U.S. patent application number 10/231062 was filed with the patent office on 2003-01-16 for hemoglobin-haptoglobin complexes.
This patent application is currently assigned to Hemosol, Inc.. Invention is credited to Adamson, J. Gordon, Moore, M.S. Celine, Wodzinska, Jolanta M..
Application Number | 20030013642 10/231062 |
Document ID | / |
Family ID | 4162380 |
Filed Date | 2003-01-16 |
United States Patent
Application |
20030013642 |
Kind Code |
A1 |
Adamson, J. Gordon ; et
al. |
January 16, 2003 |
Hemoglobin-haptoglobin complexes
Abstract
Construct-complexes of a hemoglobin, a hepatocyte modifying
substance bound to the hemoglobin, and a haptoglobin bound to the
hemoglobin, are provided, for administration to mammalian patients.
The construct-complex may be formed ex vivo, or a
hemoglobin-hepatocyte modifying substance combination may be
administered to the patient so that haptoglobin in the mammalian
body bonds thereto to form the construct-complex in vivo. Disorders
of the liver may be diagnosed and treated using construct-complexes
described herein.
Inventors: |
Adamson, J. Gordon;
(Georgetown, CA) ; Wodzinska, Jolanta M.;
(Brampton, CA) ; Moore, M.S. Celine; (Georgetown,
CA) |
Correspondence
Address: |
NIXON & VANDERHYE P.C.
8th Floor
1100 North Glebe Road
Arlington
VA
22201-4714
US
|
Assignee: |
Hemosol, Inc.
|
Family ID: |
4162380 |
Appl. No.: |
10/231062 |
Filed: |
August 30, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10231062 |
Aug 30, 2002 |
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09302351 |
Apr 30, 1999 |
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6479637 |
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Current U.S.
Class: |
424/1.69 ;
514/1.2; 514/19.8; 514/3.7; 514/4.6; 514/7.4; 530/385 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 47/62 20170801; A61K 47/6445 20170801; A61P 1/16 20180101 |
Class at
Publication: |
514/6 ;
530/385 |
International
Class: |
A61K 038/42 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 30, 1998 |
CA |
2,236,344 |
Claims
We claim:
1. A hemoglobin construct-complex comprising a hemoglobin and a
hepatocyte modifying substance bound thereto, said
construct-complex being capable of binding to haptoglobin in vivo
in a mammalian patient to effect delivery of the hepatocyte
modifying substance to hepatocytes of the patient.
2. The construct-complex of claim 1 wherein the hepatocyte
modifying substance is bound to the hemoglobin through the
intermediary of a chemical linker.
3. The construct-complex of claim 1 wherein the hepatocyte
modifying substance is an agent capable of interacting with
hepatocytes and consequently capable of acting in vivo at the liver
of a mammalian patient, and selected from therapeutic agents,
diagnostic agents and markers.
4. The construct-complex of claim 3 wherein the hepatocyte
modifying substance is a therapeutic agent selected from
antineoplastic substances, antiviral substances, anti-inflammatory
substances, anti-parasitic substances, anti-microbial substances,
antioxidant substances, hepatoprotective agents, nucleic acids,
lipid metabolism agents, anti-toxicants, proteins and enzymes.
5. The construct-complex of claim 4 wherein the hepatocyte
modifying substance is a nucleic acid.
6. The construct-complex of claim 5 wherein the hepatocyte
modifying substance is a gene coding for a protein of interest.
7. The construct-complex of claim 4 wherein the hepatocyte
modifying substance is putrescine.
8. The construct-complex of claim 4 wherein the hepatocyte
modifying substance is primaquine.
9. The construct-complex of claim 3 wherein the hepatocyte
modifying substance is a diagnostic agent.
10. The construct-complex of claim 9 wherein the diagnostic
compound is a radiolabelled compound or a fluorescent compound.
11. The construct-complex of claim 1 wherein the hemoglobin is an
intramolecularly cross-linked human hemoglobin.
12. The construct-complex of claim 11 wherein the hemoglobin is
intramolecularly cross-linked with a cross-linking reagent which
leaves residual chemical groups available for subsequent reaction
with the hepatocyte modifying agent, either directly or through the
intermediary of a chemical linker group.
13. The construct-complex of claim 12 wherein the cross-linking
reagent is a trifunctional reagent which utilizes two of its
functional groups for intramolecular cross-linking of hemoglobin
and leaves its third functional group available for reaction with a
nucleophile.
14. The construct complex of claim 13 wherein the cross-linking
reagent is trimesoyl tris(3,5-dibromosalicylate).
15. A hemoglobin construct-complex comprising a hemoglobin, a
hepatocyte modifying substance bound to the hemoglobin, and a
haptoglobin bound to the hemoglobin.
16. The construct-complex of claim 15 formed in vivo by
administering to a mammalian patient a construct-complex as defined
in claim 1.
17. The construct-complex of claim 15 wherein the hepatocyte
modifying substance is bound to the hemoglobin through the
intermediary of a chemical linker.
18. The construct-complex of claim 15 wherein the hepatocyte
modifying substance is an agent capable of interacting with
hepatocytes and consequently capable of acting in vivo at the liver
of a mammalian patient, and selected from therapeutic agents,
diagnostic agents and markers.
19. The construct-complex of claim 18 wherein the hepatocyte
modifying substance is a therapeutic agent selected from
antineoplastic substances, antiviral substances, anti-inflammatory
substances, anti-parasitic substances, anti-microbial substances,
antioxidant substances, hepatoprotective agents, nucleic acids,
lipid metabolism agents, anti-toxicants, proteins and enzymes.
20. The construct-complex of claim 19 wherein the hepatocyte
modifying substance is a nucleic acid.
21. The construct-complex of claim 18 wherein the hepatocyte
modifying substance is a diagnostic agent.
22. The construct-complex of claim 21 wherein the diagnostic
compound is a radiolabelled compound or a fluorescent compound.
23. The construct-complex of claim 15 wherein the hemoglobin is an
intramolecularly cross-linked human hemoglobin.
24. The construct-complex of claim 23 wherein the hemoglobin is
intramolecularly cross-linked with a cross-linking reagent which
leaves residual chemical groups available for subsequent reaction
with the hepatocyte modifying agent, either directly or through the
intermediary of a chemical linker group.
25. The construct-complex of claim 24 wherein the cross-linking
reagent is a trifunctional reagent which utilizes two of its
functional groups for intramolecular cross-linking of hemoglobin
and leaves its third functional group available for reaction with a
nucleophile.
26. The construct complex of claim 25 wherein the cross-linking
reagent is trimesoyl tris(3,5-dibromosalicylate).
27. A process for treating or diagnosing hepatocytic disorders in a
mammalian patient which comprises administering to the patient an
effective amount of a construct-complex, as defined in claim 1.
28. A process for treating or diagnosing hepatocytic disorders in a
mammalian patient which comprises administering to the patient an
effective amount of a construct-complex.
29. A process for treating metastatic cells carrying haptoglobin
receptors, in a mammalian patient, which comprises administering to
the patient an effective amount of a construct-complex as defined
in claim 1.
30. A process for treating metastatic cells carrying haptoglobin
receptors, in a mammalian patient, which comprises administering to
the patient an effective amount of a construct-complex as defined
in claim 15.
Description
FIELD OF THE INVENTION
[0001] 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
[0002] 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.
[0003] Thus, Kluaer et al., U.S. Pat. No. 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.
[0004] Anderson et al., U.S. Pat. No. 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.
[0005] Haptoglobins (Hp) constitute part of the 2-globin family of
serum glycoproteins. Haptoglobins are present in mammalian plasma,
and constitute about one-quarter of the .alpha..sub.2-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 .alpha..beta.
dimers and the phenotypes are conventionally denoted Hp 1-1, Hp 2-1
and Hp 2-2. The .beta. chains are identical in all haptoglobin
phenotypes, but the .alpha. chains vary (.alpha..sup.1 and
.alpha..sup.2) The amino acid sequences of all chains are known. Hp
1-1 is composed of two .alpha..sup.1.beta. 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: (.alpha..sup.1.beta.).sub.2
(.alpha..sup.2.beta.).sub.n where n=0,1,2, . . . and
(a.sup.2.beta.).sub.m where m=3,4,5, . . . respectively.
[0006] 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.
[0007] 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.
[0008] Control and manipulation of genes and gene products are
potentially powerful means of treating various diseases and genetic
disorders. When specifically 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.
[0009] 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.
SUMMARY OF THE INVENTION
[0010] It is an object of the present invention to provide a means
and composition for specifically targeting hepatocytes or other
cells having receptors for hemoglobin-haptoglobin complexes with
therapeutically active substances or diagnostic agents.
[0011] It is a further object of the present invention to provide 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 cells.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] The construct-complex according to the present invention can
be generally represented by the formula:
(Hp).sub.a-(Hb).sub.b-(L.sub.c-A.sub.d).sub.e
[0017] where a=1 to about 10;
[0018] b=0.5 to about 10;
[0019] c=0 to about 10;
[0020] d=1 to about 20;
[0021] e=1 to about 20;
[0022] Hp is haptoglobin as described herein;
[0023] Hb is a hemoglobin as described herein;
[0024] L is a linker as described herein;
[0025] 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
[0026] FIG. 1 is a reaction scheme illustrating diagrammatically a
process for producing one embodiment of a construct-complex of the
present invention;
[0027] FIGS. 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.
[0028] FIG. 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;
[0029] FIGS. 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;
[0030] FIG. 5 is a dye fluorescence assay of the products of
Example 4;
[0031] FIG. 6 is a depiction of the gel mobility shift assay of the
products of Example 4;
[0032] FIG. 7 is a fluorescence assay of another product of Example
4;
[0033] FIG. 8 is a size exclusion chromatogram of the product of
Example 6;
[0034] FIGS. 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;
[0035] FIG. 9B shows the UV-visible spectra of the products of
Example 9;
[0036] FIG. 10 is a size exclusion chromatogram of the product of
Example 11;
[0037] FIG. 11 is a size exclusion chromatogram of the product of
Example 13;
[0038] FIG. 12 shows anion exchange chromatograms (overlaid) of
products and starting materials of Example 14;
[0039] FIG. 13 shows overlaid size exclusion chromatograms of the
products of Example 15;
[0040] FIG. 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 kkDa ORHb].
[0041] FIG. 15 shows size exclusion chromatograms of products of
Example 17;
[0042] FIGS. 16 A-D are graphical presentations of analyses of
results obtained in Example 18;
[0043] FIG. 17 is a graphical presentation of further analyses of
results obtained according to Example 18.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0044] 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.
[0045] 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 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.
[0046] 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,
.alpha.-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 inhibitors;
atrial natriuretic peptide;
.alpha..sub.2-macrofetoprotein;synthetic linear terpenoid;
putrescine; cholestyramine; .epsilon.-aminocaproic acid,;
phenylmethylsulfonyl fluoride; pepstatin; glycyrrhizin; fructose
1,6-biphosphate; and ursodeoxycholic acid.
[0047] 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.
[0048] 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 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.
[0049] Human hemoglobin, e.g. that obtained from outdated red blood
cells, and purified by the displacement chromatography process
described in U.S. Pat. No. 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. Pat. No. 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 FIG.
1, and the preparation of which is described in the aforementioned
Kluger et al. U.S. Pat. No. 5,399,671.
[0050] 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 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.
[0051] 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-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 .alpha..beta.-dimers is 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.
[0052] 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 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.
[0053] 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. Provisional Patent
Application of Kluger and Li, entitled "Hemoglobin With Chemically
Introduced Disulfide Crosslinks and Preparation Thereof", filed
Nov. 3, 1997. 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-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.
[0054] 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.
[0055] 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.
[0056] 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 hemoglobin may be cross-linked, oligomerized or
unmodified, as described above.
[0057] FIG. 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 .beta.-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.
SPECIFIC EXAMPLES
Example 1
Conjugation of TTDS Cross-Linked Hemoglobin (THb) to
Poly(L-lysine)
[0058] Poly(L-lysine) conjugates of TTDS cross-linked hemoglobin
(THb-K.sub.n) 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 .alpha.-amino group of lysine.
Polymers with an average molecular weight of 4 kDa (K.sub.4kDa),
7.5 kDa (K.sub.7.5kDa), 26 kDa (K.sub.26kDa) and 37 kDa
(K.sub.37kDa) were conjugated to THb.
[0059] TTDS (13.9 mg) in ethanol (100 .mu.L) was added to
deoxyhemoglobin (5 mL, 8.5 g/dL) in 50 mM borate pH 9.0. The
reaction mixture was stirred at 30.degree. 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.times.25 mm D) equilibrated with 50 mM borate pH 9.0. 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-K.sub.n)
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.
1 TABLE 1 Amount of poly(L-lysine) added to THb Poly(L-lysine) (mg)
K.sub.4kDa 4.2 K.sub.7.5kDa 8.0 K.sub.26kDa 27.5 K.sub.37kDa
39.3
[0060] Anion Exchange Chromatography: Crude THb-K.sub.n complexes
were analyzed using anion exchange chromatography on a SynChropak
AX-300 column (250 mm L.times.4.6 mm D, SynChrom, Inc.). A sodium
chloride gradient was used to elute various modified hemoglobins.
The effluent was monitored at 280 nm.
[0061] 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 K.sub.4kDa, K.sub.7.5kDa,
K.sub.26kDa and K.sub.37kDa respectively.
[0062] Purification of THb-K.sub.n conjugates: THb-K.sub.n
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-HCl buffer pH 8.4. Modified Hbs were
eluted with a sodium chloride gradient. The effluent was monitored
at 280 nm and pooled fractions containing THb-K.sub.n conjugates
were concentrated using an Amicon.TM. diafiltration device and a 30
kDa cutoff membrane.
[0063] Size Exclusion Chromatography: The molecular weight
distribution of purified THb-K.sub.n conjugates and their
haptoglobin complexes was determined using size exclusion
chromatography (SEC) on a Superdex.TM.-200 column (300 mm
L.times.10 mm D, Pharmacia) equilibrated and eluted with 0.5 M
magnesium chloride containing 25 mM Tris-HCl pH 7.2 at a 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.sub.n and Haptoglobin 1-1
[0064] 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.sub.n (all THb-K.sub.n concentrations
represent hemoglobin concentrations) in 50 mM sodium borate pH 9.0.
Haptoglobin (14 .mu.L) was added to THb-K.sub.n in potassium
phosphate pH 7.0 to give the following final concentrations: 0.12
mg/mL (1.22 .mu.M) haptoglobin and 0.19 mg/mL (2.9 .mu.M)
THb-K.sub.n in 25 mM potassium phosphate pH 7.0 (200 .mu.L final
volume). After incubation for 180 min. at room temperature, the
samples were analyzed using SEC.
[0065] THb-K.sub.n complexes with haptoglobin 1-1: The formation of
THb-K.sub.n complexes with haptoglobin can be followed using size
exclusion chromatography (SEC). FIG. 2A shows the composition of
the THb-K.sub.4kDa 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
(A.sub.280/A.sub.414) over the elution period indicate the relative
proportions of haptoglobin and hemoglobin in the
construct-complexes and other peaks. The absorbance ratio
(A.sub.280/A.sub.414) 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 A.sub.280/A.sub.414 ratio. FIG. 2B shows SEC of
the THb-K.sub.7kDa mixture with Hp after incubation at room
temperature for 180 min. Again, a new peak appears at 25.1 min.
with a A.sub.280/A.sub.414 ratio of 0.73, followed by haptoglobin
at 29.7 min. and THb-K.sub.7.5kDa at 35.8 min. with
A.sub.280/A.sub.414 ratio of 0.3. The analysis of the SEC of
THb-K.sub.26kDa and THb-K.sub.37kDa 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 FIGS. 2C and 2D that both THb-K.sub.26kDa and
THb-K.sub.37kDa form complexes with haptoglobin. The
A.sub.280/A.sub.414 ratio is 0.64 for THb-K.sub.26kDa-Hp and 0.69
for THb-K.sub.37kDa-Hp.
[0066] Degree of THb-K.sub.26kDa-Hp complex formation: To determine
whether all structurally different components of the THb-K.sub.n
bind to haptoglobin, THb-K.sub.26kDa 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-K.sub.26kDa in potassium phosphate pH
7.0 to give the following final concentrations: 0.74 mg/mL (7.5 mM)
haptoglobin and 0.41 mg/mL (6.4 mM) THb-K.sub.26kDa(1.2:1 molar
ratio of Hp to Hb) in 25 mM potassium phosphate pH 7.0. 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-K.sub.26kDa was bound by haptoglobin
after 24 hours. The resulting THb-K.sub.26kDa-Hp complex has a
broad molecular weight distribution ranging from 370 kDa to app.
1000 kDa (FIG. 3).
Example 3
DNA Binding to THb-K.sub.n and THb-K.sub.n-Hp. Gel Mobility Shift
Assay
[0067] Gel mobility shift assays were conducted to evaluate the
stoichiometry of binding of plasmid DNA (pCMVbeta) to the
THb-K.sub.n 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-K.sub.n 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-K.sub.26kDa-Hp complex, solutions containing from 0.4
to 6400 ng of the conjugate (this weight based on the hemoglobin
component) in 32 .mu.L of 20 mM HEPES pH 7.3 containing 150 mM NaCl
were prepared. The plasmid DNA (560 ng in 28 .mu.L 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 .mu.L) were analyzed on a 1% agarose gel containing
ethidium bromide (0.2 .mu.g/mL). The amount of conjugate which
prevented DNA entry into the gel was determined. Results are
described in the following Example.
Example 4
DNA binding to THb-K.sub.26kDa and THb-K.sub.26kDa-Hp Complex:
Thiazole Orange Fluorescence Ouenching Method
[0068] 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-K.sub.n and DNA causes
the displacement of the intercalating dye from DNA and the decrease
of total fluorescence.
[0069] The following stock solutions were used in this experiment:
0.05 mg/mL DNA (pCMVbeta), 0.010 mg/mL, THb-K.sub.26kDa or
THb-K.sub.26kDa-Hp complex, 1.75.times.10.sup.-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
.mu.L), THb-K.sub.26kDa (volumes varying from 2.5 to 60 .mu.L) and
buffer (to the final volume of 200 .mu.L) 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 FluoreCount.TM. plate reader using excitation at 485 nm and
emission at 530 nm. The THb-K.sub.26kDa-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.
[0070] The gel mobility shift assay and the fluorescence quench
assay both demonstrated that THb-K.sub.n binds to DNA. FIG. 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) THb or (B) THb-K.sub.26kDa. Lane 1 of both gels
contain DNA molecular weight markers. Hb content in other lanes:
(A2) 50 ng, (A3) 100 ng, (A$) 200 ng, (AS) 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.sub.n in
the DNA samples affected DNA migration as seen in FIG. 4. FIG. 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-K.sub.26kDa is most effective at
binding DNA. One hundred ng of THb-K.sub.26kDa (THb-K.sub.26kDa 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-K.sub.n preparations were required to bind all DNA. The results
for THb-K.sub.26kDa are in good agreement with the fluorescence
quench assay which indicated 86% of fluorescence decrease at the
same THb-K.sub.26kDa to DNA ratio. FIG. 5 shows the effect of
THb.sub.26kDa on DNA-thiazole orange fluorescence. On FIG. 5, the
amount of THb-K.sub.26kDa is based on the hemoglobin component
only.
[0071] The THb-K.sub.26kDa-Hp complex also binds DNA. It was found
that 200 ng of THb-K.sub.26kDa-Hp completely prevented 140 ng of
DNA from entering the agarose gel (THb-K.sub.26kDa-Hp to DNA
ratio=1.4, w/w). FIG. 6 shows THb-K.sub.26kDa-Hp binding to DNA by
gel mobility shift assay. One hundred and forty ng of DNA were
added to increasing amounts of THb-K.sub.26kDa-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-K.sub.26kDa-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 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-K.sub.26kDa-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-K.sub.26kDa-Hp is probably due to steric crowding in the
THb-K.sub.26kDa-Hp-DNA complex.
[0072] 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
.epsilon.-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.
[0073] The gel mobility shift and the fluorescence assays have
demonstrated that THb-K.sub.26kDa-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 Tritiated or
Non-Tritiated Lysine
[0074] A solution of L-[.sup.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 .mu.mL) and
200 .mu.L of this solution was added to deoxyhemoglobin (10 mL, 9.2
g/dL) in 50 mM borate pH 9.0. 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 (U.S. Pat. No. 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 .mu.L) was added
to the radiolabeled and the non-radiolabeled lysines each day for
three days. THb-Lys (700 .mu.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
[0075] Haptoglobin (1.61 mg/mL haptoglobin 1-1 in water, 11 .mu.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 .mu.M)
haptoglobin and 0.41 mg/mL (6.4 .mu.M) THb-Lys were made up to a
final 200 .mu.L volume at 25 mM potassium phosphate pH 7.0. Within
18 hours, the 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 (FIG. 8). The construct-complex was purified by SEC. The
column was equilibrated and eluted with phosphate-buffered saline
(PBS).
Example 7
Haptoglobin-THb-[.sup.3H]-Lys Complex
[0076] THb-[.sup.3H]-Lys (75 .mu.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-[.sup.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 Conjugate (FL-Hb)
[0077] 5-Iodoacetamido fluorescein (5-IAF, 11 mg, 21 .mu.mol)
solution in N,N-dimethylformamide (DMF, 50 .mu.L) was slowly added
to oxyhemoglobin (60 mg/mL, 5 mL) in 50 mM potassium phosphate pH
7.0 with stirring at 4.degree. C. After three hours of reaction at
4.degree. 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 at 496
nm.
Example 9
Complexes of FL-Hb with Haptoglobin 1-1, 2-1 and Mixed Phenotype
Haptoglobin
[0078] FL-Hb (6 mg/mL in 50 mM potassium phosphate pH 7.2, 40
.mu.L) was added to haptoglobin 1-1, 2-1 or mixed phenotype
(Hp.sub.mix) (2.8 mg/mL in water, 39 .mu.L) to give the following
final concentrations: 0.6 mg/mL (6.2 .mu.M) Hp and 1.3 mg/mL (21
.mu.M) FL-Hb in 180 .mu.L final volume of 25 mM potassium phosphate
pH 7.0. 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.(FIG. 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. (FIG. 9A). FL-Hb complexes with Hp 1-1 and Hp.sub.mix were
isolated and analyzed by UV-Vis spectroscopy (FIG. 9B--UV-Vis
spectrum of haptoglobin 1-1 and Hp.sub.mix 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 80 .sub.max at 496 nm. FL-Hb complexes with Hp
2-1 and Hp.sub.mix are shown in FIGS. 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
[0079] 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 a 2:1 ratio of TTDS to Hb for 45
min at 30.degree. 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.times.10.sup.-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.times.10.sup.-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-[.sup.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-[.sup.3H]Pu
[0080] Haptoglobin (3.0 mg/mL in water, 51 .mu.L) was added to
THb-[.sup.3H]Pu (10 mg/mL in PBS pH 7.2, 20 .mu.L) to give the
following final concentrations: 1.4 mg/mL (14 .mu.M) haptoglobin
and 1.8 mg/mL (28 .mu.M) THb-[.sup.3H]Pu in a final 110 .mu.L
volume of 25 mM potassium phosphate pH 7.0. 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-[.sup.3H]Pu-Hp complex migrates as a
high molecular weight species with elution time from 20 to 28
min.(FIG. 10) and is well separated from haptoglobin band at 30
min. and THb-[(.sup.3H]Pu at 37 min. THb-[3H]Pu-Hp absorbs both at
280 nm and 414 nm (A.sub.280nm/A.sub.414nm=0.74) and has specific
radioactivity (cpm/mg Hb) similar to that of THb-[.sup.3H]Pu. The
construct-complex was purified by SEC.
Example 12
Synthesis of Cross-Linked Hemoglobin Bearing Monodansyl
Cadaverine
[0081] Purified Hb (8.0 g/dL, 100 mL, 1.25.times.10.sup.-4 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.times.10.sup.-4 moles) was added and
the mixture was stirred for 1 hour at 35.degree. 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 .about.20 mL of 0.1 M HCl adjusted to
25 mL with 50 mM borate, pH 9.0 was added to the crosslinked Hb
(0.63 g, 1.88.times.10.sup.-3 moles). After 60 hours at room
temperature, the MDC-Hb was diafiltered against 10 mM borate, pH
9.0. The product was purified by gel filtration and diafiltered
into Ringers Lactate.
Example 13
Haptoglobin 1-1 Complex with THb-MDC
[0082] THb-MDC (20 mg/mL in Lactated Ringer's solution pH 7.2, 3.5
.mu.L) was added to haptoglobin 1-1 (1.1 mg/mL in water, 200 .mu.L)
to give the following final concentrations: 1.1 mg/mL (11 .mu.M) Hp
and 0.34 mg/mL (5.4 .mu.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 (FIG. 11). This material migrates
separately from haptoglobin (30.9 min.) and absorbs at both 280 nm
and 414 nm (A.sub.280nm/A.sub.414nm=0.70). THb-MDC elutes at 37.9
min. with A.sub.280nm/A.sub.414nm=0.29. The construct-complex can
be purified by SEC.
Example 14
Synthesis of Cross-Linked Hemoglobin Bearing Primaguine
(THb-PO)
[0083] TTDS (14.0 mg) in ethanol (100 .mu.L) was added to
deoxyhemoglobin (10 mL, 58 mg/mL) in 50 mM borate pH 9.0. 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 9.0. Anion
exchange chromatography of the product (FIG. 12) indicates that
THb-PQ constitutes 68% of all hemoglobin components in the mixture.
THb-DBS conjugated with primaquine constitutes 64% of all .beta.
chains when the product is analyzed using reversed phase
chromatography.
Example 15
Haptoglobin 1-1 Complex with THb-PQ
[0084] THb-PQ (15 mg/mL in 50 mM borate pH 9.0, 67 .mu.L) was added
to haptoglobin 1-1 (4.0 mg/mL in water, 500 .mu.L) to give the
following final concentrations: 2.0 mg/mL (20 .mu.M) Hp and 1.0
mg/mL (15.7 .mu.M) 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. (FIG.
13). This material migrates separately from haptoglobin complexed
with uncross-linked hemoglobin (29.9 min.) and haptoglobin (30.6
min.) and absorbs at both 280 nm and 414 nm
(A.sub.280/A.sub.414nm=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-O-Raffinose-Hb] and Haptoalobin-[64
kDa-O-Raffinose-Hb] Complexes
[0085] HbA0 was crosslinked and polymerized using oxidized
raffinose (OR) according to the procedure of Pliura (U.S. Pat. No.
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.degree. C., then
analyzed by size exclusion chromatography under dissociating,
non-denaturing elution conditions (0.5 M MgCl.sub.2, 25 mM Tris pH
7.4). FIG. 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.
Example 17
Binding of Modified Human Hb ([.sup.3H]-NEM-Hb) to Rat Haptoglobin
in Plasma
[0086] 1 mCi of .sup.3H-N-ethylmaleimide ([.sup.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
.mu.Ci/mg Hb. RP HPLC analysis after 24 hours at 4.degree. C.
indicated incorporation of the majority of the radiolabel into a
modified beta peak. After 47 hr, a 15-fold excess (over .beta.Cys93
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
(.sup.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
.mu.g Hb/mL serum. 0.5 and 2.0 equivalents of .sup.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 MgCl.sub.2, 25 mM Tris pH 7.4
(FIG. 15). In the .sup.3H-NEM-Hb preparation, all radioactivity was
associated with a 32 kDa peak. At 0.5 eq. .sup.3H-NEM-Hb, all
radioactivity appeared in the Hb-haptoglobin peak (31 minutes). At
2.0 eq., haptoglobin is saturated and excess .sup.3H-NEM-Hb remains
unbound (41 minutes). 7.3% of the radioactivity combining with
plasma components appears in a high MW peak at .about.22 minutes.
These findings demonstrate that all components of the modified
human Hb, .sup.3H-NEM-Hb, are capable of binding rat haptoglobin in
plasma.
Example 18
Biodistribution of Modified Hb and Haptoglobin Complexes in Rat
[0087] 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 HbA.sub.0 modified with
tritium-labeled N-ethylmaleimide ([.sup.3H]-NEM-Hb):
[.sup.3H]-NEM-Hb alone in Ringer's lactate, and [.sup.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 FIG. 16.
This shows radioactivity contents, indicative of dose percentages.
FIG. 16A shows the percentage of dose in plasma. FIG. 16B shows the
percentage of dose in liver. FIG. 16C shows the percentage of dose
in liver+plasma. FIG. 16D shows the liver/plasma concentrations.
Star designations (* and **) show differences (p<0.05) within
treatment groups at different times. Crosses (.dagger. and
.dagger-dbl.) show differences (p<0.05) within time points for
different treatment groups.
[0088] 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 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.
2TABLE 2 Areas under distribution curves for plasma and liver in
rat. AUC* AUC* (ug Hb .multidot. min/mL/dose) (ug Hb .multidot.
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
[0089] 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.
* * * * *