Note: Descriptions are shown in the official language in which they were submitted.
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HEMOGLOBIN-HAPTOGLOBIN COMPLEXES
FIELD OF THE INVENTION
This invention relates to protein complexes and
use thereof in medical applications. More specifically, it
relates to complexes of hemoglobin compounds with
therapeutic substances such as drugs, genes etc. which have
a therapeutic action on specific parts and/or organs of the
body, and means for targeting such complexes to specific
body parts and body organs. Also within the scope of the
invention are complexes of hemoglobin with diagnostic
substances, such as imaging agents.
BACKGROUND OF THE INVENTION AND PRIOR ART
The use of hemoglobin and modified hemoglobin as
a drug delivery means has been proposed previously.
Hemoglobin, as a natural component of red blood cells,
present and circulating throughout the body in relatively
large quantities, has well-established bioacceptability and
the potential to deliver drugs throughout the body.
Thus, Kluger et al., U.S. Patent 5,399,671
describe a hemoglobin compound which has been.cross-linked
to effect intramolecular stabilization of the tetrameric
structure thereof, but which contains a residual functional
group on the cross-linker residue to which drugs for
delivery can be covalently attached.
Anderson et al., U.S. Patent 5,679,777, describe
complexes of hemoglobin compounds and polypeptide drugs, in
which the polypeptide drug is bound to a globin chain
through a disulfide linkage to a cysteine unit inherent in
or genetically engineered into the globin chain.
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Haptoglobins (Hp) constitute part of the a2-
globin family of serum glycoproteins. Haptoglobins are
present in mammalian plasma, and constitute about one-
quarter of the a2-globulin fraction of human plasma. Each
individual has one of three phenotypic forms of
haptoglobin, of close structural and chemical identity.
Haptoglobins are composed of multiple a(3 dimers and the
phenotypes are conventionally denoted Hp 1-1, Hp 2-1 and Hp
2-2. The (3 chains are identical in all haptoglobin
phenotypes, but the a chains vary (al and a2) The amino
acid sequences of all chains are known. Hp 1-1 is composed
of two a1R dimers and has a molecular weight of about 98
kDa. The structure of Hp 2-1 and Hp 2-2 can be written as
follows: (al(3) 2 (a2R)õ where
n = 0,1,2,... and (a2(3)m where m = 3,4,5,... respectively.
Delivery of drugs to a patient suffering from a
disease or disorder affecting primarily one body part or
one body organ is best accomplished by choosing a delivery
method which targets the part or organ in need of treatment
with a high degree of specificity. Such a delivery system
makes most effective use of the active drug, so as to
reduce the necessary dosage level, and reduces side effects
of the drug.
One function of haptoglobin is to bind
extracellular hemoglobin, arising from red blood cell
lysis, to form essentially irreversible haptoglobin-
hemoglobin complexes which are recognized by specific
receptors on hepatocytes in the liver. In this way,
hemoglobin is targeted to the liver for metabolism.
Control and manipulation of genes and gene
products are potentially powerful means of treating various
diseases and genetic disorders. When specifically
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introduced into the cells, genes can use the host cell
biosynthetic machinery for the expression of the therapeutic
biomolecules they encode. For successful gene therapy, one must
devise a successful method of in vivo gene delivery. One such
technique developed in recent years is receptor-mediated
delivery. This has the advantage of high specificity of delivery
to the cells which express the targeted receptor.
The specific targeting of low molecular weight
therapeutic and diagnostic agents to tissues is enhanced greatly
through the use of receptor-mediated delivery. Diagnostic agents
such as fluorescent or radiolabeled substances indicate the
location and quantity of cells bearing the targeted receptors
when such agents are administered as complexes with ligands for
those receptors. These complexes are also useful in
characterizing the binding and transport properties of receptors
on cells in culture. Such information is useful in detection of
and/or design of therapy for tissues containing the cells being
recognized, either in vitro or in vivo.
SU4MARY OF THE INVENTION
In one aspect the present invention provides a means
and composition for specifically targeting hepatocytes or other
cells having receptors for hemoglobin-haptoglobin complexes with
therapeutically active substances or diagnostic agents.
In a further aspect the present invention provides a
novel complex of a substance selected to exert a beneficial
effect on a mammalian patient's liver, in vivo, and a substance
which specifically targets hepatic
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cells.
The present invention describes haptoglobin-
hemoglobin construct-complexes to which hepatocyte-
modifying agents are attached. Such haptoglobin-hemoglobin
construct-complexes serve as effective hepatocyte-targeting
vehicles for the attached agents, for delivery of specific
hepatocyte-modifying agents (drugs, diagnostics, imaging
compounds, etc) to the liver, and to other cells having the
appropriate hemoglobin-haptoglobin receptors.
The expression "construct-complex" is used herein
to refer to the combination of haptoglobin with hemoglobin
to which a bioactive, therapeutic or diagnostic agent is
attached. The present invention provides construct-
complexes composed of a hemoglobin compound, a haptoglobin
and a hepatocyte-modifying substance of interest such as a
drug, a diagnostic agent or a gene. In one aspect of the
present invention, the construct-complex is prepared
extracorporeally and then administered to the patient. In
another aspect, a complex of hemoglobin-hepatocyte
modifying substance is prepared extracorporeally,
administered to the patient, and forms the construct-
complex of haptoglobin-hemoglobin-hepatocyte modifying
substance with haptoglobin which is naturally present in
the patient's serum. In a further aspect, the patient's
haptoglobin level may be supplemented by haptoglobin
administration, a known procedure, either before, during or
after administration of the hemoglobin-hepatocyte modifying
substance-construct-complex. In any case, the construct-
complex specifically targets and binds as a ligand to the
hepatocyte receptors, owing to the presence of the
haptoglobin and hemoglobin portions of the construct-
complex.
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The construct-complexes of the present invention,
formed ex vivo or in vivo, target any cells having
receptors for Hb-Hp complexes, and this includes metastases
arising from primary hepatoma. It is normally difficult to
identify and treat metastases because of the systemic
distribution and small size of such cancers. Secondary
hepatic metastases, i.e. hepatoma cells outside the liver
which have such receptors are targeted by the construct-
complexes of the present invention, as well as cells of the
liver, and should be regarded as "hepatocytes" as the term
is used herein.
Further, the construct-complexes of the present
invention may exert beneficial effects on neighboring
cells, if the hepatocyte modifying substance is, for
example, a drug which is active towards neighboring cells
even if they are not hepatocytes. They may also modulate
or initiate the activity of other therapeutic or diagnostic
agents delivered by other methods for hepatocyte
modification, such as prodrugs, enzymes or genes coding for
enzymes and requiring activation to cause an effect.
Agents effecting such action resulting in hepatocyte
modification or effect on other agents or cells are
hepatocyte modifying agents according to this invention.
The construct-complex according to the present
invention can be generally represented by the formula:
(HP)a - (Hb) b - (Lc - Ad)e
where a = 1 to about 10;
b = 0.5 to about 10;
c = 0 to about 10;
d = 1 to about 20;
e = 1 to about 20;
Hp is haptoglobin as described herein;
Hb is a hemoglobin as described herein;
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L is a linker as described herein;
and A is a hepatocyte modifying agent as described
herein,
in which the stoichiometry of Hp to Hb in the complex is
dictated by the available number of binding sites on the
two proteins, but is generally of the order of 1:05 to 1:2.
BRIEF REFERENCE TO THE DRAWINGS
Figure 1 is a reaction scheme illustrating
diagrammatically a process for producing one embodiment of
a construct-complex of the present invention;
Figures 2A, 2B, 2C and 2D are size exclusion
chromatography results, in the form of plots of absorbance
at 280 nm and 414 nm against elution time, indicating the
molecular weight distribution of the four products of
Example 2. Complexes were formed using poly(L-lysine) of
molecular weight (A) 4 kDa, (b) 7.5 kDa, (C) 26 kDa and (D)
37 kDa.
Figure 3 is a similar plot, for the product
complex utilizing 26 kDa poly(L-lysine) after 24 hours
incubation with haptoglobin, produced in Example 2;
Figure 4A - 4B are depictions of gel mobility
shift assays of DNA in the presence of (A) THb and (B) THb-
poly(L-lysine) produced according to Example 4;
Figure 5 is a dye fluorescence assay of the
products of Example 4;
Figure 6 is a depiction of the gel mobility shift
assay of the products of Example 4;
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Figure 7 is a fluorescence assay of another
product of Example 4;
Figure 8 is a size exclusion chromatogram of the
product of Example 6;
Figures 9A, 9C and 9D show size exclusion
chromatograms of the products of Example 9, utilizing (A)
haptoglobin 1-1, (C) haptoglobin 2-1, (D) haptoglobin 2-2;
Figure 9B shows the UV-visible spectra of the
products of Example 9;
Figure 10 is a size exclusion chromatogram of the
product of Example 11;
Figure 11 is a size exclusion chromatogram of the
product of Example 13;
Figure 12 shows anion exchange chromatograms
(overlaid) of products and starting materials of Example
14;
Figure 13 shows overlaid size exclusion
chromatograms of the products of Example 15;
Figure 14 shows size exclusion chromatography
elution profiles with detection at 280 (solid lines) and
414 nm (broken lines) for products of Example 16; (A)
haptoglobin 1-1, (B) 64 kDa ORHb, (C) haptoglobin-[64-kDa
ORHb], (D) >64 kDa ORHb, (E) haptoglobin-[>64 kDa ORHb].
Figure 15 shows size exclusion chromatograms of
products of Example 17;
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Figures 16 A-D are graphical presentations of
analyses of results obtained in Example 18;
Figure 17 is a graphical presentation of further
analyses of results obtained according to Example 18.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
A wide range of hepatocyte modifying substances
may be used in complexes of the present invention. These
can be therapeutic agents, diagnostic agents, markers or
the like capable of interacting with hepatocytes and
consequently capable of acting in vivo at the liver. They
can be designed for treatment of normal liver cells or such
cells undergoing metastases. Thus, the hepatocyte-
modifying substances can be antineoplastic substances
(doxorubicin, daunorubicin, ricin, diphtheria toxin,
diphtheria toxin A, for example), antiviral substances
(ara-AMP, trifluorothymidine, interferon, antisense
oligonucleotides, ribavirin, cytarabin, acyclovir,
didonosine, vidarabine, adefovir, zalcitabine, lamivudine,
fialvridine, and other nucleoside analogs, for example),
anti-inflammatory substances, anti-parasitic substances,
antimicrobial substances, antioxidant substances,
hepatoprotective agents, imaging and diagnostic agents,
nucleic acids and their compounds for effecting gene
therapy, agents effecting lipid metabolism, anti-toxicants,
proteins, enzymes, enzyme and prodrug combinations, and the
like.
Examples of diagnostic agents useful in
construct-complexes in this invention include radiolabeled
lysine and putrescine, and the fluorescent compounds
monodansyl cadaverine and fluorescein. Low molecular
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weight therapeutic agents can also be selectively targeted
to the cells to minimize side effects at non-targeted
tissues and vascular clearance. Examples of therapeutic
agents in this application include putrescine, a modulator
of cell growth and activity, and primaquine, an anti-
malarial substance.
More specifically, hepatocyte modifying
substances which can be used in construct-complexes
according to the present invention include agents for
treating or preventing hepatic fibrosis, a dynamic process
from chronic liver damage to cirrhosis, and for treating or
preventing other chronic liver disorders including viral
hepatitis and alcoholic and cryptogenic liver diseases.
These hepatocyte modifying substances include
cytoprotective drugs such as S-adenosyl-L-methioine,
prostaglandin E1,E2,I2 and their analogues, colchicine and
silymarin, all of which have been demonstrated to be
effective in protecting the liver from damage and having
anti-fibrotic properties. Other liver protectant substances
which are hepatocyte modifying substances within the scope
of this invention include free radical scavengers/anti-
peroxidants such as glutathione, SA 3443 (a cyclic
disulphide), S-adenosylmethionine, superoxide dismutase,
catalase, a-tocopherol, vitamin C, deferoxamine,
(+)cianidanol-3, mannitol, tryptophan, pantetheine,
pantotheinic acid, cystamine, cysteine, acetylcysteine,
folinic acid, uridine monophosphate, zinc sulphate,
schizandrin B and kopsinine; lipoxygenase inhibitors such
as the aforementioned prostaglandins and their analogs
dimethyl PGE, misoprostol and enisoprost, and prostacyclin
PGI2 and its analog iloprost; calcium channel blockers such
as trifluoroperazine, verapamil, nifedipine and related
dihydropyridine compounds, and dilitiazem; proteinase
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inhibitors; atrial natriuretic peptide; a2-
macrofetoprotein;synthetic linear terpenoid; putrescine;
cholestyramine; e-aminocaproic acid,; phenylmethylsulfonyl
fluoride; pepstatin; glycyrrhizin; fructose 1,6-
biphosphate; and ursodeoxycholic acid.
The hemoglobin compound useful as a component of
the complexes of the present invention can be substantially
any hemoglobin compound providing the necessary degree of
biocompatibility for administration to a patient or animal,
the necessary sites for attachment of the hepatocyte
modifying substance of interest, and having sufficient
binding affinity for haptoglobin. Within these
limitations, it can be a naturally occurring hemoglobin
from human or animal sources. It can be a modified natural
hemoglobin, e.g. an intramolecularly cross-linked form of
hemoglobin to minimize its dissociation into dimers, an
oligomerized form or a polymerized form. It can be a
hemoglobin derived from recombinant sources and techniques,
with its naturally occurring globin chains or such chains
mutated in minor ways. It can be comprised of subunits or
fragments of Hb, or derivatives thereof, which have
affinity for haptoglobin. It can be a hemoglobin in which
individual amino acids of the globin chains have been
removed or replaced by site specific mutagenesis or other
means. Certain modifications which are known to decrease
the affinity of hemoglobin for binding to haptoglobin are
preferably avoided in hemoglobin compounds used in the
present invention.
One type of preferred hemoglobin compounds are
those which comprise hemoglobin tetramers intramolecularly
cross-linked to prevent their dissociation into dimers, and
which leave functional groups available for chemical
reaction with the hepatocyte modifying substance, either
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directly or through a chemical linker molecule. Such
hemoglobin compounds have the advantage that they provide a
known, controlled number of reactive sites specific for the
therapeutic substance of interest, so that an accurately
controlled quantity of the therapeutic substance can be
attached to a given amount of hemoglobin compound. They
also have the added advantage that they avoid utilizing
sites on the globin chains for linkage to the
therapeutically active substance, so as to minimize
conformation disruption of the globin chains and minimize
interference with the hemoglobin-haptoglobin binding and
with binding of the construct-complex to the receptor
protein on a hepatocyte cell.
Human hemoglobin, e.g. that obtained from
outdated red blood cells, and purified by the displacement
chromatography process described in U.S. Patent 5,439,591
Pliura et al. is one preferred raw material for preparation
of the hemoglobin product for use in the complex of the
present invention. This material may be cross-linked with
a trifunctional cross-linking agent as described in
aforementioned U.S. Patent 5,399,671, Kluger et al., namely
a reagent which utilizes two of its functional groups for
intramolecular cross-linking between subunits of the
hemoglobin tetramer, and leaves its third functional group
available for subsequent reaction with a nucleophile. A
specific example of such a cross-linking reagent is
trimesoyl tris(3,5-dibromosalicylate), TTDS, the chemical
formula of which is given in the attached Figure 1, and the
preparation of which is described in the aforementioned
Kluger et al. U.S. Patent 5,399,671.
When cross-linked hemoglobin, i.e. stabilized
tetrameric hemoglobin is used as a component of the
complex, the hepatocyte modifying substance is bound to the
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hemoglobin, either directly or through a chemical linker or
spacer, and then this complex may be administered to the patient
so that the haptoglobin-hemoglobin binding takes place in vivo.
The entire construct-complex, (haptoglobin-hemoglobin-hepatocyte
modifying substance) can, if desired, be formed extracorporeally
and then administered to the patient, and this can under some
circumstances lead to better control of the amounts of active
substance finally being delivered to the hepatocytes. However,
such a procedure is not normally necessary, save for those
exceptional patients having zero or low levels of haptoglobin,
e.g. in conditions of acute hemolysis. Such patients can be
administered haptoglobin before, during and/or after
administration of the construct-complex of the invention.
Usually, however, there is sufficient haptoglobin in the
patient's plasma to form the construct-complex in situ and effect
its delivery to the hepatocytes. Preparation of the two-part
complex and administration of that to the patient, to form the
three-part complex in situ is generally cheaper and less
complicated.
Use of intramolecularly crosslinked hemoglobins will
give rise to high molecular weight polymers containing more than
one hemoglobin and/or haptoglobin owing to the presence of two
binding sites on each of these proteins. There may be
advantages to using non-intramolecularly crosslinked hemoglobin
as a component of the construct-complexes of the present
invention. Such a hemoglobin, with a hepatocyte-modifying
substance bound to it, will dissociate into dimeric hemoglobin of
approximate molecular weight 32 kDa, and two such dissociated
dimeric hemoglobin products bind to a single molecule of
haptoglobin to give a complex according to the present invention.
The formation of high molecular weight haptoglobin-hemoglobin
complexes is thus avoided. Haptoglobin binding to a8-dimers is
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generally a much faster reaction than haptoglobin binding to
crosslinked hemoglobin. The lower molecular weight complexes
resulting from the use of non-crosslinked hemoglobin may show
improved hepatocyte receptor binding and uptake.
Where hemoglobin of a form which will dissociate into
dimers is used as a component of the present invention, or where
hemoglobin dimers themselves are used, for example, where the
dimers have been modified such that they cannot reform 64 kDa
hemoglobin, it is preferred to form the construct-complex
according to the invention extracorporeally, and then to
administer the finished construct-complex to the patient, so as
to avoid the risks attendant on administering to the body of a
molecular species of too small a molecular weight, namely,
clearing the drug too rapidly through excretion. Administration
of Hb dimers bearing therapeutic or diagnostic agents may be
possible without prior binding to haptoglobin in cases where
complex formation in vivo is adequate prior to clearance of the
modified dimer.
A further example of a hemoglobin compound useful in
construct-complexes of the present invention is dimeric
hemoglobin bearing a modifying group containing thiol, preferably
a terminal side chain thiol, of the type described in U.S. Patent
Application No. 5,399,671 dated March 21, 1995 of Kluger et al.
Hepatocyte modifying substances can be ligated to such dimeric
hemoglobin, either by direct reaction with the exposed thiol, or
by direct reaction with an activated form of the thiol, or by
mixed disulfide formation, or through a linker molecule.
Construct-complexes of this type are made extracorporeally and
administered to a patient in this form. The hemoglobin-
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hepatocyte modifying substance conjugate can also be
administered for in vivo Hp binding. The use of
dissociable hemoglobin (32 kDa molecular weight) has the
advantage over the use of cross-linked hemoglobin tetramers
in that they provide an exposed dimer-dimer interface which
facilitates haptoglobin binding.
The construct-complexes of the present invention
may also utilize hemoglobin which has been modified in a
manner which results in impaired nitric oxide binding. Such
modified hemoglobins are known in the art. Reduced NO
binding may reduce the tendency of the hemoglobin to effect
modifications to a patient's blood pressure upon
administration, an effect which has been noted with some
hemoglobins, even in small amounts.
In forming the construct-complex, it may be
necessary to interpose between the reactive site on the
hemoglobin chosen and the hepatocyte modifying substance, a
chemical linker or a spacer group. This depends upon the
nature of the available chemical group on hemoglobin for
linking, and on the chemical groups available on the
hepatocyte modifying compound, for this purpose. For
example, a polycationic segment such as polylysine is
appropriately attached to the electrophilic site of the
TTDS modified hemoglobin to provide a binding site for DNA
through electrostatic interactions. Linear polymers of
lysine provide appropriate cationic segments for this
purpose.
A construct-complex according to a preferred
embodiment of the present invention comprises a haptoglobin
molecule, which may be haptoglobin 1-1 or any other
phenotype, bonded to one or more molecules of a hemoglobin
compound by means of strong non-covalent interaction. The
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hemoglobin may be cross-linked, oligomerized or unmodified,
as described above.
Figure 1 diagrammatically illustrates the
chemical steps involved in preparing a cross-linked
hemoglobin, for reaction with a linker and/or agent, and
with haptoglobin to form a construct-complex according to
various embodiments of the invention. TTDS-is reacted with
hemoglobin, whereupon two of the three 3,5-
dibromosalicylate groups leave. Primary amine groups at
Lys-82 and (3-Lys-82 on the hemoglobin are bonded by an
amide linkage to the cross-linker, forming an
intramolecularly cross-linked and stabilized tetrameric
hemoglobin with the third dibromosalicylate group intact
and available for further reaction. In the second step,
the cross-linked hemoglobin is reacted with the agent or a
linker (in the case of Example 1, polylysine) necessary for
later attachment of the agent. In other cases, the
hepatocyte modifying substance, or active agent, takes the
place of the polylysine in the scheme of Fig. 1, to form
the construct. The complex is then ready for administration
to the patient to form a construct-complex in situ, or
alternatively haptoglobin can be reacted with the complex
so formed extracorporeally, so that the haptoglobin binds
to the hemoglobin portion of the complex to form the three
part complex ready for administration to the patient.
Alternatively, the TTDS-modified hemoglobin with a linker
attached can be reacted with haptoglobin and agent attached
as a final step. After administration, the construct-
complex will bind to the hepatocytes, where the
haptoglobin-hemoglobin mediates binding to the selective
receptors thereof and allows the hepatocyte-modifying
substance to be delivered to and enter into the hepatocyte
utilizing the hepatocyte receptors selective for
haptoglobin-hemoglobin complex.
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SPECIFIC EXAMPLES
Example 1 - Conjugation of TTDS cross-linked hemoglobin
(THb) to poly(L-lysine):
Poly(L-lysine) conjugates of TTDS cross-linked
hemoglobin (THb-Kr,) were synthesized by adding poly(L-
lysine) to THb-DBS (TTDS cross-linked hemoglobin with one
unhydrolyzed 3,5-dibromosalicylate functionality) at 1:1
molar ratio to promote formation of conjugates in which
only one molecule of hemoglobin is attached to a single
poly(L-lysine) chain. The poly(L-lysine) used in this
experiment is a linear polymer with an amide linkage
between the carboxyl group and the a-amino group of lysine.
Polymers with an average molecular weight of 4 kDa (K4kDa)'
7.5 kDa (K7,SkDa), 26 kDa (K26kDa) and 37 kDa (K37kDa) were
conjugated to THb.
TTDS (13.9 mg) in ethanol (100 UL) was added to
deoxyhemoglobin (5 mL, 8.5 g/dL) in 50 mM borate pH 9Ø
The reaction mixture was stirred at 30 C under nitrogen for
45 min. The hemoglobin was then charged with CO (the
solution was kept on ice) and the excess of the cross-
linking reagent was removed by passing the hemoglobin
solution through a Sephadex G-25 column (200 mm L x 25 mm
D) equilibrated with 50 mM borate pH 9Ø The resulting
hemoglobin solution (3.6 g/dL) was again charged with CO.
Poly(L-lysine) solutions were prepared in 50 mM borate pH
8.0 and added to hemoglobin (3.6 g/dL, 1.9 mL) as indicated
in Table 1 below. The molar ratio of poly(L-lysine) to
hemoglobin was 1:1 for all four polymers. The THb-poly(L-
lysine) conjugates (THb-Kn) were sealed in serum bottles,
recharged with CO and left at room temperature for two
days. Hemoglobin concentrations in these samples were
determined using Drabkin's reagent.
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Table 1
Poly(L-lysine) Amount of poly(L-lysine) added to
THb
(mg)
K4kDa 4.2
K7.5kDa 8.0
K26kDa 27.5
K37kDa 39.3
Anion Exchange Chromatography: Crude THb-K,
complexes were analyzed using anion exchange chromatography
on a SynChropak AX-300 column (250 mm L x 4.6 mm D,
SynChrom, Inc.). A sodium chloride gradient was used to
elute various modified hemoglobins. The effluent was
monitored at 280 nm.
By the time of analysis all unreacted THb-DBS had
hydrolyzed to give THb. The reaction resulted in a mixture
of products all of which, as expected, migrated before the
THb on the anion exchange chromatography media. The yields
were calculated by adding the peak areas of the early
eluting peaks and comparing them to the total peak area.
Yields of poly(L-lysine) modified hemoglobin calculated in
this way were: 37, 37, 81 and 84% for K4kDa, K7.5kDa, K26kDa and
K37kDav respectively.
Purification of THb-Kn conjugates: THb-K,,
conjugates were separated from unconjugated THb by anion
exchange chromatography on a POROS HQ/50 column (52 mm L,
14 mm D) equilibrated with 25 mM Tris-HC1 buffer pH 8.4.
Modified Hbs were eluted with a sodium chloride gradient.
The effluent was monitored at 280 nm and pooled fractions
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containing THb-K,, conjugates were concentrated using an
AmiconTM diafiltration device and a 30 kDa cutoff membrane.
Size Exclusion Chromatography: The molecular
weight distribution of purified THb-K, conjugates and their
haptoglobin complexes was determined using size exclusion
chromatography (SEC) on a SuperdexTM-200 column (300 mm L x
mm D, Pharmacia) equilibrated and eluted with 0.5 M
magnesium chloride containing 25 mM Tris-HC1 pH 7.2 at a
10 flow rate of 0.4 mL/min. The effluent was monitored at 280
nm and 414 nm. Hemoglobin to poly(L-lysine) stoichiometry
ranged from 1:1, using 4 kDa poly(L-lysine), to
heterogeneous constructs with stoichiometries up to 4:1
using the higher molecular weight poly(L-lysine) linkers,
according to corresponding elution times with molecular
weight standards. No unmodified THb was present. These
constructs were stable under the high salt conditions of
chromatography.
Example 2 - Complex formation between THb-K, and haptoglobin
1-1
The following stock solutions were used for the
preparation of the complexes: 1.74 mg/mL haptoglobin 1-1
(Hp) in water and 1.0 mg/mL solutions of the THb-K,, (all
THb-Kn concentrations represent hemoglobin concentrations)
in 50 mM sodium borate pH 9Ø Haptoglobin (14 pL) was
added to THb-Kn in potassium phosphate pH 7.0 to give the
following final concentrations: 0.12 mg/mL (1.22 pM)
haptoglobin and 0.19 mg/mL (2.9 pM) THb-K, in 25 mM
potassium phosphate pH 7.0 (200 pL final volume). After
incubation for 180 min. at room temperature, the samples
were analyzed using SEC.
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THb-Kõ complexes with haptoglobin 1-1: The
formation of THb-K,, complexes with haptoglobin can be
followed using size exclusion chromatography (SEC). Figure
2A shows the composition of the THb-K4kDa mixture with Hp
after incubation at room temperature for 180 min. A new,
high molecular weight peak appears at 25.5 min. Plots of
the ratio of absorbance at 280 and 414 nm (A280/A414) over the
elution period indicate the relative proportions of
haptoglobin and hemoglobin in the construct-complexes and
other peaks. The absorbance ratio (A,80/A414) throughout the
new peak is 0.9 indicating that both haptoglobin and
hemoglobin components are present in this complex.
Haptoglobin 1-1 migrates at 29.7 min. and is easily
identified by high A280/A414 ratio. Figure 2B shows SEC of
the THb-K7.SkDa mixture with Hp after incubation at room
temperature for 180 min. Again, a new peak appears at 25.1
min. with a A280/A414 ratio of 0.73, followed by haptoglobin
at 29.7 min. and THb-K7.5kDa at 35.8 min. with A280/A414 ratio
of 0.3. The analysis of the SEC of THb-K26kDa and THb-K37kDa
complexes with haptoglobin is more complicated due to their
broad molecular weight distribution. The results are
presented in Figs. 2D and 2D respectively. It is evident
from Figure 2C and 2D that both THb-K26kDa and THb-K37kDa form
complexes with haptoglobin. The A280/A414 ratio is 0.64 for
THb-K26kDa-Hp and 0.69 for THb-K37kDa-HP
Degree of THb-K26kDa-Hp complex formation: To
determine whether all structurally different components of
the THb-Kõ bind to haptoglobin, THb-K26kDa was incubated with
a 15% excess of haptoglobin for various lengths of time and
then analyzed using SEC. The following stock solutions were
used for the preparation of the complex: 1.74 mg/mL
haptoglobin 1-1 in water and 7.4 mg/mL solutions of THb-
K26kDa in potassium phosphate pH 7.0 to give the following
final concentrations: 0.74 mg/mL (7.5 mM) haptoglobin and
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0.41 mg/mL (6.4 mM) THb-K26koa (1.2:1 molar ratio of Hp to
Hb) in 25 mM potassium phosphate pH 7Ø After incubation
at room temperature for various lengths of time, the
mixtures were analyzed using SEC. The progress of the
reaction was followed by monitoring the disappearance of
haptoglobin peak on a SEC profile. 85% of the THb-K26koa was
bound by haptoglobin after 24 hours. The resulting THb-
K26koa-Hp complex has a broad molecular weight distribution
ranging from 370 kDa to app. 1000 kDa (Fig. 3).
Example 3 - DNA Binding to THb-Kn and THb-Kõ-Hp. Gel
Mobility Shift Assay:
Gel mobility shift assays were conducted to
evaluate the stoichiometry of binding of plasmid DNA
(pCMVbeta) to the THb-Kõ conjugates. This gel
electrophoretic method is based on the observation that the
migratory properties of the DNA are altered upon binding
protein. Neither proteins nor DNA-protein complexes in
which protein constitutes a significant part of their mass
enter 1% agarose gels. If mixtures with an increasing THb-
Kõ to DNA ratio are analyzed, it is observed that the DNA
band disappears at and above the ratio that corresponds to
the stoichiometry of the complex. For each of the four
conjugates and for the THb-K26koa-Hp complex, solutions
containing from 0.4 to 6400 ng of the conjugate (this
weight based on the hemoglobin component) in 32 pL of 20 MM
HEPES pH 7.3 containing 150 mM NaCl were prepared. The
plasmid DNA (560 ng in 28 /.2L of 20 mM HEPES pH 7.3
containing 150 mM NaCl) was added dropwise to each sample
and the mixtures were incubated for 1 hour at room
temperature. The samples (15 pL) were analyzed on a 1%
agarose gel containing ethidium bromide (0.2 pg/mL). The
amount of conjugate which prevented DNA entry into the gel
was determined. Results are described in the following
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Example.
Example 4 - DNA binding to THb-K26kDa and THb-K26kDa-Hg
complex: Thiazole Orange Fluorescence Quenching Method:
This dye fluorescence assay is based on the
observation that a DNA intercalating dye (thiazole
orange)is fluorescent only if bound to DNA. Complex
formation between THb-Kn and DNA causes the displacement of
the intercalating dye from DNA and the decrease of total
fluorescence.
The following stock solutions were used in this
experiment: 0.05 mg/mL DNA (pCMVbeta), 0.010 mg/mL, THb-
K26kDa or THb-K26kDa-Hp complex, 1.75 x 10-6 M thiazole orange
(0.1 mg/mL solution in 1% methanol was diluted 190 times
with water), 20 mM HEPES pH 7.3 containing 0.15 M NaCl.
Plasmid DNA (10 pL), THb-K26kDa (volumes varying from 2.5 to
60 pL) and buffer (to the final volume of 200 pL) were
mixed in a generic 96 well plate and incubated for 2.5
hours at room temperature. Sample containing thiazole
orange in HEPES buffer was also prepared and used as a
background control. Fluorescence was measured on a Packard
FluoreCountTM plate reader using excitation at 485 nm and
emission at 530 nm. The THb-K26kDa-Hp complex was prepared
as described above and used without purification. It was
diluted with 20 mM HEPES pH 7.3 containing 0.15 M NaCl to
give a final concentration of 0.010 mg Hb/mL.
The gel mobility shift assay and the fluorescence
quench assay both demonstrated that THb-Kn binds to DNA.
Figure 4A (left) and 4B (right) are depictions of gel
mobility shift assays of haptoglobin-hemoglobin-DNA
conjugates produced according to Example 4. One hundred
and forty ng of DNA were added to increasing amounts of (A)
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THb or (B) THb-K26kDa. Lane 1 of both gels contain DNA
molecular weight markers. Hb content in other lanes: (A2)
50 ng, (A3) 100 ng, (A$) 200 ng, (A5) 400 ng, (A6) 800 ng,
(A7) 1600 ng, (A8) empty, (B2) 25 ng, (B3) 50 ng, (B4) 100
ng, (B5) 200 ng, (B6) 400 ng, (B7) 800 ng, (B8) DNA only.
As regards the gel mobility shift assay, increasing the
proportion of THb-K, in the DNA samples affected DNA
migration as seen in Fig. 4. Figure 4A shows the migratory
properties of DNA after incubation with increasing amount
of THb ranging from 50 to 1600 ng of protein. In this
concentration range THb does not bind DNA, since no change
in DNA migration can be detected. THb-K26kDa is most
effective at binding DNA. One hundred ng of THb-K26kDa (THb-
K26kDa to DNA ratio = 0.7, w/w) completely prevents the DNA
from entering the agarose gel (Fig. 4B). Approximately 400
ng of the other THb-Kõ preparations were required to bind
all DNA. The results for THb-K26kDa are in good agreement
with the fluorescence quench assay which indicated 86% of
fluorescence decrease at the same THb-K26kDa to DNA ratio.
Fig. 5 shows the effect of THb26kDa on DNA-thiazole orange
fluorescence. On Fig. 5, the amount of THb-K26kDa is based on
the hemoglobin component only.
The THb-K26kDa-Hp complex also binds DNA. It was
found that 200 ng of THb-K26kDa-Hp completely prevented 140
ng of DNA from entering the agarose gel (THb-K26kDa-Hp to DNA
ratio = 1.4, w/w). Fig. 6 shows THb-K26kDa-Hp binding to DNA
by gel mobility shift assay. One hundred and forty ng of
DNA were added to increasing amounts of THb-K26kDa-Hp: 25 ng
(lane 2), 50 ng (3), 100 ng (4), 200 ng (5), 400 ng (6),
800 ng (7), weights based on the hemoglobin component.
Molecular weight standards were loaded in lane 1 and 140 ng
of DNA in lane 8. At the same THb-K26kDa-Hp to DNA ratio
(1.4:1 w/w) the fluorescence assay indicates only 42% of
fluorescence decrease and 81% fluorescence decrease at 2.8
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ratio. The fluorescence assay is shown in Fig. 7 (the
weight of the conjugate is based on the hemoglobin
component thereof only). Comparison of the gel mobility
shift assays for THb-K26kDa-Hp indicates that approximately
twice as much protein-bound poly(L-lysine) is required to
prevent DNA from migrating into the gel when the
haptoglobin complex is used. Since the amount of
hemoglobin conjugated poly(L-lysine) was identical in both
experiments, the decreased DNA binding ability of THb-K26kDa-
Hp is probably due to steric crowding in the THb-K26kDa-HP-
DNA complex.
In these examples, there has been synthesized and
characterized a construct having all the necessary
components for in vivo targeted gene delivery to human
hepatocytes through haptoglobin receptors. Poly(L-lysine)
was conjugated to the TTDS cross-linked hemoglobin to
provide a site for binding DNA through electrostatic
interactions of its positively charged E-amine groups with
the negative charges of phosphate groups on DNA. It has
been previously demonstrated that when more than 90% of
DNA's negative charges are neutralized, the linear DNA
strand is compacted into a toroid structure, a form which
is more stable and more amenable to internalization by
cells. Optimal gene expression has been reported for the
DNA to poly(L-lysine) ratios which result in electroneutral
complexes.
The gel mobility shift and the fluorescence
assays have demonstrated that THb-K26kDa-Hp complex binds the
plasmid DNA thus completing the assembly of a construct
potentially capable of delivering oligonucleotides by
haptoglobin receptor-mediated endocytosis.
Example 5 - Synthesis of crosslinked hemoglobin bearing
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tritiated or non-tritiated lysine
A solution of L-[3H]-lysine was evaporated under a
stream of nitrogen to obtain 59.5 nmole (5 mCi) of solid
material. 59.5 nmole of non-radiolabeled L-lysine was
prepared in a similar manner. TTDS (39.8 mg) was dissolved
in ethanol (270 /mL) and 200 L of this solution was added to
deoxyhemoglobin (10 rL, 9.2 g/dL) in 50 mM borate pH 9Ø The
reaction mixture was stirred at room temperature under
nitrogen for one hour, then oxygenated. Excess cross-linker
was removed from half of the mixture by gel filtration and
then the solution was CO charged and frozen, giving
crosslinked Hb with an activated ester on the crosslinker
(THb-DBS, 62 mg/mL) as described by Kluger (US 5,399,671).
Unreacted crosslinker was removed from the other half of the
crude reaction mixture by gel filtration using 0.1 M L-
lysine/L-lysine hydrochloride elution buffer (pH 9.0). The
eluate was CO charged and left at room temperature overnight.
Using this process, lysine became conjugated to the linker via
the activated ester, giving THb-Lys. Freshly thawed THb-DBS
(29.5 nmole, 30.5 L) was added to the radiolabeled and the
non-radiolabeled lysines each day for three days. THb-Lys
(700 L) was then added to both mixtures and the products
desalted. Completion of the reaction was confirmed by anion
exchange chromatography.
Example 6 - Haptoglobin-THb-Lys complex:
Haptoglobin (1.61 mg/mL haptoglobin 1-1 in water,
11 L) was added to THb-Lys (38 mg/mL in 50 mM sodium
borate pH 9.0) to give the following final concentrations:
0.68 mg/mL (6.9 M) haptoglobin and 0.41 mg/mL (6.4 M)
THb-Lys were made up to a final 200 L volume at 25 mM
potassium phosphate pH 7Ø Within 18 hours, the
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haptoglobin-THb-Lys complex was observed by SEC as a high
molecular weight species, with absorption at 280 and 414
nm, eluting separately from native haptoglobin and the
original THb-Lys product (Figure 8). The construct-complex
was purified by SEC. The column was equilibrated and
eluted with phosphate-buffered saline (PBS).
Example 7 - Haptoglobin-THb-{3HI-Lys complex:
THb-[3H]-Lys (75 L, 41 mg/mL, 0.657 Ci/mmole)
was added to a solution of partially purified haptoglobin
1-1 (0.273 mL, 3.7 mg/mL) in PBS pH 7.4. The mixture was
incubated at room temperature overnight. The THb-[3H]-Lys-
Hp complex was purified using SEC equilibrated and eluted
with PBS pH 7.4. Radioactivity was associated primarily
with a high molecular weight species identified by SEC,
having absorption at 280 and 414 nm and eluting separately
from native haptoglobin and the original THb-Lys product,
and with a retention time corresponding to the non-
radiolabeled product of Example 6.
Example 8 - Synthesis of fluorescein-hemoglobin conluaate
(FL-Hb) :
5-Iodoacetamido fluorescein (5-IAF, 11 mg, 21
mol) solution in N,N-dimethylformamide (DMF, 50 L) was
slowly added to oxyhemoglobin (60 mg/mL, 5 mL) in 50 mM
potassium phosphate pH 7.0 with stirring at 4 C . After
three hours of reaction at 4 C, the excess of 5-IAF was
removed by extensive dialysis against 50 mM potassium
phosphate pH 7.2 until no 5-IAF could be detected in the
dialysate. The UV-visible absortion spectrum of the
product showed a characteristic fluorescein absorption band
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at 496 nm.
Example 9 - Complexes of FL-Hb with haptoglobin 1-1, 2-1
and mixed phenotype haptoglobin:
FL-Hb (6 mg/mL in 50 mM potassium phosphate pH
7.2, 40 L) was added to haptoglobin 1-1, 2-1 or mixed
phenotype (Hpmix) (2.8 mg/mL in water, 39 L) to give the
following final concentrations: 0.6 mg/mL (6.2 M) Hp and
1.3 mg/mL (21 M) FL-Hb in 180 L final volume of 25 mM
potassium phosphate pH 7Ø The mixture was analyzed by
SEC after incubation at room temperature for 10 min. FL-Hb
complex with haptoglobin 1-1 migrates at 33 min.(Figure 9A
- overlaid SEC chromatograms of Hp 1-1 and Hp 1-1 complex
with Fl-Hb) and can be clearly distinguished from
haptoglobin by its absorbance at 414 nm. FL-Hb migrates at
42.9 min. (Figure 9A). FL-Hb complexes with Hp 1-1 and
HPmix were isolated and analyzed by UV-Vis spectroscopy
(Figure 9B - UV-Vis spectrum of haptoglobin 1-1 and Hpmix
complexes with FL-Hb, the arrow indicates the band
characteristic of fluorescein) and fluorimetry. This
material shows fluorescence with excitation at 480 nm and
emission at 520 nm, and a characteristic absorption band
for fluorescein with Xx at 496 nm. FL-Hb complexes with
Hp 2-1 and Hpmix are shown in Figures 9C and 9D,
respectively. The construct-complexes were purified by SEC
eluted with PBS buffer.
Example 10 - Synthesis of cross-linked hemoglobin bearing
tritiated putrescine:
200 mL of purified Hb was diafiltered into 50 mM
borate buffer pH 9.0, then deoxygenated and the
concentration adjusted to 7.1 g/dL. Hb was crosslinked at
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a 2:1 ratio of TTDS to Hb for 45 min at 30 C and then
desalted using 50 mM borate pH 9.0 buffer yielding a final
concentration of 3.1 g/dL. 1.43 mL of the desalted Hb was
added to each of two 1 mL aliquots of radiolabeled
putrescine (1 mCi/mL, 6.94 x 10-5 mmol/mL) and reacted at
room temperature for 1.5 hours (10:1 Hb:putrescine ratio).
0.9 mg of cold putrescine (40 fold excess over radiolabeled
putrescine) was reacted with 17 mL of the THb-DBS at a
ratio of 1.5:1 THb-DBS:putrescine. 5 mL of this solution
was added to each of the two reactions and mixed overnight
at room temperature. Both mixtures were then added to
freshly crosslinked and desalted THb-DBS (5.3 x 10-5 moles)
and reacted at room temperature for 1.5 hours. A 20 fold
excess of cold putrescine (172 mg) was then added and
reacted overnight. The THb-[3H]Pu was then diafiltered into
Ringers Lactate. The specific activity was 1.5 Ci/mole, 90
mg/mL.
Example 11 - Haptoglobin 1-1 complex with THb-(3H1Pu:
Haptoglobin (3.0 mg/mL in water, 51 L) was added
to THb-[3H]Pu (10 mg/mL in PBS pH 7.2, 20 L) to give the
following final concentrations: 1.4 mg/mL (14 M)
haptoglobin and 1.8 mg/mL (28 M) THb-[3H]Pu in a final 110
pL volume of 25 mM potassium phosphate pH 7Ø The mixture
was analyzed by SEC after incubation at room temperature
for 2 hours. Fractions (0.4 mL) of the effluent were
collected and analyzed by scintillation counting. THb-
[3H]Pu-Hp complex migrates as a high molecular weight
species with elution time from 20 to 28 min.(Figure 10) and
is well separated from haptoglobin band at 30 min. and THb-
[3H] Pu at 37 min. THb- [3H] Pu -Hp absorbs both at 280 nm and
414 nm (A280nm/A414nm = 0.74) and has specific radioactivity
(cpm/mg Hb) similar to that of THb-[3H]Pu. The construct-
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complex was purified by SEC.
Example 12 - Synthesis of cross-linked hemoglobin bearing
monodansyl cadaverine:
Purified Hb (8.0 g/dL, 100mL, 1.25 x 10-1 moles)
was diafiltered into 50 mM borate buffer, pH 9.0, then
oxygenated and deoxygenated. A deoxygenated solution of
TTDS (2 fold molar excess over Hb, 0.26 g, 2.5 x 10-' moles)
was added and the mixture was stirred for 1 hour at 35 C,
then charged with CO. Ion exchange chromatography at this
time indicated only a small amount of unreacted Hb (1.7%).
A 15-fold molar excess of monodansylcadaverine (MDC) in -20
mL of 0.1 M HC1 adjusted to 25 mL with 50 mM borate, pH 9.0
was added to the crosslinked Hb (0.63 g, 1.88 x 10-3 moles).
After 60 hours at room temperature, the MDC-Hb was
diafiltered against 10 mM borate, pH 9Ø The product was
purified by gel filtration and diafiltered into Ringers
Lactate.
Example 13 - Haptoglobin 1-1 complex with THb-MDC:
THb-MDC (20 mg/mL in Lactated Ringer's solution
pH 7.2, 3.5 L) was added to haptoglobin 1-1 (1.1 mg/mL in
water, 200 L) to give the following final concentrations:
1.1 mg/mL (11 M) Hp and 0.34 mg/mL (5.4 M) THb-MDC. The
mixture was analyzed by SEC after incubation at room
temperature for 24 hours. THb-MDC complex with haptoglobin
migrates as a high molecular weight species with elution
time from 21 to 29 min (Figure 11). This material migrates
separately from haptoglobin (30.9 min.) and absorbs at both
280 nm and 414 nm (A280nm/A414nm = 0.70) . THb-MDC elutes at
37.9 min. with A280nm/A414nm = 0.29. The construct-complex can
be purified by SEC.
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Example 14 - Synthesis of cross-linked hemoglobin bearing
primaquine (THb-PO):
TTDS (14.0 mg) in ethanol (100 ,zL) was added to
deoxyhemoglobin (10 mL, 58 mg/mL) in 50 mM borate pH 9Ø
The reaction mixture was stirred at room temperature under
nitrogen for one hour. The excess of the cross-linker was
then removed by gel filtration eluted with 50 mM borate pH
9.0 and the product (THb-DBS, 43 mg/mL) was charged with
CO. Primaquine diphosphate (0.5 g, 1.1 mmol) was dissolved
in 50 mM borate pH 9.0 (10 mL) and the pH of the resulting
solution was adjusted to 8.5 with 10 M NaOH (primaquine
partially precipitated). THb-DBS (10 mL) was added to
primaquine and the reaction mixture was stirred in the dark
at room temperature overnight. The product was then
filtered and the filtrate dialyzed extensively against 50
mM borate pH 9Ø Anion exchange chromatography of the
product (Figure 12) indicates that THb-PQ constitutes 68%
of all hemoglobin components in the mixture. THb-DBS
conjugated with primaquine constitutes 64% of all 0 chains
when the product is analyzed using reversed phase
chromatography.
Example 15 - Haptoglobin 1-1 complex with THb-PO:
THb-PQ (15 mg/mL in 50 mM borate pH 9.0, 67 /.cL)
was added to haptoglobin 1-1 (4.0 mg/mL in water, 500 /.tL)
to give the following final concentrations: 2.0 mg/mL (20
M) Hp and 1.0 mg/mL (15.7 AM) THb-PQ. The mixture was
analyzed by SEC and anion exchange chromatography after
incubation at room temperature for 21 hours. THb-PQ
complex with haptoglobin migrates as a high molecular
weight species with elution time from 21 to 29 min. (Figure
13). This material migrates separately from haptoglobin
complexed with uncross-linked hemoglobin (29.9 min.) and
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haptoglobin (30.6 min.) and absorbs at both 280 nm and 414
nm (A2Bonm/A414nm 0 . 70) . Anion exchange chromatography
indicated that all unmodified hemoglobin and 74% of both
THb-PQ and THb have reacted with Hp. This result is in
good agreement with the SEC analysis which indicates that
78% of hemoglobin has reacted with Hp.
Example 16 - Haptoglobin-(poly-0-raffinose-Hbl and
haptoglobin-164kDa-0-raffinose-Hbl complexes
HbAO was crosslinked and polymerized using
oxidized raffinose (OR) according to the procedure of
Pliura (US patent 5,532,352). Molecular weight species
greater than 64 kDa, representing polymerized Hb (>64 kDa
OR-Hb), where separated from 64 kDa species (64 kDa OR-Hb)
by size exclusion chromatography. Hb preparation were
combined separately with human haptoglobin 1-1 in water to
a final concentration of 0.2 mg Hb/mL and 0.125 mg
haptoglobin/mL (final Hb:Hp approximately 2.2:1). The
mixtures were incubated for one hour at 22 C, then analyzed
by size exclusion chromatography under dissociating, non-
denaturing elution conditions (0.5 M MgCl2, 25 mM Tris pH
7.4). Figure 14, which shows size exclusion chromatography
elution profiles with detection at 280 (solid lines) and
414 nm (broken lines), indicates binding of the modified
hemoglobins with haptoglobin. Incubation of the modified
hemoglobins with haptoglobin results in high molecular
weight species which do not correspond to either the
modified hemoglobin or haptoglobin, and which have
absorption at 414 nm indicating hemoglobin content.
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Example 17 - Binding of modified human Hb (f3H1-NEM-Hb) to
rat haptoglobin in plasma
1 mCi of 3H-N-ethylmaleimide ([3H]-NEM) in
pentane was evaporated in 0.5 mL phosphate buffer, and 25
mg of Hb in 1 mL buffer was added giving a final NEM:Hb
ratio of 0.06:1, or 37 4Ci/mg Hb. RP HPLC analysis after
24 hours at 4 C indicated incorporation of the majority of
the radiolabel into a modified beta peak. After 47 hr, a
15-fold excess (over (3Cys93 thiol) of non-radiolabeled NEM
was added. Salts and unbound NEM were removed by gel
filtration, and the final concentration adjusted to 10.2 mg
Hb/mL. A small portion of this material (3H-NEM-Hb) was
then combined with rat serum containing haptoglobin to
determine if all radiolabeled components bound to Hp. The
Hb-binding capacity of the rat serum was adjusted to 670 pg
Hb/mL serum. 0.5 and 2.0 equivalents of 3H-NEM-Hb, based on
Hb-binding capacity, were combined with serum and analyzed
by size exclusion chromatography eluted under dissociating,
non-denaturing conditions using 0.5 M MgC12, 25 mM Tris pH
7.4 (Figure 15). In the 3H-NEM-Hb preparation, all
radioactivity was associated with a 32 kDa peak. At 0.5
eq. 3H-NEM-Hb, all radioactivity appeared in the Hb-
haptoglobin peak (31 minutes). At 2.0 eq., haptoglobin is
saturated and excess 3H-NEM-Hb remains unbound (41 minutes).
7.3% of the radioactivity combining with plasma components
appears in a high MW peak at -22 minutes. These findings
demonstrate that all components of the modified human Hb,
3H-NEM-Hb, are capable of binding rat haptoglobin in plasma.
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Example 18 - Biodistribution of modified Hb and haptoglobin
complexes in rat.
The ability of Hp to target modified Hb to the
liver was measured in a radioisotope biodistribution study.
Two test articles were prepared from purified human HbAo
modified with tritium-labeled N-ethylmaleimide ([3H]-NEM-
Hb) : [3H] -NEM-Hb alone in Ringer's lactate, and [3H] -NEM-Hb
complexed to a slight excess of rat haptoglobin in rat
plasma. Three treatment groups were analyzed; (A) normal
rats received the modified Hb-haptoglobin complex in
plasma, (B) normal rats received the modified Hb only
(approximately twice the Hb-binding capacity of the rat),
and (C) haptoglobin-depleted rats received the modified Hb
only. Approximately 3 mg of Hb were administered to
conscious Sprague-Dawley rats in each case. Liver and
plasma samples were collected at 30, 60 and 120 minutes
post-administration and radioactivity counted after
solubilization and quenching. Values were converted to
percentages of total dose and concentration/dose, and
various analyses are shown in Figure 16. This shows
radioactivity contents, indicative of dose percentages.
Figure 16A shows the percentage of dose in plasma. Figure
16B shows the percentage of dose in liver. Figure 16C shows
the percentage of dose in liver + plasma. Figure 16D shows
the liver/plasma concentrations. Star designations (* and
**) show differences (p<0.05) within treatment groups at
different times. Crosses (t and t) show differences
(p<0.05) within time points for different treatment groups.
Plasma retention was highest in group A, and both
groups A and B were higher than group C. The greatest
difference in plasma content was at 120 minutes at which
time group A plasma contained 3 times the radioactivity of
group C and 3.5 times that of group B. Liver content in
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groups A and B was higher than in group C at all time
points. At 30 minutes, groups A and B had approximately
20% of the total dose in the liver compared to 11% in group
C. Liver content was the same at 30 and 60 minutes in
groups A and B, and declined by the 120 minute time point.
By 120 minutes, group A and B liver contents were 5- and 2-
fold higher than group C, respectively. Groups A and B
contained 60% of the dose in the plasma and liver
compartments at 30 minutes, compared with roughly half that
amount in the Group C animals. Liver to plasma
concentration/dose ratios increased with time in all
groups, with liver concentration approximately 4 times that
of plasma in groups A and B by 120 minutes, roughly twice
the ratio of group C at the same time. The improvement in
plasma retention and liver targeting is further
demonstrated by comparison of mean combined liver and
plasma contents between groups, presented in Fig. 17,
namely the ratios of mean combined liver and plasma
percentages of total dose. Shaded bars are derived from
Group A/C, solid bars from Group B/C, and open bars from
group A/B. Group A and B combined liver and plasma contents
were consistently greater than in group C, with group A
having a combined content 4 times greater than in group C
at 120 minutes. Areas under the distribution curves were
calculated without extrapolation to time zero (Table 2) and
indicated that liver uptake in groups A and B was
approximately twice that of group C. The data overall
demonstrate a greater ability to concentrate product in the
liver when Hp is present, either in a pre-formed complex
with the modified Hb, or in the form of endogenous Hp where
it is capable of forming a complex with administered Hb.
There is also a clear indication that plasma retention of
Hb conjugates is increased through combination with
haptoglobin, such that a drug conjugate would be available
for tissue uptake for a greater length of time.
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Table 2: Areas under distribution curves for plasma and
liver in rat.
AUC* AUC*
(ug Hb=min/mL/dose) (ug Hb=min/g/dose)
Group Plasma Liver
A 370.0 455.3
B 284.2 510.4
C 163.0 234.0
*dose = ug Hb/g body weight
Thus it has been demonstrated that agents can be
conjugated to both 32 kDa hemoglobin dimer and to 64 kDa
intramolecularly cross-linked Hb, using either attachment
to side chain functionalities, to an intramolecular cross-
linker or to a secondary linker attached to the
intramolecular cross-linker. All of these constructs bound
to haptoglobin. There has further been demonstrated the
selective targeting of such a construct-complex, formed in
vivo or ex vivo, to the liver and the extension of
circulating half-life.
