triple-plasmid" transfection methods in which 293 cells are
co-transfected with three plasmids: pAAV with Lac Z, a second
plasmid with Rep and Cap genes, and a third plasmid that encodes
adenovirus proteins that mediate the "rescue" step (see Monahan and
Samulski, J. Virol. 61, 3096-3950 (2000)). With this strategy, the
293 cells do not have to be infected with adenovirus to "rescue"
the recombinant AAV particles, thus avoiding adenoviral
contamination of the final AAV preparation. AAV.about.Lac Z is
isolated from the 293 cell tissue culture with ammonium sulfate
precipitation and density gradient purification in accordance with
known techniques.
[0099] To attach the AAV.about.Lac Z to the platelet surface, the
platelets are incubated for three hours with the
platelet.about.anti-caps- id IgG conjugates prepared as above.
Excess AAV.about.Lac Z is removed with two centrifugation washes.
The platelet/AAV.about.Lac Z conjugates are then frozen,
lyophilized and stored at -20.degree. C. The freeze-dried
platelet/AAV.about.Lac Z conjugates are prepared so that upon
rehydration, the platelet and AAV.about.Lac Z concentrations will
respectively be 1.times.10.sup.8 platelets/ml and 1.times.10.sup.12
AAV.about.Lac Z vectors/ml.
EXAMPLE 7
[0100] Characterization of Platelet/AAV.about.Lac Z Conjugates
Surface density of AAV.about.Lac Z on the platelets. The amount of
anti-capsid protein antibody and AAV.about.Lac Z that is attached
to the platelet surface is quantified by subjecting samples to
SDS-PAG electrophoresis and Western analysis. Anti-rabbit IgG is
used to detect the anti-AAV, while anti-Capsid VP 1 is used to
probe for AAV proteins. The object is to attach approximately
100,000 AAV.about.Lac Z vectors to each platelet. If a lower
stoichiometry is obtained by any particular procedure the
concentration of the antibody can be adjusted upwards, and/or the
incubation time for the platelet reaction with AAV.about.Lac Z may
be increased.
[0101] The effect of Lac Z attachment on platelet function. The
effect of platelet surface modification with AAV.about.Lac Z on RL
platelet function may be ascertained by performing aggregation
assays and Baumgardner analysis in accordance with known techniques
(Khandelwal et al., FASEB J 11, 1812 (1997); Bode et al. J Lab.
Clin. Med. 133, 200-211 (1999)).
[0102] The effect of AAV.about.Lac Z attachment on transduction
efficiency. The effect of surface attachment on AAV.about.Lac Z
transduction efficiency is estimated by incubating the
AAV.about.Lac Z conjugates with porcine splenic macrophages and
then probing for gene transfer by staining with X-gal. Control
experiments may be performed with titers of AAV.about.Lac Z that
are equivalent to the number of virus particles delivered with the
platelets.
[0103] An important issue in the development of AAV as a gene
therapy tool is difficulties in obtain high titer preparations
(e.g., see Monahan and Samulski, supra). By immuno-absorbing
AAV.about.Lac Z to the surface of the platelet, a very high local
concentrations of the probe will be produced.
[0104] The coupling of AAV to platelets may affect the function of
both the viral and platelet systems. If the performance of the
platelets in Baumgardner and aggregation assays is measurably
affected by the probe attachment, the surface density of the
AAV.about.Lac Z may be be reduced. If platelet-bound AAV is found
to transduce splenic macrophages with a dramatically reduced
efficiency, the surface density of the viral vector may be
increased. Alternatively, other viral tethering strategies may be
utilized.
EXAMPLE 8
Reporter Gene Delivery to Sites of Vascular Injury
[0105] The ability of RL platelet/AAV.about.Lac Z conjugates to
transduce the Lac Z gene into sites of vascular injury will be
investigated. In this example atherosclerotic vessels in pigs on an
atherogenic diet are injured, RL platelet/AAV.about.Lac Z
conjugates are inufused, and the animals continjued on an
atherogenic diet to produce additional plaque development at the
sites of injury. This design models the sequence of events that
occur when a vessel is reoccluded with atherosclerotic plaque after
the endothelium is perturbed by atherosclerotic plaque rupture
and/or angioplasty. A detailed description of the experiment
follows. This system is useful in testing drugs such as statins or
the like, or dietary intervention, for activity in treating or
relieving atherosclerosis in a subject.
[0106] Induction of atherosclerosis. Four Lpb.sup.1/1 genotype
30-40 kg (young adult) male pigs are used. The Lpb.sup.1/1
(Lpg=L(lipoprotein), b(apoB100), p(pig)) is a polymorph genotype
that has been bred in a closed colony at the Francis Owen Blood
Research Laboratory at UNC-Chapel Hill. Inherited in an autosomal
co-dominant fashion, the Lpb.sup.1/1 animals develop a stable
degree of hypercholesterolemia on an atherogenic diet and
consistently develop moderately severe atherosclerosis that
correlates closely with dietary cholesterol levels (Nichols et al.,
Am. J. Pathyol. 140, 403-415 (1992)). Two pigs are placed on a high
cholesterol diet and two remain on a standard low-cholesterol
feed.
[0107] Vascular injury and treatment with RL platelet/Lac Z
constructs. After two months on the control or atherogenic diet,
the animals are anesthetized and portions of the right femoral
artery surgically isolated. Sites of vascular injury are formed by
crushing the artery with a Goldblatt clamp at two cm intervals.
Five wounds are established over a 10 cm length of vessel.
Immediately after establishing the wounds, 10.sup.9 AAV.about.Lac Z
modified platelets are rehydrated in 10 ml sterile saline and
infused into the peripheral ear vein. The surgical incisions are
repaired, and then the animals maintained on control or
high-cholesterol diets for two additional months.
[0108] Post-mortem analysis of tissues. After the post-surgery two
months on the control or high-cholesterol diet, the animals are
euthanized. The injured portion of the femoral artery, as well as
similar portions of the contralateral vessel, are histochemically
examined with X-gal staining to probe for the expression of the Lac
Z gene product galactosidase. Spleen, lung, cardiac and liver
tissue may also be analyzed for expression of the transgene.
EXAMPLE 9
Coagulation Protein Conjugation and Delivery to a Vascular Wound
Site
[0109] The preparation of a coagulation protein-platelet conjugate
can utilize a heterobifunctional cross-linker such as SANPAH
(N-succinimidyl 6-[4"-axido-2'-nitrophenylamino]hexanoate) with a
primary amine reactive and photoactivatable moieties for covalent
attachment. Various coagulation proteins can be used, including
recFVIIa (Novo Nordisk product), FVII or FVIIa and related mutants
from various expression systems, as well as coagulation proteins
that generate FVIIa, such as FXIIa or FXa. To prepare a RL
platelet-redFVIIa conjugate, a stock solution of 100 mM SANPAH is
prepared in anhydrous DMSO, then diluted 1/100 into 1 mg/ml
recFVIIa (e.g., from Novo Nordisk) in phosphate buffered saline (10
mM phosphate, 150 mM NaCl, pH=7.4) and allowed to incubate in the
dark for 1 hr. Unreacted SANPAH is removed by dialyzing the mixture
overnight vs. PBS or gel filtration on Sepharose CL4B.
[0110] Platelets are subjected to aldehyde stabilization and then
freed from excess paraformaldehyde as detailed in U.S. Pat. No.
5,651,966, and then the SANPAH-recFVIIa is conjugated to the cells.
The SANPAH-recFVIIa and the fixed platelets are then mixed for
100,000 platelets/ul and 0.1 mg/ml SANPAH-protein conjugate in PBS,
and then exposed to visible range light from a standard fluorescent
source for 1 hour to achieve photocoupling of the SANPAH-recFVIIa
conjugate to the platelet surface. Unreacted protein is then
separated from the platelets with chromatography on Sepharose
CL-2B, and then the recFVIIa-platelet conjugates are lyophilized as
detailed in U.S. Pat. No. 5,651,966.
EXAMPLE 10
Coupling Of Recombinant Factor VIIa to Surface of Lyophilized
Platelets Through Native Binding Sites
[0111] Preparation of fluorescein Isothiocyanate (FITC) labeled
recombinant factor VIIa (rFVIIa)-4.8 mg rFVIIa (NovaNordisk,
infusion grade) rehydrated with 4.8 ml dist. H.sub.2O for
[rFVIIa]=20 uM. rFVIIa was dialyzed overnight vs.phosphate buffered
saline (PBS), then incubated for 30 min at r.t. with 40 uM FITC.
The reaction mixture was dialyzed overnight vs PBS, then overnight
again vs. citrated saline to obtain rFVIIa-FITC.
[0112] Surface attachment of rFVIIa-FITC to rehydrated, lyophilized
platelets--Lyophilized platelets were hydrated with distilled water
for 1.89.times.10.sup.9cells/ml. Platelets and rFVIIa-FITC were
mixed and incubated at room temperature for 30 minutes at varying
concentrations ranging from 0 to 10 uM rFVIIa-FITC and 0 to
1.89.times.10.sup.9cells/ml. The platelets were centrifugally
washed once with saline, then diluted into PBS+2% paraformaldehyde
for flow cytometric analysis.
[0113] rFVIIa-FITC bound to lyophilized platelets in a homogeneous
manner based on the symmetrical flow-cytometric histogram (see FIG.
5, "Flow cytmometric analysis of rFVIIa-FITC bound to RL
platelets).
[0114] The degree of binding of rFVIIa-FITC to lyophilized
platelets was an increasing function of total rFVIIa-FITC
concentration (see FIG. 6, "rFVIIa binding to RL:platelets is
concentration dependent").
[0115] These results show that significant amounts of rFVIIa can be
attached to lyophilized platelets through simple co-incubation at
super-physiological concentrations.
[0116] The activity of rFVIIa that is surface-bound to lyophilized
platelets was analyzed by measuring the ability of these
preparations to catalyze prothrombin to thrombin conversion. RL
platelet-rFVIIa was prepared by incubating rFVIIa (10 uM, 3 uM, 1
uM 0.3 or 0 uM) with RL platelets (1.times.10.sup.5/ul) in citrated
saline with 10 mM CaCl.sub.2 for one hour. RL platelets-rFVIIa
particles were centrifically washed once to remove unbound rFVIIa,
then the preparations (as well as control buffer or similar
concentrations of rFVIIa alone) were diluted {fraction (1/10)} into
normal or factor IX deficient plasma that contained the
fluorometric thrombin substrate D-phe-pro-arg-ANSNH. The time for
initiation and maximal rate of thrombin substrate generation was
measured. The results in FIG. 7 show that RL platelet-bound rFVIIa
is at least as active as free rFVIIa on a molar basis (See FIG. 7,
"RL platelet-rFVIIa and free rFVIIa catalyze Ia generation in a
similar manner".
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[0143] The foregoing is illustrative of the present invention, and
is not to be construed as limiting thereof. The invention is
defined by the following claims, with equivalents of the claims to
be included therein.
* * * * *