Note: Descriptions are shown in the official language in which they were submitted.
CA 02626680 2008-04-23
Peritoneal dialysis composition and method usable during and after
peritonitis.
Field of the invention
The invention generally relates to peritone-
al dialysis solutions, especially those used in the
practice of continuous ambulatory peritoneal dialysis,
or CAPD.
Background of the Invention
Peritoneal dialysis periodically infuses
sterile aqueous solution into the peritoneal cavity.
Diffusion exchange takes place between the solution
and the bloodstream across the natural body m.embranes.'
The diffusion removes the waste products that.the kid-
neys normally excrete. The waste products typically
consist of solutes like sodium and chloride-ions, and
the other compounds normally excreted through the kid-
neys like as urea, creatinine, and water. The diffu-
sion of water across the peritoneal membrane during
dialysis is called ultrafiltration.
The inflammation of the peritoneum, called
CA 02626680 2008-04-23
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peritonitis, is an undesired complication of
peritoneal dialysis. The inflammation may lead to
loss of mesothelial cells and the excessive growth of
fibrous connective tissue in the peritoneum membrane,
called fibrosis. These reactions can lead to the loss
of ultrafiltration during dialysis.
In addition, peritonitis may lead to in-
creased protein loss in the patient, with the patient
not feeling well enough to eat to replace this loss.
To make up for the reduction in normal
ultrafiltration rates, peritoneal dialysis patients
experiencing peritonitis often receive hypertonic
dialysis solutions, typically containing glucose as_an
osmotic solute. However, the use of hypertonic solu-
tions for these purposes may be counterproductive.
Due to their low pH, high osmolarity, and the presence
of glucose, the solutions may inhibit the necessary
regeneration of mesothelial cells. They also may lead
to the growth of fibroblasts, causing fibrosis.
To enhance the patient's anabolic state and
replace protein loss experienced during peritonitis,.
conventional dialysis solutions also may include mix-
tures of nutritionally essential amino acids (like
methionine, tryptophan, and isoleucine) and nutrition-
ally non-essential amino acids (like glycine and ala-
nine). However, the presence of these amino acids may
be counterproductive, too. Many of these amino acids
can inhibit the proliferation of inesothelial cells.
Therefore, there is a need for peritoneal
dialysis solutions that can be used during and immedi-
ately after peritonitis without potentially
counterproductive effects. The solutions would
promote the replacement of inesothelial cells, minimize
the formation of fibroblasts, and counter the atten-
dant loss of ultrafiltration that peritonitis often
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causes.
Summary of the Invention
The invention provides improved peritoneal
dialysis solutions that can be used during and after
episodes of peritonitis to protect the patient against
the inflammatory reactions of peritonitis,. fibrosis,
and the loss of ultrafiltratibn. The invention also
provides improved peritoneal dialysis solutions that
can be used after episodes of peritonitis to at least
partially restore ultrafiltration characteristics lost
due to peritonitis.
One aspect of the invention provides perito-
neal dialysis solutions that maintain a positive ni-
trogen balance during peritonitis without significant-
ly inhibiting the proliferation of inesothelial cells.
This aspect of the invention replaces at least some
individual amino acids in the dialysis solution with
amino acids in their dipeptide form.
The inventors have discovered that certain
amino acids stunt the proliferation of inesothelial
cells. By using these amino acids in their dipeptide
form instead, significant improvements in the
proliferation of mesothelial cells occur. In a
preferred embodiment, at least some amino acids like
methionine, tryptophan, or isoleucine appear in their
dipeptide form (for example, glycine-tryptophan) to
achieve this beneficial effect.
The inclusion in a peritoneal dialysis solu-
tion of amino acids in dipeptide form with other es-
sential and non-essential amino acids enhances the
anabolic state of the patient suffering peritonitis.
In addition, the solution does not unduly inhibit the
regeneration of mesothelial cells that is necessary to
the patient's healing process.
Another aspect of the invention supplements
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peritoneal dialysis solution with compounds that act
as -scavengers of free radicals present within the
peritoneal cavity. The inventors have discovered that
the free radicals released by peritoneal cells during
peritonitis can injure mesothelial and endothelial
cells and may otherwise case disfunction of the peri-
toneal membrane. The presence in a peritoneal dialy-
sis solution of compounds that scavenge these free
radicals decreases the injury that.the peritoneum
might otherwise suffer during peritonitis. In a pre-
ferred embodiment, the scavengers are vitamin E,
procysteine, superoxide dismutase, or chondroitin sul-
fate.
The inventors have also discovered that use
of a dialysis solution containing chondroitin sulfate
also beneficially changes the permeability of the
peritoneal membrane during subsequent dialysis using
conventional solutions. Chondroitin sulfate enharices
the subsequent ultrafiltration characteristics of the
peritoneal membrane using conventional dialysis solu-
tion. It also decreases the absorption of glucose and
transperitoneal loss of proteins with no change in
urea diffusion. Chondroitin sulfate therefore serves
not only as a free radical scavenger to minimize cel-
lular injury caused by inflammation during peritoni-
tis, but it can be used after an episode of peritoni-
tis to at least partially restore loss of ultrafiltra-
tion characteristics caused by peritonitis.
Another aspect of the invention includes the
degradation products of hyaluronic acid as an additive
to a peritoneal dialysis solution to enhance the re-
generation of the peritoneal mesothelium without fi-
brosis. The inventors believe that these degradation
products, principally oligosaccarides, will increase
the proliferation of endothelial cells without affect-
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ing fibroblasts growth.
Used alone or in combination, these additive
compounds make possible the formulation of peritoneal
dialysis solutions specifically tailored for use during
and immediately after the development of peritonitis.
These additive compounds, used alone or in
combination in peritoneal dialysis solutions, can enhance
the regeneration of mesothelial cells and prevent the
growth of fibroblasts. They can improve the nutritional
status of the patient during peritonitis. They can
actively decrease the degree of damage occurring during
inflammation of the peritoneal membrane. They can restore
the peritoneal membrane to its pro-peritonitis condition.
In accordance with an aspect of the present
invention, there is provided a peritoneal dialysis
solution comprising:
physiological salts in concentrations
sufficient to affect the removal of solutes by diffusion
from the patient's blood across the peritoneal membrane
into the solution, and
a mixture of amino acids sufficient to
contribute to protein synthesis and a positive nitrogen
balance, wherein tryptophan is present in a glycine-
tryptophan form.
In accordance with another aspect of the
present invention, there is provided a peritoneal
dialysis solution for use during an episode of
peritonitis comprising:
physiological salts in concentrations
sufficient to affect the removal of solutes by diffusion
from the patient's blood across the peritoneal membrane
into the solution, a mixture of amino acids sufficient to
maintain a positive nitrogen balance, at least one of the
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amino acids being present in a dipeptide form, and
at least one additive compound selected from
the group consisting of:
at least one compound to scavenge free radicals
generated by activated peritoneal macrophages during
peritonitis, and
the degradation products of hyaluronic acid to
enhance the regeneration of the peritoneal mesothelium
without fibrosis.
In accordance with another aspect of the
present invention, there is provided a use, as a
peritoneal dialysis agent, of a solution, comprising
physiological salts concentrations sufficient to effect
the removal of solutes by diffusion from the patient's
blood across the peritoneal membrane into solution, and a
mixture of amino acids sufficient to contribute to
maintain a positive nitrogen balance, at least one of the
amino acids being present in a dipeptide form, in
combination with at least one additional additive
selected from the group consisting of:
at least one compound that scavenges free
radicals generated by peritoneal macrophages activated by
peritonitis,
chondroitin sulfate in a concentration
sufficient to change the permeability of the peritoneal
membrane during subsequent dialysis using solutions free
of chondroitin sulfate, and
the degradation products of hyaluronic acid to
enhance the regeneration of the peritoneal mesothelium
without fibrosis.
In accordance with another aspect of the
present invention, there is provided a use of a solution,
comprising physiological salts in concentrations
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sufficient to effect the removal of solutes by diffusion
from the patient's blood across the peritoneal membrane
into the solution, as a peritoneal dialysis agent in
combination with a mixture of amino acids sufficient to
maintain a positive nitrogen balance, wherein tryptophan
is present in a glycine-tryptophan form.
The many features and the advantages of the
invention will become even more apparent after reading
the following detailed description, associated drawings,
and claims.
Brief Description of the Drawings
Fig. 1 is a chart showing the mean value in 86 Rb
uptake by human mesothelial cells (HMC) through different
pathways after being cultured for 7 days in medium with
high concentrations (90 mM) of glucose, glycerol, and
mannitol, expressed as a % of control where the HMC were
cultured in normotonic medium; and
Fig. 2 is a graph showing the accumulation of
86Rb during 72 hours in HMC exposed to different high
glucose concentrations, expressed as a % of control where
the HMC were cultured in normotonic medium.
Description of the Preferred Embodiments
After an episode of peritonitis, the CAPD
patient typically receives a hypertonic peritoneal
dialysis solution containing glucose. The intent is to
counteract the loss of ultrafiltration that fre-
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quently occurs during peritonitis.
However, hypertonic peritoneal dialysis so-
lutions with glucose may actually interfere with the
regeneration of inesothelial' cells and thereby inter-
fere with the patient's recovery from the inflammatory
effects of peritonitis. Such solutions also may en-
courage the growth fibroblasts and could contribute to
peritoneal fibrosis.
The inventors have experimentally determined
that potassium (measured with its analog 86Rb) enters
human mesothelial cells (HMC) through three different
pathways:
(1) through an active channel that the glu-
coside ouabain blocks in a dose dependent way, which
corresponds to the activity of the Na-K-ATPase pump in
plasmalemma;
(2) through another active channel that the
diuretic drug furosemide blocks in a dose dependent
way, but that is not blocked by ouabain; and
(3) through a passive channel that neither
ouabain or furosemide block.
The inventors have also experimentally
determined that about 60% of 86Rb transport occurs
through active Channel (1), the Na-K-ATPase pump;
about 29% through active Channel (2); and about 11%
through passive Channel (3).
As the following Example demonstrates, expo-
sure of HMC to hyperosmolal medium modifies the trans-
port of 86Rb into the cells through all three pathways.
EXAMPLE 1
This study evaluated the mechanisms regulat-
ing transport of potassium from the extracellular
space into HMC in in vitro culture.
HMC were isolated from omentum following the
method described in Van Bronswwijk et al., "Cytotoxic
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Effects of Commercial Continuous Ambulatory Periton.eal-
Dialysis (CAPD) Fluids and of Bacterial Exoproducts on
Human Mesothelial Cells in Vitro," Perit Dial Inte n,
1989 (9): 197-202.
The HMC were seeded 'into 75. cm2 culture
flasks and grown to confluency. Then, the HMC were
harvested with trypsin-EDTA solution and seeded into
96-well culture plates and there again grown to
confluency. The study used the confluent mesothelial
monolayers cultured in the 96-well plates.
HMC were incubated in the culture medium for
7 days with various osmotic solutions (90 mM and more
of glucose or glycerol or mannitol). After incuba-
tion, potassium analog 86Rb was added to the medium.
The uptake of 86Rb by HMC was measured and compared
with the uptake in control HMC not.exposed to osmotic
solutes (the control HMC having been cultured in a
normotonic medium).
As Fig. 1 shows, transport through the pas-
sive Channel (3) increases in HMC exposed chronically
(over 7 days) to high concentration of all the osmotic
solutes (90 mM). Mannitol also stimulated active
transport through Channel (1), but glucose and glycer-
ol both decreased Channel (1) transport. All solutes
decreased active transport through Channel (2) as
well.
As Fig. 2 shows, the intracellular accumula-
tion of 86Rb in HMC exposed for 72 hours to increased
concentrations of glucose diminished proportionally to
the glucose concentration, compared to the accumul.a-
tion in the control HMC.
The study demonstrates that chronic exposure
of HMC to high glucose concentration (90 mM) decreases
the activity of the Na-K-ATPase pump (i.e., Channel
(1)), which is the main pump responsible for
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intracellular potassium accumulation. The activity of
Channel (2) also decreases as a result to exposure of
HMC to high glucose concentration.
In effect, this decreased capacity of HMC to
take up potassium results in diminished accumulation
of potassium in HMC. This may, in turn, cause cellu-
lar disfunction like those associated with diabetic
disorders resulting from reduced Na-K-ATPase activity.
See Greene et al., "Sorbitol, Phosphoinodsitides and
Sodium - Potassium - ATPase in the Pathogenesis of
Diabetic Complications," N En J Med 1987; 316: 599-
606; and Yorek et al., "The Effect of Increased Glu-
cose Levels on Na-K Pump Activity in Cultured Neuro-
blastoma Cells," J Neurochem 1988; 51:605-610. HMC
potassium depletion also may produce severe metabolic
abnormalities such as deranged protein synthesis. See
Lubin, "Intracellular Potassium and Control of Protein
Synthesis," Fed Proc 1964; 23: 994-997.
High concentration of glycerol, but not man-
nitol, produces the same effect as glucose. This sug-
gests that the decrease in Na-K-ATPase pump activity
depends upon the metabolism of the osmotic solute in-
side the cell, since both mannitol influx and metabo-
lism in HMC are probably small.
The study also shows that chronic exposure
of HMC to all osmotic solutes increases the passive
permeability (via Channel (3) ) of the plasmalemma to
86Rb. This may be due to a "washout" of structural
components of the plasmalemma, causing increased leak-
age and loss of intracellular metabolic substrates.
This, too, can lead to cellular disfunction.
Thus, increased extracellular tonicity due
to high glucose concentration may cause HMC potassium
loss both through diminished active influx, mostly by
reduced Na-K-ATPase activity, and through an outflux
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of ion-rich water ("wash-out") from the cells due to
a negative osmotic gradient. Also see Moreno et al.,
"Increase in Serum Potassium Resulting from- the Admin-
istration of Hypertonic Mannitol and Other Solutions,"
J Lab Clin Med 1969; 73: 291-294.
The presence of these disfunctions does not
promote the regeneration of mesothelial cells
necessary to the healing process during and after a
peritonitis episode.
The peritoneal dialysis solutions that em-
body the features of the invention are specially for-
mulated for patients for use during and immediately
after episodes of peritonitis. The solutions promote:
the healing process to avoid or at least minimize the
injury and adverse physiological effects of peritoni-
tis upon the dialysis regime of the patient.
Like conventional peritoneal dialysis solu-
tions, the solutions that embody the features of the
invention include:
(1) physiological salts such as sodium
chloride, calcium chloride and sodium acetate in ap-
propriate concentrations to maintain a normal electro-
lyte profile. Typical concentrations are from 116 to
140 mEq/liter of sodium; 0 to 6 mEq/liter of calcium,
and 100 to 144 mEq/liter of chloride.
(2) lactate or bicarbonate in appropri-
ate concentrations to maintain a physiologically tol-
erable pH of between about 5 to about 7.4. Typical
concentrations are from 30 to 45 mEq/liter of lactate;
and
(3) glycerol or glucose polymers at a
concentration (at least 0.5 percent by weight) suffi-
cient to generate the necessary osmotic pressure to
remove water from the patient through ultraf iltration.
According to the invention, the solutions
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contain one or more of the following additives:
(4) a mixture of essential and non-es-
sential amino acids to serve as a source of supplemen-
tal nitrogen for the support of protein synthesis for
the patient and to counterbalance the protein that the
patient loses because of peritonitis. According to
this aspect of the invention, at least some of these
amino acids are present in their dipeptide form to
promote the proliferation of inesothe.lial cells lost
during peritonitis.
(5) a compound that scavenges free
radicals produced by peritoneal cells that causes
peroxidation of the-peritoneum;
(6) chondroitin sulphate to restore at
least a portion of transperitoneal transport lost due
to peritonitis;
(7) compounds consisting of the degra-
dation products of hyaluronic acid to enhance the re-
generation of the peritoneal mesothelium without fi-
brosis.
The following sections describe the benefits
associated with each Additive (4) to (7).
AMINO ACID ADDITIVE (4)
A preferred embodiment of the amino acid
additive (4) comprises, based on one liter of
solution, about 0.1 to 10 mM each of the nutritionally
essential amino acids methionine, tryptophan,
isoleucine, valine, leucine, lysine, histidine, threo-
nine, and phenylalanine, at least some of which are
present in their dipeptide form. When present as
dipeptides, these amino acids do not inhibit
mesothelial cell proliferation as much as they do when
present as individual amino acids.
The most preferred embodiment includes at
least tryptophan in its dipeptide form (glycine-tryp-
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tophan, or gly-trp), as the individual amino 'acid
tryptophan inhibits mesothelia-l cell proliferation
more than an other individual amino acid.
The mixture also includes about 0.1 to 10 mM
each of arginine, alanine, proline, glycine, serine,
tyrosine, cysteine (cystine), and other individual,
nutritionally non-essential amino acids as required to
maintain a positive nitrogen balance in.the patient.
The following Example illustrates the
benefits of using amino acids in a dialysis solution,
of which certain are in their dipeptide.form.
EXAMPLE 2
This study evaluated the toxicity of.a mix-
ture of essential and non-essential amino acids on the
proliferation of HMC in conditions simulating perito-
neal dialysis.
HMC prepared as described in Example 1 were
exposed to essential and nonessential amino acids.
Adverse effects were measured in terms of the impact
upon cell proliferation (as measured by incorporation
of 3H-thymidine) and the release of LDH from cell cy-.
toplasm.
All the amino acids evaluated inhibited the
proliferation of HMC when present. Tryptophan exhib-
ited the most inhibition effect.
When HMC are exposed to tryptophan in a con-
centration of 5 mM for.24 hours, their proliferation
is reduced by 82%, compared to the proliferation of
control HMC cells not exposed to tryptophan. After 24
hours of exposure, tryptophan (5 mM) also increased
the leakage of LDH from the mesothelial monolayer HM.C
by 740%, compared to the control HMC cells.
In contrast, after 24 hours of exposure to
dipeptide tryptophan (gly-trp) in concentration of 5
mM, proliferation of HMC decreased by only 30% and LDH
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release increased by only 180%, compared to the
control-HMC cells.
In another experiment, growing HMC were ex-
posed to two mixtures each having a high concentration
of amino acids (1.1%). One amino acid mixture con-
tained tryptophan. The other amino acid mixture con-=
tained gly-trp instead of tryptophan. The concent-
ration of both amino acid mixtures was progressively
decreased by dilution down to 0.22% in 6 hours. The
HMC were incubated for 18 additional hours in media
with the low (0.22%) concentration.
The mixture of amino acids containing tryp-
tophan reduced 3H-thymidine incorporation by 30%, com-
pared to the control HMC not exposed to any amino acid
mixture. The mixture of amino acids_in which the gly-
trp replaced the tryptophan reduced 3H-thymidine in-
corporation by only 17%. The inclusion of the dipep-
tide form of the amino acid in the mixture reduced the
undesired effect by about 50%.
FREE RADICALS SCAVENGER ADDITIVE (5)
Peritonitis activates peritoneal macrophages
(as proved by others). The activation of the
macrophages leads to the increased generation of free
radicals. The inventors believe that the increased
generation of free radicals causes peritoneal
peroxidation.
According to this aspect of the invention,
the use of compounds that scavenge free radicals in
peritoneal dialysis solutions minimizes or alleviates
peritoneal peroxidation during episodes of
peritonitis.
The inventors have also shown that the
increased presence of free radicals also injures meso-
thelial cells in the peritoneum. The free radicals
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probably also injure endothelial cells, too. The free
radicals can depolymerize hyaluronic acid and/or col-
lagen in interstitium, causing disfunction of the
peritoneal membrane.
According to this aspect of the invention,
these undesired effects of' peritonitis also can be
minimized or lessened by supplementing the dialysis
solution with free radical scavengers.
EXAMPLE 3
In one experiment, exposure to normal saline
in the peritoneal cavities of rats for over 6 days
increased peroxidation of the peritoneal membrane, as
measured by the concentration of malondialdehyde in
the animal's omentum: 8.12+/-0.51 uM/100 ug tissue
(n=7) in controls not infused with saline, compared to
11.36+/-1.07 uM/100 ug tissue (n=12) in rats infused
with saline. In another experiment, one group of rats
(n=22) was infused over 6 days with :saline sup-
plemented with vitamin E(0.1 g%). Another control
group of rats (n=18) was infused over 6 days with sa-
line alone. In rats infused with the vitamin E-sup-
plemented saline, the concentration of malondialdehyde
in the omentum (and therefore the severity of perito-
neal peroxidation) was lower (4.53+/-0.30 uM/100 ug
tissue) than in the- rats infused with saline alone
(9.38+/-0.90 uM/100 ug tissue).
In other in vitro experiments, free radicals
generated by an xanthine-xanthine oxidase system were
observed to injure mesothelial cells. The injury was
prevented by using vitamin E(0.1 o to 1.0 %) and
chondroitin sulphate (0.1 %).
In another experiment, the xanthine-xanthine
oxidase system was added to dialysis solution (2.5%
dextrose). The solution was infused into the
peritoneal cavities of rats. The increased presence
CA 02626680 2008-04-23
14
of the free radicals generated by the infused oxidase
system caused loss of ultrafiltration and increased
glucose absorption, the same physiological effects
observed during episodes of peritonitis. This result
further links the increased presence of free radicals to
the inflammatory effects and injury of peritonitis.
The addition of free radical scavenger vitamin E (0.01%)
reduced or totally reversed the adverse effects caused by
the free radicals generated by the xanthine-xanthine
oxidase system. The free radical scavenger would have the
same beneficial effect in the increased presence of the
free radicals during peritonitis.
In a preferred embodiment, the scavengers are
selected from the group consisting of vitamin E,
procysteine, superoxide dismutase, and chondroitiri
sulfate and are present in concentrations of about 0.01
to 0.5 g%.
TRANSPORT RESTORATION ADDITIVE (6)
Peritonitis can adversely alter peritoneal
transport, leading to a reduction of ultrafiltration.
According to this aspect of the invention, the peritoneal
dialysis solution includes chondroitin sulphate to change
or restore transperitoneal transport after an episode of
peritonitis.
EXAMPLE 4
Saline supplemented with chondroitin sulphate
(0.2. g%) was infused into the peritoneal cavity of rats
over a period of six days. Then, conventional 2.5%
Dianeal peritoneal dialysis solution (sold by Baxter
Healthcare Corporation, Deerfield, Illinois) was infused
into the peritoneal cavities of the rats.
The chronic exposure to the chondroitin sul-
phate modified the permeability of the peritoneal mem-
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brane during the subsequent dialysis with conventional
dialysis solution. The net ultrafiltration measured
after a dwell period of four hours was more than the
ultrafiltration measured before exposure to the
chondroitin sulphate. Also, absorption of glucose
from the dialysate *and transperitoneal loss of pro-
teins decreased, with no change in urea diffusion,
when compared to these same transport parameters mea-
sured before chronic exposure to the chondroitin sul-
phate.
This Example illustrates the benefits of,
using chondroitin sulphate in a dialysis solution to
restore transperitoneal transport after an episode of
peritonitis.
In a preferred embodiment, the chondroitin
sulphate is present in a concentration of about 0.01
to 0.5 g%.
REGENERATION ADDITIVE (7)
Wound healing during fetal life is charac-
terized by healing without fibrosis or scar formation.
It is believed that this healing process is at least
partly mediated by the high concentration of hyaluron-
ic acid in a fetal wound extramural matrix. By
increasing the concentrations of hyaluronic acid in
the extracellular fluids of adults, the healing of
wounds without fibrosis is enhanced.
Other studies have shown that the hyaluronic
acid is not responsible by itself. It is believed
that its degradation products (oligosaccharides) that
are the active agents in promoting fibrosis-free wound
healing. In in vitro experiments, oligosaccharides
products of the degradation of hyaluronic acid in-
crease the proliferation of endothelial cells without
effect on fibroblasts growth.
According to this aspect of the invention,
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peritoneal dialysis solution includes degradation
products of hyaluronic acid to enhance the
regeneration of the peritoneal mesothelium without
fibrosis.
The dialysis solutions containing one or
more of the additives (4) to (7), when sterile, may be
used as the peritoneal dialysis solution in a conven-
tional CAPD procedure, using the techniques and equip-
ment developed and sold by the Baxter Healthcare Cor-
poration, Deerfield, Illinois.
The above description and Examples are for
illustrative purposes only. They are not intended to
limit the scope of the inventions, as defined in the
following claims.
