U.S. patent application number 09/921663 was filed with the patent office on 2002-01-24 for reactively modified therapeutic compounds having an extended lifetime in vivo.
Invention is credited to Pouletty, Christine, Pouletty, Philippe.
Application Number | 20020009441 09/921663 |
Document ID | / |
Family ID | 26835615 |
Filed Date | 2002-01-24 |
United States Patent
Application |
20020009441 |
Kind Code |
A1 |
Pouletty, Philippe ; et
al. |
January 24, 2002 |
Reactively modified therapeutic compounds having an extended
lifetime in vivo
Abstract
First and second compounds are provided, where the first
compound is administered to a mammalian host into blood for
covalent bonding to blood components, where the components have an
extended lifetime in the blood stream. The first compound comprises
an active functionality and an agent of interest or a first binding
entity. A second compound may be subsequently administered to the
patient, which comprises a second binding entity, complementary to
the first binding entity and an agent of interest. By virtue of
binding to long-lived blood components, the half-life of the agent
of interest is greatly extended in vivo.
Inventors: |
Pouletty, Philippe;
(Atherton, CA) ; Pouletty, Christine; (Atherton,
CA) |
Correspondence
Address: |
Michael R. Ward
Morrison & Foerster LLP
425 Market Street
San Francisco
CA
94105-2482
US
|
Family ID: |
26835615 |
Appl. No.: |
09/921663 |
Filed: |
August 3, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09921663 |
Aug 3, 2001 |
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09539766 |
Mar 31, 2000 |
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09539766 |
Mar 31, 2000 |
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08477900 |
Jun 7, 1995 |
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6103233 |
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08477900 |
Jun 7, 1995 |
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08237346 |
May 3, 1994 |
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5612034 |
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08237346 |
May 3, 1994 |
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08137821 |
Oct 15, 1993 |
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Current U.S.
Class: |
424/130.1 ;
514/10.1; 514/10.3; 514/10.8; 514/10.9; 514/11.4; 514/11.5;
514/11.8; 514/11.9; 514/12.4; 514/12.5; 514/13.3; 514/13.6;
514/14.1; 514/15.8; 514/19.1; 514/2.8; 514/20.1; 514/20.9; 514/3.8;
514/5.9; 514/7.4; 514/7.5; 514/7.7; 514/9.9 |
Current CPC
Class: |
A61K 38/00 20130101;
A61K 47/6901 20170801; C07K 14/70514 20130101; A61K 39/385
20130101; C07K 16/08 20130101; A61K 47/6849 20170801; C12N
2740/16122 20130101; C07K 16/16 20130101; C07K 16/34 20130101; A61K
2039/505 20130101; A61P 43/00 20180101; B82Y 5/00 20130101; C07K
16/44 20130101; A61K 2039/625 20130101; A61K 47/6839 20170801; C07K
14/005 20130101; A61K 47/6898 20170801; A61K 2039/6056
20130101 |
Class at
Publication: |
424/130.1 ;
514/2 |
International
Class: |
A61K 039/395; A61K
038/00; A61K 038/17 |
Claims
What is claimed is:
1. A method for extending the lifetime of a biologically active
agent of interest, said method comprising: administering to the
vascular system of a mammalian host a first compound, said first
compound comprising (i) a reactive functional group which reacts
with proteins to form stable covalent bonds and (ii) either a
biologically active agent of interest or a first binding entity
which is a member of a specific binding pair consisting of said
first binding member and a complementary second binding member,
whereby said reactive functional group reacts with at least one of
mobile protein and cellular components of the vascular system to
produce modified vascular components; wherein when said first
compound comprises said first binding entity, the additional step
of administering a second compound comprising said second binding
entity and a biologically active agent of interest, wherein said
biologically active agent of interest is added in an amount to
achieve its biological function over an extended period of
time.
2. A method according to claim 1, wherein said first compound
comprises said biologically active agent of interest.
3. A method according to claim 1, wherein said agent of interest is
an immunoglobulin or binding fragment thereof.
4. A method according to claim 1, wherein said agent of interest is
a synthetic peptide.
5. A method according to claim 1, wherein said protein is a
glycoprotein.
6. A method according to claim 1, wherein said agent of interest is
a naturally occurring compound.
7. A method according to claim 1, wherein said agent of interest is
a synthetic organic compound of less than about 5 kDa.
8. A method according to claim 1, wherein said agent of interest is
an immunogen.
9. A method according to claim 1, wherein said reactive functional
group is a carboxylate ester which reacts with amines in an aqueous
medium to form amides.
10. A method for extending the lifetime in a mammalian host of an
immunogen for enhancing the immune response, said method
comprising: administering to the vascular system of a mammalian
host a first compound, said first compound comprising (i) a
reactive functional group which reacts with proteins to form stable
covalent bonds and (ii) a first binding entity which is a member of
a specific binding pair consisting of said first binding member and
a complementary second binding member, whereby said reactive
functional group reacts with at least one of mobile protein or
cellular components of the vascular system to produce modified
vascular components; adminstering a second compound comprising said
second binding entity and an immunogen; whereby said host mounts an
immune response to said immunogen with the production of
antibodies.
11. A blood composition comprising conjugates of immunoglobulins
and serum albumin covalently bonded to a biologically active agent
as the major protein conjugates in said blood composition, said
conjugates resulting from the addition of a first compound, said
first compound comprising a reactive functional group which reacts
with proteins to form stable covalent bonds and either a
biologically active agent of interest or a first binding entity
which is a member of a specific binding pair consisting of said
first binding member and a complementary second binding member, to
blood in vivo.
12. A blood composition according to claim 11, wherein said
biologically active agent is biotin.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 08/137,821, filed Oct. 15, 1993, which application was a
continuation-in-part of application Ser. No. 08/070,092, filed May
27, 1993, which was a continuation-in-part of application Ser. No.
07/592,214, filed Oct. 3, 1990.
INTRODUCTION
TECHNICAL FIELD
[0002] The field of this invention is agent, particularly
therapeutic agent, delivery in a mammalian host.
BACKGROUND
[0003] Delivery of therapeutic agents to a mammalian host can
frequently be as important as the activity of the drug in providing
effective treatment. For the most part, drugs are delivered orally,
frequently initially at a dosage below the therapeutic dosage and
by repetitive administration of the drug, the dosage is raised to a
therapeutic level or a level exceeding the therapeutic level. In
many cases, the fact of having a dosage above therapeutic level
provides for adverse effects, since most drugs are not only
effective for the intended purpose, but frequently have adverse
side effects. Various proposals have been made to avoid these
problems, such as slow-release capsules, depots, pumps, and the
like. These various approaches have numerous short comings for
general applications where one wishes to maintain the presence of a
therapeutic agent at a therapeutic dosage for an extended period.
Invasive procedures are frequently undesirable, requiring surgery
for introduction of the delivery device, followed by subsequent
removal. Where the delivery device is placed on the skin, the agent
must be capable of transport across the skin at the desired rate.
Slow release particles have a limited time span and when introduced
into the blood stream will be rapidly phagocytosed.
[0004] For those therapeutic agents which must be administered by
injection, the need to have repetitive injections is particularly
undesirable. The need in many cases for self administration is
particularly problematical and in many instances may require
trained individuals for the administration. There is, therefore, a
serious need for methodologies which would allow for extended
administration of therapeutic agents, particularly in the blood
stream, which can be easily administered and efficacy maintained
for extended periods of time.
SUMMARY OF THE INVENTION
[0005] Methods and compositions are provided for delivery of agents
in the blood stream of mammalian hosts, by initially providing a
bolus of a first compound comprising a chemically reactive group
and a first entity, which may be the active agent or a first
binding entity. The first compound will react with active
functionalities of blood components, so as to provide
functionalized blood components in relation to the proportion of a
particular blood component to the total amount of blood components,
and the reactivity of the functionalities of the blood component.
After a few days, the population of functionalized blood components
will be related, as well, to the half-life of the blood component,
making immunoglobulins a major functionalized component.
[0006] At any time, where the first entity is a binding entity, a
second compound may be introduced into the blood stream, where the
second compound comprises a second binding entity, which
specifically binds to the first binding entity, and an agent of
interest. By varying the linking assemblage between the blood
component and the agent of interest, the rate of release of the
agent from the blood component may be controlled. The agent of
interest is maintained in the host blood stream based on the
lifetime of the long lived blood component, as modified, to provide
a substantially extended lifetime for the agent of interest.
DESCRIPTION OF SPECIFIC EMBODIMENTS
[0007] Methods and compositions are provided for delivery of
agents, particularly therapeutic agents, where the agents have an
extended half-life in the blood stream. The invention comprises
using from one to two compounds: a first compound comprising a
chemically reactive entity which is capable of forming covalent
bonds with functionalities present on proteins, joined by a
covalent bond or first linking group to an agent of interest or a
first binding entity, which binding entity is capable of binding
specifically to a reciprocal binding member, where the two members
define a specific binding pair; and, when the first compound
comprises a first binding entity, a second compound which comprises
the reciprocal binding member of the first binding entity and an
agent of interest joined by a linking group.
[0008] By introducing the first compound into the blood of the
host, particularly the blood stream, the chemically reactive group
will react with available functionalities of blood components, so
as to create a population of vascular functionalized blood
components. During the lifetime of the functionalized blood
components, as appropriate, the second compound may be added, which
will bind to the first binding entity. By linking the agent of
interest to a long-lived blood component, a long-lived depot of the
agent of interest is achieved. The life-time at a useful dosage
will usually be at least 10 days, more frequently 15 days or
more.
[0009] The primary mobile blood components which react are red
blood cells, immunoglobulins, such as IgM and IgG, serum albumin,
transferrin, p90 and p38, where the IgG and serum albumin have the
longer half-lives.
1 Mol. wt., kDa Conc., mg/ml Half-life, days IgM 600 1 5 IgG 160 10
23* transferrin 74-82 2.5 10 serum albumin 67 40 18 *IgG.sub.3 has
a half-life of 8 days Usually, by day 5, IgG, serum albumin and red
blood cells will be at least about 60 mole %, usually at least
about 75 mole %, of the conjugated components in blood, with IgG,
IgM (to a substantially lesser extent) and serum albumin being at
least about 50 mole %, usually at least about 75 mole %, more
usually at least about 80 mole %, of the non-cellular conjugated
components.
[0010] The first compound will comprise the active functionality, a
linking group, and the agent of interest or first binding entity.
The functionalities which are available on proteins are primarily
amino groups, carboxyl groups and thiol groups. While any of these
may be used as the target of the reactive functionality, for the
most part, bonds to amino groups will be employed, particularly
formation of amide bonds. To form amide bonds, one may use a wide
variety of active carboxyl groups, particularly esters, where the
hydroxyl moiety is physiologically acceptable at the levels
required. While a number of different hydroxyl groups may be
employed, the most convenient will be N-hydroxysuccinimide, and
N-hydroxy sulfosuccinimide, although other alcohols, which are
functional in an aqueous medium such as blood, may also be
employed. In some cases, special reagents find use, such as azido,
diazo, carbodiimide anhydride, hydrazine, dialdehydes, thiol
groups, or amines to form amides, esters, imines, thioethers,
disulfides, substituted amines, or the like. Usually, the covalent
bond which is formed should be able to be maintained during the
lifetime of the agent of interest, unless it is intended to be the
agent release site.
[0011] A large number of bifunctional compounds are available for
linking to entities. Illustrative entities include: azidobenzoyl
hydrazide,
N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyidithio)propionamide),
bis-sulfosuccinimidylsuberate, dimethyladipimidate,
disuccinimidyltartrate, N-.gamma.-maleimidobutyryloxysuccinimide
ester, N-hydroxy sulfosuccinimidyl-4-azidobenzoate, N-succinimidyl
[4-azidophenyl]-1,3'-dithiopropionate, N-succinimidyl
[4-iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl
4-[N-maleimidomethyl]cyclohexane-1-carboxylate.
[0012] When one or both of the linking groups is permanent, the
linking group(s) will not be critical to this invention and any
linking group which is convenient, physiologically acceptable at
utilized doses, and fills the requirements of the molecule, such as
being stable in the blood stream, effectively presenting the agent
of interest or first binding entity, allowing for ease of chemical
manipulation, and the like, may be employed. The linking group may
be aliphatic, alicyclic, aromatic, or heterocyclic, or combinations
thereof, and the selection will be primarily one of convenience.
For the most part, any heteroatoms will include nitrogen, oxygen,
sulfur or phosphorus. Groups which may be employed include
alkylenes, arylenes, aralkylenes, cycloalkylenes, and the like.
Generally the linking group will be of from 0-30, usually 0-10,
more usually of from about 0-6 atoms in the chain, where the chain
will include carbon and any of the heteroatoms indicated above. For
the most part, the linking group will be straight chain or cyclic,
since there will normally be no benefit from side groups. The
length of the linking group will vary, particularly with the nature
of the agent of interest and the first binding entity, since in
some instances, the agent of interest or the first binding entity
may naturally have a chain or functionality associated with it. In
some instances, amino acids, generally from 1-3 amino acids may
serve as the linking chain, particularly where the carboxyl group
of the amino acid may be the reactive functionality. Thus, the
amino group may serve to bond to the agent of interest or first
binding entity.
[0013] The length of the arms may be used to provide for
flexibility, rigidity, polyfunctionality, orientation, or other
characteristic for improved function of the molecule. The covalent
linking group may be a functionality which has an unequal affinity
for different blood proteins, or which has a high affinity for a
given protein epitope or sequence, such as an IgG or albumin
epitope.
[0014] The first binding entity will generally be a small molecule,
where the molecule is likely to minimize any immune response. Thus,
for the most part, the first binding entity will be haptenic,
usually below about 1 kD and generally more than about 100 D
preferably less than about 600 D. Any physiologically acceptable
molecule may be employed, where there is a convenient reciprocal
binding member. Thus, of particular interest is biotin, where
avidin may be the reciprocal binding member, but other molecules
such as metal chelates, molecules mimicking a natural epitope or
receptor or antibody binding site, also may find use, where the
reciprocal binding member may be an antibody or a fragment thereof,
particularly a Fab fragment, an enzyme, a naturally occurring
receptor, or the like. Thus, the first binding entity may be a
ligand for a naturally occurring receptor, a substrate for an
enzyme, or a hapten with a reciprocal receptor.
[0015] The manner of producing the first compound will vary widely,
depending upon the nature of the various elements comprising the
first compound. The synthetic procedures will be selected so as to
be simple, provide for high yields, and allow for a highly purified
product. Normally, the reactive functionality will be created as
the last stage, for example, with a carboxyl group, esterification
to form an active ester will be the last step of the synthesis,
unless one wishes to deprotect some functionality of the agent of
interest or the first binding entity as the last step.
[0016] Usually, the first compound when it comprises the first
binding entity will have a molecular weight of at least about 200 D
and not more than about 2.5 kD, usually not more than about 1.5 kD
and frequently less than about 1 kD.
[0017] Illustrative compounds include N-hydroxysuccinimidyl biotin
ester, N-hydroxysulfosuccinimidyl biotin ester,
N-hydroxysulfosuccinimidyl ester of N-biotinyl 6-aminohexanoic
acid, N-hydroxysulfosuccinimidyl ester of N-biotinyl 4-butyryl,
3-aminopropyl disulfide, and the like. A large number of water
soluble biotin derivatives for functionalizing proteins are
available and to the extent that such compounds have linkers which
are physiologically acceptable, these compounds may find
application in this invention.
[0018] The first compound will usually be administered as a bolus,
but may be introduced slowly over time by transfusion using metered
flow, or the like. Alternatively, although less preferable, blood
may be removed from the host, treated ex vivo, and returned to the
host. The first compound will be administered in a physiologically
acceptable medium, e.g. deionized water, phosphate buffered saline,
saline, mannitol, aqueous glucose, alcohol, vegetable oil, or the
like. Usually a single injection will be employed although more
than one injection may be used, if desired. The first compound may
be administered by any convenient means, including syringe, trocar,
catheter, or the like. The particular manner of administration,
will vary depending upon the amount to be administered, whether a
single bolus or continuous administration, or the like. For the
most part the administration will be intravascularly, where the
site of introduction is not critical to this invention, preferably
at a site where there is rapid blood flow, e.g. intravenously,
peripheral or central vein. Other routes may find use where the
administration is coupled with slow release techniques or a
protective matrix. The intent is that the first compound be
effectively distributed in the blood, so as to be able to react
with the blood components.
[0019] For the most part, reaction will be with mobile components
in the blood, particularly blood proteins and cells, more
particularly blood proteins and red cells. By "mobile" is intended
that the component does not haave a fixed situs. For the most part,
reaction will be with plasma proteins, such as the immunoglobulins,
particularly IgM and IgG, albumin, ferritin, and to a lesser degree
other proteins which are present in substantially reduced amount.
There may also be reaction with platelets, endothelial cells and
white blood cells. There will, therefore, initially be a relatively
heterogeneous population of functionalized proteins and cells.
However, for the most part, the population within a few days will
vary substantially from the initial population, depending upon the
half-life of the functionalized proteins in the blood stream.
Therefore, usually within about three days or more, IgG will become
the predominant functionalized protein in the blood stream. This
means that after a few days, the agent of interest will be
conjugated to, or the second compound will, for the most part,
become conjugated with and bound to IgG.
[0020] In many situations, one may use a single first compound
comprising the first binding entity, once such compound has been
thoroughly tested in hosts, particularly human hosts, since its
physiology will be well established, its pharmacokinetics will be
established, and its safety over an extended period of time may be
also established. In some instances the first compound will be
physiologically and/or therapeutically active, where it may find
use independent of addition of the second compound. However, to the
extent that there may be idiosyncratic individuals, or that chronic
administration of the first compound may result in some immune
reaction, it may be desirable to have more than one first compound
to be used for administration. However, since the role of the first
compound may be somewhat restricted when used in combination with a
second compound, it is not necessary that one develop numerous
alternatives, although there will be numerous alternatives which
will be useful for the same purpose.
[0021] For the most part, the half-life of the first compound will
be at least about five days, more usually at least about 10 days
and preferably 20 days or more. The period for providing an
effective concentration may be much longer, since one may introduce
a substantial excess of the first compound so that even after two
or three half-lives, there may still be a useful amount of the
first compound in the blood stream.
[0022] Generally, it will be satisfactory to have the agent of
interest as part of the first compound. However, there will be
situations where it will be desirable to use the combination of the
first and second compounds. For example, if one wishes to have a
relatively homogeneous population of components carrying the agent
of interest, one can wait till the short lived blood components
have dissipated, before adding the second compound. Particularly,
where the initial concentration of the first compound may be high,
it may be desireable to add the second compound subsequently which
will primarily bind to the first compound, rather than react with a
variety of blood components. In some instances the synthesis of the
first compound with the agent of interest may be difficult, due to
the highly functionalized nature of the agent of interest. This
could be particularly true with oligopeptides which are synthesized
and require removal of protecting groups at the end of the
synthesis. Also, where the agent of interest is to be released from
the blood component, it may be easier to design a combination of
first and second compounds which allow for efficient release,
rather than depending on the linking group of the first
compound.
[0023] The dosage of the first compound will depend upon whether it
comprises the agent of interest and will therefore be dependent on
the adverse effects of the agent of interest, its activity when
bound to blood components, the time necessary to reduce the free
concentration of the first compound containing the agent of
interest, the dosage necessary for therapeutic activity, the
indication being treated, the sensitivity of the agent to blood
enzymes, the route and mode of administration, and the like. As
necessary, the dosage may be determined empirically, initially
using a small multiple of the therapeutic dosage normally
administered, and as greater experience is obtained, enhancing the
dosage. Where the agent of interest is used to elicit an immune
response, the agent of interest will be an antigen, and will not
rely on the conjugation to the blood component for enhancing the
immune response. In this case, relatively large dosages may be
employed, where one is interested in producing antibodies as a
product, where the treated host will normally be a domestic or
laboratory animal. Where the agent of interest is acting as a
vaccine, the dosage may be smaller and may be determined
empirically. For the most part, as a vaccine the dosage will
generally be in the range of about 10 ng to 100 .mu.g. Normally,
any additional dose will be administered after the original dose
has been diminished by at least 50%, usually at least 90%, of the
original dose.
[0024] As compared to the first compound, in some ways, the second
compound may be much more sophisticated. This compound will have a
second binding entity, which will be determined by the nature of
the first binding entity. As already indicated, this entity may
take numerous forms, particularly as binding proteins, such as
immunoglobulins and fragments thereof, particularly Fab, Fv, or the
like, particularly monovalent fragments, naturally occurring
receptors, such as surface membrane proteins, enzymes, lectins,
other binding proteins, such as avidin or streptavidin, or the
like.
[0025] Particularly where the first binding entity is used with a
second compound, the first binding entity will be naturally found
at low concentration, if at all, in the host blood stream, so there
will be little if any competition between the first binding entity
and naturally occurring compounds in the blood for the second
binding entity. The second binding entity should not bind to
compounds which it may encounter in the blood or associated cells.
Therefore, enzymes which are used will usually be active on
substrates which do not appear in the blood. Lectins which are used
will usually bind to sugars which do not appear in the blood and
are not present on endothelial or other cells which line the
vascular system.
[0026] For the most part, the second binding entity will be a
protein, although other molecules which can provide for relatively
high specificity and affinity may also be employed. Combinatorial
libraries afford compounds other than proteins which will have the
necessary binding characteristics. Generally, the affinity will be
at least about 10.sup.-6, more usually about 10.sup.-8, e.g.
binding affinities normally observed with specific monoclonal
antibodies. Of particular interest is avidin or streptavidin,
although other receptors of particular interest include receptors
for steroids, TSH, LH, FSH, or their agonists, as well as sialic
acid and viral hemagglutinins, and superantigens. The second
binding entity will usually be a macromolecule, generally of at
least about 5 kD, more usually of at least about 10 kD and usually
less than about 160 kD, preferably less than about 80 kD, which may
be mono- or divalent in binding sites, usually monovalent.
[0027] One or more agents of interest may be present in the first
or second compound, usually multiple agents of interest being bound
through first or second linking groups to a central core, e.g. a
protein, nucleic acid, polysaccharide, or other multifunctional
entity. Not only does a linking group serve to covalently bond the
agent of interest, but it also serves to determine whether the
agent of interest remains bound to the blood component or if
released, the manner and rate of release. Therefore, the nature of
the linking group will vary widely depending upon its role.
[0028] Where the agent of interest is to be retained bound to the
blood component, any of a wide variety of convenient linking
groups, including a bond, may be employed. Thus, the same types of
linking groups employed in the first compound will find acceptance
for the second compound. However, where the linking group is to be
cleaved and release the agent of interest, the linking group will
vary depending upon the nature of the agent of interest, the
desired rate of release, the valency or the functionality on the
agent of interest which is to be released, and the like. Thus,
various groups may be employed, where the environment of the blood,
components of the blood, particularly enzymes, activity in the
liver, or other agent may result in the cleavage of the linking
group with release of the agent of interest at a reasonable
rate.
[0029] Functionalities which may find use include esters, either
organic or inorganic acids, particularly carboxyl groups or
phosphate groups, disulfides, peptide or nucleotide linkages,
particularly peptide or nucleotide linkages which are susceptible
to trypsin, thrombin, nucleases, esterases, etc., acetals, ethers,
particularly saccharidic ethers, or the like. Generally, the
linking group for cleavage will require at least two atoms in the
chain, e.g. disulfide, and may require 50 atoms, usually not more
than about 30 atoms, preferably not more than about 20 atoms in the
chain. Thus, the chain may comprise an oligopeptide,
oligosaccharide, oligonucleotide, disulfide, organic divalent
groups which are aliphatic, aromatic, alicyclic, heterocyclic or
combinations thereof, involving esters, amides, ethers, amines, or
the like. The particular linking group will be selected in
accordance with physiological acceptance, desired rate of cleavage,
synthetic convenience, and the like.
[0030] The disclosed therapeutic methods are applicable to a broad
range of target entities, both host derived and foreign (meaning
exogenous or non-host), which may be present in the blood and have
a deleterious physiological effect, due to an undesirably high
effective concentration, or as in the case of neoplastic cells,
being present in any amount. Host derived cellular target entities
include, (with parenthetical examples of clinical indication): T
cells or subsets, such as CD2+, CD7+, CD4+, CD8+, CD25 + or LFA1+
cells (autoimmune disease, alloreactivity and inflammation), B
cells or subsets such as pre-B cells, such as CD5+, IgE+, IgM+ etc.
(B cell lymphoma, xenograft, autoimmunity, anaphylaxy), leukocytes,
such as macrophages and monocytes (inflammation, myelomonocytic
leukemia), other leukocytes such as neutrophils, basophils, NK
cells, eosinophils, or allo- or xeno-reactive leukocytes, etc.
(inflammation, anaphylaxis), stem cells such as CD34+ cells
(polycythemia), malignant cells (malignancies; CALLA) or infected
cells, particulaly HIV infected host cells, or the like.
[0031] Host derived non-cellular target entities include soluble
HLA, class I and class II, and non-classical class I HLA (E, F and
G) for modulating immunoregulation, soluble T or B cell surface
proteins, cytokines, interleukins and growth factors, such as
individually IL1-16, soluble IL2 receptor, M-CSF, G-CSF, GM-CSF,
platelet derived growth factor, alpha, beta, and gamma interferons,
TNF, NGFs, arachidonic acid metabolites such as prostaglandins,
leukotrienes, thromboxane and prostacyclin for cardiovascular
diseases, immunoglobulins such as total IgE for anaphylaxy,
specific anti-allergen IgE, auto- or allo-antibodies for
autoimmunity or allo- or xenoimmunity, Ig Fc receptors or Fc
receptor binding factors, erythropoietin, angiogenesis factors,
adhesion molecules, MIF, MAF, complement factors, PAF aceter, ions
such as calcium, potassium, magnesium, aluminum, iron, etc, enzymes
such as proteases, kinases, phosphatases, DNAses, RNAses, lipases
and other enzymes affecting cholesterol and other lipid metabolism,
esterases, dehydrogenases, oxidases, hydrolases, sulphatases,
cyclases, transferases, transaminases, atriopeptidases,
carboxylases and decarboxylases and their natural substrates or
analogs, superoxide dismutase, hormones such as TSH, FSH, LH,
Thyroxine (T4 and T3), renin, insulin, apolipoproteins, LDL, VLDL,
dehydroepiandrosterone, cortisol, aldosterone, estriol, estradiol,
progesterone, testosterone, dehydroepiandrosterone (DHEA) and its
sulfate (DHEA-S), calcitonin, parathyroid hormone (PTH), human
growth hormone (hGH), vasopressin and antidiuretic hormone (ADH),
prolactin, ACTH, LHRH, THRH, VIP, cathecolamines (adrenaline,
vanillylmandelic acid, etc.), bradykinins and corresponding
prohormones, metabolites, ligands or natural cell or soluble
receptors thereof, cofactors including atrionatriuretic factor
(ANF), vitamins A, B, C, D, E and K, serotonin, coagulation
factors, e.g. thrombin, fibrin, fibrinogen, Factor VIII, Factor XI,
von Willebrand factor, tissue plasminogen activator, or other
factors, complement activation factors, LDL and ligands thereof,
uric acid, etc..
[0032] Foreign target entities include drugs, especially drugs
subject to abuse such as heroin and other opiates, PCP,
barbiturates, cocaine and derivatives thereof, benzodiazepins,
etc., poisons, toxins such as heavy metals like mercury and lead,
chemotherapeutic agents, paracetamol, digoxin, free radicals,
arsenic, bacterial toxins such as LPS and other gram negative
toxins, Staphylococcus toxins, Toxin A, Tetanus toxins, Diphtheria
toxin and Pertussis toxins, plant and marine toxins, virulence
factors, such as aerobactins, radioactive compounds or pathogenic
microbes or fragments thereof, including infectious viruses, such
as hepatitis A, B, C, E and delta, CMV, HSV (type 1, 2 & 6),
EBV, varicella zoster virus (VZV), HIV-1, -2 and other
retroviruses, adenovirus, rotavirus, influenzae, rhinovirus,
parvovirus, rubella, measles, polio, reovirus, orthomixovirus,
paramyxovirus, papovavirus, poxvirus and picornavirus, prions,
protists such as plasmodia, toxoplasma, filaria, kala-azar,
bilharziose, entamoeba histolitica and giardia, and bacteria,
particularly gram-negative bacteria responsible for sepsis and
nosocomial infections such as E. coli, Acynetobacter, Pseudomonas,
Proteus and Klebsiella, but also gram positive bacteria such as
staphylococcus, streptococcus, etc. Meningococcus and Mycobacteria,
Chlamydiae, Legionnella and Anaerobes, fungi such as Candida,
Pneumocystis carini, and Aspergillus, and Mycoplasma such as
Hominis and Ureaplasma urealyticum.
[0033] The agent of interest may be varied widely, including
naturally occurring compounds, synthetic compounds, and
combinations thereof. Thus, a wide variety of compounds may be of
interest, where the drug binds to a surface membrane receptor, so
as to induce a signal. Alternatively, antagonists may be employed
which will reduce the level of effective binding of the naturally
occurring ligand. As agonists or antagonists, peptides and proteins
include hormones, e.g. glucagon, insulin, TSH, LH. FSH, cytokines,
e.g. IL-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16,
colony stimulating factors, e.g. G-, M- and GM-, immunoglobulins to
specific ligands, where the ligand may be any hapten or antigen of
interest, blood proteins, anticoagulants, e.g. heparin, and the
like. The immunoglobulins are of particular interest, since they
can provide for a wide variety of therapies, associated with
infectious diseases, oncology, inflammation, immunological
disorders, toxicology, and the like. The antibodies may be
allogeneic or xenogeneic, particularly mouse or rabbit, monoclonal
or polyclonal, particularly monoclonal, and may be modified
antibodies, where the constant region, by itself, or in conjunction
with the conserved framework segments in the variable region are
modified to conserved human sequences.
[0034] Other compounds of interest, may be sugars, particularly
oligosaccharides, which may bind to lectin domains to inhibit
binding of naturally occurring substances, e.g. leukocyte binding
to high endothelial venules, or other trafficking, nucleic acids,
synthetic organic compounds, which are known to have therapeutic
activity, such as antibiotics, antihypertensive agents,
anti-coagulants, analgesics, hormones, chemotherapeutic agents,
immunosuppressive or immunoregulatory agents, enzymes, vasoactive
drugs, anti-inflammataory drugs, anti-histamines, etc.
[0035] Of particular interest are synthetic drugs, particularly
drugs which act as impermeant drugs, such as antibiotics,
adrenergic agonists and antagonists, cardiovascular drugs,
antiproliferative drugs, diuretics, as well as some of the drugs
listed above.
[0036] For the most part, the second compound will be administered
in a bolus, where the concentration will depend upon the particular
nature of the agent of interest. The dosage may be much higher than
normal dosage for a particular agent, where the binding of the
agent to the linking group substantially inactivates the agent. In
this manner, the agent is only active upon release. Where the agent
remains bound to the linking group, the dosage will be dependent
upon the effective dosage of the agent as complexed with the first
compound. The second compound may be administered substantially in
the same manner as the first compound.
[0037] Because of the extended delivery time or availability of the
subject agents, the subject invention may be used in a wide variety
of situations. The second agent may be an antibody to a toxin,
where there is concern about nosocomial infection in a hospital or
other situation where disease may be spread. The subject invention
may be employed before surgery so as to ensure that a level of drug
is maintained during and subsequent to the surgery without
requiring repetitive administration, avoiding the disturbance of
the patient. For example, one may use anticlotting agents, where
the nature of the surgery and indication is susceptible to the
formation of clots. One may use inhibitors of leukocyte homing to
prevent perfusion injury. One may use the subject invention with
cardiovascular drugs, where a patient is particularly susceptible
during an extended period to myocardial infarction. Other
treatments which will benefit from long term availability of drugs
include hormonotherapy, infertility therapy, immunosuppressive
therapy, neuroleptic therapy, drug of abuse prophylaxy, treatment
of diseases caused by infectious agents, treatment of hemophilia,
and the like.
[0038] By conjugating a biologically active agent of interest to
IgG in the blood of a mammalian host, many advantages ensue, in
providing for a new activity for the immunoglobulins, while
retaining many of the desirable features of the immunoglobulins, in
addition to the extended life-time. For example, the
immunoglobulins will usually still have F.sub.c effector function,
such as its role in complement fixation, or the action of antibody
dependent cytotoxic cells, effect on inactivation and secretion,
and the like. Similarly, serum albumin and other long lived serum
proteins can act to inactivate target entities and aid in their
rapid elimination. Thus, the blood components to which the agent of
interest is bound impart their physiological activities to the
activity of the agent of interest. In this way cellular targets may
be inactivated or eliminated by having immunoglobulins directed to
a cellular or soluble target or by coating the cellular or soluble
target with blood proteins.
[0039] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
[0040] I. Two rabbits were injected with a solution of
N-succinimidyl biotin ester (NHS-biotin)(5 or 50 mg in 200 .mu.l or
1 ml of DMSO) i.v. on day 0. Blood samples were taken at initially
hourly, followed by daily intervals and the samples analyzed for
the presence of biotin by gel efectrophoresis (SDS-PAGE) employing
60 .mu.g of sample, detecting the proteins to which biotin is bound
by avidin-peroxidase conjugate, using a luminescent substrate (ECL
kit, Amersham). For biotin bound to red blood cells, the cells were
isolated by centrifugation, washed, lysed by hypotonic lysis, and
the proteins separated by SDS-PAGE in the same manner as the plasma
proteins. For the plasma proteins, the electrophoresis was run
under reducing and non-reducing conditions, where the IgM and IgG
were reduced to sub-units under the reducing conditions.
[0041] The gels for the plasma proteins of one of the rabbits
showed that biotin was bound to IgM, IgG, p90, p75 (transferrin),
serum albumin, and p38. The duration for serum albumin for
detection in the gel electrophoresis under reducing conditions was
9 to 12 days for the serum albumin, and up to 33 days for IgM and
IgG, while for the non-reducing conditions, where the time for
exposure was substantially less, bands could be observed for the
serum albumin for 2 days and for the IgG for 9 days. With the red
blood cells, a number of proteins could be observed for 12 days,
the next point being 33 days, at which time no bands could be
observed. The major labled component had an electrophoretic pattern
suggesting Band 3.
[0042] II. To demonstrate that the subject invention can be used to
enhance the immune response to a heterologous protein, after
adminstration of the biotin conjugate as described above, 250 .mu.g
or 1 mg of egg white avidin was injected intravenously into each of
two rabbits. Within about 2 days the titer began to rise rapidly
and by 10 days a high anti-avidin titer was observed by ELISA,
using avidin coated to microiplates, as evidenced by an OD value of
about 600. By comparison, when avidin was administered as described
above to rabbits to which the biotin conjugate had not been
previously administered, there was substantially no production of
antibodies until an intravenous booster injection of 50 .mu.g of
avidin was made on day 6, at which time there was a rapid increase
in anti-avidin titer, approximating the titer observed with the
biotin conjugate modified rabbits by day 12.
[0043] It is evident from the above results, that the subject
invention provides for extended half-lives of agents for use in
mammalian patients. Thus, agents which allow for prophylaxis or
therapy can be administered to a patient, so as to become bound to
long-lived components of blood where the lifetime of the agent is
substantially extended. In this manner, frequency of administration
can be substantially diminished, continued therapeutic benefit over
extended periods of time is provided, the patient need not be
repetitively disturbed for repeated administrations, and one can be
assured that the agent is retained at a therapeutic dosage during
the period of treatment.
[0044] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0045] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
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