Method and composition for treatment of renal failure with antibodies and
their equivalents as partial or complete replacement for dialysis
United States Patent: 7,504,106
Issued: March 17, 2009
Boris (Pawtucket, RI), Millstein; Ellen (LaJolla, CA), Skurkovich; Simon
Appl. No.: 11/375,378
Filed: March 14, 2006
Training Courses --Pharm/Biotech/etc.
A method for treating patients with renal
failure includes administering to them an effective amount of antibody or
of a functional equivalent thereof to at least two of urea, creatinine,
tumor necrosis factor alpha, interferon gamma, interleukin 6 and
interleukin 1 beta. Soluble cytokine receptors also can be employed. The
method can be used as a supplement to or as partial or complete
replacement for dialysis. A pharmaceutical composition includes antibody
or functional equivalent thereof to urea, creatinine, or both; antibody,
functional equivalent or soluble cytokine receptor to tumor necrosis
factor alpha, interferon gamma, interleukin 6, interleukin 1 beta or any
combination thereof The composition can be included in a kit.
Description of the
SUMMARY OF THE INVENTION
A need continues to exist, therefore, for methods of treating renal disease
and in particular for treatments that can be provided in conjunction with or
as a replacement for renal or peritoneal dialysis.
Generally the invention relates to a method and composition for treating
renal disease. In further aspects, the invention relates to a process for
making the composition and to a kit suitable in administering the treatment.
The treatment disclosed herein can be administered as a supplement to
dialysis or as partial or complete replacement of dialysis. In some
patients, the treatment can be used to delay the onset of kidney failure.
In one embodiment of the invention a method for treating renal disease
includes selecting a patient suffering from renal disease and administering
to the patient an antibody component, wherein a first portion of the
antibody component is to a compound selected from the group consisting of
urea, creatinine and any combination thereof, and wherein a second portion
of the antibody component is to a cytokine selected from the group
consisting of tumor necrosis factor alpha, interferon gamma, interleukin 6,
interleukin 1 beta, and any combination thereof.
In another embodiment of the invention, a method for treating a patient
suffering from renal disease includes administering to the patient effective
amounts of antibodies, functional equivalents of antibodies or soluble
cytokine receptors to at least two compounds selected from the group
consisting of urea, creatinine, tumor necrosis factor alpha, interferon
gamma, interleukin 6, and interleukin 1 beta.
In a further embodiment of the invention, a method for treating renal
insufficiency or renal failure includes determining in a patient an elevated
level of urea, creatinine or both and determining in the patient an elevated
level of a cytokine selected from the group consisting of tumor necrosis
factor alpha, interferon gamma, interleukin 6, and interleukin 1 beta. An
antibody component is administered to the patient to reduce said elevated
In yet another embodiment of the invention, a method for treating a patient
suffering from renal disease includes administering to the patient an
effective amount of antibody, or of a functional equivalent thereof, to a
compound selected from the group consisting of urea, creatinine and any
combination thereof, and an effective amount of antibody to a cytokine, an
effective amount of a functional equivalent of the antibody, or an effective
amount of a soluble receptor to the cytokine, or any combination thereof,
wherein the cytokine is selected from the group consisting of tumor necrosis
factor alpha, interferon gamma, interleukin 6, interleukin 1 beta, and any
In one aspect, the invention is directed to a method for treating a patient
suffering from renal disease which includes administering to the patient an
effective amount of a vaccine for producing endogenous antibody to a
compound selected from the group consisting of urea, creatinine, or to a
cytokine selected from the group consisting of tumor necrosis factor alpha,
interferon gamma, interleukin 6, interleukin 1 beta, and any combination
In another aspect, the invention is directed to a method for treating a
patient suffering from a renal disease and includes administering to the
patient an effective amount of an antibody or of a functional equivalent
thereof to urea, creatinine or a combination thereof.
In a further aspect, the invention is directed to a method for treating
renal disease, the method including administering to a patient suffering
from renal disease an effective amount of a functional equivalent of an
antibody or of a soluble cytokine receptor to tumor necrosis factor alpha,
interferon gamma, interleukin 6 or interleukin 1 beta.
In still another aspect, the invention is directed to a method for treating
renal disease, wherein the method includes administering to a patient
suffering from renal disease an effective amount of antibody to interleukin
6, interleukin 1 beta or any combination thereof.
In other embodiments of the invention the patient also receives an antibody,
functional equivalent thereof, or a soluble cytokine receptor to at least
one of interleukin 10 and interleukin 13.
The invention also is directed to a pharmaceutical composition and a method
for its manufacture. The pharmaceutical composition includes effective
amounts of antibody or a functional equivalent thereof to urea, creatinine,
or both and of antibody, functional equivalent thereof or soluble cytokine
receptor to tumor necrosis factor alpha, interferon gamma, interleukin 6,
interleukin 1 beta or any combination thereof.
In a specific example, the pharmaceutical composition includes effective
amounts of antibody to urea, creatinine, tumor necrosis factor alpha,
interferon gamma, interleukin 6 and interleukin 1 beta. Functional
equivalents of one or more antibodies or soluble cytokine receptors also can
In other examples, the composition can include antibody, a functional
equivalent thereof, and/or a soluble cytokine receptor to at least one and
preferably both of interleukin 10 or interleukin 13.
In one aspect, a method for manufacturing a preferred pharmaceutical
composition includes combining effective amounts of antibody, functional
equivalent thereof or a soluble cytokine receptor to urea, creatinine, tumor
necrosis factor alpha, interferon gamma, interleukin 6 and interleukin 1
In yet other aspects, the invention is directed to a kit, for instance a kit
that includes an effective amount of antibody, or functional equivalent
thereof, to a compound selected from the group consisting of urea,
creatinine and any combination thereof and an effective amount of antibody,
functional equivalent thereof, or a soluble cytokine receptor to a cytokine
selected from the group consisting of tumor necrosis factor alpha,
interferon gamma, interleukin 6, interleukin 1 beta, and any combination
In one example, the kit includes effective amounts of antibody, functional
equivalent thereof or, if applicable, cytokine soluble receptor, to urea,
creatinine, tumor necrosis factor alpha, interferon gamma, interleukin 6,
interleukin 1 beta and, optionally, to interleukin 10 and interleukin 13.
The invention has several advantages. It is believed, for example, that the
method of treatment can result in a reduction and possibly in the
elimination of dialysis requirements and/or improvements in quality of life.
The invention can provide for more pathogenetic treatment by removing
hyperproduced cytokines and not just urinary retention products. Compared to
dialysis, the method of treatment disclosed herein is more convenient and
easier to provide. In some patients, the treatment described herein also may
delay the onset of kidney failure.
Practicing the invention also allows customized treatment, depending on the
specific cytokines that are elevated in a patient's circulation.
Accordingly, a decrease in the incidence of side effects is expected.
The use of functional equivalents to the antibodies described herein can
result in improvements in formulation and manufacturing, thereby reducing
treatment costs. Functional equivalents also can be used to reduce or
minimize side effects. Some of the advantages associated with the use of
soluble cytokine receptors include specificity, high affinity and low
In some aspects, the invention is particularly beneficial in managing ESRD
patients who do not respond to erythrorpoietin therapy.
The above and other features of the invention including various novel
details of construction and combinations of parts, and other advantages,
will now be more particularly described and pointed out in the claims. It
will be understood that the particular method and composition embodying the
invention are shown by way of illustration and not as a limitation of the
invention. The principles and features of this invention may be employed in
various and numerous embodiments without departing from the scope of the
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention generally relates to the treatment of renal disease and can be
practiced in patients that include human as well as other mammalian
subjects, for example, farm or wild animals, companion animals, laboratory
or experimental animals, zoo animals, sports animals and others.
As used herein, the term "treatment" refers to an intervention performed in
order to alter or prevent the development or pathology of a disorder or
disease, for instance, to prevent or slow down the progression of a disease,
or to lessen its severity. The term refers to both, therapeutic, as well as
prophylactic or preventive measures.
As used herein, the term "renal disease" refers to acute or chronic renal
failure, and other types of renal impairment or insufficiency as known in
the medical arts. In some cases, the treatment is provided to a patient in
an advanced stage of renal disease. In specific aspects of the invention,
the treatment is provided as a supplement to dialysis. In other aspects, the
treatment is provided as a partial or complete substitute to dialysis. The
treatment also can be undertaken to delay the onset of kidney failure.
In addition to renal disease, the patient can also suffer from other
diseases or underlying conditions such as diabetes, hypertension, shock,
overwhelming infection, poisoning, heavy metal intoxication, cardio-vascular
abnormalities, endocrine, hormonal or metabolic disturbances and other
medical problems. The treatment also can benefit patients exhibiting
surgery-related complications affecting kidney function.
Candidates for the treatment disclosed herein can be selected based on
clinical diagnosis, laboratory evaluations, or both.
In most patients with stable chronic renal disease (CRD), for example, the
total body contents of Na.sup.+ and H.sub.2O are increased modestly. The
underlying etiologic disease process may itself disrupt glomerulotubular
balance and promote Na.sup.+ retention (e.g., glomerulonephritis), or
excessive Na.sup.+ ingestion may lead to cumulative positive Na.sup.+
balance and attendant extracellular fluid volume (ECFV) expansion. Such ECFV
expansion contributes to hypertension, which in turn accelerates further the
progression of nephron injury. As long as water intake does not exceed the
capacity for free water clearance, the ECFV expansion will be isotonic and
the patient will remain normonatremic.
Hyponatremia is an uncommon complication in predialysis patients, and water
restriction is only necessary when hyponatremia is documented. In the CRD
patient who is not yet on dialysis but has clear evidence of ECFV expansion,
administration of loop diuretics coupled with restriction of salt intake are
the mainstays of therapy. The combination of loop diuretics with metalozone,
which inhibits the Na.sup.+Cl.sup.- cotransporter of the distal convoluted
tubule, can sometimes overcome diuretic resistance. When the GFR falls to <5
to 10 mL/min per 1.73 m.sup.2, even high doses of combination diuretics are
ineffective. ECFV expansion under these circumstances usually means that
dialysis is indicated.
In CRD, the decline in GFR is not necessarily accompanied by a concomitant
and proportionate decline in urinary K.sup.+ excretion. In addition, K.sup.+
excretion in the gastrointestinal tract is augmented in patients with CRD.
However, hyperkalemia may be precipitated in a number of clinical
situations, including constipation, augmented dietary intake, protein
catabolism, hemolysis, hemorrhage, transfusion of stored red blood cells,
metabolic acidosis, and following the exposure to a variety of medications
that inhibit K.sup.+ entry into cells or K.sup.+ secretion in the distal
nephron. In addition, certain etiologies of CRD may be associated with
earlier and more severe disruption of K.sup.+ secretory mechanisms in the
distal nephron, relative to the reduction in GFR. Most important are
conditions associated with hyporeninemic hypoaldosteronism (e.g., diabetic
Acidosis is a common disturbance during the advanced stages of CRD. Although
in a majority of patients with CRD the urine can be acidified normally,
these patients have a reduced ability to produce ammonia. Hyperkalemia
further depresses urinary ammonium excretion. The combination of
hyperkalemia and hyperchloremic metabolic acidosis (known as type IV renal
tubular acidosis, or hyporeninemic hypoaldosteronism) is most
characteristically seen in patients with diabetes or in those with
predominantly tubulointerstitial disease. Treatment of the hyperkalemia
frequently improves the acidosis as well.
Adjustments in dietary intake and use of loop diuretics, occasionally in
combination with metalozone, may be needed to maintain salt and hence
extracellular fluid volume balance. Occasional patients with salt-wasting
states need to be given sodium-rich diets or sodium supplements. Water
restriction is indicated only if there is a demonstrated propensity to
hyponatremia. Intractable ECFV expansion, despite dietary restriction and
diuretic use, indicates the need to initiate renal replacement therapy.
Hyperkalemia often responds to dietary restriction of potassium, avoidance
of potassium-containing or -retaining medications, and to the use of
diuretics if they are also indicated for management of sodium balance.
Potassium-binding resins taken with cathartics can promote gastrointestinal
potassium losses and thus are useful as temporizing measures in the
treatment or avoidance of hyperkalemia in CRD patients. However, the need
for such treatment over a prolonged period, in the absence of other
reversible causes of hyperkalemia, usually signifies the need to initiate
renal replacement therapy.
Patients with renal disease often exhibit elevated levels of non-protein
nitrogen compounds. Urea, for example, is a waste product of protein
metabolism and can be measured as blood urea nitrogen (BUN). In the course
of renal disease, damaged kidneys generally are less able to clear urea from
the blood stream and high BUN levels are observed.
Another non-protein nitrogen compound, creatinine, is a waste product formed
in the enzymatic conversion of creatine by creatine amidohydrolase. As with
BUN, patients having impaired renal function often exhibit increased
creatinine serum concentrations.
Patient BUN and creatinine levels can be determined by routine laboratory
procedures as known in the clinical or veterinary arts.
In many cases, renal disease, e.g., renal insufficiency or renal failure,
also is associated with increased levels in at least one of tumor necrosis
factor alpha (TNF-.alpha.), interferon gamma (INF-.gamma.), interleukin 6
(IL-6) or interleukin 1 beta (IL-1.beta.). ESDR patients who do not respond
to erythropoietin often exhibit high levels of interleukin 10 (IL-10) and/or
interleukin 13 (IL-13).
Compounds such as TNF-.alpha., INF-.gamma., IL-6, IL-1.beta., IL-10 and
IL-13, generally are referred to herein as "cytokines". Most cytokines are
not produced constitutively, but transiently after stimulation and are not
normally found in the serum in a bioactive form. Cytokines can play a role
in the generation of an immune response and are intercellular mediators
secreted by lymphocytes and/or macrophages and other cells. In turn, some
cytokines such as, for instance, interferons, are capable of inducing other
Cytokine levels can be measured, for example, by collecting a blood sample,
separating plasma, for instance by centrifugation, and detecting cytokines,
by a standard assay, e.g., enzyme linked immunosorbent assay (ELISA) and
many other standard methods.
The presence of elevated levels of non-protein nitrogen compounds and/or
cytokines can be established by comparing levels found in a patient with
control levels, for instance with levels known to be present in normal
subjects. In normal adult human subjects, for example, levels of creatinine
generally are in the range of from about 0.8 to about 1.2 mg/dL and levels
of urea nitrogen (BUN) in the range of from about 8 to about 21 mg/dL.
Levels of non-protein nitrogen compounds and/or cytokines can be measured
and/or monitored prior to, during and after the treatment described herein.
In one example, the invention aims at reducing elevated levels of one or
more non-protein nitrogen compound(s) and/or cytokine(s) in a patient
suffering from renal disease through a treatment that includes antibodies,
functional equivalents thereof, and, if applicable, soluble cytokine
receptors. As used herein, the term "antibody component" refers to any
combination of antibodies, functional equivalents thereof and, if
applicable, one or more soluble cytokine receptors.
In a preferred aspect of the invention, the treatment includes antibody to
at least two compounds selected from the group consisting of urea,
creatinine, TNF-.alpha., INF-.gamma., IL-6, and IL-1.beta.. Functional
equivalents of antibodies as well as soluble cytokine receptors also can be
employed. In another aspect, an antibody component includes a portion
targeting one or more non-protein nitrogen compounds and another portion
targeting a cytokine selected from the group consisting of TNF-.alpha.,
INF-.gamma., IL-6, IL-1.beta. and any combination thereof.
The antibody component can further include a third portion targeting IL-10
and/or IL-13. Providing antibodies, functional equivalents thereof and/or
soluble cytokine receptors to one or both of IL-10 and IL-13 is particularly
helpful in managing ESRD patients who do not respond to erythropoietin
As used herein, the term "antibody" refers to monoclonal antibodies,
polyclonal antibodies, multispecific antibodies, chimeric, human, including
spontaneously produced antibodies derived from a subject's blood, humanized
monoclonal and camelid antibodies.
Monoclonal antibodies are obtained from a population of substantially
homogeneous antibodies, with individual antibodies being identical except
for possible naturally-occurring mutations that may be present in minor
amounts. Monoclonal antibodies are highly specific, being directed against a
single antigenic site. In contrast to polyclonal antibody preparations,
which include different antibodies directed against different determinants (epitopes),
each monoclonal antibody is directed against a single determinant on the
antigen. In addition to their specificity, monoclonal antibodies can be
synthesized to be uncontaminated by other antibodies.
Chimeric antibodies are monoclonal antibodies in which a portion of the
heavy and/or light chain is identical with or homologous to corresponding
sequences of antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the chain(s)
is identical with or homologous to corresponding sequences of antibodies
derived from another species or belonging to another antibody class or
subclass. See, e.g., U.S. Pat. No. 4,816,567 and Morrison et al., Proc.
Natl. Acad.Sci. USA 81: 6851-6855 (1984).
Chimeric antibodies for use herein include primatized antibodies comprising
variable domain antigen-binding sequences derived from a non-human primate
(e.g., Old World Monkey, Ape, etc.), and human constant region sequences.
Humanized monoclonal antibodies are chimeric antibodies that contain minimal
sequence derived from a non-human antibody. In many cases, humanized
antibodies are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by residues from a
hypervariable region of a non-human species (donor antibody) such as mouse,
rat, rabbit or non-human primate, having the desired antibody specificity,
affinity, and capability. In some cases, framework region (FR) residues of
the human immunoglobulin are replaced by corresponding non-human residues.
Humanized antibodies may also comprise residues that are not found in the
recipient antibody or in the donor antibody. These modifications can further
refine antibody performance. A humanized antibody optionally can also
comprise at least a portion of an immunoglobulin constant region (Fc),
typically that of a human immunoglobulin. Further details are described by
Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature
332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
Other suitable antibodies for use in the treatment disclosed herein are
camelid antibodies, derived from camelid species. Camelid antibodies differ
from those of most other mammals in that they lack a light chain, and thus
comprise only heavy chains with complete and diverse antigen binding
capabilities (Hamers-Casterman et al., 1993, Nature, 363:446-448). Such
heavy-chain antibodies tend to be smaller than conventional mammalian
antibodies, often are more soluble than conventional antibodies, and further
demonstrate an increased stability compared to some other antibodies.
Camelid species include, but are not limited to Old World camelids, such as
two-humped camels (C. bactrianus) and one humped camels (C. dromedarius).
The camelid family further includes New World camelids including, but not
limited to llamas, alpacas, vicuna and guanaco. Camelid species for the
production of antibodies and for other uses are available from various
sources, including but not limited to, Camello Fataga S. L. (Gran Canaria,
Canary Islands) for Old World camelids, and High Acres Llamas (Fredricksburg,
Tex.) for New World camelids.
Multispecific antibodies can be specific to different epitopes of a single
molecule or can be specific to epitopes on different molecules. Such
antibodies can be monospecific, bispecific, trispecific or can have greater
multispecificity. Methods for designing multispecific antibodies are known
in the art. See, e.g., Millstein et al. (1983) Nature 305:537-539; Kostelny
et al. (1992) J. Immunol. 148:1547-1553; WO 93/17715.
Antibodies and antibody production are further described, for instance, by
Harlow et al., 1999, "Using Antibodies: A Laboratory Manual", Cold Spring
Harbor Laboratory Press, NY; Harlow et al., 1989, "Antibodies: A Laboratory
Manual", Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad.
Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).
To produce antibodies suitable for use in the present invention, a cytokine
or antigen may be modified or administered in an adjuvant in order to
increase the peptide antigenicity. Methods of increasing the antigenicity of
a polypeptide are known in the art. Such procedures include coupling the
antigen with a heterologous protein (such as globulin or .beta.-galactosidase)
or through the inclusion of an adjuvant during immunization.
Antibodies suitable in the treatment dislosed herein also can be antibodies
that are spontaneously produced by a subject. For example, it can be
determined by laboratory testing that a patient suffering from renal disease
produces antibodies to one or more of urea, creatinine, TNF-.alpha.,
INF-.gamma., IL-6, IL-1.beta., IL-10 or IL-13. The antibodies can be
isolated from the patient's blood and used in treating the patient.
Procedures for separating endogenously produced antibodies from a subject's
blood are known in the art. Patient's plasma is fractionated by ethanol
precipitation of the proteins according to Cohn Method 6 and Oncley Method
9, and the immunoglobulin is purified. Each milliliter contains 50.+-.10 mg
immunoglobulin, primarily IgG, and trace amounts of IgA and IgM; 50 mg
sucrose; and 10 mg Albumin (Human). The sodium content is 20-30 mEq per
liter, i.e., 0.4-0.6 mEq per 20 mL or 1.0-1.5 mEq per 50 mL.
Several techniques for preparing monoclonal antibodies are known in the art
(Campbell, A. M., "Monoclonal Antibody Technology: Laboratory Techniques in
Biochemistry and Molecular Biology," Elsevier Science Publishers, Amsterdam,
The Netherlands (1984); St. Groth et al, J. Immunol. Methods 35:1-21 (1980).
For example, in one embodiment an antibody capable of binding to INF-.gamma.
is generated by immunizing an animal with natural, synthetic or recombinant
Monoclonal antibodies can be prepared, for instance, by a hybridoma
methodology such as described by Kohler et al., Nature 256: 495 (1975), or
they may be produced using recombinant DNA methods in bacterial or
eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).
Monoclonal antibodies can also be isolated from phage antibody libraries
using, for instance, the techniques described in Clackson et al., Nature
352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991).
For example, spleen cells from the immunized animals are removed, fused with
myeloma cells, such as SP210-Ag14 myeloma cells, and allowed to become
monoclonal antibody producing hybridoma cells. A hybridoma is an
immortalized cell line which is capable of secreting a specific monoclonal
antibody. Any one of a number of methods known in the art can be used to
identify the hybridoma cell which produces an antibody with the desired
characteristics. These include screening the hybridomas with an ELISA assay,
western blot analysis, or radioimmunoassay (Lutz et al., Exp. Cell Res.
175:109-124 (1988)). Hybridomas secreting the desired antibodies are cloned
and the class and subclass are determined using procedures known in the art.
See, e.g., "Monoclonal Antibody Technology: Laboratory Techniques in
Biochemistry and Molecular Biology", supra).
For polyclonal antibodies, antibody-containing antiserum is isolated from
the immunized animal and is screened for the presence of antibodies with the
desired specificity using one of the above-described procedures. Polyclonal
antibodies raised from animals immunized with specific antigens (IFNs, TNF,
etc.) can be used after the isolation of the active fraction (e.g., IgG) or
isolated Fab fragment.
In a specific example, the production is that of polyclonal sera from
camelid species. The production method is similar to the production of
polyclonal sera from other animals such as sheep, donkeys, goats, horses,
mice, chickens, rats, and the like. Isolation of camelid antibodies from the
serum of a camelid species can be performed by many methods known in the
art, including but not limited to ammonium sulfate precipitation, antigen
affinity purification, Protein A and Protein G purification, and the like.
A camelid species can be immunized with a desired antigen, for example IFN-.gamma.,
IL-1, a TNF-.alpha. peptide, or fragment thereof, using techniques known in
the art. The whole blood can then be drawn from the camelid and sera can be
separated using standard techniques. The sera can then be absorbed onto a
Protein G-Sepharose column (Pharmacia, Piscataway, N.J.) and washed with
appropriate buffers, for example 20 mM phosphate buffer (pH 7.0). The
camelid antibody can then be eluted using a variety of techniques known in
the art, for example 0.15M NaCl, 0.58% acetic acid (pH 3.5). The efficiency
of the elution and purification of the camelid antibody can be determined by
various methods, including SDS-PAGE, Bradford Assays, and the like. The
fraction that is not absorbed can be bound to a Protein A-Sepharose column
(Pharmacia, Piscataway, N.J.) and eluted using, for example, 0.15M NaCl,
0.58% acetic acid (pH 4.5).
In addition, camelid antibodies can be expressed from nucleic acid, by
methods known in the art, as described, for instance, in U.S. Pat. Nos.
5,800,988; 5,759,808; 5,840,526, and 6,015,695. Briefly, cDNA can be
synthesized from camelid spleen mRNA. Isolation of RNA can be performed
using methods and compositions, such as TRIZOL (Gibco/BRL, La Jolla,
Calif.). Total RNA can be isolated from tissues using the guanidium
isothiocyanate method detailed in, for example, Sambrook et al. (1989,
"Molecular Cloning, A Laboratory Manual", Cold Spring Harbor, N.Y.). Methods
for purification of mRNA from total cellular or tissue RNA are known in the
art, and include, for example, oligo-T paramagnetic beads. cDNA synthesis
can then be obtained from mRNA using mRNA template, an oligo dT primer and a
reverse transcriptase enzyme, available commercially from a variety of
sources, including Invitrogen (La Jolla, Calif.). Second strand cDNA can be
obtained from mRNA using RNAse H and E. coli DNA polymerase I, according to
techniques known in the art.
Antibodies also can be produced using polyclonal antibody libraries, such as
described, for instance, in U.S. Pat. No. 6,335,163, issued to Sharon on
Jan. 1, 2002, the teachings of which are incorporated herein by reference in
their entirety. Further techniques that can be used are described in
International Patent Application Publication No. WO 2004061104, "Method for
Manufacturing Recombinant Polyclonal Proteins", published on Jul. 22, 2004
and International Patent Application Publication No. WO 2005042774, "Method
for Linking Sequences of Interest", published on May 12, 2005. The teachings
of both international publications are incorporated herein by reference in
In specific examples, antibodies are prepared using Symphage.TM., a phage
display technology for generating and screening libraries for recombinant
polyclonal antibodies, available from Symphogen A/S, Denmark. Other
techniques available from Symphogen A/S, that can be employed include:
Sympress.TM., a manufacturing technology for the production of a recombinant
polyclonal antibody (rpAb) in a single bioreactor. The rpAb constitute a
large diversity of individual antibody members (produced in the same
bioreactor), a diversity which is maintained from batch to batch; and
Symplex.TM., a technique for high throughput isolation of original pairs of
antibody heavy and light chain variable region genes from human donor blood.
In further aspects of the invention, antibodies are produced endogenously by
an "immunization" procedure whereby administration of a "vaccine", e.g., an
antigen, elicits an antibody response to the antigen. For example, the use
of an anti-interferon-alpha vaccine, specifically inactivated recombinant
IFN-alpha-2b, aimed at counteracting overproduction of IFN-alpha in AIDS
patients is described, for example, by A. Gringeri et al. in the article
with the title "Absence of Clinical, Virological, and Immunological Signs of
Progression in HIV-1-Infected Patients Receiving Active
Anti-Interferon-alpha Immunization: a 30-Month Follow-Up Report", J. Acquir.
Immune Defic. Syndr. Hum. Retrovirol., 13(1), pp 55-67 (1996).
The treatment disclosed herein also can be practiced using one or more
"functional equivalent(s)" of an antibody. Such a functional equivalent is
capable of specifically binding to, or destroying, the same antigenic
determinant as the antibody, thereby neutralizing the molecule, e.g.,
antibody-like molecules, such as single chain antigen binding molecules.
Functional equivalents of an antibody include antibody fragments, antibody
variants, antibody derivatives and/or antibody analogs that exhibit the
desired biological activity.
The phrase "fragment" or "analog" of an antibody refers to a compound having
qualitative biological activity in common with the full-length antibody.
The term "antibody mutant" refers to an amino acid sequence variant of an
antibody wherein one or more of the amino acid residues have been modified.
Such mutants necessarily have less than 100% sequence identity or similarity
(homology) with the amino acid sequence having at least about 75% amino acid
sequence identity with the amino acid sequence of either the heavy or light
chain variable domain of the antibody, alternatively at least about 80%
amino acid sequence identity, alternatively at least about 85% amino acid
sequence identity, alternatively at least about 90% amino acid sequence
identity, alternatively at least about 95% amino acid sequence identity or
alternatively at least about 96%, 97%, 98% or 99% amino acid sequence
As used herein, the phrase "biologically active fragment" refers to a part
of the complete molecule which retains all or some of the catalytic or
biological activity possessed by the complete molecule, especially activity
that allows specific binding of the antibody to an antigenic determinant.
The term "analog" refers to peptides, and in particular polypeptides,
derivatives, allelic or species variants that have the desired biological
activity. The peptides and polypeptides act on the same target as antibodies
and can be used to inhibit binding of a cytokine to its receptor. A
"variant" or "allelic or species variant" of a protein refers to a molecule
substantially similar in structure and biological activity to the protein.
Thus, if two molecules possess a common activity and may substitute for each
other, it is intended that they are "variants," even if the composition or
secondary, tertiary, or quaternary structure of one of the molecules is not
identical to that found in the other, or if the amino acid or nucleotide
sequence is not identical.
The term "derivative" includes functional and chemical derivatives, for
instance fragments, segments, variants or analogs of a molecule. A molecule
is a "chemical derivative" of another, if it contains additional chemical
moieties not normally a part of the molecule. Such moieties may improve the
molecule's solubility, absorption, biological half life, and the like, or
they may decrease toxicity of the molecule, eliminate or attenuate
undesirable side effects of the molecule, and the like. Moieties capable of
mediating such effects are disclosed in Remington's Pharmaceutical Sciences
(1980). Procedures for coupling such moieties to a molecule are well known
in the art.
As used herein, the term "functional equivalent" of an antibody also refers
to enzymes that act on the same target as antibodies.
Functional equivalents such as, for instance, antibody fragments, can be
identified using phage display techniques combined with synthetic libraries.
Soluble cytokine receptors are known to regulate inflammatory or immune
events and are believed to function as agonists or antagonists of cytokine
signaling. Receptors that bind cytokines are typically composed of one or
more integral membrane proteins that bind the cytokine with high affinity
and transduce this binding event to the cell through the cytoplasmic
portions of the certain receptor subunits. Cytokine receptors have been
grouped into several classes on the basis of similarities in their
extracellular ligand binding domains. For example, the receptor chains
responsible for binding and/or transducing the effect of interferons are
members of the class II cytokine receptor family, based upon a
characteristic 200 residue extracellular domain.
In some examples, the treatment is practiced using a combination of
different types of antibody or equivalents thereof targeting the same
compound, e.g., a cytokine. For instance, the treatment can employ a mixture
of a monoclonal antibody targeting INF-.gamma. as well as a biologically
active fragment of an antibody also targeting INF-.gamma.. In other
examples, the treatment includes a monoclonal antibody targeting
INF-.gamma., a biologically active fragment of an antibody also targeting
INF-.gamma. as well as a soluble INF-.gamma. receptor.
In preferred aspects of the invention, the treatment employs a combination
or antibodies, preferably targeting those compounds found at elevated levels
in patients suffering from renal disease. A patient can receive an antibody
to a non-protein nitrogen compound in combination with an antibody to a
cytokine. More specifically, an antibody or its functional equivalent
targeting at least one non-protein nitrogen compound, e.g., urea and/or
creatinine, can be combined with one and preferably more than one antibody,
its/their functional equivalent(s) and/or one or more soluble cytokine
receptor(s) targeting at least one cytokine selected from TNF-.alpha.,
INF-.gamma., L-6, and IL-1.beta..
A combination that includes antibodies to both creatinine and urea, together
with antibodies targeting TNF-.alpha., INF-.gamma., IL-6 and IL-1.beta. is
preferred. This "cocktail" or combination can further include an antibody to
at least one of IL-10 and IL-13. Functional equivalents of the antibodies
and/or soluble receptors to the cytokines also can be employed.
Specific antibodies, functional equivalents of antibodies and soluble
cytokine receptors suitable for practicing the invention are further
Both creatinine and urea are haptens and they can be bound to a protein
carrier (e.g., gelatine). Methods of binding haptens with carrier proteins
are described, for example, by Obermeier F & Pick E. Wein Klin Wschr., 1903,
Bd 16, S. 659-7; and Idem Ibid 1904 Bd 17, S. 265. In other examples, the
amino acid valine has been used as a coupling agent between a carrier
protein and a hapten, as described by Kaverzneva E & Kiseleva V.
Biochemistry, 1966, 6, p 204; Skurkovich S & Milonova T. Journal of
Microbiology, Epidemiology & Immunolgy, 1974, 1, p 89.
Several antibodies to urea and/or creatinine have been developed
specifically for laboratory assays designed to detect creatinine and/or urea
in serum samples. A creatinine-specific antibody is described, for example,
in U.S. Pat. No. 4,485,177 issued on Nov. 27, 1984, and U.S. Pat. No.
4,578,361, issued on Mar. 25, 1986, both to Siedel et al; the teachings of
U.S. Pat. Nos. 4,485,177 and 4,578,361 are incorporated herein by reference
in their entirety. This creatinine specific antibody forms a hapten-antibody
complex with creatinine and can be prepared by raising an antiserum with a
conjugate of creatinine and a protein suitable for antiserum formation,
connected via an aliphatic or araliphatic carboxylic bridge member.
Creatinine-specific antibodies have been generated and used for highly
sensitive and specific immunochemical creatinine determinations. Creatinine
was derivatized at N3 and coupled to KLH carrier protein. On the basis of
this immunogen, monoclonal antibodies were developed by hybridoma
technology. Antibodies from various clones have been characterized with
BIAcore 2000 with respect to the dissociation constant and specificity.
Antibodies of clone B90-AH5 exhibited the lowest dissociation constant (0.74
microM) and the highest specificity for creatinine, as described by Benkert
A, Scheller F, Schossler W, Hentschel C, Micheel B, Behrsing O, Scharte G,
Stocklein W, Warsinke A., "Development of a Creatinine ELISA and an
Amperometric Antibody-Based Creatine Sensor with a Detection Limit in the
Nanomolar Range", Anal Chem. Mar. 1, 2000;72(5):916-21.
Antibodies targeting urea are described by Rohde M, Schenk J A, Heymann S,
Behrsing O, Scharte G, Kempter G, Woller J, Hohne W E, Warsinke A, Micheel
B., in an article entitled "Production and Characterization of Monoclonal
Antibodies Against Urea Derivatives", Appl Biochem Biotechnol., October
1998; 75(1), pp. 129-37. A panel of monoclonal antibodies was generated
against the urea-based hapten
N-(2-N-chloroacetylaminobenzyl)-N'-4-chlorophenylurea as a tool for building
up sensitive immune assays to detect urea derivatives and to screen them for
catalytic antibodies (Abs). Eleven hybridomas were obtained that produced
Abs reactive to the hapten. All Abs were of IgG class. Cross reactivities of
the Abs to different haptens were examined, especially to a possible
transition-state analog and four of the hybridomas, namely R2-DA10/F7,
R2-GE7/H2, R2-HC2/A5, R2-HD6/F7, produced Abs crossreactive with the
transition-state analog. From the 11 hybridomas, hybridoma B76-BF5 was
chosen for further characterization. Compared to the other Abs, B76-BF5
showed the strongest binding and had a rather restricted specificity.
Polyclonal antibodies targeting TNF-.alpha. can be obtained by immunizing
goats, or other animals, with recombinant human TNF-.alpha. ("r-Hu-TNF-.alpha."),
and isolating the IgG from the animals, as described, for example in U.S.
Pat. No. 6,333,032, issued to Skurkovich et al. on Dec. 25, 2001, the
teachings of which are incorporated herein by reference in their entirety.
Examples of other suitable antibodies targeting TNF-.alpha. include but are
not limited to those currently known in the clinical and medical arts, such
as, for example, a chimeric human-mouse monoclonal IgG1 antibody directed
against TNF-.alpha. developed by Centocor and known clinically as infliximab
(Remicade.TM.), and a fully human antibody directed against TNF-.alpha.,
developed by Abbott and known clinically as adelimumab (Humira.TM.).
An example of a functional equivalent of an antibody targeting TNF-.alpha.
is anti-TNF-.alpha. antibody fragment labeled CDP 870, from Celltech Group
plc, UK. CDP 870 has been reported in Phase III clinical trials in Europe
for rheumatoid arthritis and Crohn's disease.
An example of a vaccine directed against autologous TNF-.alpha. was reported
by A. M. Waterson et al. in an article with the title "Phase I Study of
TNFalpha AutoVaccine in Patients with Metastatic Cancer", Cancer Immunol.
Immunother. 54(9), pp. 848-57 (2005). The vaccine used consisted of two
recombinant TNF-.alpha. proteins, with specific peptides replaced by foreign
immunodominant T cell epitopes from tetanus toxoid.
Receptors to TNF and their use are described, for instance, in U.S. Pat.
Nos. 5,395,760; 5,945,397; 6,201,105; 6,572,852, to Smith et al. and U.S.
Pat. No. 5,605,690 to Jacobs et al., the teachings of which are incorporated
herein by reference in their entirety.
A specific example of a soluble receptor binding to TNF-.alpha. is a dimeric
fusion protein consisting of the extracellular ligand-binding portion of the
human 75 kilodalton (p75) TNF receptor linked to the Fc portion of human
IgG1. It is known clinically as etenercept (Enbrel.TM.). Innitially
developed by Immunex, Enbrel.TM. can be currently obtained from Amgen.
Antibodies that can be used to target human INF-.gamma. are disclosed, for
instance, in U.S. Pat. No. 6,333,032, issued to Skurkovich et al. on Dec.
To prepare the anti-INF-.gamma. antibody, adult rabbits are immunized with a
purified INF-.gamma. in a fluid medium, including, e.g., Freund's adjuvant.
For an initial period, the rabbits receive several subcutaneous injections,
for instance at day 1, 4, 14, and 43. Once monthly injections are then
administered for the following several months, e.g., six months. The serum
is drawn from the rabbits when the desired titer is reached and is used to
isolate and purify IgG. In addition, polyclonal antibodies targeting
INF-.gamma. can be obtained, for example, by immunizing goats with r-Hu IFN-.gamma.
and isolating the IgG from the animals.
Fontolizumab (HuZAF.TM.) also can be employed. HuZAF.TM. is a humanized
monoclonal antibody that binds to IFN-.gamma.. It is made by Protein Design
Labs and is being investigated in Crohn's disease.
Examples of polypeptides that can be used to inhibit the binding of human
INF-.gamma. to cellular receptors and the biological activity of INF-.gamma.
are described in U.S. Pat. No. 5,451,658, "Antagonists of Human Gamma
Interferon", issued to Seelig on Sep. 19, 1995, the teachings of which are
incorporated herein by reference in their entirety. Some of the antagonists
described bind to specific regions of gamma interferon which are believed to
be involved in interactions between the interferon and its receptors. Other
antibody antagonists are anti-idiotypic antibodies which appear to compete
directly with gamma interferon for binding to the cellular receptors.
An example of a soluble interferon-gamma receptor fragment and techniques
for obtaining it are described in U.S. Pat. No. 5,578,707, "Soluble
Interferon-Gamma Receptor Fragment", issued to Novick et al. on Nov. 26,
1996, the teachings of which are incorporated herein by reference in their
Examples of polyclonal or monoclonal anti-IL-6 antibodies include those
available, for instance, from R&D Systems, Inc. of Minneapolis, Minn., such
as monoclonal and/or polyclonal antibodies targeting human, canine, porcine
or equine IL-6.
Human, feline and other polyclonal and/or monoclonal anti-IL-1.beta.
antibodies are available commercially from R&D Systems, Inc. of Minneapolis,
Minn. A functional equivalent, a PEGylated anti-IL-1.beta. antibody
fragment, labeled CDP 484 (Celltech Group, PLC, UK) has been reported in
preclinical trials for inflammatory disease.
In some cases, antibodies, equivalents thereof, or soluble cytokine
receptors, targeting at least one of IL-10 or IL-13, also can be employed to
treat patients suffering from renal disease and are particularly useful in
treating ESDR patients who are resistant to erythropoietin treatment. In one
aspect of the invention, the treatment includes an antibody targeting human
IL-10 as well as an antibody targeting IL-13. Specific examples include
those available commercially from ProSci Incorporated of San Diego, Calif.
The antibodies, their functional equivalent(s), and/or soluble cytokine
receptors preferably are provided according to an administration route,
schedule and dosage regiment suitable to the patient being treated, the
severity of elevated levels, other underlying conditions, synergistic
effects, the specific formulation, drug bioavailability curves, presence
and/or severity of adverse reactions and other factors known in the medical
and pharmaceutical arts.
The antibodies or functional equivalents described herein can be
administered parenterally or by other routes. Specific examples of suitable
administration routes include but are not limited to intravenous,
intramuscular, subcutaneous, intranasal, ocular, pulmonary, vaginal, rectal,
transdermal, oral and others known in the art.
The entire antibody component can be administered by a single route, using,
for instance a composition such as the one described below. In other aspects
of the invention, different routes are employed to deliver the antibodies or
their functional equivalents. For instance, an antibody to creatinine can be
administered by a route that is different from that used to deliver one or
more of the antibodies targeting cytokines.
In one embodiment the entire antibody component is administered at the same
time. Administration can be daily or can be at higher or lower frequency. In
specific examples the antibody component is administered 1 to 3 times a week
The administration of some of the antibodies, equivalents thereof or soluble
cytokine receptors also can be staggered with respect to that of others. For
instance, a patient can receive a functional equivalent to INF-.gamma. two
or three times a week, and antibodies to creatinine and to urea, both of
which can be administered once daily or simultaneously or at twelve hours
intervals of one another.
In many cases, the antibody component is administered in conjunction with a
dialysis regimen. For a majority of patients with chronic renal failure,
between 9 and 12 h of dialysis is required each week, usually divided into
three equal sessions. However, the dialysis dose must be individualized.
Recently there has been much interest in the possibility that more frequent
dialysis may be associated with improved outcomes in patients with acute or
chronic renal failure.
In a preferred aspect of the invention, administering the antibody component
results in essentially eliminating the need for dialysis. Thus,
administering an antibody component two or three times per week can lead to
either no need of dialysis or making it necessary on an infrequent basis,
such as once every two to four weeks.
In another aspect of the invention, administration of the antibody component
results in a decrease in dialysis frequency. Thus, administering antibody
component once weekly can lead to decreasing dialysis frequency to once or
twice per week.
In a further aspect of the invention, administration of the antibody
component is tailored to increase or maximize the beneficial effects of
dialysis. In one example, a patient undergoing dialysis on a schedule of
three times per week, receives the entire antibody component on a schedule
of once per week.
Without wishing to be bound by a particular mechanism or interpretation of
the invention, it is believed that one of the reasons for continuing to
administer dialysis is to address accumulation of potassium and development
of metabolic acidosis. However, even if dialysis is continued, it is
believed that administering the antibody component, as described herein,
results in a reduction in dialysis frequency.
The methods and compositions described herein preferably provide the
antibodies, their functional equivalents or soluble cytokine receptors in
effective amounts. As used herein, the term "effective amount" refers to an
amount effective at dosages and for periods of time necessary to achieve the
desired therapeutic or prophylactic result. For instance, the desired result
may be to reduce the level of a non-protein nitrogen compound or a cytokine,
e.g., IL-6 by at least 50%. Preferably, the desired result is to reduce the
level by at least 80%. In most preferred cases, the desired result is a
level within a normal range for that compound.
A "therapeutically effective amount" may vary according to factors such as
the stage of the disease, age, sex, and weight of the individual, and the
ability of the antibody to elicit a desired response in the individual.
A "prophylactically effective amount" refers to an amount effective at
dosages and for periods of time necessary to achieve the desired
prophylactic result. Since a prophylactic dose is used in subjects prior to
or at an earlier stage of disease, the prophylactically effective amount
often is less than the therapeutically effective amount.
Synergistic effects can result in lower effective amounts being
administered. Such effects can be obtained by a specific combination of
antibodies targeting one or more compounds selected from urea, creatinine
and cytokines. Synergistic effects also can be observed with the use of
adjuvants and/or other compounds.
Homeopathic approaches also can be employed and the invention can be carried
out using effective amounts that encompass amounts established by
An effective amount of an antibody, functional equivalent thereof or soluble
cytokine receptor is that amount which is effective, upon single or multiple
dose administration to a patient, to bind to, neutralize or inhibit at least
a portion of the compound, e.g., urea, creatinine and/or, cytokine, causing,
directly or indirectly, the high levels observed in the patient suffering
from renal disease. In preferred embodiments, the effective amount is that
amount which is effective, upon single or multiple dose administration to a
patient, to bring non-nitrogen compounds and/or cytokine levels to within a
For example, the effect of administering an effective amount of antibody to
TNF-.alpha., IFN-.gamma., IL-6 and/or IL-1.beta. to a patient suffering from
renal failure results in lowering the levels of these compounds. The
reduction can be quantitatively determined in terms of reduced fluid
activity level of one or more of the elevated cytokines, i.e., TNF-.alpha.,
IFN-.gamma., IL-6 and/or IL-1.beta., or all four. The lowering of the
cytokine activity level may be measured directly in the treated patient, or
the reduction in cytokine activity level may be projected from clinical
studies in which dose regimens useful in achieving such reduction are
An effective amount of the antibody, its functional equivalent or soluble
cytokine receptor can be determined by the use of known techniques and/or by
observing results obtained under analogous circumstances.
In determining dosage, a number of factors are considered, including, but
not limited to: the species of mammal; its size, age, and general health;
the specific disease involved; the degree of or involvement or the severity
of the disease; the response of the individual patient; route of
administration and desired schedule; bioavailability characteristics of the
preparation being administered; the use of concomitant medication; and other
In oral administration, the dose of one antibody or functional equivalent
thereof can be in the range of from about 0.1 to about 10.0 mg/kg patient
body weight per day. For injectable formulations a suitable starting dose
may be around 0.1 mg/kg patient body weight per day. The dose may be
increased or decreased depending on factors such as response to treatment,
patient tolerance and so forth.
The antibodies, their functional equivalents and/or soluble cytokine
receptors can be formulated as a pharmaceutical composition. The
pharmaceutical composition can consist essentially of the antibody component
or can include additional ingredients, as further described below.
A portion of the antibody component includes an antibody or a functional
equivalent thereof to urea, creatinine or both. Another portion of the
antibody component includes an antibody, a functional equivalent thereof or
a soluble cytokine receptor to TNF-.alpha., INF-.gamma., IL-6 and
In a preferred embodiment, the antibody component targets urea, creatinine,
TNF-.alpha., INF-.gamma., IL-6 and IL-1.beta.. Effective amounts are
As an illustrative example, a suitable antibody component can include
antibodies to urea, creatinine, IFN-.gamma. and IL-6, a soluble receptor to
TNF-.alpha., and a functional equivalent of an antibody to IL-1.beta., e.g.,
an antibody fragment. The antibody component can further target at least one
of IL-10 and IL-13.
The pharmaceutical composition that consists essentially of or that includes
the antibody component can be prepared as a solution, can be lyophilized, or
dried. The composition can be provided in gels, solutions, suspensions,
emulsions, microemulsions, high viscosity fluids, syrups, chewing gum,
nanoparticles, powders, caplets, tablets, suppositories, enemas, injections,
infusions, inhalation formulations, e.g., aerosols or powders, and so forth.
The pharmaceutical composition can include additives, for example carriers,
excipients, adjuvants, stabilizers and others. Physiologically acceptable
compounds are preferred. The additives can be a solid, semi-solid, or liquid
materials which can serve as a vehicle or medium for the active ingredient(s).
Examples of suitable carriers, excipients, or stabilizers include but are
not limited to buffers such as phosphate, citrate, histidine and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium chloride;
phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides; proteins, such
as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine,
histidine, arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating agents such
as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein
complexes); and/or non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
Other materials that can be employed include, for instance, binders such as
microcrystalline cellulose, starch paste, gum tragacanth or gelatin;
excipients such as starch or lactose, disintegrating agents such as alginic
acid, corn starch and the like; lubricants such as magnesium stearate;
glidants such as colloidal silicon dioxide; sweetening agents such as
sucrose or saccharin may be added, or a flavoring agent such as peppermint,
methyl salicylate or orange flavoring, of the types usually used in the
manufacture of medical preparations; coatings such as sugar, shellac, or
other enteric coating agents; dyes and coloring agents; diluents, sterile
solutions or normal saline; and other materials.
The proportion and nature of carriers, excipients, adjuvants, stabilizers,
etc. can be determined by the solubility and chemical properties of the
compound selected, the chosen route of administration, and standard
The pharmaceutical composition can have sustained-release properties.
Suitable examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody component,
which matrices are in the form of shaped articles, e.g., films, patches,
microcapsules and so forth.
Examples of sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and
.gamma.-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable
lactic acid-glycolic acid copolymers such as the LUPRON DEPOT.TM. (injectable
microspheres composed of lactic acid-glycolic acid copolymer and leuprolide
acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release of
molecules for over 100 days, certain hydrogels release active ingredients
for shorter time periods.
When encapsulated antibodies remain in the body for a long time, they may
denature or aggregate as a result of exposure to moisture at 37.degree. C.
Rational strategies can be devised for stabilization depending on the
mechanism involved. For example, if the aggregation mechanism is discovered
to be intermolecular S--S bond formation through thio-disulfide interchange,
stabilization may be achieved by modifying sulfhydryl residues, lyophilizing
from acidic solutions, controlling moisture content, using appropriate
additives, and developing specific polymer matrix compositions.
Optionally, the pharmaceutical composition can include other active
ingredients such as erythropoietin, vitamins, pain killers, antibiotics,
antiviral and other drugs, steroids, potassium blockers, anti-acidosis
compounds, and so forth.
In a specific example, the antibody component is added to a formulation
suitable for supplying subcutaneous fluids to a veterinary subject such as a
cat or dog suffering from chronic renal failure. In other examples, the
pharmaceutical composition includes a drug that reduces a cytokine level,
such as, for instance, pentoxyfilline.
Preferably, the antibody component is present in the composition in an
amount in the range of from about 10 to about 99% by weight of the
pharmaceutical composition. The amount may depend upon the particular type
of formulation and preferably is such that a suitable dosage will be
obtained. The amount of antibody component present in a composition, e.g., a
caplet, tablet, powder, gel or solution can be determined by a suitable
assay such as ELISA or by other techniques.
The pharmaceutical composition can be prepared by combining antibodies,
functional equivalents of antibodies and/or soluble cytokine receptors in
one formulation. The process is not limited to any particular order of
adding ingredients. One or more ingredients can be added simultaneously and
sequential additions also can be carried out. Laboratory, pilot plant and
commercial operations can be employed. Mixing, spray drying, emulsifying,
purifying, compounding, and many other additional steps known in the fields
of drug synthesis and manufacture also can be used to produce the
One or more of the antibodies discussed herein, equivalents thereof, or
soluble cytokine receptors can be provided in a kit. In one example, the
entire antibody component is present in the kit in one composition, e.g., as
gel caplets. The antibody component can be present in a pharmaceutical
composition and in amounts such as described above. The kit can include the
antibody component to be administered, arranged according to an
administration schedule. Thus tablets or caplets can be provided in blisters
or pouches arranged on one or more sheets, with rows and columns labeled to
facilitate tracking a desired administration schedule.
In other examples, the kit includes separate antibodies, functional
equivalents thereof, and/or soluble cytokine receptors to at least some of
urea, creatinine, TNF-.alpha., INF-.gamma., IL-6, IL-1.beta., L-10 and
IL-13. Color coding and/or text can be employed to distinguish antibodies
specific to each of the non-protein nitrogen compound(s) and/or cytokine(s)
The kit can further comprise an applicator, for example, a pipette, a
syringe, a dropper, a spray, an inhaler, a nebulizer, enema equipment and
others known in the art.
Claim 1 of 11 Claims
1. A method for treating renal failure in
a patient suffering with advanced stage renal disease, the method
comprising: a. selecting a patient suffering from renal failure in
advanced stage renal disease, wherein said renal failure is associated
with elevated levels of one or more compounds selected from the group
consisting of urea and creatinine and one or more cytokines selected from
the group consisting of tumor necrosis factor alpha, interferon gamma,
interleukin 6 and interleukin 1 beta; and b. administering to the patient
antibodies to the one or more of said compounds associated with said renal
failure and antibodies or soluble cytokine receptors to the one or more of
said cytokines associated with said renal failure thereby reducing levels
of the selected one or more compounds and the selected one or more
cytokines in the patient.
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